Patent Application
20220380739
The invention relates generally to the field of molecular biology, biotechnology or bioprocess engineering for the production and use of a modified caspase-2, specifically a circularly permuted caspase-2. The invention further relates to the production and isolation of recombinant protein constructs, specifically using modified caspase-2 for the maturation of recombinant fusion proteins or polypeptides comprising a caspase recognition site.
Despite all the recent advances in biotechnology the production of proteins is still challenging due to their diverse characteristics. Protocols usually have to be optimized for every protein, which is especially problematic for large scale production.
The diverse characteristics of proteins makes their purification challenging and impede a general protocol. This is why proteins are often fused to tags with special binding properties. Already the first human proteins recombinantly expressed in E. coli, somatostatin [1] and insulin [2], were fusion proteins. Even today protein tags are still widely used in recombinant protein production, not only to facilitate purification and detection but also to enhance expression and solubility. Popular tags to stabilize expression and increase solubility are for example GST (Glutathione S-transferase), MBP (Maltose-binding protein), SUMO (Small ubiquitin-related modifier), or DsbA (Protein disulfide isomerase I). Tags for affinity purification include among others His, HA (hemaglutinin antigen), Strep II, and FLAG tag.
However, as versatile and useful tags are, as difficult can be their removal. For many—especially medical—applications a tag-free protein is essential. Tags can influence the structure and characteristics of proteins and therefore also alter the response to immunogens or trigger an immune reaction themselves [3]. Especially for biopharmaceutical applications a protease which efficiently cleaves tags from the product is essential [4].
A variety of proteases for tag removal are available. They all cleave at defined recognition sequences which are inserted between the tag and the protein of interest. Usually the protease has a tag itself and is subsequently removed in a second purification step. The most commonly used are endopeptidases like factor Xa, thrombin, TEV (tobacco etch virus protease), and enterokinase. However, all of these proteases have one or several downsides: they cleave inefficiently or unspecifically, do not accept all residues in the P1′ position, leave overhang residues at the N-terminus, or they need special buffer conditions that are not favorable to the target protein. Another major drawback for industrial application is the high cost of these proteases [5].
Despite the fact, that caspases have been studied more intensively than other protease classes, and have a quite high specificity, they have hardly ever been considered for biotechnological purposes like tag cleavage.
Caspase is the acronym for cysteinyl aspartate-specific protease, a class of proteases that is defined by a conserved catalytic cysteine and their strong preference to cleave their substrates after aspartate residues [6]. The first caspase was described in the early 1990s, since then a total of fifteen have been discovered in mammalia, thirteen of which are found in humans [7]. They are well known for their role in regulated cell death [8] and inflammatory reactions [9]. More recently it has been discovered, that they are also involved in other processes like cell differentiation [10], cell cycle regulation [11], and maybe even cell motility [12]. MacKenzie and Clark investigated the role of dimerization in the ability of caspases to form fully functional proteases and describe that dimerization is necessary for active site formation because both caspase monomers contribute residues that enable the formation of a fully functional active site (MacKenzie and Clark, Adv Exp Med Biol. (2012); 747:55-73).
In recent years, reversed caspases, where the small subunit of the caspase is N-terminal to the large subunit of the caspase have been developed (U.S. Pat. No. 6,379,950).
Srinivasa et al. for example describe recombinant caspases 3 and 6 precursors, which are constitutively active and have their small subunit preceding their large subunit (Srinivasa et al. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology (1998), 273(17):10107-10111).
Circular Permutation may provide potential benefits by reorganizing the polypeptide chain of a protein, however, by connecting the native protein termini via a covalent linker and introducing new ends through the cleavage of an existing peptide bond, circular permutation can also perturb local tertiary structure and protein dynamics, as well as introduce possible quaternary structure changes and problems (Yu and Lutz, Trends in Biotechnology (2011), 29(1):18-25).
WO2009/044988A1 describes a method of producing a caspase, using a recombinant caspase expression vector capable of being over-expressed without cytotoxicity because the auto-activation recognition site of caspase is replaced with a non-cysteine protease recognition site to nullify the auto-activation activity during the mass-expression in E. coli.
Three systems employing caspases for tag removal have been published, which are difficult to compare because different fusion proteins, buffers, substrate to enzyme ratios, and incubation temperatures were used.
Caspase-3 and an engineered caspase-3 with uncleavable propeptide (but wild-type order of subunits) have been used to cleave GST tags from fusion proteins. The modified caspase was able to achieve complete tag cleavage at 25° C. in about three hours (molar caspase to substrate ratio 1:80, mass ratio 1:100) [13]. In another system which also uses caspase-3 to cleave GST tags (caspase to substrate mass ratio 1:200) processing was complete to over 90% in 45 min, but incubation was at 30° C. [14].
A system with caspase-6 has also been published, it is more effective and manages complete cleavage of substrates in about thirty minutes (molar caspase to substrate ratio 1:500) [15].
Even though these caspase-based tag cleaving systems have been published more than ten years ago, they have not been adopted into the common repertoire of protein purifications. Use of the caspase-3 based system has only been published once, for the expression of interleukins [16]. The caspase-6 system also has been used by merely two other groups, both from the Indian Institute of Immunology, for the purification of Mycobacterium tuberculosis [17] and Helicobacter pylori proteins [18].
EP1597369B1, for example, discloses a method of protein production using a fusion protein, comprising a protein of interest and a protease recognition site, wherein a protease, such as a caspase, is used to cleave the fusion protein at the recognition site.
U.S. Pat. No. 7,604,980B2 also uses a fusion protein comprising a protein of interest and a caspase recognition site to produce a protein of interest. In this disclosure, caspase-6 is preferably used to cleave the fusion protein.
A main reason why caspases have not become more popular in biotechnology might be the challenges during their recombinant production. Native caspases are synthesized as inactive zymogens, thus to obtain an active enzyme there are two main possibilities. The subunits can be expressed separately and then mixed after purification, which makes their production very complex [19]. Or, the procaspase is expressed which causes autocatalytic activation. This process, however, is often not complete [20], therefore enzyme activity can vary between batches [21]. Furthermore, as caspases are active in E. coli they can also cleave bacterial proteins [22] and negatively influence growth and yield. In addition, the substrate specificities of both caspase-3 [23] and caspase-6 [24] have been described as rather promiscuous. They are very likely to cleave fusion proteins at undesired sites.
Therefore, there is an urgent need for an industrially applicable platform technology which enables efficient and specific tag removal to improve purification of recombinant protein products.
In the production of recombinant proteins, processing of fusion proteins with state-of-the-art enzymes to remove tags often generates a non-authentic N- or C-terminus since these enzymes lack specificity. Such lack of specificity can also lead to unspecific cleavage or proteolytic degradation of the protein of interest.
It is the objective of the present invention to provide an improved system for the production of recombinant proteins employing a modified caspase-2, specifically a circular permuted caspase-2.
The objective is solved by the present invention.
Specifically provided herein is a modified caspase-2, specifically a circularly permuted caspase-2, with significantly improved P1′ tolerance. The caspase-2 variants provided herein are specifically used in the production of recombinant proteins to generate a protein of interest comprising an authentic N-terminus by target specific cleavage of N-terminal tags.
According to the invention, there is provided a single-chain circular permuted caspase-2 (cp caspase-2) comprising the following structure from N- to C-terminus:
wherein said cp caspase-2 comprises one or more amino acid substitutions increasing P1′ tolerance of said cp caspase-2 compared to a cp caspase-2 without said amino acid substitutions.
Specifically, the cp caspase-2 provided herein is catalytically active, specifically upon dimerization. Specifically, the cp caspase-2 described herein is catalytically active and capable of catalyzing peptide bond cleavage upon dimerization. Specifically, the cp caspase-2 described herein is a single-chain caspase-2 that does not require cleavage by initiator caspases for activation.
Specifically, the caspase-2 or cp caspase-2 provided herein is a functionally active variant of wild-type caspase-2 comprising improved P1′ tolerance. Specifically, said functionally active variant is capable of cleaving a substrate with high efficiency and specificity.
Specifically, provided herein is a single chain caspase-2 comprising the following structure from N- to C-terminus:
Specifically, said variant is of animal origin, specifically of mammalian, reptile or fish origin, more specifically from human, marsupial, iguana or cartilaginous fish, ghost shark or tasman devil origin.
According to a specific embodiment, the cp caspase-2 provided herein comprises one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or any combination thereof. Specifically, a cp caspase-2 comprising one or more of said amino acid substitutions, also referred to as “cp caspase-2 variant”, comprises improved P1′ tolerance compared to a cp caspase-2 not comprising said substitutions. Specifically, the cp caspase-2 variants provided herein comprise improved P1′ tolerance for at least one amino acid other than glycine.
According to a further specific embodiment, the cp caspase-2 provided herein comprises a propeptide of a small caspase-2 subunit (SS propeptide), fused to the N-terminus of the small subunit. Specifically, the SS propeptide comprises one or more amino acid substitutions at the C-terminus of the SS propeptide. Specifically, the SS propeptide of the cp caspase-2 described herein is modified to prevent cleavage at its C-terminus. Specifically, the SS propeptide comprises an amino acid substitution at position Asp14 of SEQ ID No. 2 or at a position functionally equivalent to Asp347 of SEQ ID No. 11, specifically Asp is substituted to Ala.
According to a specific embodiment, the SS propeptide described herein comprises the amino acid sequence of SEQ ID No. 2, wherein X can be any amino acid except D or E, specifically it is A, or a variant thereof having 1, 2, 3, 4, or 5 point mutations or deletions. Specifically, said variant is a functionally active variant. Preferably, the SS propeptide sequence comprises the amino acid sequence of SEQ ID No. 2, wherein X is not D or E.
According to a further specific embodiment, the cp caspase-2 provided herein comprises one or more linker sequences, specifically consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or even more amino acid residues. Specifically, the linker can comprise more than 20 or 30 or even more amino acids, as long as the caspase retains its functional activity as described herein. Specifically, the linker sequence comprises glycine, alanine and/or serine residues. Specifically, the linker comprises at least one glycine and serine residue, more specifically the linker is GS, GSG, GGSGG, GSGSGSGS and/or GSAGSAAGSG.
Specifically, the cp caspase-2 comprises a subunit-linker sequence, which is a linker sequence between the small subunit and the large subunit of the cp caspase-2 described herein.
According to a further specific embodiment, the cp caspase-2 provided herein comprises one or more C-terminal or N-terminal tags, specifically selected from the group consisting of affinity tags, solubility enhancement tags and monitoring tags. Specifically, any tag known in the art can be fused to the cp caspase-2.
Specifically, the affinity tag is selected from the group consisting of poly-histidine tag, poly-arginine tag, peptide substrate for antibodies, chitin binding domain, RNAse S peptide, protein A, ß-galactosidase, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, c-Myc tag, SUMO tag, E. coli thioredoxin, NusA, chitin binding domain CBD, chloramphenicol acetyl transferase CAT, LysRS, ubiquitin, calmodulin, and lambda gpV, specifically the tag is a His tag comprising one or more His, more specifically it is a hexahistidine tag.
Specifically, the solubility enhancement tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC, T3, N1, N2, N3, N4, N5, N6, N7, calmodulin-binding peptide (CBP), poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, DsbA, DsbC and thioredoxin.
Preferably, the solubility enhancement tag is selected from the group consisting of T7A3 tag and T7AC tag.
Specifically, the monitoring tag is selected from the group consisting of m-Cherry, GFP and f-Actin.
According to a specific embodiment, the cp caspase-2 described herein comprises more than one tag sequences, specifically it comprises an affinity tag and a solubility enhancement tag. Specifically, it comprises an affinity tag, a solubility enhancement tag and a monitoring tag. Specifically, it comprises more than one tag of the same functionality, specifically it comprises more than one affinity tag, more than one solubility enhancement tag and/or more than one monitoring tag, and any combination thereof. Specifically, the cp caspase-2 described herein comprises a C-terminal and an N-terminal tag, each comprising one or more tag sequences, preferably selected from affinity tag, solubility enhancement tag and monitoring tag
Specifically, the affinity tag is a hexahistidine tag and the solubility enhancement tag is a T7AC tag.
According to a further specific embodiment, the cp caspase-2 provided herein comprises a tag-linker sequence, which is a linker sequence between two tags or a tag and the small subunit, the large subunit or the SS propeptide of the cp caspase-2. Specifically, the cp caspase-2 provided herein comprises one or more N-terminal tags and optionally one or more tag-linker sequences between the tags or between a tag and the N-terminus of the small subunit or the SS propeptide. Specifically, the cp caspase-2 provided herein comprises one or more C-terminal tags and optionally one or more tag-linker sequences, which are linker sequences between the tags or between a tag and the C-terminus of the large subunit.
According to a further embodiment, herein provided is a functionally active variant of the cp caspase-2 or caspase-2, wherein
According to a specific embodiment, the cp caspase-2 provided herein comprises an N-terminal and/or C-terminal truncation of at least 1, 2, 3, 4, 5 and up to 10 or even more, as long as the caspase retains its functional activity as described herein. According to a further specific embodiment, the cp caspase-2 provided herein comprises an N-terminal and/or C-terminal extension of at least 1, 2, 3, 4, 5 and up to 10 or even more, as long as the caspase retains its functional activity as described herein. Specifically, the cp caspase-2 may comprise a truncation and an extension.
Specifically, the small subunit of the cp caspase-2 described herein comprises the amino acid sequence of SEQ ID No. 3, SEQ ID No. 91, SEQ ID No. 94, SEQ ID No. 97, SEQ ID No. 100, SEQ ID No. 103, SEQ ID No. 106, SEQ ID No. 109, SEQ ID No. 112, SEQ ID No. 115, SEQ ID No. 118 or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity. Specifically, the large subunit of the cp caspase-2 described herein comprises the amino acid sequence of SEQ ID No. 4, SEQ ID No. 90, SEQ ID No. 93, SEQ ID No. 96, SEQ ID No. 99, SEQ ID No. 102, SEQ ID No. 105, SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity.
According to a specific embodiment, the cp caspase-2 variant provided herein comprises one or more amino acid substitutions, selected from
with reference to the positions of SEQ ID No. 6, or positions functionally equivalent to positions of SEQ ID No. 6.
Specifically, selection of alternative amino acid exchanges at a given position with a high potential for resulting in similar effects as in the described selected variants is based on the categorization of all amino acids into distinct, not overlapping groups according to their hydrophobicity attributes: polar (R, K, E, D, Q, N), neutral (G, A, S, T, P, H, Y), hydrophobic (C, V, L, I, M, F, W), as determined by Stapor et al. (Stapor K, et al. Machine Learning Paradigms—Advances in Data Analytics. Tsihrintzis G A, Sotiropoulos D N and Jain L C (eds.), Springer 2019 (ISSN 1868-4394), pp 101-128).
Specifically, the cp caspase-2 provided herein comprises amino acid substitutions at positions of SEQ ID No. 6, or at positions functionally equivalent to positions of SEQ ID No. 6, selected from
wherein said cp caspase-2 has increased P1′ tolerance compared to a cp caspase-2 without the respective amino acid substitution, optionally wherein said cp caspase-2 comprises an SS propeptide comprising an amino acid substitution to A1a at position Asp14 of SEQ ID No. 2 or at a position functionally equivalent to position Asp347 of SEQ ID No. 11.
Specifically, the cp caspase-2 variant ms9 ProD comprising E105V, G171D, V225G and D282E substitutions displays excellent P1′ tolerance.
Specifically, the cp caspase-2 variant E105V G171 D comprising E105V and G171D substitutions displays excellent P1′ tolerance, which is increased compared to the cp caspase-2 variant ms9 ProD. Specifically, the highest tolerance of the cp caspase-2 variant E105V G171 D is for the amino acid residue proline.
According to a specific embodiment, the cp caspase-2 described herein comprises at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to SEQ ID No. 9 (Homo sapiens), SEQ ID No. 64 (Sarcophilus harrisii, Tasmanian Devil), SEQ ID No. 66 (Anolis carolinensisilus), SEQ ID No. 68 (Callorhinchus milii, Ghost Shark), SEQ ID No. 76 or SEQ ID No. 77 (Homo sapiens) and comprises one or more amino acid substitutions at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or any combination thereof.
Specifically, the cp caspase-2 variant described herein comprises SEQ ID No. 6 and one or more amino acid substitutions at position 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or at a position functionally equivalent to position 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6, or any combination thereof.
Specifically, the cp caspase-2 variant described herein comprises any one or more of amino acid substitutions G171 D, E105V, E172V, D282E, D282T, V225G, K83E, H185A, V255M, D285Y and D285E, with reference to the numbering according to SEQ ID No. 6.
According to a further specific embodiment, the cp caspase-2 variant described herein comprises SEQ ID No. 6 or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity with SEQ ID No. 6, and comprises amino acid substitutions E105V, G171 D, V225G, D282E, and/or D285E, with reference to the numbering of SEQ ID No. 6, wherein said cp caspase-2 has increased P1′ tolerance.
According to a further specific embodiment, the cp caspase-2 variant described herein comprises SEQ ID No. 6 or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity with SEQ ID No. 6, and comprises amino acid substitutions K83E, E105V, E172V, V255M and/or D285Y, with reference to the numbering of SEQ ID No. 6, wherein said cp caspase-2 has increased P1′ tolerance.
According to a further specific embodiment, the cp caspase-2 variant described herein comprises SEQ ID No. 6 or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity with SEQ ID No. 6, and comprises amino acid substitutions H185A and/or D282T, with reference to the numbering of SEQ ID No. 6, wherein said cp caspase-2 has increased P1′ tolerance, specifically for branched P1′ amino acid residues.
Specifically, the cp caspase-2 variant described herein comprises an amino acid sequence selected from the group consisting of SEQ ID No. 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with any one of SEQ ID No. 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192.
According to a specific embodiment, the cp caspase-2 described herein comprises a C-terminal tag and an amino acid substitution at positions 285 and 292 of SEQ ID No. 6 or at a position functionally equivalent to positions 285 and 292 of SEQ ID No. 6, specifically comprising substitutions to Glu and Ser, respectively (D285E and D292S).
Specifically, the caspase-2 or cp caspase-2 described herein is recruited by a recognition site for proteolytic cleavage, comprising 5 amino acids of the sequence P5
P4 P3 P2 P1, wherein
P1 can be any amino acid, preferably it is D or E,
P2 can be any amino acid, preferably it is A,
P3 can be any amino acid, preferably it is V,
P4 can be any amino acid, preferably it is D, and
P5 can be any amino acid, preferably it is V.
Specifically, the caspase-2 variant or cp caspase-2 variant described herein has increased specificity, specifically to the recognition site VDVAD wherein P5 is V, P4 is D, P3 is V, P2 is A and P1 is D, compared to wild-type (wt) caspase-2 comprising the amino acid sequence of SEQ ID No. 11.
Specifically, the caspase-2 or cp caspase-2 described herein recognizes or can be further modified to recognize a variety of recognition sites. According to a specific example, the caspase-2 variant or cp caspase-2 variant described herein recognizes any one or more of the recognitions sites LDESD, DVAD, DEVD, DEVE, ADVAD, VDTTD, DTTD, DVPD, VDVPD, VDQQD, or TDTSD. Preferably, the caspase-2 or cp caspase-2 described herein recognizes and has high specificity for one recognition site.
According to a further specific example, the variants of caspase-2 or cp caspase-2 described herein recognizes the recognition site DRKD, DAVD, VKVD, DTLD, EEPD, DETD, DATD, NKVD, DALD, DSVD, NAID, DKPD, IQLD, DNAD, DVVD, ENPD, DMAD, DLID, DGAD, DVKD, GYND, ELPD, DSTD, DRQD, HAVD, QERLD, LERD, MMPD, EEPD, VESID, EAMD, EDAD, EEED, AVLD, and/or EEGD.
According to a further specific example, the variants of caspase-2 or cp caspase-2 described herein recognizes the recognition site TDTSD, LDEPD, and/or KDEVD.
Specifically, the recognition site can be selected from the group consisting of DEXD (SEQ ID No. 202) and DVXD (SEQ ID No. 203), wherein X is any amino acid.
Specifically, the recognition site comprises the sequence P5 P4 P3 P2 P1, wherein P5 is V, P4 is D, P3 is Q, P2 is Q and P1 is D. Specifically, the V on position P5 can be replaced with I, Y, L, T, N, or A, and/or the D on position P4 can be replaced with S, and/or the Q on position P3 can be replaced with V, E or T, and/or the Q on position P2 can be replaced with A, S, K, V, M, or L
Testing of a recognition site library (P4-P1) for Caspase 2 resulted in the following predominant amino acids: position P4: D, V; position P3: V, E, T; position P2: S, T and position P1: D.
Specifically, the caspase-2 or cp caspase-2 described herein is capable of cleaving at a cleavage site P1/P1′, wherein P1′ can be any amino acid.
Further provided herein is a caspase-2 comprising one or more amino acid substitutions at positions 212, 431, 213, 323, 266, 409, 226, 296 or 326 of SEQ ID No. 11 or at a position functionally equivalent to any of positions 212, 431, 213, 323, 266, 409, 226, 296 or 326 of SEQ ID No. 11 or a combination thereof, also referred to as “caspase-2 variant”, and wherein said amino acid substitution increases P1′ tolerance compared to a caspase-2 which has the same sequence but does not comprise said substitutions. In other words, the caspase-2 that the caspase-2 variant is compared to has an identical sequence as the caspase-2 variant, except that it does not comprise any of the amino acid substitutions at positions 212, 431, 213, 323, 266, 409, 226, 296 or 326 of SEQ ID No. 11, or at a position functionally equivalent to any of positions 212, 431, 213, 323, 266, 409, 226, 296 or 326 of SEQ ID No. 11, which increase the P1′ tolerance according to the invention.
Specifically, the caspase-2 variant provided herein comprises improved P1′ tolerance for at least one amino acid other than glycine compared to a caspase-2 not comprising the respective amino acid substitution.
Specifically, the caspase-2 variant described herein comprises at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to SEQ ID No. 11, SEQ ID No. 89, SEQ ID No. 92, SEQ ID No. 95, SEQ ID No. 98, SEQ ID No. 101, SEQ ID No. 104, SEQ ID No. 107, SEQ ID No. 110, SEQ ID No. 113 or SEQ ID No. 116 and comprises one or more amino acid substitutions at positions 409, 431, 212, 213, 266, 296, 226, 323 or 326 of SEQ ID No. 11 or at a position functionally equivalent to any of positions 409, 431, 212, 213, 266, 296, 323 or 326 of SEQ ID No. 11 or a combination thereof.
Specifically, the caspase-2 described herein comprises at least a small caspase-2 subunit and a large caspase-2 subunit.
Specifically, the small subunit of the caspase-2 described herein comprises the amino acid sequence of SEQ ID No. 3, SEQ ID No. 91, SEQ ID No. 94, SEQ ID No. 97, SEQ ID No. 100, SEQ ID No. 103, SEQ ID No. 106, SEQ ID No. 109, SEQ ID No. 112, SEQ ID No. 115, SEQ ID No. 118, or a functionally active variant thereof comprising at least at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity. Specifically, the large subunit of the caspase-2 described herein comprises the amino acid sequence of SEQ ID No. 4, SEQ ID No. 90, SEQ ID No. 93, SEQ ID No. 96, SEQ ID No. 99, SEQ ID No. 102, SEQ ID No. 105, SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity.
According to a specific embodiment, the caspase-2 provided herein comprises one or more linker sequences, specifically consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or even more amino acid residues. Specifically, the linker can comprise more than 20 or 30 or even more amino acids, as long as the caspase retains its functional activity as described herein. Specifically, the linker sequence comprises glycine, alanine and/or serine residues. Specifically, the linker comprises at least one glycine and serine residue, more specifically the linker is GS, GGSGG and/or GSAGSAAGSG.
Specifically, the caspase-2 comprises a subunit-linker sequence, which is a linker sequence between the small subunit and the large subunit of the caspase-2 described herein.
According to a further specific embodiment, the caspase-2 provided herein comprises one or more C-terminal or N-terminal tags, specifically selected from the group consisting of affinity tags, solubility enhancement tags and monitoring tags described herein. Specifically, any tag known in the art can be fused to the caspase-2.
According to a specific embodiment, the caspase-2 provided herein comprises an N-terminal and/or C-terminal truncation of at least 1, 2, 3, 4, 5 and up to 10 or even more, as long as the caspase retains its functional activity as described herein. According to a further specific embodiment, the caspase-2 provided herein comprises an N-terminal and/or C-terminal extension of at least 1, 2, 3, 4, 5 and up to 10 or even more, as long as the caspase retains its functional activity as described herein. Specifically, the caspase-2 may comprise a truncation and an extension.
According to a specific embodiment, the caspase-2 variant provided herein comprises one or more amino acid substitutions, selected from
with reference to the positions of SEQ ID No. 11, or positions functionally equivalent to positions of SEQ ID No. 11.
Specifically, the caspase-2 variant provided herein comprises amino acid substitutions at positions of SEQ ID No. 11, or at positions functionally equivalent to positions of SEQ ID No. 11, selected from
Further provided herein is a method of producing a caspase variant, specifically a caspase-2, even more specifically the circular permuted caspase-2, comprising increased P1′ tolerance.
Specifically, a wild-type cp caspase-2 or a cp caspase-2 variant as described herein, or a functionally active variant thereof, is produced by a method comprising the steps of
Specifically, the nucleic acid sequence encoding the caspase-2 described herein is operably linked to a promoter. Specifically, the promoter is an inducible or a constitutive promoter. Specifically, the promoter is selected from the group consisting of T7, lac, tac, trc, lacUV5, trp, phoA, pL, XylS/Pm regulator/promoter system, Pm-promoter, Pm-promoter variants, araBAD, T3, T5, T4, T7A1, T7A2, T7A3, hybrid promoters and the strong constitutive HCD promoter. Specifically, the promoter is associated with one or more lac operators or respective other operators or regulators or further regulation elements or the promoter is not associated with such regulatory elements. In a preferred embodiment, the promoter/regulator is a promoter/regulator selected from the group consisting of T7 promoter/operator, XylS/Pm regulator/promoter, functionally active variants of the Pm promoter, araBAD promoter/operator, T5, T7A1, T7A2, T7A3 promoter/operator, phoA promoter/regulator, and the trp promoter/operator system. Specifically, the promoter/regulator is of the T7 promoter/operator system.
Specifically, the host cell is a eukaryotic or a prokaryotic microbial host cell. Specifically, host cells are selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells, preferably the host cells are bacterial or yeast cells selected from the group consisting of E. coli, Pseudomonas sp., Bacillus sp., Streptomyces sp., Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Kluyveromyces sp. and Hansenula sp.
Even more specifically, the host cell is of an E. coli B or K strain, such as but not limited to BL21 or HMS174. Specifically, the host cell has integrated in its genome a nucleotide sequence encoding the T7 RNA polymerase and is capable of constitutive of inducible expression of the caspase-2 described herein. as According to a specific example, the host cell is an E. coli BL21 (DE3), or HMS 174 (DE3) cell or a cell derived from BL21 (DE3), or HMS 174 (DE3) comprising a deletion of at least one essential lambda phage protein.
The caspase-2 or cp caspase-2 described herein may comprise a tag sequence, within its sequence or fused to its N- or C-terminus.
The circular permuted caspase-2 can optionally have an affinity tag, preferably fused to its N- or C-terminus, preferably the affinity tag is a 6 His Tag. In a preferred embodiment the 6 His tag is N-terminal. Specifically, in order to increase the expression of soluble cp caspase-2 the cp caspase-2 is fused with a solubility enhancement tag at its C- or N-terminus. In a preferred embodiment, the solubility enhancement tag is N-terminal to the cp caspase-2. Preferably the solubility tag is based on highly charged peptides of bacteriophage genes. Exemplary solubility tags and their sequences are listed in Table 1 of U.S. Pat. No. 8,535,908 B2. Specifically, the solubility tag is selected from the group consisting of the tags, T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC, T3, N1, N2, N3, N4, N5, N6, N7, calmodulin-binding peptide (CBP), DsbA, DsbC, poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin tag, preferably it comprises a T7AC or a T7A3 tag. According to a specific example, the tag is a modified T7A3 tag, herein referred to as T7AC (SEQ ID No. 43). Preferably, one or more T7A3 (SEQ ID No. 37) and/or T7AC (SEQ ID No. 43) tags or functional variants thereof having 1-5 amino acid substitutions, additions, dilutions or the like, are used.
Specifically, the caspase produced according to the method described herein has one or more affinity tags and one or more solubility enhancement tags fused to its N-terminus with or without linker sequences between the tags or between a tag and the N-terminus of the cp caspase-2. Specifically, said caspase has a T7AC or a T7A3 tag and a 6 His tag fused to its N-terminus, whereas from N- to C-terminus the 6 His Tag is the first and the T7AC or T7A3 tag is the second tag, or the T7AC or T7A3 tag is the first and the 6 His tag is the second tag.
It has surprisingly been found that production of a cp caspase-2 described herein can be significantly improved using a solubility enhancement tag, such as T7A3 or T7AC, fused to the N-terminus of the enzyme. Use of such tag significantly increases the titer of the enzyme by about 2.5-fold or more.
According to a specific embodiment, the cp caspase-2 produced according to the method described herein, thus comprises the following elements fused to its N-terminus, in the order from N- to C-terminus:
According to a further specific embodiment, the cp caspase-2 produced according to the method described herein, comprises the following elements fused to its N-terminus, in the order from N- to C-terminus:
Specifically, the expression cassette for expression of the cp caspase-2 comprises the nucleotide sequence encoding the cp caspase-2 under control of a promoter. Optionally, the expression cassette further comprises the nucleotide sequences encoding the affinity tag, in a preferred embodiment the 6 His tag and/or a nucleotide sequence encoding the T7AC or T7A3 tag and nucleotide sequences encoding linker sequences between the tags and/or between a tag and the cp-caspase-2. In another embodiment, the expression cassette is flanked by two sequences homologous to a sequence in the genome of the host cell, preferably a microbial cell, more preferably a bacterial cell, more preferably E. coli, for integration of the expression cassette by homologous recombination into the genome of the host cell.
Specifically, the cell is transformed by a vector comprising the expression cassette. Specifically, the cp caspase-2, with or without tags as described herein, is expressed from one or more plasmids or from one or two copies of a nucleic acid sequence integrated in the genome of the host cell.
Specifically, the cp caspase-2 with or without tags as described herein, can be produced by cultivation of the host cell and induction of expression by addition of an inducer, such as e.g. IPTG when using the T7 promoter/operator system, in a bioreactor (fermenter).
Specifically, culturing of step (iii) of the method to produce a cp caspase-2 as described herein comprises a fed-batch phase for expression of the cp caspase-2 comprising a specific growth rate and induction of expression, preferably using IPTG.
Specifically, culturing of step (iii) of the method to produce a cp caspase-2 as described herein comprises a fed-batch phase for expression of the cp caspase-2, said fed batch phase specifically comprising a specific growth rate, μ of about 0.01-0.1 h−1, and induction of expression of the cp caspase-2 by addition of IPTG at a concentration of about 0.01-1.5 μmol/g of actual CDM (cell dry mass). Specifically, concentration of IPTG of μmol/g of actual CDM means the concentration of IPTG in the fermenter at a certain time point during the feed phase related to the CDM in g at that certain time point.
According to a specific embodiment, growth rate μ is about 0.01-0.07 h−1, preferably it is about 0.01-0.03 h−1 or 0.01-0.05 h−1 or 0.02-0.05 h−1 or 0.03-0.05 h−1 or 0.03-0.07 h−1 or 0.05-0.07 h−1, preferably it is any of about 0.03, 0.05 or 0.07 h−1.
According to a further specific embodiment, the IPTG concentration is about 0.1-1.5 μmol/g or 0.1-1.3 μmol/g or 0.2-1.3 μmol/g or 0.3-1.3 μmol/g or 0.5-1.3 μmol/g of actual CDM, preferably it is about 0.5-0.9 μmol/g actual CDM or about 0.9-1.3 μmol/g actual CDM, preferably it is about 0.5, 0.9 or about 1.3 μmol/g CDM.
According to yet a further specific embodiment, culturing of step (ii) further comprises a first fed-batch phase for the production of biomass, prior to the fed-batch phase for the expression of the cp caspase-2, said first fed-batch phase comprising a growth rate, μ of about 0.05-0.5 h−1 or 0.05-0.4 h−1 or 0.07-0.3 h−1. Specifically, the growth rate μ is about 0.1-0.3 h−1 or 0.1-0.2 h−1, or 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20 h−1. preferably about 0.13-0.21 h−1, even more preferably about 0.16-0.18 h−1 and most preferably it is about 0.17 h−1. Specifically, said first fed-batch phase is followed by a second fed-batch phase for expression of the recombinant protein, preferably started by addition of an inducer of expression, such as IPTG, and typically comprising a lower growth rate.
Specifically provided herein is a fusion protein comprising the following structure from N- to C-terminus:
Specifically, P1′ is the N-terminal amino acid of the protein of interest (POI).
Specifically, the tag sequence of the fusion protein described herein further comprises one or more tags selected from the group consisting of affinity tags, solubility enhancement tags and monitoring tags. Specifically, any tag with any function known in the art can be fused to the POI. Specifically, the fusion protein further comprises one or more linker sequences. Specifically, the fusion protein comprises a caspase recognition site comprising 5 amino acids of the sequence P5 P4 P3 P2 P1, and a cleavage site P1/P1′, wherein P1′ is the N-terminal amino acid of the POI.
According to a specific embodiment, the fusion protein provided herein comprises the cp caspase-2 or caspase 2 described herein within its sequence. Specifically, the fusion protein comprises the cp caspase-2 or caspase 2 described herein fused to the N- or the C-terminus of the fusion protein.
Specifically, such fusion protein is used to produce a POI comprising an authentic N-terminus by cleavage of the fusion protein at the N-terminus of the POI using the cp caspase-2 or caspase-2 described herein.
In one embodiment, the POI comprises an N-terminal tag which comprises at least a caspase recognition site, wherein the C-terminal amino acid of the recognition site, P1, is the last (C-terminal) amino acid residue of the tag and the N-terminal amino acid of the POI is the P1′ residue of the caspase cleavage site. In this embodiment, the tag sequence including the recognition site is released from the POI through proteolytic cleavage by the caspase, generating a POI comprising an authentic N-terminus.
Further provided herein are methods of producing a protein or polypeptide of interest (POI) using the cp caspase-2 or caspase-2 described herein. Specifically, the cp caspase-2 described herein is used for the production of a POI comprising an authentic N-terminus
Specifically provided herein are methods of producing a POI comprising an authentic N-terminus, using the fusion protein described herein and the caspase-2 or the cp caspase-2 described herein.
Specifically provided herein are methods of producing a POI comprising an authentic N-terminus, using the fusion protein described herein, wherein the fusion protein comprises the caspase-2 or the cp caspase-2 described herein at its N- or C-terminus.
Specifically, the fusion protein used in the methods described herein comprises the following structure from N- to C-terminus:
wherein said recognition site is specifically recognized by the caspase-2 or the cp caspase-2 described herein. Specifically, P1′ is the N-terminal amino acid of the POI.
Specifically provided herein is a method of producing a POI in vivo.
Specifically, the in vivo method of producing a POI comprising an authentic N-terminus comprises the steps of:
Specifically, the host cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells, preferably the host cells are bacterial or yeast cells selected from the group consisting of E. coli, Pseudomonas sp., Bacillus sp., Streptomyces sp., Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Kluyveromyces sp. and Hansenula sp.
According to a specific embodiment, the fusion protein and the caspase described herein are under transcriptional control of different promoters and the expression of the caspase is induced after expression of the fusion protein. According to a different embodiment, the fusion protein and the caspase are under transcriptional control of the same promoter, specifically they are expressed at the same time.
Specifically, the caspase comprises an N- or C-terminal tag, which may be used to separate the caspase and the POI.
According to a further specific embodiment, the caspase described herein is part of the fusion protein expressed in the host cell and cleaves the fusion protein releasing a POI comprising an authentic N-terminus in the host cell.
Specifically, the fusion protein, the POI and/or the caspase are isolated using a column, specifically a chromatography column, more specifically an immobilized metal affinity chromatography column (IMAC).
Specifically provided herein is a method of producing a POI in vitro.
Specifically, the in vitro method of producing a protein of interest (POI) comprising an authentic N-terminus comprises the steps of:
Specifically, the method of producing a POI as described herein, comprises the steps of:
Specifically, the caspase used in such method comprises at its N- or C-terminus an affinity tag identical or similar to the affinity tag of the fusion protein. Specifically, the caspase, the cleaved affinity tag and any un-cleaved fusion protein are removed in step vi. using said affinity tag.
Specifically, said fusion protein is purified using the tag, for example using affinity chromatography, more specifically immobilized metal affinity chromatography. Specifically, the captured fusion protein is released and the N-terminal tag is removed in solution or in immobilized enzyme reactor, wherein the caspase is immobilized in a column or on a carrier.
According to a further specific embodiment, the fusion protein and the caspase are bound on a column.
Specifically, the method of producing a POI comprising an authentic N-terminus using a column comprises the following steps:
Specifically, the caspase comprises a tag sequence, specifically an affinity tag, to allow separation of the caspase from the POI after cleavage.
In a specific embodiment, the caspase-2 or cp caspase-2 described herein comprises an affinity tag and is immobilized on a solid support or column and the fusion protein is brought into contact with the immobilized caspase. Specifically, the fusion protein is brought into contact with the caspase immobilized in a column by flowing the fusion protein through the column. Specifically, the cleaved tag is separated from the POI using the affinity tag in the tag sequence.
In a further specific embodiment, the caspase and the fusion protein comprise an identical N-terminal affinity tag, allowing immobilization of the fusion protein and the caspase on the solid support. Upon cleavage of the POI by the caspase, the POI is released from the column and the tag sequence is retained in the column as well as the caspase and optional uncleaved fusion protein.
Specifically, the solid support is a column, specifically a chromatography column, more specifically an immobilized metal affinity chromatography column (IMAC) or an activated NHS column allowing immobilization of a polypeptide through amine coupling.
Specifically, a flow-through reactor is used comprising immobilized caspase-2, cp caspase-2 or fusion protein described herein. Specifically, the flow-through reactor is a plug flow reactor.
Further provided herein is an isolated nucleotide sequence encoding the caspase-2 or cp caspase-2 described herein.
Further provided herein is a vector comprising the isolated nucleotide sequence described herein, specifically said vector is a bacterial expression vector. More specifically the vector is a plasmid. In another embodiment, the vector is a linear vector flanked with homology regions for homologous integration of the nucleotide sequence encoding the caspase-2 or cp caspase-2 described herein into the chromosome of the host cell.
Further provided herein is an expression cassette comprising the nucleotide sequence operably linked to regulatory elements such as promoter, operator, terminator and the like. Specifically, said regulatory elements are one or more promoters or expression enhancing elements.
Further provided herein is a host cell or a host cell line expressing the caspase-2 or cp caspase-2 described herein, wherein the host cells are selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells, preferably the host cells are bacterial or yeast cells selected from the group consisting of E. coli, Bacillus sp., Streptomyces sp., Saccharomyces sp., Schizosaccharomyces sp., Kluyveromyces sp. and Pichia sp.
According to a specific embodiment, the expression cassette and the host cell or host cell line described herein are comprised in an expression system. Further provided herein is an expression system comprising the expression cassette and the host cell or host cell line described herein.
Further described herein is the use of the caspase-2 or cp caspase-2 described herein for the in vivo cleavage of a substrate in a non-human organism. Specifically, the non-human organism is a prokaryotic organism, specifically it is E. coli.
Further provided herein is a kit, comprising
The kit may optionally further comprise chromatography material for affinity chromatography, preferably an IMAC (immobilized metal affinity chromatography) material, preferably Ni-NTA (Ni— Nitrilotriacetic acid) chromatography material, preferably pre-packed in a chromatography column.
Further optionally comprised in the kit is a plasmid comprising the nucleotide sequence from 5′ to 3′ encoding an affinity tag, preferably a 6 His tag, optionally a linker sequence and a nucleotide sequence coding for the recognition site, preferably VDVAD. Via a multiple cloning site, a DNA sequence encoding a POI can be inserted into the plasmid directly fused to the nucleotide sequence encoding the recognition site.
Further described herein is a pharmaceutical composition comprising the caspase-2 or cp caspase-2 provided herein and optionally one or more excipients.
Further described herein is use of the caspase-2 or cp caspase-2 provided herein for preparing a pharmaceutical composition.
Specifically, the caspase-2 or cp caspase-2 described herein is provided for use in the treatment of a disease. Specifically, the caspase-2 or cp caspase-2 described herein is provided for use in the treatment of cancer, osteoporosis, Alzheimer's disease, Parkinson's disease, inflammatory disease, or auto-immune diseases, specifically via proteolytically attacking respective disease relevant proteins.
Specifically, the caspase-2 or cp caspase-2 described herein is provided for the manufacture of a medicament for the treatment of cancer, Alzheimer's disease, Parkinson's disease or inflammatory disease. Further provided herein is a protein tag for enhanced expression of a POI, comprising a solubility enhancement tag and the amino acid sequence VDVAD (SEQ ID NO: 45). Specifically, the sequence VDVAD is at the C-terminus of the protein tag described herein, specifically directly linked to the N-terminus of the POI.
It has been surprisingly found that by including the amino acid sequence VDVAD in a protein tag, the expression of a protein of interest fused to the protein tag can be significantly increased. Importantly, this increase in expression persists, despite addition of a histidine tag sequence to the protein tag. Using a histidine affinity tag, such as 6-His, typically decreases the expression rate of a protein of interest significantly. The inventors have surprisingly found that by including the sequence VDVAD in the protein tag this effect is reversed, and increased expression titers can be provided.
According to a specific embodiment, the solubility enhancement tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T3, N1, N2, N3, N4, N5, N6, N7, T7AC, calmodulin-binding peptide (CBP), DsbA, DsbC, poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin tag. Specifically, the solubility enhancement tag is T7AC or T7A3.
According to a further specific embodiment, the protein tag described herein further comprises a histidine tag sequence, preferably comprising 1-20 histidine residues, even more preferably it is a 1-His, 2-His, 3-His, 4-His, 5-His, 6-His, 7-His, 8-His, 9-His, 10-His, 11-His, 12-His, 13-His, 14-His, 15-His, 16-His, 17-His, 18-His, 19-His or 20-Histag sequence.
Specifically, the solubility enhancement tag is located at the N-terminus of the protein tag described herein.
Specifically, the histidine tag sequence is located at the N-terminus of the protein tag described herein.
According to a specific embodiment, the protein tag described herein further comprises one or more linker sequences comprising one or more amino acid residues. Specifically, said linker sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acid residues. Specifically, the linker can comprise more than 20 or 30 or even more amino acids, as long as the caspase retains its functional activity as described herein. Specifically, the one or more amino acid residues of the linker sequence are any of the naturally occurring amino acids or derivatives thereof, preferably selected from the group consisting of G, S, T, N, A. Specifically, the linker sequence comprises glycine, alanine and/or serine residues. Specifically, the linker comprises at least one glycine and serine residue, more specifically the linker is GS, GSG, GGSGG, GSGSGSG and/or GSAGSAAGSG.
Specifically, said one or more linker sequences are located between the VDVAD sequence and the solubility enhancement tag or the histidine tag sequence.
According to a specific embodiment, the protein tag described herein further comprises a signal peptide at its N-terminus. Signal peptides are known to the person skilled in the art, and comprise for example those described by Choi and Lee, Appl Microbiol Biotechnol (2004); 64:625-635 or Karyolaimos et al. Frontiers in Microbiology (2019); 10:1-11. Specifically, the signal peptide is selected from the group consisting of ompA (outer membrane protein A), DsbA (Thiol:disulfide interchange protein), MalE (maltose-binding protein), PelB (pectate lyase B) from Erwinia carotovora, PhoA (alkaline phosphatase), OmpC (outer-membrane protein C), OmpF (outer-membrane protein F), OmpT (protease VII), Endoxylanase from Bacillus sp., LamB (A receptor protein), Lpp (murein lipoprotein), LTB (heat-labile enterotoxin subunit B), PhoE (outer-membrane pore protein E), and Stll (heat-stable enterotoxin 2).
Using a signal sequence, also herein referred to as signal peptide, leader sequence or leader peptide, such as the ompA signal peptide, which guides the protein through the inner membrane into the periplasma of bacteria, e.g, E. coli, which has been fused to the N-terminus of the protein tag described herein allows successful production of the POI and the fusion proteins described herein in the periplasma of E. coli. This is surprising since expression enhancers are usually located at the N-terminus of the whole fusion protein (respectively the expression construct, respectively the gene encoding the fusion protein), as described herein. As demonstrated in Example 10.2, and
Specifically, the protein tag described herein comprises one of the following structures from N- to C-terminus:
Specifically, the protein tag described herein comprises one of the following structures from N- to C-terminus:
Further provided herein is a fusion protein comprising the protein tag described herein and a POI. Specifically, the N-terminus of the POI is fused to the C-terminus of said protein tag. Even more specifically, the N-terminus of the POI is directly fused to the C-terminus of the protein tag, which C-terminus is the sequence VDVAD, i.e., the N-terminal amino acid of the POI is directly linked to the C-terminal D of the VDVAD sequence of the protein tag.
With regard to the POI there is no limitation. The POI may be any polypeptide, including e.g. the caspases described herein.
Further also provided herein is a method of producing a POI, comprising the steps of:
According to a specific embodiment, the method of producing a POI as described herein comprises the following steps:
Specifically, the promoter is selected from the group consisting of T7 promoter/operator, XylS/Pm regulator/promoter or variants of the Pm promoter, araBAD promoter/operator, T5, T7A1, T7A2, T7A3 promoter/operator, phoA promoter/regulator and the trp promoter/operator system.
Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to standard handbooks, such as Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990), and Janeway et al, “Immunobiology” (5th Ed., or more recent editions, Garland Science, New York, 2001).
The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wild-type) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells and modified proteins including enzymes, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.
The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.
The term “about” as used herein refers to the same value or a value differing by +/−5% of the given value.
As used herein, amino acids refer to twenty naturally occurring amino acids encoded by sixty-one triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges:
The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity:
Alanine: (Ala, A) nonpolar, neutral;
Asparagine: (Asn, N) polar, neutral;
Cysteine: (Cys, C) nonpolar, neutral;
Glutamine: (Gln, Q) polar, neutral;
Glycine: (Gly, G) nonpolar, neutral;
Isoleucine: (Ile, I) nonpolar, neutral;
Leucine: (Leu, L) nonpolar, neutral;
Methionine: (Met, M) nonpolar, neutral;
Phenylalanine: (Phe, F) nonpolar, neutral;
Proline: (Pro, P) nonpolar, neutral;
Serine: (Ser, S) polar, neutral;
Threonine: (Thr, T) polar, neutral;
Tryptophan: (Trp, W) nonpolar, neutral;
Tyrosine: (Tyr, Y) polar, neutral;
Valine: (Val, V) nonpolar, neutral; and
Histidine: (His, H) polar, positive (10%) neutral (90%).
The “positively” charged amino acids are:
Arginine: (Arg, R) polar, positive; and
Lysine: (Lys, K) polar, positive.
The “negatively” charged amino acids are:
Aspartic acid: (Asp, D) polar, negative; and
Glutamic acid: (Glu, E) polar, negative.
Caspases are the key enzymes in the initiation and execution of apoptosis and inflammation, hence their activity has to be tightly controlled. Although the sequences of caspases do differ (e.g. human caspase-1 and -2 have only 27% amino acid identity and 52% similarity), their active sites and tertiary structure are highly conserved. All caspases are synthesized as relatively inactive single-chain zymogens (procaspases), which comprise a prodomain (2-25 kDa), as well as a large and a small subunit of 17-21 kDa and 10-13 kDa respectively. The executioner caspases (caspases-3, -6, -7) and caspase-14 have a short, while all other caspases have a long prodomain. To get fully active, wild-type caspases first need to dimerize through hydrophobic interactions, then their intersubunit linker is cut and the prodomain is removed by proteolytic cleavages after aspartate residues. A main difference between the activation of executioner and initiator caspases is that the latter are already active after dimerization and the autocatalytic separation of their subunits is only necessary for stabilization. Active wild-type caspases are homodimers of heterodimers. Each heterodimer consists of a large and small subunit derived from a single protein chain. The enzyme is formed by a central twelve-stranded β-sheet, to which each of the four subunits contributes. From this core four loops protrude which contain the active site and form the binding pockets. In all caspases the catalytic center is in the large subunit. The substrate recognition site is formed by amino acids from both subunits, though the small subunit contributes the main residues which are responsible for differing substrate specificity between caspases. The cleavage of the inter-subunit linker causes a rearrangement of the active site loops, allowing the binding pockets to form and to make the active cysteine solvent accessible.
As used herein the term “recognition site” or “caspase recognition site” refers to an amino acid sequence of at least 3, preferably at least 4 or 5, amino acid residues of a substrate, which is specifically recognized by the caspase-2 or cp caspase-2 described herein. Specifically, the at least three substrate amino acids which are targeted and bound by the caspase provided herein and which form the recognition site are termed P3-P1 or P3 P2 P1, P4-P1 or P4 P3 P2 P1 for a recognition site comprising 4 substrate amino acids, P5-P1 or P5 P4 P3 P2 P1 for a recognition site comprising 5 substrate amino acids, P6-P1 or P6 P5 P4 P3 P2 P1 for a recognition site comprising 6 substrate amino acids, P7-P1 or P7 P6 P5 P4 P3 P2 P1 for a recognition site comprising 7 substrate amino acids, and so on. As described herein, the caspase provided herein interacts with its substrate in a target-specific manner by specifically recognizing and binding the recognition site comprising at least 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues comprised in the sequence of the substrate. The recognition site amino acid residues occupy specific pockets on the caspase, numbered with the matching S designation (S1, S2, S3, S4, S5 etc; S1′, S2′ etc), each of which may be constructed of several amino acid residues. The objective of this interaction mode, which almost always binds the cleavage region in an extended peptide conformation, is to align the substrate accurately into register with the catalytic machinery.
Specifically, the caspase-2 or cp caspase-2 provided herein is not limited to the recognition site of wild-type caspase-2, VDVAD (SEQ ID No. 45). Further provided herein are variants of caspase-2, which target recognition sites other than VDVAD with high specificity and efficiency. Specifically provided herein are caspase-2 variants which target any one or more of the recognition sites described herein.
Preferably, the caspases described herein have high specificity towards a single recognition site, but embodiments are envisioned wherein a caspase recognizes more than one recognition site, for example for cleavage of one protein at multiple sites or for simultaneous cleavage of different proteins comprising different recognition sites.
Using the selection method described herein, caspase-2 variants can be selected which specifically recognize any one or more recognition sites. Specifically, any of the caspase variants described herein, comprising any one or more of the amino acid substitutions increasing P1′ tolerance as described herein can be subjected to the selection method described herein. In such a way, for example, caspase-2 variants comprising increased P1′ tolerance and target specificity towards a certain recognition site can be selected.
According to a further specific embodiment, recognition site specificity of the caspase variants described herein can be influenced, by introduction of amino acid substitutions, additions or deletions which are known to increase or decrease specificity to a certain recognition site.
Specifically, the caspase-2 or cp caspase-2 described herein recognizes a recognition site comprising the sequence XDXXD (SEQ ID No. 201), wherein X can be any amino acid. Specifically, the recognition site can be selected from the group consisting of DEXD (SEQ ID No. 202) and DVXD (SEQ ID No. 203), wherein X is any amino acid.
According to a specific example, the caspase-2 or cp caspase-2 described herein recognizes any one or more of the recognitions sites LDESD (SEQ ID No. 204), DVAD (SEQ ID No. 205), DEVD (SEQ ID No. 206), DEVE (SEQ ID No. 207), ADVAD (SEQ ID No. 208), VDTTD (SEQ ID No. 209), DTTD (SEQ ID No. 210), DVPD (SEQ ID No. 211), VDVPD (SEQ ID No. 212), VDQQD (SEQ ID No. 213), or TDTSD (SEQ ID No. 214).
According to a further specific example, the caspase-2 or cp caspase-2 described herein recognizes the recognition site DRKD (SEQ ID No. 215), DAVD (SEQ ID No. 216), VKVD (SEQ ID No. 217), DTLD (SEQ ID No. 218), EEPD (SEQ ID No. 219), DETD (SEQ ID No. 220), DATD (SEQ ID No. 221), NKVD (SEQ ID No. 222), DALD (SEQ ID No. 223), DSVD (SEQ ID No. 224), NAID (SEQ ID No. 225), DKPD (SEQ ID No. 226), IQLD (SEQ ID No. 227), DNAD (SEQ ID No. 228), DVVD (SEQ ID No. 229), ENPD (SEQ ID No. 230), DMAD (SEQ ID No. 231), DLID (SEQ ID No. 232), DGAD (SEQ ID No. 233), DVKD (SEQ ID No. 234), GYND (SEQ ID No. 235), ELPD (SEQ ID No. 236), DSTD (SEQ ID No. 237), DRQD (SEQ ID No. 238), HAVD (SEQ ID No. 239), QERLD (SEQ ID No. 240), LERD (SEQ ID No. 241), MMPD (SEQ ID No. 242), EEPD (SEQ ID No. 243), VESID (SEQ ID No. 244), EAMD (SEQ ID No. 245), EDAD (SEQ ID No. 246), EEED (SEQ ID No. 247), AVLD (SEQ ID No. 248), and/or EEGD (SEQ ID No. 249).
According to yet a further specific example, the caspase-2 or cp caspase-2 described herein recognizes the recognition site TDTSD, LDEPD (SEQ ID No. 250), and/or KDEVD (SEQ ID No. 251).
As used herein the term “cleavage site” refers to the amino residues P1/P1′ wherein cleavage occurs at the residue of the amino terminal scissile bond P1 and the one to the carboxy-terminal side P1′.
Proteolytic cleavage of the substrate happens after the P1 residue. Specifically, the amino acids following the P1 residue are referred to as P1′-P4′ residues, also termed the prime side. The prime side of the substrate is important for substrate processing, specifically the P1′ residue. The P1′-P4′ residues can under certain circumstances influence binding by steric hindrance. The P1′ residue is close to the active site and in particular branched (e. g. leucine or valine) and polar amino acids (e. g. threonine or aspartate) in this position can compete for space with the catalytic cysteine and negatively influence the cleavage.
Wild-type caspases have a high preference for aspartate in the P1 position. The P2 and P3 positions are less selective and a variety of residues is accommodated, although many caspases have the highest activity with a glutamate residue at the P3 position. The P4 position is crucial for distinction between caspase classes: Inflammatory caspases and caspase-14 prefer hydrophobic residues, initiator caspases and caspase-6 aliphatic residues, and executioner caspases as well as wild-type caspase-2 favor aspartate. The prime side positions of substrates have not been investigated as intensively, although studies have shown that the P1′ site has an influence on cleavage, as certain residues can reduce the activity up to 1000-fold. All wild-type caspases prefer substrates with small residues (glycine, serine, alanine), but large hydrophobic amino acids (phenylalanine, tyrosine) are also surprisingly well tolerated. Most likely the P1′ site is not necessary for efficient binding of the substrate, but certain residues can hinder it. The prime sites further away (P2′-P4′) from the cleavage site have little influence. However, whether a substrate is cleaved by a caspase or not, does not only depend on the mere presence or absence of a recognition site, as many proteins are processed at non-canonical sites. Secondary and tertiary structures of the substrate are very important for recognition. In vivo proteins are preferably cleaved at solvent accessible loops, but a significant amount is also cleaved within α-helices.
Specifically, wildtype caspase-2 highly prefers a glycine residue at the P1′ site.
According to a specific embodiment, variants of cp caspase-2 and/or caspase-2 can be selected for increased P1′ tolerance as described herein. Specifically, functionally active variants of cp caspase-2 having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 6, preferably at least 85, 90 or 95% sequence identity with SEQ ID No. 6, comprise improved tolerance for P1′ residues other than glycine. According to a further specific embodiment, caspase-2 variants as described herein can be selected for specific cleavage of recognition sites other than VDVAD.
Hence there are no limitations regarding the substrate. Substrates of the caspase provided herein can be any protein or polypeptide, a naturally occurring protein or polypeptide naturally comprising a recognition site specifically targeted by the caspase described herein, or heterologous proteins or polypeptides engineered to comprise a recognition site within their sequence or at or near their N-terminus or C-terminus.
According to a specific embodiment, the substrate comprises a protein of interest as described herein.
Caspase-2 was first described as apoptotic protein in 1994, due to its similarity with CED-3, a cell death protein in Caenorhabditis elegans, and human caspase-1. Procaspase-2 consists of a CARD followed by a large and a small subunit (see
The substrate binding site of wild-type caspase-2 is mainly formed by three protein loops. The first loop (residues 212-221 of SEQ ID No. 11, large subunit) interacts with the prime site of the substrate (P1′-P4′), while the second loop (residues 373-382 of SEQ ID No. 11, small subunit) binds to the whole substrate (P5-P4′), and the third loop (residues 419-431 of SEQ ID No. 11, small subunit) interacts with the recognition site (P5-P1). Wild-type caspase-2 has a near absolute requirement for aspartate residues on both P1 and P4 positions of the recognition site, while many residues are accepted in the P2 and P3 positions. The S1 pocket is positively charged and the substrate residue stabilized by two arginine residues. The S4 pocket is also deep and narrow and therefore very specific. Likewise, the P3 and P5 residues bind to their unique pockets, only the P2 residue is not bound individually. Wild-type caspase-2 is unique in recognizing a pentapeptide and not a tetrapeptide like all other caspases. VDVAD is considered the preferred cleavage site of wild-type caspase-2.
As used herein, the term “wild-type” generally refers to a phenotype, genotype, or gene that predominates in a natural population of organisms or strain of organisms in contrast to that of natural or recombinant mutant variants. In other words, “wild-type” refers to the form or forms of a gene commonly occurring in nature in a given species. The term “wild-type” with respect to caspase-2 and cp caspase-2 as used herein, refers to amino acid or nucleotide sequences of caspase-2, or domains thereof such as e.g. small and large subunit, originating from different species and commonly occurring in nature.
Within the context of this invention, it should be understood that the term “caspase” generally refers to “caspase-2” and functionally active variants thereof.
The term “cp-caspase-2” as used herein refers to a circular permuted caspase-2, as described herein, which is a single chain caspase-2 comprising the small subunit N-terminal of the large subunit of a caspase-2 as further described herein. “cp-caspase-2” comprises the small subunit and large subunit of caspases 2 originating from different species as well as functionally active variants thereof. Specifically, wild type caspase-2 of the different species comprises several, specifically 4, domains.
The terms “wild-type caspase-2” or “wt caspase-2” and “wild-type cp caspase-2” or “wt cp caspase-2” encompass wild-type caspase-2 sequences originating from different species and functionally active variants thereof. Wild-type caspase-2 described herein, may comprise one or more amino acid substitutions, deletions and/or insertions, which are conservative modifications and do not alter the enzyme's protease function. The wild-type caspase-2 and wild-type cp caspase-2 as described herein, do not comprise amino acid substitutions increasing P1′ tolerance.
The terms “caspase-2 variant” and “cp caspase-2 variant” as used herein refer to variants of the wild-type caspase-2 or wild-type cp caspase-2 which have increased proteolytic activity, specifically increased P1′ tolerance, and comprise specific amino acid substitutions as described herein.
The terms “caspase-2” and “cp caspase-2” encompass both the wild-type version of the enzyme, circularly permuted or not circularly permuted, and the variant version of the enzyme, circularly permuted or not circularly permuted, comprising increased P1′ tolerance as described herein, unless otherwise specified.
The boundaries of the small subunit and the large subunit, as well as the other domains, are identified either experimentally by amino acid sequence analysis of the mature caspase or by inspection of structural homology (e.g., the conserved Asp-X cleavage site, in human for example Asp14 of SEQ ID No. 2 or Asp347 of SEQ ID No. 11). For exemplary purposes, the Table below presents the boundaries of the 4 domains including prodomain (CARD), large Subunit (LS), intervening sequence (small subunit propeptide), and small subunit (SS) of caspase-2 in different species. The amino acid positions of the table below refer to the amino acid positions in SEQ ID Nos. 11, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116.
The caspase described herein comprises at least a portion of the caspase-2 small subunit and at least a portion of the caspase-2 large subunit. In preferred embodiments, the propeptide of the small caspase-2 subunit is also present. The prodomain (CARD) is generally not required for enzyme activity and is normally released in vivo. Caspases of the present invention optionally have a prodomain or portion thereof. The propeptide of the small subunit is optional for inclusion in the cp-caspase-2. Although it is preferred that both subunits are derived from the same species, combinations of subunits from different species may be used.
As noted above, a portion of the large subunit and a portion of the small subunit may be used in the caspase described herein, but when designing the caspase-2 and/or the cp caspase-2 described herein, active sites are preferably not deleted.
Caspase-2 is unique in the caspase-family in that it comprises the following consensus sequence (SEQ ID NO: 277):
Caspase-2 is an initiator caspase, while caspase-3 and caspase-6 are effector caspases. The structure of caspase-2 is stabilized by a disulfide bond and wt caspase-2 is the only caspase with a recognition site comprising 5 amino acid residues (Grinshpon et al., A C. Biochem J. 2019; 476(22):3475-3492).
In a preferred embodiment, the caspase-2 variants described herein, that are not circularly permuted, comprise at least a portion of a small caspase-2 subunit and at least a portion of a large caspase-2 subunit and amino acid substitutions at any one or more of positions 212, 431, 213, 323, 266, 409, 226, 296 or 326 of SEQ ID No. 11, or at a position functionally equivalent to positions 212, 431, 213, 323, 266, 409, 226, 296 or 326 of SEQ ID No. 11. Specifically, said caspase-2 variant comprises improved P1′ tolerance, specifically for amino acids other than glycine in the P1′ position, compared to the respective wildtype caspase-2.
The person skilled in the art will readily understand that the respective wildtype caspase-2 is a protein comprising the amino acid sequence of the caspase-2 from which the caspase-2 variant originates. For example, a caspase-2 variant as described herein which is of human origin, comprises improved P1′ tolerance compared to human wildtype caspase-2 comprising SEQ ID No. 11. According to a further specific example, a caspase-2 variant as described herein which is of ghost shark origin and comprises amino acid substitutions at any one or more of positions 369, 391, 174, 187, 227, 257, 284 or 287 of SEQ ID No. 113, comprises improved P1′ tolerance compared to ghost shark wildtype caspase-2 comprising SEQ ID No. 113. According to a further specific example, a caspase-2 variant as described herein which is of Tasmanian devil origin and comprises amino acid substitutions at any one or more of positions 386, 408, 189, 190, 203, 243, 273, 300, or 303 of SEQ ID No. 95, comprises improved P1′ tolerance compared to Tasmanian devil wildtype caspase-2 comprising SEQ ID No. 95.
Additionally, the caspase-2 variants described herein can comprise the amino acid sequence of the homologous wild-type caspase 2 of several different species, such as but not limited to Mouse, Sheep, Tasman Devil, Chicken, Anolis, Alligator, Xenopus, Danio, Ghost Shark, Sea Squirt or any other species, as shown in SEQ ID Nos. 11, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116 and
Circular permutation (CP) has first been discovered in natural proteins in 1979. Circularly permuted (cp) proteins arise by covalent linkage of native N- and C-terminus and the introduction of new termini by cleavage elsewhere in the protein. In nature this either happens by duplication/deletion or fission/fusion events at the gene level. The new variants have an altered order of amino acids but maintain the same tertiary structure. Despite one published variant, an uncleavable reversed caspase-3, all described reverse variants still cleave themselves at the intersubunit linker, to make their structure more similar to the wild-type variants. Circularly permuted, constitutively active forms of caspase-7 and -14 have been published but all of them cleave their intersubunit linker. It was not expected that circular permutation of caspase-2 would be successful. As the structure of the N-terminus of the large subunit has not been completely determined, it was unclear if the two subunits of caspase-2 could be joined. It was thus surprising to see that the circular permuted variants of caspase-2 provided herein were active and it was even more surprising that they depicted significantly improved characteristics, in particular higher P1′ tolerance, higher specificity, higher catalytic efficiencies, increase in heat tolerance and tolerance to chaotropic conditions and significantly improved manufacturability over wild-type caspase-2.
As used herein the term “circular permuted caspase-2” or “cp caspase-2” refers to a modified variant of caspase-2 comprising an altered order of amino acids, specifically, the order of amino acids is altered compared to order of amino acids in wildtype caspase-2. The cp caspase-2 referred to herein is a protease in which a small subunit of a caspase-2 is N-terminal to a large subunit of a caspase-2. Specifically, the amino acid order is altered by linkage of the native N-terminus of the LS and the C-terminus of the SS and the introduction of new termini by cleavage elsewhere in the protease. The cp caspase-2 provided herein comprises the following structure from N- to C-terminus: a small caspase-2 subunit, or a functionally active variant thereof, covalently linked, either via a linker or directly, to a large caspase-2 subunit, or a functionally active variant thereof. The structure of the cp caspase-2 described herein is exemplified in
Hence a caspase-2 was designed whose scaffold was changed by circular permutation, i.e. covalent ligation of the wild type N- and C-termini and intramolecular cut of the protein backbone at a different position to create new N- and C-termini, which leads to swapped domains, i.e. exchange of the positioning of the small and large caspase subunits, and which are active without the need of processing steps as in wild-type executioner or apoptotic caspases.
Circular permuted caspase-2 as described herein includes wildtype caspase-2 sequences without amino acid alterations, albeit in altered order, and circular permuted variants of wildtype caspase-2, which differ from wildtype caspase-2 sequences in one or more amino acid substitutions, deletions, additions and the like and comprise increased proteolytic activity, specifically increased P1′ tolerance.
Variants of caspase and cp-caspase genes provided herein may be engineered from natural variants (e.g., polymorphisms, splice variants, mutants), synthesized or constructed. Many methods have been developed for generating mutants (see, generally, Sambrook et al., Supra; Ausubel, et al., Supra). Briefly, preferred methods for generating a few nucleotide substitutions utilize an oligonucleotide that spans the base or bases to be mutated and contains the mutated base or bases. The oligonucleotide is hybridized to complementary single stranded nucleic acid and second strand synthesis is primed from the oligonucleotide. The double-stranded nucleic acid is prepared for transformation into host cells, typically E. coli, but alternatively, other prokaryotes, yeast or other eukaryotes may be used. Standard screening and vector growth protocols are used to identify mutant sequences and obtain high yields. Similarly, deletions and/or insertions of the caspase-2 or cp caspase-2 genes may be constructed by any of a variety of known methods, such as discussed herein. For example, the gene can be digested with restriction enzymes and re-ligated such that a sequence is deleted or re-ligated with additional sequences such that an insertion or large substitution is made. Other means of generating variant sequences may be employed with methods known in the art. Verification of variant sequences is typically accomplished by restriction enzyme mapping, sequence analysis, or probe hybridization.
Specifically, the cp caspases of the present invention are generated by rearranging the gene sequence of a caspase-2 gene such that the nucleic acid sequence encoding the small subunit precedes (is 5′ to) the nucleic acid sequence encoding the large subunit.
Specifically, the wild-type cp caspase-2 or cp caspase-2 variant described herein is of animal origin, specifically it is of mammalian, reptile or fish origin. Specifically, is derived from Human (SEQ ID No. 11), Mouse (SEQ ID No. 89), Sheep (SEQ ID No. 92), Tasmanian Devil (SEQ ID No. 95), Chicken (SEQ ID No. 98), Anolis (SEQ ID No. 101), Aligator (SEQ ID No. 104), Xenopus (SEQ ID No. 107), Danio (SEQ ID No. 110), Ghost Shark (SEQ ID No. 113), or Sea Squirt (SEQ ID No. 116) caspase-2. Preferably, the cp caspase-2 described herein is derived from human, marsupial, iguana, Tasmanian devil, ghost shark or cartilaginous fish caspase-2.
According to a specific embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises a sequence having more than 80 or 90%, specifically at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity compared to an active site, e.g. comprising SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49 and SEQ ID No 50. Preferably, the cp caspase-2 described herein has at least 90, 95% or more sequence identity with SEQ ID Nos. 46-50.
According to a further specific embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 64 (Sarcophilus harrisii, tasman devil), SEQ ID No. 66 (Anolis carolinensisilus) or SEQ ID No. 68 (Callorhinchus milii, ghost shark).
According to a preferred embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises the amino acid sequence of SEQ ID No. 9 or is a functional variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 9. Specifically, the cp caspase-2 described herein has the amino acid sequence of SEQ ID No. 9, or it is a functional active variant thereof comprising one or more amino acid substitutions or deletions, preferably comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions or the like.
According to a further preferred embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises the amino acid sequence of SEQ ID No. 6 or is a functional variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 6. Specifically, the cp caspase-2 described herein has the amino acid sequence of SEQ ID No. 6, or it is a functional active variant thereof comprising one or more amino acid substitutions or deletions, preferably comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions or the like.
According to a further preferred embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises the amino acid sequence of SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76 or SEQ ID No. 77 or is a functional variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76 or SEQ ID No. 77. Specifically, the cp caspase-2 described herein has the amino acid sequence of SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76 or SEQ ID No. 77, or it is a functional active variant thereof comprising one or more amino acid substitutions or deletions, preferably comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions or the like.
According to a specific embodiment, the caspase-2 variant described herein comprises one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83,185, 255, or 285 of SEQ ID No. 6 such as positions 409, 431, 212, 213, 266, 226, 296, 323 or 326 of SEQ ID No. 11.
The term “functionally equivalent” as used in respect of amino acid substitutions herein refers to amino acids at positions corresponding to the position in the sequence of caspase-2 of a different species. Specifically, by “functionally equivalent” it is meant that variants of the caspase-2 or cp caspase-2 described herein comprise an amino acid substitution at a position considered to concur with the substitutions described herein numbered with respect to SEQ ID No. 6 and that have the same functional role in the variant. Specifically, an amino acid substitution at a position functionally equivalent to the amino acid substitutions described herein confer improved P1′ tolerance upon the variant.
Generally, functionally equivalent substitution mutations occur at homologous amino acid positions in the amino acid sequences of caspase-2. Hence, use herein of the term “functionally equivalent” also encompasses mutations that are “positionally equivalent” or “homologous” to a given mutation, regardless of whether or not the particular function of the mutated amino acid is known. It is possible to identify positionally equivalent or homologous amino acid residues on the basis of sequence alignment and/or molecular modelling.
By way of example, the residues shown in the table 63 below are identified as positionally equivalent and/or functionally equivalent to positions 171, 105, 172, 282, 225, 83, 185, 255, and 285 of SEQ ID No. 6. It will be readily known by one of ordinary skill in the art how to identify positionally equivalent and/or functionally equivalent positions for the amino acid substitutions described herein in caspase-2 sequences of other species.
According to a specific example, homologues of the caspase-2 and the cp caspase-2 described herein are constructed analogue to the caspase-2 or cp caspase-2 of human origin. For example, using the wildtype sequence of caspase-2 in the respective species, such as e.g. Tasmanian devil caspase-2 (Sarcophilus harrisii, UniProtKB14 ID G3VQP7) and Ghost shark caspase-2 (Callorhinchus milii, UniProtKB14 ID V9KZT1), the caspase-2 subunits are determined (see
Surprisingly, the amino acid substitutions described herein confer improved P1′ tolerance upon the caspase-2 and cp caspase-2 described herein. As used herein, the term “improved P1′ tolerance” refers to increased proteolytic activity of a caspase regarding one or more P1′ residues. For example, while wildtype human caspase-2 prefers glycine in the P1′ position, the caspases and cp caspases described herein are capable of proteolytic cleavage at a cleavage site comprising a P1′ residue other than glycine with increased activity compared to wildtype caspase-2 or to a cp caspase-2 not comprising the amino acid substitutions described herein. Specifically, the caspase-2 variants described herein comprise improved P1′ tolerance compared to the respective wildtype caspase-2. The person skilled in the art will readily understand that the respective wildtype caspase-2 is a protein comprising the amino acid sequence of the caspase-2 from which the caspase-2 variant originates. For example, a caspase-2 variant as described herein which is of human origin, specifically a cp caspase-2 as described herein, e.g. a cp caspase-2 comprising SEQ ID No. 70, comprises improved P1′ tolerance compared to human cp caspase-2 comprising SEQ ID No. 6. Specifically, the caspases of the present invention comprise at least 5, 10, 25, 50, 75 or 100% or more increase in proteolytic activity for at least one amino acid residue in the P1′ position compared to a cp caspase-2 not comprising the amino acid substitutions described herein.
As described above, the cp caspase-2 and the caspase-2 provided herein comprise at least a small caspase-2 subunit and a large caspase-2 subunit.
The term “small caspase-2 subunit” as used herein, refers to a small subunit, derived from caspase-2, which is covalently linked to a large caspase-2 subunit also derived from caspase-2, optionally the two subunits are linked via a linker sequence comprising one or more and up to 12 amino acids or more, as long as the remaining cp caspase 2 is still a functional active variant of caspase 2 or cp caspase 2. According to a specific example, the small subunit of cp caspase-2 is derived from wild-type caspase-2 spanning amino acid residues 348 to 452 of the amino acid sequence of wild-type caspase-2 (SEQ ID No. 11). Specifically, the small caspase-2 subunit comprises the amino acid sequence of SEQ ID No. 3 or a variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 3.
Specifically, a variant of the small caspase-2 subunit described herein is functionally active, when direct or indirect fusion or combination with a large caspase-2 subunit or variant thereof results in a functionally active caspase-2 variant.
Specifically, the small subunit of the cp caspase-2 described herein comprises the amino acid sequence of SEQ ID No. 3, SEQ ID No. 91, SEQ ID No. 94, SEQ ID No. 97, SEQ ID No. 100, SEQ ID No. 103, SEQ ID No. 106, SEQ ID No. 109, SEQ ID No. 112, SEQ ID No. 115, SEQ ID No. 118, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity.
The term “modified small caspase-2 subunit pro-peptide” as used herein refers to the pro-peptide of the small subunit of caspase-2, which has been modified at its C-terminus. According to a specific example, the pro-peptide of the small subunit of cp caspase-2 is derived from wild-type caspase-2 spanning amino acid residues 334 to 347 of the amino acid sequence of wild-type caspase-2 (SEQ ID No. 11), comprising an amino acid substitution or deletion of at least one residue at its C-terminus. Specifically, the pro-peptide of the small subunit as described herein comprises the amino acid sequence of SEQ ID No. 2, wherein X can be any amino acid, preferably it is not D and preferably it is not E, even more preferably it is A, or a variant thereof having 1, 2 or 3 amino acid substitutions or 1, 2 or 3 amino acid deletions or additions.
The term “large caspase-2 subunit” as used herein, refers to a large subunit, derived from caspase-2, which is covalently linked to a small caspase-2 subunit also derived from caspase-2, optionally linked via a linker sequence. According to a specific example, the large subunit of cp caspase-2 is derived from wild-type caspase-2 spanning amino acid residues 170 to 333 of the amino acid sequence of wild-type caspase-2 (SEQ ID No. 11). Specifically, the large caspase-2 subunit comprises the amino acid sequence of SEQ ID No. 4 or a variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID No. 4.
Specifically, a variant of the large caspase-2 subunit described herein is functionally active, when direct or indirect fusion to or combination with a small caspase-2 subunit or variant thereof results in a functionally active caspase-2 variant.
Specifically, the large subunit of the cp caspase-2 described herein comprises the amino acid sequence of SEQ ID No. 4, SEQ ID No. 90, SEQ ID No. 93, SEQ ID No. 96, SEQ ID No. 99, SEQ ID No. 102, SEQ ID No. 105, SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or a functionally active variant thereof comprising at least at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity.
Further provided herein is a functionally active variant of the cp caspase-2 as described herein, which is essentially identical to the cp caspase-2 described above, but differs from its polypeptide or the nucleotide sequence, respectively, in that it is derived from a homologous sequence of a different species.
As used herein the term “catalytically active” refers to the ability of the caspase described herein to catalyze the hydrolysis of the substrate's peptide bond. Caspases are endopeptidases capable of forcing formation of a tetrahedral intermediate by promotion of a cysteine residue to act as a nucleophile in order to cleave its substrate. Specifically, the cp caspase-2 described herein is catalytically active and is capable of specifically cleaving its substrate at the caspase recognition site as described herein. Specifically, the cp caspase-2 described herein is catalytically active upon dimerization, comprising two single chain cp caspase-2 units as described herein. Specifically, the cp caspase-2 described herein is catalytically active irrespective of proteolytic cleavage of its subunits or pro-peptide, more specifically its small caspase-2 subunit pro-peptide is not cleaved at its C-terminus. Therefore, the cp caspase-2 described herein is not a zymogen, since it does not require activation through cleavage, neither through an activating enzyme nor through autocatalytic cleavage.
Activation of wild-type caspase-2 requires cleavage at the C-terminus of the large subunit and consequent separation of the small and large subunit. In mature wild-type caspase-2, the pro-peptide of the small subunit is removed by cleavage at its C-terminus. Surprisingly, the cp caspase-2 described herein, does not require cleavage between the subunits for activation. Despite modifications that prevent cleavage between the subunits as well as separation of the C-terminus of the propeptide of its small subunit, single-chain cp caspase-2 as described herein is catalytically active.
Specifically, two single-chain cp caspases-2 as described herein dimerize via covalent linkage, specifically via one or more disulfide bonds. Specifically, dimerization, however, can also be independent of disulfide bond linkage.
Specifically, the catalytic efficiency of a protease is defined as the rate of hydrolysis and can be determined using the Michaelis-Menten equation (kcat/KM). The Michaelis constant, KM, is equal to the substrate concentration at which the enzyme converts substrates into products at half its maximal rate and hence is related to the affinity of the substrate for the enzyme. The catalytic constant (kcat) is the rate of product formation when the enzyme is saturated with substrate and therefore reflects the enzyme's maximum rate. The rate of product formation is dependent on both how well the enzyme binds substrate and how fast the enzyme converts substrate into product once substrate is bound. An equation with a low KM value indicates a large binding affinity, as the reaction will approach Vmax, the maximal rate of the reaction, more rapidly. An equation with a high KM indicates that the enzyme does not bind as efficiently with the substrate, and Vmax will only be reached if the substrate concentration is high enough to saturate the enzyme. The catalyst rate constant (kcat) measures the number of substrate molecules turned over by enzyme per second. The reciprocal of kcat is then the time required by an enzyme to turn over a substrate molecule. The higher the kcat is, the more substrates get turned over in one second. When kcat is divided by KM, a measure of enzyme efficiency is obtained. The enzyme efficiency can be increased as kcat has high turnover and a small number of KM.
Specifically, a comparison of catalytic efficiency constants is used as a measure of the preference of an enzyme for different substrates, i.e. substrate specificity. The higher the specificity constant, the more the enzyme “prefers” that substrate.
Specifically, catalytic activity of the caspase-2 or cp caspase-2 described herein can be measured by examining cleavage of the caspase substrate. Specifically, cleavage activity of the caspase described herein can be examined by methods well known in the art. According to a specific example but not limited thereto, cleavage of the caspase substrate can be examined by eye on an SDS-PAGE gel or by densitometric scanning. Specifically, catalytic activity of the caspase described herein is analyzed with SDS-PAGE to separate cleaved and uncut substrate from the caspase and band intensities of cleaved substrate are determined to evaluate the percentage of cleavage product at a specific time point. Specifically, caspase and substrate are mixed and at timed intervals samples are taken and the reaction is stopped. Preferably, to standardize the process only samples with about 50% of cleaved substrate are used.
According to a further specific example, cleavage activity of the caspases described herein is determined using a Förster resonance energy transfer (FRET) assay.
According to another specific example but not limited thereto, cleavage of the caspase substrate can be examined by measuring the increase in fluorescence when a peptide substrate, encompassing a recognition sequence for the caspase described herein, a fluorophore and a quencher, is cleaved by said caspase. Specifically, caspase and substrate are mixed at defined concentrations and the increase in fluorescence is monitored for a certain time. This fluorescence increase can be used to calculate the rate of product generation, which is then used to fit a Michaelis-Menten kinetic. The resulting Michaelis-Menten parameters kcat and KM can be used to define the catalytic efficiency of the caspase.
The term “single-chain” as used herein refers to a polypeptide comprising a linear chain of amino acids. A protein contains at least one long polypeptide, specifically a polypeptide comprising a linear chain of more than 100 amino acids. Short polypeptides, containing less than 20-30 residues are commonly called peptides, or sometimes oligopeptides. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. Specifically, as used herein, the term “single-chain” refers to a protein which is active irrespective of proteolytic cleavage within its amino acid sequence.
In the fully mature wild-type caspase-2 the small subunit is reduced from a p14 to a p12 chain by cleavage after the recognition site CEESD (residues 343 to 347 of SEQ ID No. 11, residues 17 to 21 of SEQ ID No. 6). The pro-peptide of the small subunit of wild-type caspase-2 is thus separated from the small subunit by proteolytic cleavage after the recognition site CEESD. According to a specific embodiment, the C-terminal amino acid of the small subunit pro-peptide of the cp caspase-2 is modified to prevent separation of the pro-peptide of the small subunit of cp caspase-2 from the small subunit. Specifically, amino acid residue 21 of SEQ ID No. 6 is substituted with any amino acid but aspartic acid (D) or glutamic acid (E). Specifically, amino acid residue 21 of SEQ ID No. 6 is selected from the group consisting of alanine (A), arginine (R), asparagine (N), cysteine (C), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V). Specifically, the C-terminal aspartic acid of the propeptide of the small subunit of cp caspase-2 is substituted with any amino acid residue but aspartic acid or glutamic acid, preferably with alanine, to ensure expression of the cp caspase-2 described herein as a single protein chain.
As described herein, the caspases of the invention may be used to produce a protein of interest (POI) comprising an authentic N-terminus. The term “authentic N-terminus” as used herein refers to the desired N-terminus of a protein to be produced using the means provided herein. In other words, a protein comprises an authentic N-terminus if it comprises the N-terminus that was designed to be generated by the method of recombinant protein production described herein. The authentic N-terminus may be the N-terminus naturally occurring in the protein that is to be produced, or it may be designed artificially, i.e. an N-terminal sequence not naturally occurring in said protein. In a specific example, the P1′ residue is the N-terminal amino acid of the POI and cleavage by the caspase described herein generates an authentic N-terminus.
Low cleavage efficiencies of substrates with sub-optimal P1′ residues or recognition sites can be an issue for applications, in particular large-scale applications, where an authentic N-terminus of the product is desired. Specifically, the caspase described herein has such high activity and efficiency that substrates with all P1′ residues are still cleaved within a reasonable time frame, even for large scale processes. Histidine, for example, is tolerated fifty times less than glycine, still the cp caspase-2 described herein is capable of cleaving 90% of a substrate comprising histidine at the P1′ site at 25° C. within two hours. According to a further specific example, when the concentration of cp caspase-2 is increased, even 50% of a substrate with an isoleucine P1′ residue can be cleaved within two hours.
According to a specific example, variants of caspase-2 comprising improved P1′ tolerance of increased specificity for a predetermined recognition site as described herein can be produced by screening for cp caspase-2 variants capable of efficiently cleaving substrates comprising amino residues at their P1′ site which are not well tolerated by cp caspase-2 such as for example branched (Thr, Leu, Val, Ile) and acidic (Asp, Glu) residues as well as Gln and Pro.
For example, a circularly permuted catalytic subunit of aspartate transcarbamylase (cpATCase) which harbors its new N-terminus in a beta strand located in the interior of the protein is used for the selection of variants of the caspase described herein comprising desired characteristics such as for example increased P1′ tolerance or different or improved recognition site specificity. The respective E. coli gene is named pyrB, the gene product of which forms a complex quaternary structure with the regulatory subunit pyrl in a stoichiometry of 3 regulatory subunit dimers and 2 catalytic subunit trimers. This cp enzyme is used to detect specific proteases via the growth of E. coli, because fusion of any stretch of amino acids towards this new N-terminus renders the enzyme inactive as it can no longer fold properly due to space limitations in the interior of the protein. However, if a protease is provided that can exactly cleave off this additional stretch of amino acids, the enzyme gets reactivated. As this is an essential enzyme of the pyrimidine synthesis in E. coli, it is possible to use this reactivation for applying a strong selection pressure. An E. coli mutant that lacks the original ATCase (e.g. by deleting pyrB and pyrl) and carries a plasmid encoding a cpATCase, e.g. cp-pyrB and pyrl provided on a single vector, that is inhibited by a N-terminal fusion sequence harboring a protease recognition site is provided. Thus the E. coli mutant becomes a pyrimidine auxotroph strain which can only survive in media supplemented with pyrimidines or when the cells are complemented with a vector encoding ATCase. The cpATCase can be activated by catalytic (in vivo) cleavage of the N-terminal fusion sequence. If a respective protease is provided via an additional plasmid, the E. coli can grow. Thereby, proteases can be selected that specifically recognize the recognition sites in the N-terminal fusion and/or that have increased tolerance for specific P1′ residues, such as e.g. proline (P).
According to a specific embodiment, the caspase-2 or cp caspase-2 described herein comprises significantly improved specificity for a recognition site other than VDVAD, compared to wild-type caspase-2.
The term “linker” as used herein refers to any amino acid sequence that does not interfere with the function of elements being linked. Linkers may connect e.g., nucleotide sequences, or amino acid sequences. Linkers can be used between the small and large subunit of cp caspase-2 or between caspase-2 or cp caspase-2 and N-terminal or C-terminal tags or between tag sequences. Linkers can also be used in the fusion protein described herein. The linkers may be used to engineer appropriate amounts of flexibility. Preferably, the linkers are short, e.g., 1-20 nucleotides or amino acids or even more and are typically flexible. Amino acid linkers commonly used consist of a number of glycine, serine, and optionally alanine, in any order. Such linkers usually have a length of at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids, as required. Preferably, the linker comprises 1 to 12 amino acid residues, preferably it is a short linker. Preferably the linker is a GS, GGSGG (SEQ ID NO: 278), GSAGSAAGSG (SEQ ID NO: 279), (GS)n, GSGSGSG (SEQ ID NO: 280), GSG or GGGGS (SEQ ID NO: 281) linker or any combination thereof. In some embodiments, the linker comprises one or more units, repeats or copies of a motif, such as for example GS, GSG or G4S.
According to a specific embodiment, the caspase described herein and/or the fusion protein as described herein comprises one or more N-terminal and/or C-terminal tag sequences. Such tag sequence may comprise any number of amino acids of more than 2, 5 or 10 amino acids and up to 20, 50, 100, 200 or more amino acids. Specifically, tag sequences used herein may be any tag sequence known to the person skilled in the art. Specifically, tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags. Specifically, any tag with any function known in the art can be fused to caspase2 or cp-caspase-2.
Affinity tags are amino acid sequences that can be used for example for the purification of proteins where they are attached to (fusion proteins with affinity tag e.g. at its N-terminus). These affinity tags have high affinity to appropriate ligands of a solid support, like chromatography resins or directly to the resins. By selectively binding of the fusion protein having the affinity tag to the particular resin the fusion protein and/or the caspase (caspase-2, cp cspase-2) can be purified highly effective by only one chromatography step. According to a specific embodiment, affinity tag sequences used herein are selected from histidine (His) tag, specifically a poly-histidine tag, arginine-tag, specifically a poly-arginine tag, peptide substrate for antibodies, chitin binding domain, RNAse S peptide, protein A, ß-galactosidase, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, or c-Myc tag or any other tag known to be useful for the efficient purification of a protein it is fused to. Preferably, the tag is a His tag comprising one or more H, specifically a hexahistidine tag. Specifically, fusion proteins comprising a poly-, or hexa-histidine tag (His-tag) can be captured and purified by IMAC, preferably using a Ni-NTA chromatography material.
Solubility enhancement tags can be fused C- or N-terminal to a POI and/or the caspase (caspase-2, cp cspase-2, wild-type or variant) described herein. Solubility enhancement tags can increase the titer of the soluble fusion protein and/or the caspase (caspase-2, cp cspase-2) when expressed in a host cell, e.g. a bacterial cell, e.g. E. coli significantly, e.g. in the cytosol of E. coli, compared to expression of the proteins without the tag. According to a further specific embodiment, solubility enhancement tag sequences used herein are selected from calmodulin-binding peptide (CBP), poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin or any other tag known to improve the solubility of the protein it is fused to e.g. during expression in a host cell. Preferably the solubility tag is based on highly charged peptides of bacteriophage genes, for example such as those listed in U.S. Pat. No. 8,535,908 B2. Specifically, the solubility enhancement tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC T3, N1, N2, N3, N4, N5, N6, N7, calmodulin-binding peptide (CBP), poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, DsbA, DsbC and thioredoxin.
Preferably, the solubility enhancement tag is selected from the group consisting of T7A3 tag and T7AC tag. According to a specific example, the tag is a modified T7A3 tag, herein referred to as T7AC (SEQ ID No. 43). Preferably, one or more T7A3 (SEQ ID No. 37) and/or T7AC (SEQ ID No. 43) tags or functional variants thereof having 1-5 amino acid substitutions, additions, dilutions or the like, are used.
According to a further specific embodiment, the monitoring tag sequence used herein is m-Cherry, GFP or f-Actin or any other tag useful for detection or quantification of the caspase and/or the fusion protein during production of the caspase and/or the fusion protein including fermentation, isolation and purification by simple in-situ, inline online or atline detectors, like UV, IR, Raman, Fluorescence and the like.
The caspase described herein and the fusion protein described herein may comprise any number of tag sequences in any order and any combination. Specifically, the caspase described herein and the fusion protein described herein may comprise one or more tag sequences of the same functionality, for example more than one affinity tag, e.g. two or more T7AC tags, or of different functionality, e.g. a T7AC affinity tag and an m-Cherry monitoring tag. Specifically, the caspase or the fusion protein described herein may comprise an affinity tag, a solubility enhancement tag and a monitoring tag in any order, optionally separated by linker sequences. For example, the caspase or the fusion protein described herein may comprise an affinity tag and a solubility enhancement tag, wherein the affinity tag preferably is a hexahistidine tag and the solubility enhancement tag preferably is a T7AC tag. According to a further example, the tag sequences may be separated by a linker sequence as described herein and said linker sequence may optionally comprise a recognition site for specific cleavage by the caspase described herein.
The term “functional variant” or “functionally active variant” also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, an allelic variant, or also referred to as homologue, is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or more nucleotides or amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide. Specifically, a functional variant may comprise a substitution, deletion and/or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, or a combination thereof, which substitutions, deletions and/or additions are conservative modifications and do not alter the enzyme's function. Specifically, a functional variant as described herein comprises no more than or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, deletions and/or additions, which are conservative modifications and do not alter the enzyme's function. Specifically, a functionally active variant as described herein comprises up to 15, preferably up to 10 or 5, amino acid substitutions, deletions and/or additions, which are conservative modifications and do not alter the enzyme's function.
Specifically, a functionally active variant described herein comprises at least 5% or at least 10, 20, 30 or 40, 50, 60, 70, 80 or 90% or even more of the proteolytic activity of cp caspase-2 comprising SEQ ID No. 6 for the recognition site VDVAD, wherein glycine (G) is in the P1′ position. Specifically, functionally active variants described herein comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 90% or more of the proteolytic activity of cp caspase-2 comprising SEQ ID No. 6 for the recognition site VDVAD of the substrate VDVAD-E2 (SEQ ID No. 33). Specifically, the proteolytic activity is determined using a Förster resonance energy transfer (FRET) assay.
Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence, e.g. by one or more point mutations, wherein the sequence alterations retain or improve a function of the unaltered polypeptide or the nucleotide sequence, when used in combination of the invention. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.
A point mutation is particularly understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the non-engineered amino acid sequence in the substitution or exchange, deletion or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.
The term “sequence identity” as used herein is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%.
Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50%) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.
Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry. Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different organisms or species.
To determine the % complementarity of two complementary sequences, one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.
“Percent (%) identity” with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
For purposes described herein, the sequence identity between two amino acid sequences can be determined using NCBI BLAST, specifically NCBI BLAST+2.9.0 program version (Apr. 2, 2019).
“Percent (%) identity” with respect to a nucleotide sequence e.g., of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).
According to a specific embodiment, the caspase provided herein is used for the production of a mature and/or functional protein or polypeptide of interest. Specifically described herein is a process for the production of a mature protein or polypeptide by producing it as a fusion protein comprising an N-terminal fusion sequence, wherein the fusion sequence comprises an engineered recognition site specifically recognized by the caspase described herein and wherein upon proteolytic cleavage by the caspase a mature and/or functional protein of interest is released.
Fusion protein strategies for enhancing expression level, improving solubility and facilitating purification of a protein of interest have been around since 1983 and before. However, these strategies are not used widely and adaptation of a fusion protein strategy for large-scale process development is difficult in regard of the specificity, activity, availability and purity of the protease enzyme used. The specificity needs to be high enough to at least allow a number of proteins to be cleaved only at the engineered cleavage site in the connecting linker sequence. The activity of the enzyme needs to be high enough to allow sufficient cleavage in a short period of time. This avoids hold-up time during the production and minimizes degradation of the protein of interest during incubation. The protease needs to be available at low cost, so an efficient expression system and a low-cost production method are necessary. The protease should also be sufficiently pure, especially free of even trace contamination of non-specific proteases from the host organism. No protease will fit these requirements for all possible proteins of interest, therefore an easy and effective way of adapting proteases to different POIs is necessary.
Specifically described herein is a method for producing a POI having a predetermined N-terminal amino acid residue, comprising: expressing the POI in a host cell as a fusion protein, wherein the N-terminus of the POI is fused to fusion sequence comprising a caspase recognition site, the fusion protein being specifically cleavable by the cp caspase-2 described herein at the junction of the linker with the N-terminal amino acid residue of the POI. Specifically, the host cell does not express an endogenous functional protease capable of cleaving the fusion protein at the recognition site. According to the method specifically described herein the fusion protein is isolated from the host cell and the fusion protein is contacted with an extract containing the cp caspase-2 described herein which cleaves the fusion protein exactly at the junction of the linker and the N-terminal amino acid residue of the POI, thereby producing a mature POI. Specifically, said extract comprising the caspase is derived from cells which produce said caspase by recombinant DNA methods.
With regard to the protein or polypeptide of interest (POI) there are no limitations. More specifically, the protein may either be a polypeptide not naturally occurring in the host cell, i.e. a heterologous protein, or else may be native to the host cell, i.e. a homologous protein to the host cell, but is produced, for example, upon integration by recombinant techniques of one or more copies of the nucleic acid sequence encoding the homologous POI into the genome or chromosome of the host cell, or by recombinant modification of the promoter sequence controlling the expression of the gene encoding the POI. According to a further example, the POI can also be expressed in a host using a vector, more specifically a plasmid. The POI can be a monomer, dimer or multimer, it can be a homomer or heteromer. Examples for proteins that can be produced by the method of the invention are, without limitation, enzymes, regulatory proteins, receptors, growth factors, hormones, peptides, e.g. peptide hormones, cytokines, membrane or transport proteins. The POIs may also be antigens as used for vaccination, vaccines, antigen-binding proteins, immune stimulatory proteins, interleukins, interferons, allergens, full-length antibodies or antibody fragments or derivatives or affinity scaffolds. Antibody derivatives may be for example, but not limited to single chain variable fragments (scFv), Fab fragments or single domain antibodies or camelid antibodies or heavy chain antibodies or derivatives thereof such as VHH fragments or the like.
As used herein the term “fusion protein” refers to a POI comprising at its N- or C-terminus an engineered fusion sequence comprising a caspase recognition site as described herein. Specifically, the fusion sequence described herein comprises at least one caspase recognition site, one or more tag sequences as described herein and optionally one or more linker sequences as described herein. According to a specific example, the fusion protein comprises one or more tag sequences, optionally linked via linker sequences, one or more caspase recognition sites and one or more POIs.
According to a specific embodiment, the fusion protein provided herein comprises a first part, comprising one or more tag sequences optionally linked via linker sequences, a second part, comprising a recognition site for target-specific proteolytic cleavage using the cp caspase-2 described herein and a third part, comprising a POI. Specifically, the fusion protein described herein may comprise each part more than once and in different order. For example, the fusion protein provided herein may comprise a first part comprising a tag sequence, a second part comprising a caspase recognition site, another first part comprising the same or a different tag sequence, another second part comprising the same or a different recognition site and a third part comprising a POI. According to a further example, the fusion protein described herein may comprise more than one POI separated by one or more fusion sequences comprising one or more recognition sites.
The cp caspase-2 or caspase-2 itself as described herein can be part of a fusion protein as the POI or part of the fusion sequence to e.g. facilitate production of the caspase itself.
The fusion protein described herein is encoded by a heterologous gene which is engineered in such a way that it is translated into protein by a host organism. As a host organism, any living cell or organism applies. Living cells or organisms can be of prokaryotic or eukaryotic nature. Common cells that serve as hosts for expression of recombinant genes are e.g. Escherichia coli, Bacillus species, Streptomyces species, Yeast strains such as Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces or Hansenula strains, insect cells, mammalian cell lines, plant cells. Expression hosts can also be at the level of a multicellular organism such as transgenic plants, sheep, goat, cow, chicken and rabbit, whereby the product can be isolated either from organs or from body fluids such as milk, blood or eggs. Alternatively, the gene can be translated into protein using cell free translation systems, possibly coupled to an in vitro transcription system. These systems provide all steps necessary to obtain protein from DNA by supplying the necessary enzymes and substrates in an in vitro reaction. In principle, any living cell or organism can provide the necessary enzymes for this process and extraction protocols for obtaining such enzyme systems are known in the art. Common systems used for in vitro transcription/translation are extracts or lysates from reticulocytes, wheat germ or Escherichia coli.
According to a specific embodiment, the fusion protein is isolated and purified before cleavage with the cp caspase-2 described herein. The physicochemical features of the fusion sequence, comprising one or more tag sequences, can be used for uniform, streamlined and highly specific purification of the fusion protein. The characteristics of the fusion sequence towards adsorption chromatographic medium, or specific affinity purification methods should be considered. For example, tag sequences can be included that increase the binding to ion exchange columns (e.g. poly-arginine), hydrophobic interaction columns (e.g. poly-phenylalanine), or immobilized metal chelating chromatography (e.g. poly-histidine). Other non-limiting examples are fusion protein or domains that have an affinity for a substrate or ligand e.g. maltose binding protein MBP, glutathione S transferase GST, protein A, biotinylated peptides or domains, chitin binding domains CBD. Further non-limiting examples are the use of tag sequences that increase solubility at higher temperatures (e.g. thioredoxin), or will reversibly precipitate at certain conditions. A purification scheme based on the properties of the fusion sequence will most probably be applicable to the complete fusion protein. A combination of such specific purification methods can be used if the fusion sequence comprises different tag sequences with different functionalities, or when they show a different selective behavior on different chromatography media.
According to a specific embodiment the number of steps needed in the maturation of the fusion protein, subsequent removal of the enzyme and removal of the fusion sequence cut from the fusion protein can be reduced. If an affinity tag is incorporated in the fusion sequence, the same affinity tag can be fused, e.g. by recombinant DNA technology, to the caspase. Using this strategy, the fusion protein can be captured on a solid support, for example a chromatographic column, and then incubated with the cp caspase-2 described herein fused to an affinity tag that shows affinity for the same solid support. After an appropriate incubation time, the liquid phase of the reaction vessel will contain the protein of interest, while both the fusion part and the enzyme are adsorbed on the solid phase.
According to a further specific embodiment, cleavage of the fusion protein is induced in vivo. Cleavage in the cell has the advantage that no post-productional processing is needed. However, the advantage of a specific affinity purification based on the properties of the fusion part is lost in this case. Specifically, two alternative strategies can be applied. First, the caspase may be induced at the same time as the fusion protein, e.g. using an expression cassette comprising both the caspase and the fusion protein, or by engineering a fusion protein including the caspase as part of the fusion protein, or by using expression vectors comprising the caspase and the fusion protein under separate promoters which are induced at the same time. The latter can be realized by using the same promoter in two transcriptional cassettes, or by using two promoters that are induced with the same inducer (e.g. IPTG/lactose), or by using two promoters, that are inducible with different agents, whereby both agents are added at the same time. Alternatively, the caspase enzyme can be induced at a different time point than the onset of production of the fusion protein. The caspase can be produced before or more preferably after the production onset of the fusion protein. In the latter case, the protein of interest will more likely fold to a soluble, active protein.
The terms “mature form” or “mature protein” of interest refer to the polypeptide of interest in its desired form, without pre-peptides, leader sequences or fusion sequences. Preferably, in its mature form the protein is starting with the amino terminal amino acid or ending with the carboxy terminal amino acid of the POI occurring under its biological active or functional form. Specifically, the mature protein comprises an authentic N- or C-terminus, which is the desired N- or C-terminus.
Further provided herein is a method of producing a cp caspase-2 or a fusion protein as described herein. The cp caspase-2 produced according to the method described herein may comprise SEQ ID No. 6 or comprises amino acid substitutions with reference to SEQ ID No. 6. Specifically, said cp caspase-2 may be derived from wild type caspase-2.
Specifically, the fusion protein comprises a POI, which may be a caspase-2 as described herein, and a protein tag as described herein. Use of the protein tag as described herein significantly increases expression of the fusion protein and improves production of the POI.
Specifically, the method of producing a cp caspase-2 described herein, allows more efficient production of the caspase and the caspase produced according to said method comprises improved characteristics, such as e.g. improved P1′ tolerance or improved target specificity.
According to a specific example, but not limited thereto, wild-type or variant caspase-2 or cp caspase-2 or a fusion protein as described herein is produced in a fermentation process comprising 2 phases. Specifically, the 2 phases comprise:
i.Biomass production: For biomass production to a certain concentration of biomass, the first fed-batch phase can be performed with an exponential feed (exponential substrate feed) at a specific growth rate (μ) of 0.05-0.5 h−1 or 0.05-0.4 h−1 preferably at a μ of 0.07-0.3 h−1 or 0.1-0.3 h−1 or 0.1-0.2 h−1, even more preferably at a μ of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20 h−1. preferably about 0.13-0.21 h−1, even more preferably about 0.16-0.18 h−1 and most preferably it is about 0.17 h−1 Also, any other feed mode appropriate for the formation of a certain amount of biomass can be applied, such as but not limited to step-feed, linear increasing feed, or constant feed. The substrate feed can be controlled by increasing pump speed according to the exponential growth algorithm, X=X0*eμt, with superimposed feedback control of weight loss in the substrate tank. Specifically, the substrate feed comprises glucose or glycerin or any other carbon-source and optionally comprises Ca2+, Mg2+ and/or trace elements. In a preferred embodiment, the first fed-batch phase was performed for 0.5-2.5 generations, more preferred for 0.7-2.3 generations.
ii. In a second feed-phase with an exponential feed (exponential substrate feed) at a specific growth rate (μ) a lower growth rate, a μ of 0.01-0.1 h−1 or 0.01-0.07 h−1, preferably a μ of 0.01-0.03 h−1 or 0.01-0.05 h−1 or 0.02-0.05 h−1 or 0.03-0.05 h−1 or 0.03-0.07 h−10.05-0.07 h−1, and even more preferably a μ of about 0.03, 0.05 or 0.07 h−1, can be applied. For adaption to the low growth conditions, the cells can initially be grown at the low μ without induction, e.g. for about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50 generations. Subsequently an inducer, e.g. IPTG for the T7 promoter/operator system can be added. Isopropyl β-d-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and is used to induce protein expression where the gene is under the control of the lac operator. Induction can be done with different or varying IPTG concentrations ranging from 0.01-1.5 or 0.1-1.5 μmol/g CDM (cell dry mass) more preferably 0.1-1.3 or 0.2-1.3 or 0.3-1.3 or 0.5-1.3 μmol/g CDM even more preferably ranging from about 0.5-0.9 μmol/g CDM or about 0.9-1.3 μmol/g actual CDM, preferably it is about 0.5, 0.9 or about 1.3 μmol/g CDM. for one or two or even more generations. Specifically, the fed-batch phases are performed at 30° C.
Thus, induction can be performed as follows: Induction starts with fed-batch phase by adding feed medium including IPTG (so called “over feed” induction, table 20) to achieve a final IPTG concentration as described above in μmol IPTG/g theoretical CDM at the end of the fermentation.
In another embodiment IPTG corresponding to the CDM at induction time (μmol/g DCM), can be injected into the reactor and then IPTG calculated to the actual CDM can be fed into the fermenter within the feed medium. To that end the needed IPTG can be transferred into the feed bottle calculated to the IPTG needed until the theoretical CDM at the end of fermentation. Thus, the IPTG concentration related to the theoretical CDM is constant throughout the whole fermentation.
The produced caspase or fusion protein can be isolated by cell disintegration e.g. by high pressure homogenization, centrifugation of the cell debris, concentration of the supernatant by tangential flow micro-filtration or the like. Further purification can be done by chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, isoelectric focusing, mixed mode chromatography reversed phase high performance chromatography, tangential flow microfiltration, depth filtration, ammonium sulphate, -chloride, -citrate precipitation heat precipitation, solubilization, crystallization, centrifugation or the like. Specifically, when the cp caspase-2 comprises an affinity tag, it can be purified highly effectively by only one chromatography step, which is an affinity chromatography step. Preferably, the affinity tag is a 6 His tag and the affinity chromatography is an IMAC, more specifically a Ni-NTA chromatography.
Specifically, using said method the cp caspase-2 with or without tags and/or linkers as described herein can be produced. Specifically, the cp caspase-2 produced according to the method described herein comprises significantly improved specificity for the recognition site VDVAD (SEQ ID No. 45) compared to wild-type caspase-2. Specifically, a cp caspase-2 comprising the exemplary amino acid sequence of SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 13, SEQ ID No. 35, SEQ ID No. 39 or SEQ ID No. 41 recognizes and cleaves substrates comprising the recognition site VDVAD with significantly improved specificity compared to wild-type caspase-2. Such increased specificity has the distinct advantage that it leads to a significant reduction of off-target effects and avoids proteolytic cleavage of the target substrate or other proteins within the host at sites other than the recognition site. Specifically, the cp caspase-2 described herein is at least 2 times, preferably at least 3 times, more specific for the recognition site VDVAD than wild-type caspase-2.
Further provided herein is a method of producing a POI using the protein tag described herein. Specifically, the POI is fused to the protein tag and cloned into an expression vector under operable linkage to a promoter, which may be an inducible promoter. Said expression vector is integrated into a host cell and the host cell is cultured under conditions allowing expression of the fusion protein, optionally following a growth phase for the accumulation of biomass before the recombinant protein is expressed. The POI may be produced employing a fed-batch process as described herein, comprising an expression phase as described herein and optionally a growth phase as described herein.
According to a specific embodiment of the method of producing a POI as described herein, the fusion protein is contacted with a caspase-2 or cp caspase-2 as described herein after expression, to produce a POI comprising the desired N-terminus, i.e. the natural or designed N-terminus without any unwanted tags attached. Specifically, the fusion protein is contacted with the caspase enzyme after isolation of the fusion protein from the host cell culture.
After production of the POI according to the method described herein, the POI may be further modified, purified and/or formulated.
The methods described herein specifically refer to the production of heterologous compounds. Such term used with respect to a nucleotide or amino acid sequence or protein, refers to a compound which is either foreign, i.e. “exogenous”, such as not found in nature, to a given host cell; or that is naturally found in a given host cell, e.g., is “endogenous”, however, in the context of a heterologous construct, e.g., employing a heterologous nucleic acid, thus “not naturally-occurring”. The heterologous nucleotide sequence as found endogenously may also be produced in an unnatural, e.g., greater than expected or greater than naturally found, amount in the cell. The heterologous nucleotide sequence, or a nucleic acid comprising the heterologous nucleotide sequence, possibly differs in sequence from the endogenous nucleotide sequence but encodes the same protein as found endogenously. Specifically, heterologous nucleotide sequences are those not found in the same relationship to a host cell in nature (i.e., “not natively associated”). Any recombinant or artificial nucleotide sequence is understood to be heterologous.
As used herein the term “host cell” refers to one or more cells which can be used in the methods described herein. Typically, the term refers to viable cells, capable of growing in a cell culture, into which a heterologous nucleic acid sequence or amino acid sequence is introduced. Specifically, the host cells are selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells. Mammalian cells used in accordance with the present disclosure typically are human or rodent cells, such as mouse, rat or hamster cells, such as for example Chinese Hamster Ovary (CHO) cells. Preferably the host cells are bacterial or yeast cells selected from the group consisting of E. coli, Pseudomonas sp., Bacillus sp., Streptomyces sp., Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Kluyveromyces sp. and Hansenula sp.
The term “expression” is understood in the following way. Nucleic acid molecules containing a desired coding sequence of an expression product such as e.g., a fusion protein as described herein or a cp caspase-2 as described herein may be used for expression purposes. Hosts transformed or transfected with these sequences are capable of producing the encoded proteins. In order to effect transformation, the expression system may be included in a vector; however, the relevant DNA may also be integrated into the host chromosome. Specifically, the term refers to a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular polypeptide or protein. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, one or more nuclear localization signals (NLS) and one or more expression cassettes.
“Expression vectors” or “vectors” as used herein are defined as DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism. To obtain expression, a sequence encoding a desired expression product, such as e.g. the fusion protein described herein or the cp caspase-2 described herein, is typically cloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art. The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the expression product is to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the expression product. In addition, a preferred promoter for administration can be a weak promoter. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements and lac repressor response elements. Expression vectors comprise the expression cassette and additionally usually comprise an origin for autonomous replication in the host cells or a genome integration site, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring resistance to antibiotics such as zeocin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.
An “expression cassette” refers to a DNA coding sequence or segment of DNA coding for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct”.
The term “vector” as used herein includes autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Specifically, the term “vector” or “plasmid” refers to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
Expression products, such as the caspase-2 or cp caspase-2 described herein, can be expressed from an autonomously replicating nucleotide sequence, or from nucleotide sequences stably integrated into the genome of a host cell.
Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al.).
According to specific embodiments, the fusion protein or the cp caspase-2 described herein are expressed as inclusion body. Methods for the purification of recombinant proteins expressed as inclusion bodies are well known in the art. Typically, 70 to 80% of recombinant proteins expressed in bacteria, such as e.g. E. coli, are contained in inclusion bodies. Specifically, the purification of the expressed proteins from the inclusion bodies requires two main steps: extraction of inclusion bodies from the bacteria, for example via cell lysis followed by affinity purification, followed by solubilization and optionally refolding of the purified inclusion bodies.
Further described herein is a pharmaceutical composition comprising the cp caspase-2 or caspase-2 provided herein. According to a specific embodiment, such pharmaceutical composition comprising the cp caspase-2 or its variants as described herein is used for the treatment of for example cancer, Alzheimer's disease, Parkinson's disease or inflammatory disease. Specifically, the pharmaceutical composition described herein further comprises pharmaceutically acceptable carriers or excipients, such as for example bulking agents, when used for diagnosis or therapy. These pharmaceutical compositions can be administered in accordance with the present invention as a bolus injection or infusion or by continuous infusion. Pharmaceutical carriers suitable for facilitating such means of administration are well-known in the art.
Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are physiologically compatible with a caspase provided by the invention. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof.
Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., Remington's Pharmaceutical Sciences (Gennaro, A R, ed., Mack Publishing Co, 1985). Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.
Exemplary formulations as used for parenteral administration include those suitable for subcutaneous, intramuscular or intravenous injection as, for example, a solution, emulsion or suspension.
The caspase-2 or cp caspase-2 described herein is specifically administered at a therapeutically effective amount, meaning a quantity or activity sufficient to effect beneficial or desired results, including clinical results, when administered to a subject, e.g. a patient suffering from cancer. As such, an effective amount or synonymous quantity thereof depends upon the context in which it is being applied. An effective amount is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit such diseases or disorders.
The amount of the compound that will correspond to such an effective amount will vary depending on various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
The caspase-2, variants and dimers thereof described herein are particularly provided in the isolated form, which are substantially pure, meaning free of other proteins or enzymes. Still, such isolated enzyme may be comprised in a combination preparation, containing a combination of the isolated cp caspase-2, e.g., with at least one other enzyme or protein or antibody, such as monoclonal antibodies or antibody fragments. The term “substantially pure” or “purified” as used herein shall refer to a preparation comprising at least 50% (w/w), preferably at least 60%, 70%, 80%, 90%, or 95% of a compound, such as a caspase or a POI. Purity is measured by methods appropriate for the compound (e.g., chromatographic methods, polyacrylamide gel electrophoresis, HPLC analysis, and the like).
The following items are particular embodiments described herein.
1. A single-chain circular permuted caspase-2 (cp caspase-2) comprising the following structure from N- to C-terminus:
2. The cp caspase-2 of item 1 comprising one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or any combination thereof.
3. The cp caspase-2 of item 1 or 2, comprising a propeptide of a small caspase-2 subunit (SS propeptide), fused to the N-terminus of the small subunit.
4. The cp caspase-2 of item 3, wherein the SS propeptide comprises one or more amino acid substitutions at the C-terminus of the SS propeptide.
5. The cp caspase-2 of item 3 or 4, wherein the SS propeptide comprises an amino acid substitution at position Asp14 of SEQ ID No. 2 or at a position functionally equivalent to Asp347 of SEQ ID No. 11, specifically Asp is substituted to Ala.
6. The cp caspase-2 of any one of items 1 to 5, further comprising one or more linker sequences, specifically consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues.
7. The cp caspase-2 of item 6, wherein the linker sequence comprises glycine and/or serine residues, more specifically the linker is GS, GGSGG, GSAGSAAGSG, (GS)n, GSG or G4S.
8. The cp caspase-2 of items 6 or 7, wherein the linker sequence is a subunit-linker sequence between the small subunit and the large subunit.
9. The cp caspase-2 of any one of items 1 to 8, comprising one or more C-terminal or N-terminal tags, specifically selected from the group consisting of affinity tags, solubility enhancement tags and monitoring tags.
10. The cp caspase-2 of item 9, wherein the affinity tag is selected from the group consisting of poly-histidine tag, poly-arginine tag, peptide substrate for antibodies, chitin binding domain, RNAse S peptide, protein A, ß-galactosidase, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, c-Myc tag, SUMO tag, E. coli thioredoxin, NusA, chitin binding domain CBD, chloramphenicol acetyl transferase CAT, LysRS, ubiquitin, calmodulin, and lambda gpV, specifically the tag is a His tag comprising one or more His, more specifically it is a hexahistidine tag.
11. The cp caspase-2 of item 9, wherein the solubility enhancement tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T3, N1, N2, N3, N4, N5, N6, N7, T7AC, calmodulin-binding peptide (CBP), DsbA, DsbC, poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin.
12. The cp caspase-2 of item 9, wherein the monitoring tag is selected from the group consisting of m-Cherry, GFP and f-Actin.
13. The cp caspase-2 of any one of items 9 to 12, comprising more than one tags, specifically comprising an affinity tag and a solubility enhancement tag.
14. The cp caspase-2 of item 13, wherein the affinity tag is a hexahistidine tag and the solubility enhancement tag is a T7AC or a T7A3 tag.
15. The cp caspase-2 of any one of items 6 to 14, wherein the linker sequence is a tag-linker sequence, linking two tags or linking a tag and the small subunit, the large subunit or the SS propeptide of the cp caspase-2.
16. The cp caspase-2 of any one of items 1 to 15, comprising one or more N-terminal tags and optionally one or more tag-linker sequences between the tags or between a tag and the N-terminus of the small subunit or the SS propeptide.
17. The cp caspase-2 of any one of items 1 to 16, comprising one or more C-terminal tags and optionally one or more tag-linker sequences, which are linker sequences between the tags or between a tag and the C-terminus of the large subunit.
18. A functionally active variant of the cp caspase-2 of any one of items 1-17, wherein
19. A functionally active variant of the cp caspase-2, comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to the cp caspase-2 of any one of items 1 to 18.
20. The functionally active variant of item 19, comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to SEQ ID No. 9, 6, 14, 15, 16, 80, 88, 25, 26, 27, 28, 29, 30, 35, 39, 41, 64, 66, 68, 73, 74, 75, 76, 77, 81, 82, 83, 84, or 85.
21. The cp caspase-2 of any one of items 1 to 20, wherein the
4, SEQ ID No. 90, SEQ ID No. 93, SEQ ID No. 96, SEQ ID No. 99, SEQ ID No. 102, SEQ ID No. 105, SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or functionally active variants thereof having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity.
22. The cp caspase-2 of any one of items 1 to 21, comprising
23. The cp caspase-2 of any one of items 1 to 22, comprising one or more amino acid substitutions, selected from
with reference to the positions of SEQ ID No. 6, or positions functionally equivalent to positions of SEQ ID No. 6.
24. The cp caspase-2 of any one of items 1 to 22 comprising amino acid substitutions at positions of SEQ ID No. 6, or at positions functionally equivalent to positions of SEQ ID No. 6, selected from the group consisting of
wherein said cp caspase-2 has increased P1′ tolerance compared to a cp caspase-2 without the respective amino acid substitution, optionally wherein said cp caspase-2 comprises an SS propeptide comprising an amino acid substitution to A1a at position Asp14 of SEQ ID No. 2 or at a position functionally equivalent to position Asp347 of SEQ ID No. 11.
25. The cp caspase-2 of any one of items 1 to 22, comprising SEQ ID No. 6 and one or more amino acid substitutions at position 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6 or at a position functionally equivalent to position 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID No. 6, or any combination thereof.
26. The cp caspase-2 of item 25, comprising any one or more of amino acid substitutions G171 D, E105V, E172V, D282E, D282T, V225G, K83E, H185A, V255M, D285Y and D285E.
27. The cp caspase-2 of any one of items 1 to 26, comprising an amino acid sequence selected from the group consisting of SEQ ID No. 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with any one of SEQ ID No. 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192.
28. The cp caspase-2 of any one of items 1 to 27, comprising a C-terminal tag and an amino acid substitution at positions 285 and 292 of SEQ ID No. 6 or at a position functionally equivalent to positions 285 and 292 of SEQ ID No. 6, specifically comprising substitutions to Glu and Ser (D285E and D292S).
29. The cp caspase-2 of any one of items 1 to 28, wherein said cp caspase-2 is recruited by a recognition site for proteolytic cleavage, comprising 5 amino acids of the sequence P5 P4 P3 P2 P1, wherein
P1 can be any amino acid, preferably it is D or E,
P2 can be any amino acid, preferably it is A,
P3 can be any amino acid, preferably it is V,
P4 can be any amino acid, preferably it is D, and
P5 can be any amino acid, preferably it is V.
30. A method of producing a circular permuted caspase-2 (cp caspase-2) comprising the steps of
31. The method of item 30, wherein the cp caspase-2 is the cp caspase-2 of any one of items 1 to 29.
32. The method of item 30 or 31, wherein the promoter is selected from the group consisting of T7 promoter/operator, XylS/Pm regulator/promoter or variants of the Pm promoter, araBAD promoter/operator, T5, T7A1, T7A2, T7A3 promoter/operator, phoA promoter/regulator and the trp promoter/operator system.
33. The method of any one of items 30 to 32, wherein the cp caspase-2 comprises an solubility enhancement tag, selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC, T3, N1, N2, N3, N4, N5, N6,N7, calmodulin-binding peptide (CBP), DsbA, DsbC, poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin tag, preferably it comprises a T7AC or a T7A3 tag.
34. The method of any one of items 30 to 33, wherein the cp caspase-2 comprises an affinity tag, preferably a His tag, and even more preferably a 6-His tag.
35. The method of any one of items 30 to 34, wherein the host cell is a eukaryotic or prokaryotic host cell, preferably a yeast cell or a bacterial cell, and even more preferably an E. coli cell.
36. The method of any one of items 30 to 35, wherein the cp caspase-2 comprises an N-terminal tag comprising an affinity tag, preferably a His tag and even more preferably a 6-His tag, and a solubility enhancement tag, preferably T7AC or T7A3.
37. The method of item 36, wherein the cp caspase-2 further comprises a linker between the affinity tag and the solubility enhancement tag.
38. The method of items 36 or 37, wherein the cp caspase-2 comprises the following elements fused to its N-terminus, in the order from N- to C-terminus:
39. The method of items 36 or 37, wherein the cp caspase-2 comprises the following elements fused to its N-terminus, in the order from N- to C-terminus:
40. The method of any one of items 30 to 39, wherein culturing of step (iii) comprises a fed-batch phase for expression of the cp-caspase-2, said fed batch phase specifically comprising a growth rate, μ of about 0.01-0.1 h−1, and induction of expression of the cp caspase-2 by addition of IPTG at a concentration of about 0.01-1.5 μmol/g actual CDM (cell dry mass).
41. The method of item 40, wherein the growth rate μ is about 0.03-0.07 h−1, preferably it is about 0.05-0.07 h−1 or 0.03-0.05 h−1, preferably it is any of about 0.03, 0.05 or 0.07 h−1.
42. The method of item 40 or 41, wherein the IPTG concentration is about 0.5-1.3 μmol/g CDM, preferably it is about 0.5-0.9 μmol/g CDM or about 0.9-1.3 μmol/g CDM, preferably it is about 0.5, 0.9 or about 1.3 μmol/g CDM.
43. The method of any one of items 40 to 42, wherein culturing of step (iii) further comprises a first fed-batch phase for the production of biomass, prior to the fed-batch phase for the expression of the cp caspase-2, said first fed-batch phase comprising a growth rate, μ of about 0.07-0.3 h−1.
44. The method of item 43, wherein the growth rate μ is about 0.1-0.2 h−1, preferably about 0.13-0.21 h−1, even more preferably about 0.16-0.18 h−1 and most preferably it is about 0.17 h−1.
45. The method of any one of items 30 to 44, wherein the cp caspase-2 is purified using affinity chromatography, preferably IMAC.
46. A cp caspase-2 obtained by the method of any one of items 30 to 45.
47. A method of producing a protein of interest (POI) comprising an authentic N-terminus, comprising the steps of:
48. A method of producing a protein of interest (POI) comprising an authentic N-terminus, comprising the steps of:
49. The method of item 47 or 48, wherein the fusion protein comprises a caspase recognition site comprising 5 amino acids of the sequence P5 P4 P3 P2 P1, and a cleavage site P1/P1′, wherein P1′ is the N-terminal amino acid of the POI.
50. The method of item 47 or 48, wherein the fusion protein and the cp caspase-2 are under transcriptional control of different promoters and wherein the expression of the cp caspase-2 is induced after expression of the fusion protein.
51. The method of any one of items 47 to 50, wherein the fusion protein comprises the cp caspase-2 of any one of items 1 to 29, specifically wherein the fusion protein comprises the cp caspase-2 of any one of items 1 to 29 at its N- or C-terminus and wherein the fusion protein comprises the following structure from N- to C-terminus:
wherein P1′ is the N-terminal amino acid of said POI and said cp caspase-2 specifically recognizes said recognition site.
52. The method of any one of items 47 to 51, comprising the steps of:
53. The method of item 52, wherein the cp caspase-2 comprises at its N- or C-terminus an affinity tag identical to the affinity tag of the fusion protein and wherein the cp caspase-2 is removed in step vi. using said affinity tag.
54. The method of item 52 or 53, comprising the steps of
55. The method of item 54, wherein the cp caspase-2 and the fusion protein comprise an identical affinity tag, allowing binding of the fusion protein and the caspase on the solid support and release of the POI upon cleavage by the caspase.
56. The method of item 54 or 55, wherein the solid support is a column, specifically a chromatography column, more specifically an immobilized metal affinity chromatography column (IMAC).
57. The method of any one of items 47 to 56, wherein a flow-through reactor comprising immobilized cp caspase-2 of any one of items 1 to 29 is used.
58. An isolated nucleotide sequence encoding the cp caspase-2 of any one of items 1 to 29.
59. A vector comprising the nucleotide sequence of item 58, specifically it is a bacterial expression vector.
60. An expression cassette comprising the nucleotide sequence of item 58 operably linked to regulatory elements.
61. A host cell or a host cell line expressing the cp caspase-2 of any one of items 1 to 29, wherein the host cells are selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells, preferably the host cells are bacterial or yeast cells selected from the group consisting of E. coli, Pseudomonas sp., Bacillus sp., Streptomyces sp., Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Kluyveromyces sp. and Hansenula sp.
62. An expression system comprising the vector of item 59 or the expression cassette of item 60 and a host cell of item 61.
63. Use of the cp caspase-2 of any one of items 1 to 29 for the in vivo cleavage of a substrate in a non-human organism.
64. The use of item 63, wherein the non-human organism is a prokaryotic organism, specifically it is E. coli.
65. Use of the cp caspase-2 of any one of items 1 to 29 for the production of a protein of interest (POI).
66. The use of item 65, wherein the POI comprises an authentic N-terminus.
67. A fusion protein comprising the following structure from N- to C-terminus:
68. The fusion protein of item 67, wherein the tag sequence further comprises one or more tags selected from the group consisting of affinity tags, solubility enhancement tags and monitoring tags.
69. The fusion protein of item 68, further comprising one or more tag-linker sequences.
70. A kit comprising
71. The kit of item 70, further comprising an expression vector, comprising a polynucleotide encoding the protein tag of items 75 to 89.
72. The cp caspase-2 of any one of items 1 to 29, for use in the treatment of a disease.
73. The cp caspase-2 of any one of items 1 to 29, for use in the treatment of cancer, Alzheimer's disease, Parkinson's disease or inflammatory disease.
74. A caspase-2 comprising one or more amino acid substitutions at positions 409, 431, 212, 213, 266, 226, 296, 323 or 326 of SEQ ID No. 11 or at a position functionally equivalent to any of positions 409, 431, 212, 213, 266, 226, 296, 323 or 326 of SEQ ID No. 11 or a combination thereof, wherein said amino acid substitution increases P1′ tolerance compared to a caspase-2 comprising the same sequence but not comprising said amino acid substitutions.
75. A protein tag for enhanced expression of a POI, comprising a solubility enhancement tag and the amino acid sequence VDVAD (SEQ ID NO: 45), wherein the sequence VDVAD is located at the C-terminus of the protein tag.
76. The tag of item 75, wherein the solubility enhancement tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T3, N1, N2, N3, N4, N5, N6, N7 and T7AC.
77. The tag of item 76, wherein the solubility enhancement tag is T7AC or T7A3.
78. The tag of any one of items 75 to 77, further comprising a histidine tag sequence, preferably comprising 1-20 histidine residues, even more preferably it is a 3-His, 6-His or 9-His tag sequence.
79. The tag of any one of items 75 to 78, wherein the solubility enhancement tag is located at the N-terminus of said protein tag.
80. The tag of any one of item 78, wherein the histidine tag sequence is located at the N-terminus of said protein tag.
81. The tag of any one of items 75 to 80, further comprising one or more linker sequences comprising one or more amino acid residues.
82. The tag of item 81, wherein said one or more linker sequences are located between the VDVAD sequence and the solubility enhancement tag and/or the histidine tag sequence.
83. The tag of item 81 or 82, wherein the one or more amino acid residues of the linker sequence are any of the naturally occurring amino acids or derivatives thereof, preferably selected from the group consisting of G, S, A, T and N.
84. The tag of any one of items, 81 to 83, wherein the linker sequence is GSG.
85. The tag of any one of items 81 to 83, wherein the linker sequence is GSGSGSG.
86. The tag of any one of items 75 to 85, further comprising a signal peptide at the N-terminus of said protein tag.
87. The tag of item 86, wherein the signal peptide is selected from the group consisting of ompA (outer membrane protein A), DsbA (Thiol:disulfide interchange protein), MalE (maltose-binding protein), PelB (pectate lyase B) from Erwinia carotovora, PhoA (alkaline phosphatase), OmpC (outer-membrane protein C), OmpF (outer-membrane protein F), OmpT (protease VII), Endoxylanase from Bacillus sp., LamB (λ receptor protein), Lpp (murein lipoprotein), LTB (heat-labile enterotoxin subunit B), PhoE (outer-membrane pore protein E), and Stll (heat-stable enterotoxin 2).
88. The tag of any one of items 75 to 87, wherein the tag comprises one of the following structures from N- to C-terminus:
89. The tag of item 86 or 87, wherein the tag comprises one of the following structures from N- to C-terminus:
90. A fusion protein comprising the protein tag of any one of items 75 to 89 and a POI, wherein the N-terminus of the POI is fused to the C-terminus of said protein tag.
91. The fusion protein of item 90, wherein the N-terminus of the POI is directly fused to the C-terminus of the protein tag, which C-terminus is the sequence VDVAD.
92. A method of producing a POI, comprising the steps of:
93. The method of item 92, further comprising the following steps:
94. The method of item 92 or 93, wherein the promoter is selected from the group consisting of T7 promoter/operator, XylS/Pm regulator/promoter or variants of the Pm promoter, araBAD promoter/operator, T5, T7A1, T7A2, T7A3 promoter/operator, phoA promoter/regulator and the trp promoter/operator system.
95. The method of items 65-66, wherein the host cell is a eukaryotic or prokaryotic host cell, preferably a yeast or a bacterial cell, preferably it is an E. coli cell.
The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Different embodiments of the present invention have been described according to the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the spirit and scope of the invention. Accordingly, it should be understood that the examples are illustrative only and are not limiting upon the scope of the invention.
1.1 Escherichia coli Strains
E. coli BL21 (DE3) was used for all standard protein expressions.
For plasmid extractions and for cloning experiments E. coli strain NovaBlue (Novagen, Madison, Wis., USA) was used as a host.
1.2 Culture Media
TY (tryptone-yeast) medium (1% peptone, 0.7% yeast extract, 0.25% (w/v) NaCl).
TB medium (1.2% peptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 72 mM K2HPO4).
SOC (super optimal broth with catabolite repression) (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 and 20 mM glucose, pH 7.0).
Medium for the recovery of cells after transformation.
Optimized M9 minimal medium (50 mM Na2HPO4, 20 mM KH2PO4 10 mM NaCl, 1 mM MgSO4, 0.1 mM CaCl2, 0.4% Glucose, 20 mM NH4Cl, 0.5% (w/v) casamino acids, 10 μg/ml FeSO4, vitamins (0.001 mg/ml of each biotin, thiamine, riboflavin, pyridoxine, niacinamide). For induction 0.1 to 0.4 mM IPTG were used.
1.3 Recombinant Protein Expression
Standard Expression Protocol: Substrate proteins were expressed in TY medium, induction with 1 mM IPTG, at OD600 1.0 and executed at 37° C., 220 rpm, for 4 h.
Expression protocol for caspases: Caspases were expressed in TB medium, induction was at OD600 1.2 with 0.4 mM IPTG, 25° C., for 4 h.
1.4 Cell Lysis and Protein Purification
Substrates and caspases were purified using Immobilized Metal Affinity Purification (IMAC).
The harvested cell pellets were suspended in Tris-Buffer (50 mM Tris, 50 mM NaCl, pH 7.5), disrupted with a French press and the clarified supernatant applied to an IMAC column (HisTrap FF Crude, 1 ml, GE Healthcare). Washing was executed for five column volumes with running buffer (50 mM Tris/HCl, pH 7.4, 300 mM NaCl, 20 mM Imidazole), the fifth wash fraction had an increased imidazole concentration (40 mM). Elution was conducted for five column volumes with buffer containing 250 mM imidazole.
After affinity-chromatography imidazole and excess NaCl were exchanged to Tris-buffer with a sepharose column (HiTrap Desalting, 5 ml, GE Healthcare). All elution fractions were pooled, the concentration determined with a BCA assay, and the proteins stored in Tris-Buffer with 2 mM DTT at −80° C.
1.5 Testing of Caspases—In Vitro Cleavage Assay
The activity of purified caspases was assessed with an in vitro cleavage assay. The samples were analyzed with SDS-PAGE to separate cleaved and unprocessed substrate. The band intensities were measured with ImageQuant TL 1D software, version 8.1 (GE Healthcare) and used for statistical analysis and calculation of cleavage efficiency. To standardize the process samples with about 50% of cleaved substrate were used for calculations.
Standard conditions where defined as: enzyme to substrate mass ratio of 1:100 (1 mg/ml substrate and 0.01 mg/ml caspase, molar ratio 1:170) in caspase assay buffer (20 mM PIPES, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mm EDTA, 10 mM DTT, pH 7.2) and incubation at 25° C. For slowly proceeding reactions the caspase concentration was increased to 0.1 mg/ml (enzyme to substrate mass ratio 1:10).
cp caspase-2 (0.01 mg/ml) (SEQ ID No. 6) cleaved 50% of the substrate VDVAD-E2 with a P1′ glycine (1 mg/ml) (SEQ ID No. 33) at 25° C., in caspase assay buffer within 1 min (
By N-terminal Edman sequencing of the processed substrate, it was proven, that it was only cleaved between the VDVAD recognition site and the P1′ glycine.
For in vitro cleavages that compared the activity to commercially available caspase-2 about 0.005 mg/ml wt caspase-2 (Caspase-2 (human), recombinant, active, Enzo Life Sciences Inc.; Farmingdale (N.Y.), USA) were used to cleave 1 mg/ml VDVAD-E2 with a P1′ glycine (mass ratio 1:200, molar ratio 1:340).
2.1 Cloning of Constructs
To create specific changes like deletions, insertions, substitutions, site mutations or the like in initial proteins, caspases-2 or cp caspases-2 (e.g.: SEQ ID No. 6) in plasmid DNA, site directed mutageneses were performed.
The specific primers were designed back-to-back and used for an exponential amplification with a high-fidelity DNA Polymerase.
After amplification a KLD (kinase ligase Dpnl) reaction was performed. In this treatment the PCR product was incubated with a Kinase, a Ligase and Dpnl restriction enzyme, so that the PCR fragments were phosphorylated and ligated to a circular plasmid and the template DNA was removed. Constructs were transformed into NovaBlue heat shock cells and a fraction of the cell suspension was plated on TY agar containing the appropriate antibiotic. Successful cloning was verified by sequencing of single colonies.
All substrates and caspases were expressed and purified as described in sections 1.3 and 1.4, Example 1.
Protein and nucleotide sequences of all constructs are listed in
2.2 Caspase Substrates
Human ubiquitin-conjugating enzyme E2 L3 (E2; UniProt ID P6803612) as fusion protein was used as standard caspase substrate. A fusion protein (VDVAD-E2) with N-terminal His tag, short GSG-linker and VDVAD caspase-2 recognition site was designed. The first amino acid after the cleavage site (P1′) was a glycine (VDVAD-E2, SEQ ID No. 33). The whole protein has a size of 21.3 kDa, whereas when the tag is cleaved off, the E2 protein itself has 19.5 kDa. This difference is big enough to visualize the cleavage activity on an SDS-PAGE.
As the P1′ site is known to influence cleavage activity, E2 was expressed and purified with all twenty possible residues after the VDVAD cleavage site. E2 was also cloned with cleavage sites differing from VDVAD. All tested tag sequences fused to E2-protein are listed in Table 1.
β-galactosidase was chosen as a model protein, because due to its large size (116 kDa) it is vulnerable to unspecific cleavage. An N-terminal His tag as well as a GSG linker and the caspase-2 cleavage site VDVAD were added (SEQ ID No. 34).
Superoxide Dismutase, SOD, was used as an additional model fusion protein with an N-terminal 6 His Tag and the recognition site, VDVAD, directly fused to the N-terminus of SOD (SEQ ID No. 193).
hFGF (Human Fibroblast Growth Factor) was used to evaluate the influence of His tag and VDVAD cleavage site on protein expression. Three pET30a constructs (hFGF, 6H-hFGF, and 6H-VDVAD-hFGF (SEQ ID No. 32) were cloned.
Recombinant expressions of wild-type (hFGF), His tagged (6H-hFGF), and 6H-hFGF with caspase-2 cleavage site VDVAD (6H-VDVAD-hFGF) were compared. The expression of both variants with His tag was reduced. This effect was less pronounced in the 6H-VDVAD-hFGF variant. Total expression was significantly reduced, but, interestingly, the amount of soluble protein remained the same for 6H-hFGF and was even increased for 6H-VDVAD-hFGF compared to wild-type hFGF.
It has been described that His tags can influence the rate of both total and soluble production of recombinant proteins. The important result is, that the VDVAD sequence itself does not seem to have a negative influence on production or solubility of recombinant proteins. For proteins whose yield is reduced by a His tag, the caspase cleavage site can easily be combined with other tags.
2.3 Designed Variants of Circularly Permuted Caspase-2
Circularly permuted caspase-2: Circularly permuted caspase-2 variants (cp caspases-2) were designed. based on the sequence of human caspase-2 (UniProtKB14 ID P42575, SEQ ID No. 11) the N-terminal CARD was removed and the order of large (LS) and small subunit (SS) exchanged to create a constitutively active caspase. The SS was linked to the N-terminus of the LS via a GS-linker. Optionally the SS pro-peptide was linked to the N-terminus of the SS. In this case to ensure expression as a single chain protein, an aspartate (Asp343 in the wild-type sequence of caspase-2, Asp21 in the cp caspase-2) was mutated to alanine, to avoid cleavage of the small subunit from a p14 to a p12 chain. This resulted in the cp caspase-2 variants SEQ ID No. 9, SEQ ID No. 6 and, SEQ ID No. 76, both of the latter having additionally an N-terminal 6 His tag. The basic structures of these variants are shown in
The protein sequence was codon optimized for E. coli with the GeneArt™ online tool (Thermo Fisher Scientific). Between the small and the large subunit, a glycine-serine linker was added which also forms a BamHI restriction site. This enables the separate cloning of the subunits and facilitates the creation of chimera consisting of subunits from different caspases. The N-terminal His tag enabled IMAC-purification.
All cp-caspase-2 variants described under this chapter 2.3 were constructed based on SEQ ID No. 6, except otherwise described. The amino acid positions of the mutations indicated correspond to SEQ ID No. 6, unless explicitly stated otherwise. All variants have 6 His Tag, except otherwise described.
cp caspase-2 Stop and cp caspase-2 D285E: To test the influence of the propeptide annotated in UniProtKB14 (ID P42575) within the C-terminus of the large subunit, a truncated version was produced by deleting amino acids 286-292 in the cp caspase-2 of SEQ ID No. 6, thereby creating the cp caspase-2 Stop variant (SEQ ID No. 14), and an uncleavable variant (cp caspase-2 D285E) (SEQ ID No. 13) was created.
cp caspase-2 with C-terminal Strep tag: Strep tags were fused C-terminal to create cp caspase-2 Strep and cp caspase-2 D285E Strep variants (SEQ ID No. 15 and SEQ ID No. 16, respectively).
In SEQ ID No. 15, a Strep tag was fused to the C-terminus of the cp caspase 2 (SEQ ID No. 6), which was mutated to VDQQS (the substitution: D292S), as experiments had shown that VDQQE is recognized as a cleavage site. Despite the VDQQS mutation, the Strep tag was partially cleaved from the caspase. The cleavage product had the same size as the Stop variant (31.9 kDa), indicating that it had been cleaved at the DETD-R (between Asp285 and Arg286) and not at the VDQQS site.
Therefore, a Strep tag was added to the C-terminus of cp caspase-2 with the D285E and the E292S mutations. This variant (SEQ ID No. 16) was expressed as a single chain with 33.9 kDa. Proving that the mutation of Asp285 to Glu prevents cleavage. The C-terminal Strep-tag did not influence the cleavage activity of this variant.
cp caspase-2 D282T and cp caspase-2H185A D282T: Two cp caspase-2 variants were generated, the first with a D282T mutation and the second with an additional H185A mutation in cp caspase-2 (SEQ ID No. 6) comprising SEQ ID No. 17 and SEQ ID No. 18, respectively.
cp caspase-2 G171D, cp caspase-2 V225G, and cp caspase-2 D282E: cp caspase-2 (SEQ ID No. 6) was mutated at positions 171, 225, or 282 respectively resulting in amino acid exchanges G171D, V225G, or D282E resulting in the variants having SEQ ID No. 190, 192 and 191, respectively.
cp caspase-2 with different linkers between small and large subunit: The GS linker between small and large subunit of cp caspase-2 (SEQ ID No. 6) was mutated. Resulting variants contained no linker (cp caspase-2 Δ Linker, SEQ ID No. 73), a GGSGG linker (cp caspase-2 5 aa Linker, SEQ ID No. 74), and a GSAGSAAGSG linker (cp caspase-2 10 aa Linker, SEQ ID No. 75).
cp caspase-2 with partial and without small subunit propeptide: The propeptide of the small subunit of cp caspase-2 (SEQ ID No. 6) was mutated by site directed mutagenesis. Deletion of residues 8-22 produced a variant without propeptide (cp caspase-2 Δ SS Prop, SEQ ID No. 76, see also
cp caspase-2 with shifted circular permutation: cp caspase-2 Δ SS Prop (SEQ ID No. 76) was used to generate variants with shifted circular permutation. At the N-terminus of the small subunit three amino acids were deleted and added to the C-terminus of the large subunit. Because of possible auto-cleavage, detected when adding a Strep-tag to the C-terminal end of cp caspase-2, additionally the mutations D267E and D274S according to SEQ ID No. 76 were inserted. The resulting variant cp caspase-2 C-term +3 (SEQ ID No. 82) was expressed, purified and tested as described above.
In parallel, a variant was generated by deletion of the 3 C-terminal residues of the large subunit and insertion of those residues to the N-terminus of the small subunit of cp caspase-2 Δ SS Prop (SEQ ID No. 76). The resulting variant cp caspase-2 N-term +3 (SEQ ID No. 83) was expressed, purified and tested as described in the standard protocol in Example 1.
cp caspase-2 C203S: The variant was created by insertion of the C203S mutation in cp caspase-2 (SEQ ID No. 6) resulting in SEQ ID No. 198.
cp caspase-2 S9 C203S: The substitution C203S was inserted in cp caspase-2 S9 (SEQ ID No. 51), resulting in SEQ ID No. 199.
cp caspase-2 N85C and cp caspase-2 A86C: The variants were created by insertion of the mutations N85C (SEQ ID No. 80) and A86C (SEQ ID No. 88) in cp caspase-2 (SEQ ID No. 6).
Homologue cp Caspase-2 Variants:
The cp caspase-2 variants from different species were constructed analogue to the cp caspase-2 of human origin (SEQ ID No. 6).
Based on the sequence of Tasmanian devil caspase-2 (Sarcophilus harrisii, UniProtKB14 ID G3VQP7, SEQ ID No. 95) and Ghost shark caspase-2 (Callorhinchus milii, UniProtKB14 ID V9KZT1, SEQ ID No. 113) the N-terminal CARD was removed and the order of large and small subunit exchanged to create a constitutively active caspase. The SS was linked to the N-terminus of the LS via a GS-linker. The SS pro-peptide was linked to the N-terminus of the SS. To ensure expression as a single chain protein, an aspartate (corresponding to Asp343 in the wild-type sequence of human caspase-2, Asp21 in the cp protein) was mutated to alanine, to avoid cleavage of the small subunit propeptide.
The protein sequence was codon optimized for E. coli with the GeneArt™ online tool (Thermo Fisher Scientific). Between the small and the large subunit, a glycine-serine linker was added which also forms a BamHI restriction site. This enables the separate cloning of the subunits and facilitates the creation of chimera consisting of subunits from different caspases. The N-terminal His tag enabled IMAC-purification.
Resulting variants are Sarcophilus cp caspase-2 (SEQ ID No. 64) and Callorhinchus cp caspase-2 (SEQ ID No. 68).
Mutations at positions corresponding to (at positions functionally equivalent to) residues Glu105 and Glu172 in cp caspase-2 (SEQ ID No. 6) were inserted in Sarcophilus cp caspase-2, generating variant Sarcophilus cp caspase-2 E105V E172V (SEQ ID No. 78).
Mutations at positions corresponding to Glu105 and Gly171 in cp caspase-2 (SEQ ID No. 6) were inserted in Callorhinchus cp caspase-2, generating variant Callorhinchus cp caspase-2 E105V G171D (SEQ ID No. 79).
Additionally, the variants were cloned containing an N-terminal T7AC tag (SEQ ID No. 84, 85, 86, 87).
Functionally equivalent positions are listed in Table 2.
UniProt IDs: Human (P42575), Mouse (P29594), Sheep (W5Q8H6), Tasmanian Devil (G3VQP7), Chicken (Q98943), Anolis (H9GC58), Alligator (A0A1U8D1 G6), Xenopus (F6RDY9), Danio (Q0PKX3), Ghost Shark (V9KZT1), Sea squirt (A0A1W2WKB0)
Selection System to Detect Variants with Improved P1′ Tolerance
A selection system was used for the improvement of cp caspase-2. It is based on a circularly permuted ATCase (aspartate transcarbamylase) catalytic subunit and a pyrimidine auxotroph strain. The pyrBI operon (encoding regulatory pyrl and catalytic pyrB subunits of ATCase) was deleted in E. coli BL21(DE3), so this knock-out strain can only survive in media containing pyrimidines or when the cells are complemented with a vector encoding ATCase. A cp catalytic subunit of ATCase (cp-pyrB), which harbors its new N-terminus in the interior of the protein, is used to detect specific proteases via the growth of E. coli, because fusion of any stretch of amino acids to its N-terminus renders the enzyme inactive as it can no longer fold properly due to space limitations in the interior of the protein. However, if a protease is provided that can exactly cleave off this additional stretch of amino acids, the enzyme gets reactivated.
3.1 Design of Constructs and Caspase Mutant Libraries
Selection medium: Optimized M9 medium (see Example 1, section 1.2)
Strain: E. coli BL21(DE3) with pyrBI operon exchanged to kanamycin resistance (id est: pyrBI is deleted)
Vectors: expressions of the ATCase subunits, cp-pyrB and pyrl from pETDuet™-1 vector using T7 promoters and the ampicillin-resistance as selection marker; expressions of the diverse caspase variants from pACYCDuet™-1 vector using a T7 promoter and the chloramphenicol resistance marker. Selection protocol was performed with respective cotransformations with simultaneous use of ampicillin, kanamycin and chloramphenicol in the above selection medium.
VDVAD-cpATCase
The used pETDuet-1 plasmid (substrate plasmid), contained a pyrl gene in MCSI (SEQ ID No. 20) and cp-pyrB gene in MCSII (SEQ ID No. 21). In pyrl the potential caspase cleavage site DQVD was changed to DQVE by mutation of Asp73. A 6 His tag followed by a GSG linker and a caspase recognition site were fused to the N-terminus of cp pyrBc227 [25]. This hinders the correct folding of the enzyme and makes it inactive, but proteolytic cleavage of this tag can restore its function. The first Met of cp pyrB was deleted. The amino acid after Met is Thr. The cpATCase is still active when this residue is substituted. Only mutations to His, Lys, Phe, Tyr, and Trp render it inactive. This enables the selection for caspases with improved or altered recognition site specificity and/or improved P1′ tolerance. CpATCase constructs with 6 His-GSG-VDVAD-ΔM-X-pyrB (SEQ ID No. 22) were used for in vivo selection of altered P1′ tolerance.
Construction of Caspase Mutant Library—ep PCR and oe PCR
Mutant gene libraries of different cp caspase-2 variants were generated by error prone (ep) PCR and overlap extension (oe) PCR of vector and the mutated caspase gene. The linear DNA fragments were ligated using T4 DNA ligase. The amount of mutations can be modified by changing the Mg (II) and Mn (II) ion concentrations in the PCR buffer. The used concentrations caused in average one to three amino acid exchanges in the caspase. The cp caspase-2 variants, of which mutant libraries were made of, are indicated in Table 5 in the column “Mutated Caspase”.
3.2 Selection of Caspase Libraries
The caspase mutant libraries were transformed into E. coli BL21(DE3) ΔpyrBI electro competent cells that already contained the cpATCase plasmid with the desired protease cleavage site and P1′ residue. Selection was executed either in optimized M9 medium or on M9 agar plates at 30° C. for 24-48 h. Liquid cultures were used to enrich mutants with improved growth. IPTG concentrations in liquid culture and in agar plates between 0.025 and 1 mM were used.
Mutant libraries in E. coli BL21(DE3) ΔpyrBI cells were selected with VDVAD-cpATCase with different P1′ residues. Selections were executed with Pro, Met, Thr, and Val. Selections with P1′ Met were executed with cp ATCase without deletion of the native methionine, all other selections were executed with constructs comprising SEQ ID No. 22. Selection with Met, Thr, and Val as P1′ lead to hundreds of positive variants, thus only the largest colonies were analyzed.
All together 77 clones with a total of 263 mutations were analyzed from all selections combined. Some mutations were found several times in independent experiments. The mutations of resulting variants in comparison to SEQ ID No. 6 are shown in Table 5 below. P1′ amino acids used for selection are indicated under “P1′ cpATCase”.
Mutations of variants were analyzed and several were selected for expression and characterization by in vitro cleavages. Variants were chosen when they had been enriched in liquid culture or contained mutations that were found several times independently. Description of those variants can be found in Example 4.
cp caspase-2 S9 D285E and S9 D285: Selection of a cp caspase-2 D285E (SEQ ID No. 13) library, containing about 5,500 variants, was performed, with VDVAD-cpATCase that contained a methionine as P1′ and with an induction strength of 0.025 mM IPTG. The E105V mutation was found repeatedly among 16 analyzed clones. One selected variant with this mutation (cp caspase-2 S9 D285E, SEQ ID No. 1) was expressed, purified and tested as described in Example 1.
The selected cp caspase-2 S9 D285E was mutated to generate the cp caspase-2 S9 D285 variant (SEQ ID No. 51). The variant was expressed, purified and tested as described above (Example 1).
cp caspase-2 mS9 Pro D285E and cp caspase-2 mS9 Pro D285: The cp caspase-2 S9 D285E (SEQ ID No. 1) variant was used for a further round of mutation because of its improved P1′ tolerance. The new mutant library contained about 10,000 variants and was selected with VDVAD-ΔM-Pro-cpATCase. Selection in liquid culture enriched a variant (mS9 Pro D285E, SEQ ID No. 70) with the mutations E105V, G171 D, V225G, D282E and D285E. The caspase was expressed and purified as described above.
The selected cp caspase-2 mS9 Pro D285E (SEQ ID No. 70) was mutated to generate the cp caspase-2 mS9 Pro D285 variant (SEQ ID No. 52). The variant was expressed, purified and tested as described above.
cp caspase-2 mS9 Thr 0.8: The variant with K83E, E105V, E172V, V255M, and D285Y mutations was selected from mutated cp caspase-2 S9 D285 (SEQ ID No. 51). The new variant (SEQ ID No. 53 and SEQ ID No. 54) was enriched in liquid culture in a selection with VDVAD-Thr-cpATCase and 0.8 mM IPTG. It was expressed, purified and tested as described in Example 1.
cp caspase-2 S17: Variant with E105V, C132R, E141G, H200R, and D285E mutations that was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-cpATCase with Met as P1′ and 0.1 mM IPTG. The variant was never purified and tested in vitro, mutations at positions 105, 132 and 105 were found repeatedly in different experiments.
cp caspase-2 S20: The variant with C203Y and D285E mutations (SEQ ID No. 26) was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-cpATCase with Met as P1′ and 0.1 mM IPTG.
cp caspase-2 D285E SV4: The variant with V201A and D285E mutations (SEQ ID No. 28) was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-Val-cpATCase and 0.1 mM IPTG. The mutation V201A was found several times independently.
cp caspase-2 SV19: The cp caspase-2 SV 19 (SEQ ID No. 81) was selected from variants with mutated C-terminus with VDVAD-Val-cpATCase and 0.1 mM IPTG.
The sequence equals the consensus-sequence of 13 active variants with mutated C-terminus.
cp caspase-2 D285E SV30: The variant with E174G and D285E mutations (SEQ ID No. 30) was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-Val-cpATCase and 0.1 mM IPTG. The variant was enriched in liquid culture.
5.1 β-Galactosidase
The model substrate β-galactosidase contains four DXXD and one DXXE sites, three of which are on the surface and could be accessible to the caspase.
After incubating 1 mg/ml β-galactosidase fusion protein (with N-terminal tag including the recognition site VDVAD with 0.1 mg/ml cp caspase-2 (SEQ ID No. 6) for 24 hours, no unspecific cleavage was observed. Correct cleavage of the His tag was confirmed by N-terminal protein sequencing.
5.2 VDVAD-SOD Cleavage
5.3 VDVAD-Gly-E2 Cleavage Values of all Tested cp Caspase-2 Variants
cp caspase-2 (0.01 mg/ml) (SEQ ID No. 6) cleaved 50% of the substrate VDVAD-E2 with a P1′ glycine (1 mg/ml) at 25° C., in caspase assay buffer within 1 min. These conditions were defined as standard activity to which all other reactions were compared (
Not all tested variants cleaved the standard substrate to 50% in 1 min. A list of all cleavages with a P1′ Gly is given in Table 6.
5.4 P1′ Tolerance
Cleavage site specificity and P1′ tolerance of caspases have been studied using peptide substrates, degradome analysis, and phage libraries. Peptides are not ideal for this purpose, as structure influences the cleavage activity. Degradome studies, on the other hand, are influenced by the sequences occurring in the analyzed cells. To our knowledge, so far no study has systematically tested caspase specificity and P1′ tolerance with protein substrates. Therefore, we permuted the P1′ residue after the cleavage site in the fusion protein VDVAD-E2 (Example 2, section 2.2) to evaluate the cleavage efficiency of cp caspase-2 in dependency of the P1′ residue.
Glycine was highly preferred in the P1′ position, cleavage before all other residues was at least five-times less efficient. The group of amino acids that was reasonably well tolerated comprised small, basic, and aromatic residues, as well as Asn and Met.
Table 7 (Table 7.1 and Table 7.2) shows cleavage of E2 substrates with VDVAD recognition site and different P1′ residues by cp caspase-2 variants. Activity is given in percent of activity for cleavage of VDVAD-E2 with a P1′ glycine for each cp-caspase-2 variant. Thus Table 7 shows the P1′ tolerance of the respective cp caspase-2 variant. All values (means±standard deviation) were determined with at least three independent experiments, executed with 1 mg/ml E2. For Asp-E2, Glu-E2, Ile-E2, Pro-E2 and Val-E2 cp caspase-2 concentration was 0.1 mg/ml, for all others 0.01 mg/ml. The given values already consider these concentration differences.
Table 8 (Table 8.1 and Table 8.2) further below shows the cleavage activity of all cp caspase-2 variants for all P1′ amino acids related to the cleavage activity of the standard cp caspase-2 (SEQ ID No. 6) in %. Thus Table 8 shows the extent of increase (or decrease) of P1′ tolerance.
Taken together, these data show that variants of a cp caspase-2, comprising amino acid substitutions at any one or more of positions 83, 105, 171, 172, 185, 225, 255, 282, 285 of SEQ ID No. 6, display significantly improved P1′ tolerance for at least one amino acid. In most cases, these variants comprise significantly improved P1′ tolerance for multiple amino acids.
Furthermore, these data show that even though amino acid substitutions at positions 85, 86, 132, 141, 174, 200, 201, 203 of SEQ ID No. 6 do not improve P1′ tolerance, they do not hamper caspase activity significantly. Table 6, for example, shows that variants comprising amino acid substitutions at positions 85, 86, 132, 141, 174, 200, 201, or 203 of SEQ ID No. 6 still cleave about 50% of the substrate VDVAD-E2 within 2 or 3 minutes. These represent examples for functionally active variants of cp-caspases-2 of the present invention. Furthermore, all variants selected using the selection system as described in Example 3 and as shown in Table 24 are further examples of functionally active variants of cp-caspases-2, since they all have catalytic activity for the cleavage of the VDVAD P1′ motiv (a caspase-2 cleavage site). Otherwise the colonies/clones would not have grown.
6.1 System for In Vivo Selection of cp Caspase-2 Variants, Similar as 3.1
The selection system described in section 3.1 of Example 3 is used for the selection of caspases that tolerate different cleavage sites than VDVAD.
A gene library of 6 His-GSG-XDXXD-ΔM-Thr-pyrB (SEQ ID No. 22) cpATCase constructs was cloned with degenerate primers to insert random mutations in the caspase recognition sequence at the positions P5, P3, and P2.
E. coli BL21(DE3) ΔpyrBI cells were generated that contain the cp caspase-2 construct (SEQ ID No. 7) in a pACYCDuet vector. After transformation of the cpATCase library into the cells the selection, as described above, was executed either in M9 medium or on M9 agar plates at 30° C. for 24-48 h.
Several single colonies were sequenced and the nucleotide sequence of the cpATCase was analyzed detecting alternative cleavage sites tolerated by cp caspase-2.
The alternative cleavage sites were cloned into the substrate proteins and the activity of different cp caspase-2 variants were tested as described above in Example 1.
7.1 Design of Constructs and Selection
Saturated mutagenesis with degenerate primers, designed to create all possible 19 amino acid substitutions in the protein, was performed with cp caspase-2 S9 (SEQ ID No. 51), comprising the additional G171 D substitution, as a template. The gene library containing all 400 variants with possible combinations of mutations in positions 105 and 171 were transformed in E. coli BL21(DE3) ΔpyrBI cells that contained the VDVAD-cpATCase substrate with P1′ Thr (SEQ ID No. 22). The selection, as described in Example 3 above, was executed either in M9 medium or on M9 agar plates at 30° C. for 24-48 h.
The DNA of several single colonies was analyzed, detecting combinations of mutations in active variants.
The combinatorial mutants were expressed, purified and tested as described above in Example 1.
DEVD-E2 (SEQ ID No. 57)
DEVD is the preferred cleavage site of caspases-3 and -7. DEVD-E2 was used to evaluate the influence of the P5 residue, because the influence of the amino acids in the P2 and P3 positions on caspase-2 activity are considered insignificant. The substrate was processed 140 times slower than VDVAD-E2 (SEQ ID No. 33) by cp caspase-2 (SEQ ID No. 6) showing that the recognition of the P5 residue is very important for caspase-2 and cp caspase-2.
This is in accordance with results from fluorescent peptides [26, 24], and proves the initial assumption of this study that caspase-2 was more specific than other caspases, because of its pentapeptidic recognition site. This seems to be even more pronounced in the circularly permuted variant, as the literature only describes a 35-fold increase in activity with VDVAD over DEVD [26].
8.1 Comparison of Specificity with Wild-Type Caspase-2
The specificity of cp caspase-2 (SEQ ID No. 6) was compared with commercially available wild-type caspase-2 (human, recombinant, active Caspase-2, Enzo Life Sciences, Farmingdale, N.Y., USA). 72 U/ml of the wild-type caspase-2 were used for cleavage reactions, according to the specifications, this equals about 0.005 mg/ml enzyme, half the concentration used in standard reactions with cp caspase-2. But the wild-type caspase was even six times less active than cp caspase-2 under the same conditions (1 mg/ml VDVAD-E2 was processed to 50% in 6 min).
While the absolute activities of the enzymes might be difficult to compare, because of different purity and concentration, a clear discrepancy could be found between their specificities. Wild-type caspase-2 cleaved DEVD-E2 only 44 times slower than VDVAD-E2, while cp caspase-2 has a 140-fold preference for VDVAD over DEVD. Thus, the cp caspase-2 is three times more specific than the wild-type enzyme (
8.2: Production and Characterization of a Wild Type Caspase-2
For comparison of wild-type caspase-2 with cp-caspase-2 variants a human caspase-2 was produced.
Production of Wt Caspase-2:
Production of wt caspase-2 was performed in a 30 L (23 L net volume, 5 L batch volume) computer-controlled bioreactor (Bioengineering; Wald, Switzerland) equipped with standard control units (Siemens PS7, Intellution iFIX). The pH was maintained at a set-point of 7.0±0.05 by addition of 25% ammonia solution (w/w), the temperature was set to 37° C.±0.5° C. in the batch phase and 30° C.±0.5° C. in the fed-batch phase. To avoid oxygen limitation the DO level was held above 30% saturation by adjusting the stirrer speed and the aeration rate of the process air. The maximum overpressure in the head space was 1.1 bar.
Pre-cultures for inoculation were grown in synthetic media calculated to produce 3 g/L. For incubation 1 mL of a deep frozen MCB was aseptically transferred to 400 mL medium and cultivated in two 2000 mL shaking flasks at 37° C. and 180 rpm until an OD of approx. 4 was reached.
For cultivation, minimal media calculated to produce 64 g cell dry mass (CDM) in the batch phase and 890 g CDM during feed phase were used. The batch medium was prepared volumetrically; the components were dissolved in 8 L RO—H2O. The fed-batch medium was prepared gravimetrically; the final weight was 8.45 kg. All components for the fed-batch medium were weighed in and dissolved in RO—H2O separately. All components (obtained from MERCK), were added in relation to the theoretical grams of cell dry mass to be produced: The composition of the batch and the fed-batch medium is as follows: 94.1 mg/g KH2PO4, 31.8 mg/g H3PO4 (85%), 41.2 mg/g C6H5Na3O7*2 H2O, 45.3 mg/g NH4SO4, 46.0 mg/g MgCl2*2 H2O, 20.2 mg/g CaCl2*2 H2O, 50 μL trace element solution, and 3.3 g/g C6H12O6*H2O. The trace element solution was prepared in 5 N HCl and included 40 g/L FeSO4.*7H2O, 10 g/L MnSO4.*H2O, 10 g/L AlCl3.*6H2O, 4 g/L COCl2, 2 g/L ZnSO4.*7H2O, 2 g/L Na2MoO2.*2H2O, 1 g/L CuCl2.* 2H2O, and 0.5 g/L H3BO3. To accelerate initial growth of the population, the complex component yeast extract (150 mg/g calculated CDM) was added to the batch medium. Nitrogen level was maintained by adding 25% ammonium hydroxide solution (w/w) for pH control. Antifoam (PPG 2000) 0.5 mL/L total volume was added at the beginning.
The fed-batch phase (29 h) was performed at 30° C. with an exponential feeding strategy with a consistent growth rate of μ=0.1 h−1. The substrate feed was controlled by increasing pump speed according to the exponential growth algorithm, X=X0·eμt, with superimposed feedback control of weight loss in the substrate tank. Induction started with fed-batch phase by adding 0.5 μmol IPTG/g CDM directly to the feed-media to achieve a protein production for 4 generations. IPTG concentration was calculated with the theoretical final CDM.
In addition to standard online monitoring (pH, stirrer speed, temperature and pO2) the concentration of pO2 and O2 in the outlet air was measured with a BlueSens gas analyzer. Sampling of the standard offline process parameters started after one generation in fed-batch mode. The first sample was withdrawn from the bioreactor prior to induction. Optical density (OD600) was measured with a spectrophotometer at wavelength λ=600 nm. Samples were diluted in PBS to ensure a measurement at a linear range from 0.1 to 0.8. Cell dry mass (CDM) was determined by centrifugation of 10 mL of cell suspension for 8 min at 8500 rpm. The supernatant was discarded and cells were resuspended with RO—H2O and centrifuged. Water was discarded and cell were resuspended again with RO—H2O. Cell suspension was transferred into a beaker, which was weighted before. Beakers were dried for at least 24 h at 105° C. and weighted again. The difference in weight account for the CDM.
For the determination of the content of cp caspase-2 and variants, aliquots of approximately 1.0 mg CDM of the samples were centrifuged (10 min. at 13200 rpm); the supernatants were discarded, the insides of the tubes were carefully blotted dry and the samples were stored at −20° C. The E. coli cell mass was harvested by centrifugation at 18,590 rcf for 15 minutes and the supernatant was discarded. The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0). The cells were re suspended at a concentration of 400 g wet cell mass per L. Cell lysis was performed through high pressure homogenization at 1400 bar/140 bar with two passages with an in-line counter current chiller set to 10° C. The homogenate was centrifuged at 18,590 rcf for 2.5 hours at 4° C. The pellet was discarded and the supernatant used. Before chromatography the supernatant was filtered through a 0.22 μm membrane.
The wt caspase-2 carrying a poly-his-tag was captured using immobilized metal affinity chromatography (IMAC). The following buffers were used: equilibration buffer: 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0. Elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0.
Imidazole was added to the clarified supernatant before IMAC, to a final concentration of 20 mM imidazole. 57 CV clarified supernatant were loaded to an equilibrated Ni-Sepharose 6 Fast Flow column (50×18 mm, 35 mL). A residence time of 7 minutes was used during loading and 3 minutes for subsequent steps. After loading was completed the column was washed for 10 CV with equilibration buffer. The bound wt caspase 2 was eluted using a step gradient to 100% elution buffer for 10 CV.
The elution fractions were analyzed using SDS-PAGE and all fractions containing wt caspase-2 were used for the next purification step.
The capture eluate of wt caspase-2 was buffer exchanged before the polishing chromatography step. Tangential flow ultra-/diafiltration with a 5 kDa cut off membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. In total 5 volumes were exchanged.
The capture step used cation exchange chromatography on SP Sepharose HP (5×24 mm, 0.5 mL) using the following buffers: equilibration buffer A: 50 mM sodium citrate, pH 5.0. Elution buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0.
Buffer exchanged capture eluate was loaded on the equilibrated polishing column. The residence time was held constant at 5 minutes. The column was loaded with 37 CV of buffer exchanged capture eluate. Wt caspase-2 was eluted in a linear gradient from 0-100% B in 10 CV. The elution fractions were analyzed using Western blot and SDS PAGE and the fractions positive for the small sub unit of wt caspase-2 were combined and stored at −80° C. Before performing enzyme kinetic measurements, oxidation induced activity losses were reversed by incubating wt caspase-2 with 100 mM DTT for 15 minutes.
Characterization of Wt Caspase-2
FRET Assay
Michaelis Menten kinetic was determined for wt caspase-2 and cp caspase-2 for the following substrates: VDVADFA, VDVADGA, VDVADQA and VDVADVA, where the P1′ amino acid is indicated by bold and underlined font.
The FRET results in Table 9 show significant differences between the two proteases. cp caspase-2 exhibits catalytic efficiencies approximately one order of magnitude higher than wt caspase-2. While the Michaelis constant KM appears mostly unaffected by circular permutation, the turnover number kcat is the cause for the stark differences in catalytic efficiency kcat/KM between wt caspase-2 and cp caspase-2. The produced wt caspase-2 seems to exhibit slightly better P1′ tolerance compared to cp caspase-2 (both not comprising the amino acid substitutions for improved P1′ tolerance described herein), e.g. F as P1′ is cleaved with 2.5% catalytic efficiency in cp caspase 2 compared to 3.2% in wt caspase-2. This slight increase in P1′ (1.3 to 2.3-fold increase) is overshadowed by the, on average eleven times lower catalytic efficiency and eight times lower turnover number of wt caspase-2.
Tolerance for Elevated Temperatures
Cleavage of a heat stable model fusion tag protein, namely GFP, was used to quantify the tolerance of caspase-2 towards elevated temperatures.
The GFP cleavage results in Table 10 show comparable heat tolerance between the two proteases. The cleavage reaction with cp caspase-2 is 1.6-fold faster, than with wt caspase-2 at 25° C. This difference increases to 2.3-fold at 50° C., showcasing the increased stability of cp caspase-2 at elevated temperatures. In general, the cleavage reaction at 50° C. is 1.9 times faster for wt caspase-2 and 2.8 times faster for cp caspase 2. This is a clear benefit if a heat stable target protein has to be processed.
Tolerance to Chaotropic Conditions
Cleavage of a model fusion tag protein stable in 4 M urea, namely FGF2, was used to quantify the tolerance of caspase-2 towards chaotropic conditions.
The FGF2 cleavage results in Table 11 show comparable tolerance for chaotropic conditions between the two proteases. In order to quantify the cleavage product in the linear range, the reaction had to be stopped at differing time points. Both proteases show almost identical behavior in the presence of 4 M urea, were the reaction rate is reduced to 1.2% and 1.3% for wt caspase-2 and cp caspase-2 respectively. For this particular model protein, cp caspase-2 exhibited a 3.2-fold increased reaction rate relative to wt caspase-2.
Manufacturability
Perhaps the biggest observable difference between the two proteases, is in their ease of manufacture. In order to express the difference in manufacturability between wt caspase-2 and cp caspase-2, we calculated the amount of dry cell mass required to produce one milligram of purified enzyme. This takes into account the differences in specific protein content of the E. coli fermentation and the differences in downstream processing yields. It does not take into account differences in biomass yield between fermentations. In order to produce 1 mg of wt caspase-2, 70 g of cell dry mass (CDM) were required. For the production of cp caspase-2, only 34 mg of CDM were needed per milligram pure enzyme. This corresponds to a difference in manufacturability of a factor of 2,033.
Conclusion
FRET assay results with 4 different P1′ amino acids showed a general trend of tenfold higher catalytic efficiencies of the cp caspase-2 compared to wt caspase-2. The cleavage of non peptide substrates, showed two to three-fold faster cleavage reaction depending on the protein substrate. The circular permutation of caspase-2 has apparently lead to an increase in heat tolerance, showcased by the larger increase in turnover rate at 50° C. The tolerance to chaotropic conditions also appears slightly higher The largest differentiating factor between wt and cp enzymes is their manufacturability. While the expression level of wt caspase-2 is very low (under the limit of quantification), cp caspase-2 reaches expression levels of 80 mg specific protein content per g CDM. This also results in much lower losses during DSP, where a process yield of about 35% can be achieved for cp caspase-2.
9.1 Upstream processing of cp caspase-2 and variants For the production of cp caspase-2 and variants with and without solubility tag lab-scale fermentations were performed as described below. Different expression clones were compared regarding cell growth and soluble recombinant protein production. For final process optimization, a series of cultivation runs were conducted according to a Design of experiments (DoEs) approach.
9.1.1 Bacterial Strain, Plasmid and cp Caspase-2 and Variants
The E. coli strain BL21(DE3) [F−, fhuA2, Ion, ompT, gal, dcm, ΔhsdS λ DE3 [λ sBamHIo, ΔEcoRI-B int::(lacl::PlacUV5::T7 gene1) i21 Δnin5], purchased from Novagen, was transformed with a pET30a vector carrying the gene for cp caspase-2 or variants with and without solubility tag under the T7 promoter/operator system. The expression systems cultivated in lab-scale bioreactors are listed in Table 12.
9.1.2 Lab-Scale Fermentation of cp Caspase-2 and Variants.
9.1.2.1 Fermentation Media
For high cell density (HCD) cultivation experiments minimal media calculated to produce 80 g cell dry mass (CDM) in the batch phase and 1450 g CDM during feed phase were used. The batch medium was prepared volumetrically; the components were dissolved in 10 L RO—H2O. The fed-batch medium was prepared gravimetrically; the final weight was 10.1 kg. All components for the fed-batch medium were weighed in and dissolved in RO—H2O separately. All components (obtained from MERCK), were added in relation to the theoretical grams of cell dry mass to be produced: The composition of the batch and the fed-batch medium is as follows: 94.1 mg/g KH2PO4, 31.8 mg/g H3PO4 (85%), 41.2 mg/g C6H5Na3O7*2H2O, 45.3 mg/g NH4SO4, 46.0 mg/g MgCl2*2 H2O, 20.2 mg/g CaCl2*2 H2O, 50 μL trace element solution, and 3.3 g/g C6H12O6*H2O. The trace element solution was prepared in 5 N HCl and included 40 g/L FeSO4.*7H2O, 10 g/L MnSO4.*H2O, 10 g/L AlCl3.*6 H2O, 4 g/L COCl2, 2 g/L ZnSO4.*7H2O, 2 g/L Na2MoO2.*2 H2O, 1 g/L CuCl2.*2 H2O, and 0.5 g/L H3BO3. To accelerate initial growth of the population, the complex component yeast extract (150 mg/g calculated CDM) was added to the batch medium. Nitrogen level was maintained by adding 25% ammonium hydroxide solution (w/w) for pH control. Antifoam (PPG 2000) 0.5 mL/L total volume was added at the beginning. Pre-cultures for inoculation were grown in synthetic media calculated to produce 3 g/L).
9.1.2.2 Cultivation and Induction Conditions for Standardized Lab-Scale Fermentations
All HCD fermentations were performed in a 30 L (23 L net volume, 5 L batch volume) computer-controlled bioreactor (Bioengineering; Wald, Switzerland) equipped with standard control units (Siemens PS7, Intellution iFIX). The pH was maintained at a set-point of 7.0±0.05 by addition of 25% ammonia solution (w/w), the temperature was set to 37° C.±0.5° C. in the batch phase and 30° C.±0.5° C. in the fed-batch phase. To avoid oxygen limitation the DO level was held above 30% saturation by adjusting the stirrer speed and the aeration rate of the process air. The maximum overpressure in the head space was 1.1 bar. Foaming was suppressed by addition of 0.5 mL/L antifoam (PPG 2000 Sigma Aldrich) to the batch medium and by pulsed addition of antifoam during the fed-batch phase. The cultivation was inoculated with an overnight pre-culture. The pre-culture was set-up by inoculating 200 mL LB media with 1 mL of a deep frozen WCB in 2000 mL shake flasks. Cells were grown on an orbital shaker at 180 rpm and at 37° C. until the OD600 reached a value of approx. 4. Thereafter, batch was inoculated with the pre-culture to an initial OD600 of 0.10 and cultivated at 37° C. At the end of the batch phase as soon as cells entered the stationary growth phase, an exponential substrate feed was started. The fed-batch phase (29 h) was performed at 30° C. with an exponential feeding strategy with a consistent growth rate of μ=0.1 h−1. The substrate feed was controlled by increasing pump speed according to the exponential growth algorithm, X=X0·eμt, with superimposed feedback control of weight loss in the substrate tank. Induction started with fed-batch phase by adding 0.5 μmol IPTG/g CDM directly to the feed-media to achieve a protein production for 4 generations. IPTG concentration was calculated with the theoretical final CDM.
9.1.2.3 Cultivation and Induction Conditions for DoE Approach
Pre-cultivation and batch phase were identical to the previously described standardized fermentations. The fed-batch phases were performed at 30° C. For biomass production the first fed-batch phase was performed with an exponential feed (μ=of 0.17 h−1) for 1.72 generations. As previously described, the substrate feed was controlled by increasing pump speed according to the exponential growth algorithm, X=X0*eμt, with superimposed feedback control of weight loss in the substrate tank. In a second feed-phase a lower growth rate (0.03, 0.05 and 0.07 h−1) was adjusted resulting in a total feed time of 60.5 h, 39 h and 30 h. The calculated CDM was 70 g/L. To ensure sufficient adaption to the low growth conditions, the cells grew for 0.25 generations without induction. Then induction was performed with three different IPTG concentrations (0.5, 0.9 and 1.3 μmol/g CDM) for two generations. 9 DoE fermentations were performed.
9.1.2.4 Fermentation Monitoring
In addition to standard online monitoring (pH, stirrer speed, temperature and pO2) the concentration of pO2 and O2 in the outlet air was measured with a BlueSens gas analyzer. Sampling of the standard offline process parameters started after one generation in fed-batch mode. The first sample was withdrawn from the bioreactor prior to induction. Optical density (OD600) was measured with a spectrophotometer at wavelength λ=600 nm. Samples were diluted in PBS to ensure a measurement at a linear range from 0.1 to 0.8. Cell dry mass (CDM) was determined by centrifugation of 10 mL of cell suspension for 8 min at 8500 rpm. The supernatant was discarded and cells were resuspended with RO—H2O and centrifuged. Water was discarded and cell were resuspended again with RO—H2O. Cell suspension was transferred into a beaker, which was weighted before. Beakers were dried for at least 24 h at 105° C. and weighted again. The difference in weight account for the CDM.
For the determination of the content of cp caspase-2 and variants, aliquots of approximately 1.0 mg CDM of the samples were centrifuged (10 min. at 13200 rpm); the supernatants were discarded, the insides of the tubes were carefully blotted dry and the samples were stored at −20° C.
9.1.2.5 Determination of cp Caspase-2 and Variants in Fermentation Samples
Cell disintegration, fractionation of soluble and insoluble recombinant protein and IB dissolving: Cell disintegration was performed from fermentation samples containing approximately 1.0 mg CDM. 200 μL of cell integration buffer was added to the cell pellet and vortexed until the pellet was completely resuspended. For cell disruption 50 μL Lysozyme and 50 μL Benzonase were added and incubated while shaking at room temperature. 100 μL Triton X-100 was added and samples were incubated again while shaking. Then, samples were centrifuged at 4° C. and 13000 rpm to separate soluble proteins and inclusion bodies (IB). The supernatant was transferred into a new reaction tube for direct analysis (SDS-PAGE) or stored at −20° C.
The remaining pellet (IBs and cell debris) was washed two times by resuspending with 1 mL Tris/HCL (100 mM). After resuspending the pellet was centrifuged at 4° C. and 13000 rpm for 10 min. The supernatant was discarded. Afterwards, 400 μL IB solvent buffer was added and incubated at room temperature for 30 min. while shaking. Finally, the sample was centrifuged again and the supernatant containing dissolved IBs was used for analysis (SDS-PAGE) or stored at −20° C.
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate and analyze the recombinant proteins. Electrophoresis was performed by using precast gels with an acrylamide gradient (NuPAGE 4-12% BisTris, Thermo Fisher Scientific, Waltham, Mass., USA) and NuPAGE® MES SDS Running buffer.
Loading samples were prepared by mixing 13 μL of the supernatant (soluble fraction) or IB supernatant (insoluble fraction) with 5 μL LDS sample buffer (4×) and 2 μL NuPAGE® reducing agent (10×) and incubating the mixture in a thermos mixer at 70° C. for 10 minutes. A ready-to-use molecular weight marker (Mark12™, Unstained Standard, Invitrogen) was directly loaded as size marker. For quantification, purified T7AC_6H_cpCasp2 standards (75, 50 and 25 μg/mL) listed in Table 12, produced as described in Example 9 (see sections 9.1, and 9.2), were used. Electrophoresis settings were 200 V and 400 mA for 40 to 50 minutes in a XCell SureLock™ Electrophoresis Cell chamber (Thermo Fisher Scientific). After electrophoresis the SDS Gels were fixed in fixing solution (40% ethanol; 50% dH2O; 10% acetic acid) for 30 minutes and stained afterwards with Coomassie brilliant blue R250 staining solution for 30 minutes. Finally, the gel was decolorized in a destaining solution (25% acetic acid; 8% ethanol; 67% dH2O) for at least two hours. Gels were transferred in water and scanned with a desktop scanner, converted to grey-scale and analysed using the software ImageQuant TL (7.0). The concentration of cp caspase-2 and variants was quantified via a linear regression curve.
9.1.2.6 Comparison of Production of cp Caspase-2 and Variants with and without Solubility Tag in Fermentations with a μ=0.1 h−1 and an IPTG Concentration of 0.5 μMol IPTG/g CDM During Induction.
While overexpression of cp caspase-2 was possible in E. coli, the expression rate of soluble cp caspase-2 was generally low. In order to increase the fermentation titer, a solubility tag was added to the enzyme. The tag T7A3 (SEQ ID No. 37) is based on a highly negatively charged peptide from the T7 bacteriophage. When used on the cp caspase-2 variants we noticed autocatalytic cleavage of the tag and subsequently modified the tag, using a cleavage site prediction algorithm. The altered solubility tag was coined T7AC (SEQ ID No. 43) and was able to double the expression level of soluble cp caspase-2. For this solubility- and His-tagged enzyme, we developed a downstream process based on an IMAC capture step and a cation exchange chromatography (CEX) polishing step. With this downstream process we were able to produce highly pure (>99% protein purity by reversed phase HPLC) cp caspase-2 in the hundreds of mg scale.
For evaluation of the production of cp caspase-2 and variants with and without solubility tag (T7AC), standardized lab-scale fermentations were performed. Expression clones were compared regarding cell growth and soluble and insoluble recombinant protein production.
Comparing the production of 6H-cpCasp2D and T7AC-6H-cpCasp2D in lab-scale fermentations, we observed that the production of 6H-cpCasp2D without solubility tag lead predominantly to inclusion body formation (
Comparing the production of three cp caspase-2 variants (cp caspase-2, mS9Pro E285 and mS9 Pro D285) with and without T7AC solubility tag, it turned out that the variant itself has no influence on the performance, no significant differences in cell growth and soluble cp caspase-2 expression. By means of the T7AC solubility tag the soluble expression of all three variants was significantly improved.
Cell growth kinetics off all cultivations were almost the same (VC<4%). Only at the end of the fermentations slight deviations were observed (
9.1.2.7 DoE Approach for Process Optimization
For final process optimization, a series of cultivation runs were conducted according to a Design of experiments (DoEs) approach described previously. The production clone BL21 (DE3)(pET30a-T7AC_6H_cpCasp2) was used. The influence of different growth rates (μ=0.03, 0.05 and 0.07 h−1) and induction strengths (0.5, 0.9 and 1.3 μmol IPTG/g CDM) were investigated regarding cell growth and soluble and insoluble recombinant protein production. The results are shown in Table 18.
It was observed that the specific yield of soluble cp caspase-2 was higher at low growth rates and IB formation decreased. The calculated CDM was not reached at the end of fermentation with μ=0.03 h−1 due to too high expression levels (
Nevertheless, the highest volumetric soluble yield was reached with μ=0.03 h−1 and 0.9 or 0.5 μmol IPTG/g CDM (
This process can be applied to all cp caspase-2 variants irrespective if it includes or not mutations at positions that increase the P1 tolerance.
9.2 Downstream Processing of cp Caspase-2 and Variants
9.2.1 Downstream Processing of cp Caspase-2 without Solubility Tag
The E. coli cell mass from fermentations as described under 10.1 was harvested by centrifugation at 18,590 rcf for 15 minutes and the supernatant was discarded. The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0). The cells were resuspended at a concentration of 150 g wet cell mass per L. Cell lysis was performed through high pressure homogenization at 700 bar/70 bar with two passages. The homogenate was centrifuged at 18,590 rcf for 2 hours. The pellet was discarded and the supernatant used. Before chromatography the supernatant was filtered through a 0.22 μm membrane.
The cp caspase-2 carrying a poly-his-tag was captured using immobilized metal affinity chromatography. The following buffers were used: equilibration buffer: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0. Wash buffer: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 30% iso-propanol, pH 7.0. Elution buffer: 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.0.
Clarified supernatant was loaded to an equilibrated Ni-Sepharose 6 Fast Flow column to a capacity of ˜40 mg/mL. A residence time of 3-5 minutes was used. After loading was completed the column was washed for 5 column volumes (5 CV) with equilibration buffer, 10 CV with wash buffer and 5 CV of equilibration buffer. The bound cp caspase-2 was eluted using a linear gradient from 0-100% elution buffer in 10 CV, with a 10 CV hold step to fully elute all protein.
The elution fractions were analyzed using SDS-PAGE and all fractions containing cp caspase-2 were used for the next purification step.
The capture eluate of cp caspase-2 was buffer exchanged before the polishing chromatography step. Tangential flow ultra-/diafiltration with a 5 kDa cut off membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. In total 5 volumes were exchanged.
The capture step used cation exchange chromatography on SOURCE 30S using the following buffers: equilibration buffer A: 50 mM sodium citrate, pH 5.0. Elution buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0.
Buffer exchanged capture eluate was loaded on the equilibrated polishing. The residence time was held constant at 5 minutes. The column was loaded to a capacity of ˜100 mg/ml. cp caspase-2 was eluted in a linear gradient from 0-100% B in 20 CV. The elution fractions were analyzed using RP-HPLC as described under 10.3 and the fractions showing a purity of ˜99% were combined and stored at −80° C.
9.2.2 Downstream processing of cp caspase-2 with solubility tag
Lysis was executed as described in section 9.2.1 but cells were resuspended at a concentration of 200 g wet cell mass per L.
IMAC was executed as described above, but the column was loaded to a capacity of ˜30 mg/ml and a residence time of 2-3 minutes was chosen. After loading was completed the column was washed for 5 column volumes (5 CV) with equilibration buffer, 5 CV with wash buffer and 1 CV of equilibration buffer. The bound cp caspase-2 was eluted using a linear gradient from 0-50% elution buffer in 5 CV, followed by a gradient from 50-100% B in 1 CV, followed by a 2 CV hold step at 100% to fully elute all protein.
The elution peak fraction was used for the next purification step. Buffer exchange of the capture eluate was executed as described above.
The capture step used cation exchange chromatography on SP Sepharose High Performance using the following buffers: equilibration buffer A: 50 mM sodium citrate, pH 5.0. Elution buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0.
Buffer exchanged capture eluate was loaded on the equilibrated polishing. The residence time was held constant at 1-2 minutes. The column was loaded to a capacity of ˜50 mg/ml. The column was washed for 5 column volumes (5 CV) with 30% B, cp caspase-2 was eluted with 10 CV of 45% B and the column was stripped with 3 CV of 100% B. The elution fraction was aliquoted and stored at −80° C. The purity was determined by RP-HPLC and was ˜99% (98.6-99.4%).
9.3 Characterization of cp Caspase-2 and Variants
9.3.1 Purity Determination of cp Caspase-2 and Variants (HPLC)
Experiments were performed on a Tosoh TSKgel Protein C4-300, L×I.D. 5 cm×4.6 mm, 3 μm column with a guard column on a Waters e2695 HPLC. Mobile phase A was water with 0.15% trifluoroacetic acid (TFA) and mobile phase B was acetonitrile with 0.15% TFA. The flowrate was 1 ml/min. Temperature of the column oven was 40° C., temperature of the autosampler 10° C. The following gradient was used as shown in Table 19.
200 μL of purified cp caspase-2 (or variant) sample (˜4 g/L) was diluted with 100 μL PBS and 100 μL 2 M Dithiothreitol (DTT). 10 μl of 0.22 μm filtered sample were injected. The outlet was monitored at 214 nm and 280 nm. The HCP peaks eluted between retention times 3.8 and 9 minutes. The cp caspase-2 peaks eluted between 9.2 and 12.4 minutes. The peak areas in the 214 nm signal were used to calculate the purity of the protein of interest.
9.3.2 Quantification of Released Fusion Tag with RP-HPLC
The calibration curve was generated mixing the substrate protein, e.g. human fibroblast growth factor 2 (hFGF-2), and cp caspase-2 in a ratio 10:1 (in triplicates) and incubated for 4 hours at 25° C. while shaking. The reaction was stopped by adding formic acid to a final concentration of 0.3% or by adding cystamine to a final concentration of 10 mM. Each triplet was diluted with PBS buffer to get six different concentrations (100 μM, 46 μM, 21 μM, 10 μM, 4 μM, 2 μM).
10 μL of 0.22 μm filtered sample were injected to a reversed phase high pressure liquid chromatography (RP-HPLC) using a method outlined below. The outlet was monitored at 214 nm. The fusion tag peaks eluted between retention times 3.9 and 5.6 minutes. The peak areas in the 214 nm signal were used to calculate the quantity of the fusion tag using a linear calibration function.
Experiments were performed on a Tosoh TSKgel Protein C4-300, L×I.D. 5 cm×4.6 mm, 3 μm column with a guard column on a Waters e2695 HPLC. Mobile phase A was water with 0.15% trifluoroacetic acid (TFA) and mobile phase B was acetonitrile with 0.15% TFA. The flowrate was 1 mL/min. Temperature of the column oven was 40° C., temperature of the autosampler 10° C. The following gradient was used (Table 20):
9.3.3 Determination of Enzymatic Activity with FRET Assay
A Förster resonance energy transfer (FRET) assay for the determination of the Michaelis-Menten enzymatic activity parameters was performed in the following way.
The substrates were obtained from Bachem AG and were of the general structure of Abz-VDVAD-XA-Dap (Dnp), where all 20 amino acids were substituted for X (the P1′ position). All substrates were dissolved in 10 mM HEPES, pH 7.5 to a concentration of 750 μM. Abz means 2-Aminobenzoyl, Dap (Dnp) means α,β-diamino-propionic acid (2,4-Dinitrophenyl).
The buffer for the assay was 50 mM HEPES, 150 mM NaCl, pH 7.2.
The calibration curve was generated by incubating varying amounts of substrate (20 μM, 6.9 μM, 2.4 μM, 0.8 μM, 0.3 μM, 0.1 μM) with 72 μM cp caspase-2 D285E in phosphate buffered saline (PBS) and incubated at room temperature for up to 24 hours. 100% conversion was assumed. Fluorescence was measured in black 96 well plates on a Tecan Infinite M200 Pro plate reader. Excitation wavelength was 320 nm, emission wavelength 420 nm.
Michaelis-Menten kinetics were measured by varying substrate concentrations (200 μM, 100 μM, 50 μM, 20 μM, 10 μM) at constant enzyme concentration ([E]=1 μM). The initial slope was measured by measuring the fluorescence for 3-15 minutes (or 3 to 20 hours for proline as P1′) and calculating the slope of the initial measurement in μM product generated per second. Fluorescence was measured in black 96 well plates on a Tecan Infinite M200 Pro plate reader. Excitation wavelength was 320 nm, emission wavelength 420 nm. In the FRET assay all substrates, except for proline as P1′ showed excellent linearity for at least a few minutes.
Evaluation of the data was performed by fitting the data in the TableCurve 2D v5 software to a Michaelis-Menten kinetic:
Where v is the initial slope, Vmax is the maximum rate, KM is the Michaelis constant and [S] is the substrate concentration. The parameters Vmax and KM were fitted. kcat was calculated by dividing Vmax by the enzyme concentration [E].
An example kinetic curve can be seen in
The results are shown in Table 21 and 22 and
kcat/KM
9.3.4 Determination of Enzymatic Activity with Model Proteins
Purified model proteins carrying a fusion tag e.g. MHHHHHHGSGVDVAD (SEQ ID No. 252) fused to the N-terminus of the POI were used as the substrate for a kinetic assay. All model protein substrates were formulated in PBS. The model proteins used were human fibroblast growth factor 2 (FGF-2) which was produced as a soluble protein in the cytosol of E. coli, human tumor necrosis factor alpha (TNFa) which was produced as a soluble protein in the periplasma of E. coli, and a single chain variable fragment, BIWA4 (scFv) which was produced as an inclusion body in the cytosol of E. coli. The buffer for the digestion was PBS.
To determine a Michaelis-Menten kinetics six different concentrations (100 μM, 384 μM, 668 μM, 952 μM, 1236 μM, 1520 μM) of the model protein hFGF-2 were incubated with 1 μM of different cp caspase-2 variants (in triplicates). The reaction was stopped by adding formic acid to a final concentration of 0.1% after 45 seconds.
To determine a Michaelis-Menten kinetics five different concentrations (50, 101, 135, 220, 305 μM) of the model protein BIWA4 were incubated with 10 μM of mS9ProD/E (in triplicates). The reaction was stopped by adding formic acid to a final concentration of 0.2% after 420 seconds.
To determine a Michaelis-Menten kinetics six different concentrations (100 μM, 299 μM, 498 μM, 697 μM, 896 μM, 1093 μM) of the model protein tumor necrosis factor α were incubated with 10 μM of different cp caspase-2 variants (in duplicates). The reaction was stopped by adding formic acid to a final concentration of 0.1% after 420 seconds.
Product generation was determined using the RP-HPLG method outlined in section 9.3.2. Initial rate (v0) in μM/s of each concentration was calculated using the fusion tag peak area at the time points of the initial slope. This data was transferred to TableCurve 2D to fit a Michaelis-Menten kinetics in order to get values for Vmax and KM.
9.3.5 Protein Cleavage in Solution
The fusion proteins as described in section 9.3.4 were used substrates for a kinetic assay. All model protein substrates were formulated in PBS. The buffer for the digestion was PBS.
Product generation was determined using the RP-HPLG method outlined in section 9.3.2.
For the digestion of fusion protein, a certain concentration of fusion protein was incubated under agitation at room temperature with a defined concentration of cp caspase-2. For the digestion of hFGF-2, 2.9 g/L hFGF-2 fusion protein was incubated with 0.055 g/L cp caspase-2 or the variant mS9 Pro D285E or mS9 Pro D. The cleavage of FGF-2 fusion protein was also performed with varying concentrations of FGF-2 (2 g/L and 10 g/L) and cp caspase-2 (0.02 g/L, 0.1 g/L) and the product generation was determined over time. For the digestion of TNF-alpha, 2.4 g/L TNF-alpha fusion protein was incubated with 0.046 g/L cp caspase-2 or the variant mS9 Pro D285E. For the digestion of GFP, 9.1 g/L GFP fusion protein was incubated with 0.11 g/L cp caspase-2 or the variant mS9 Pro D285E.
Tag cleavage from FGF-2 with cp caspase-2 and variants thereof showed very fast processing. Complete removal of the tag for hFGF-2 was measured after 15 minutes for mS9 Pro D285E and mS9 Pro D and after 180 minutes for cp caspase-2 as shown in
TNF-alpha is a more difficult substrate, due to its N-terminal valine. The cleavage reaction is slower compared to FGF-2, but high yields are still possible. TNF-alpha fusion protein could be cleaved efficiently with either cp caspase-2 or mS9 Pro D285E variant, with the variant producing up to 98% cleaved protein of interest (
The cleavage of GFP fusion protein is slower, but up to 60% of GFP can be processed as shown in
9.3.6 Protein Cleavage with Immobilized Enzyme
Enzyme immobilization was performed through amine coupling. The primary amino groups of the lysine residues on the enzyme were coupled to activated NHS-groups, placed on spacer arms in the resin. The coupling forms a stable amide bond. cp caspase-2 was immobilized at the following concentrations 1 μM, 10 μM, 50 μM and 100 μM. The enzyme was diluted in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) to reach the desired concentration. For a 500 μl column, around 1.5-2 ml of resin slurry in 100% isopropanol was transferred to a 15 ml centrifuge tube. The first step was to wash the resin for removal of the isopropanol. This was done with 10 to 15 resin volumes of cold 1 mM HCl. Immediately after the washing step, the resin and the coupling buffer with enzyme were mixed using a vortex. The sample was left at 4° C. overnight for the coupling reaction. After the coupling the samples were mixed with blocking buffer (0.1 M Tris-HCl, pH 8.5) and kept in the buffer for 2 to 4 hours to block all non-reacted NHS groups in the resin. The samples were then washed alternating two buffers with high (0.1 M Tris-HCl, pH 8.5) respectively low (0.1 M HAc, 0.5 M NaCl, pH 4.7) pH using 3 medium volumes each time and repeating the procedure for 3 to 6 times. In each step, the buffer was added, the sample vortexed, thereafter centrifuged (1.000×g, 1 min, 4° C.) and the supernatant was discarded. The immobilized resin was then stored at 4° C. in either 20% EtOH or 0.01% NaN3 in 1×PBS to prevent microbial contamination before packed in columns.
To determine the kinetics and activity of the immobilized cp caspase-2, the columns were tested with different concentrations of the model protein, hFGF-2 at varying residence times in the column. The flow through from the sample application and first column wash was collected in fractions in 96 deep well plates containing 1/1000 formic acid to deactivate any leaked enzyme and to stop the reaction. The amount of product was quantified using the RP-HPLC method outlined in section 9.3.2.
The amount of cleavage varied with residence time (See
10.1 Escherichia coli Strains
E. coli BL21 (DE3) was used for all standard protein expressions and for the selection system as outlined in Example 3.
For plasmid extractions and for cloning experiments E. coli strain NovaBlue (Novagen, Madison, Wis., USA) was used as a host.
10.2 Culture Media
TY (tryptone-yeast) medium (1% peptone, 0.7% yeast extract, 0.25% (w/v) NaCl). TB medium (1.2% peptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 72 mM K2HPO4).
SOC (super optimal broth with catabolite repression) (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 and 20 mM glucose, pH 7.0). Medium for the recovery of cells after transformation.
Optimized M9 minimal medium (50 mM Na2HPO4, 20 mM KH2PO4 10 mM NaCl, 1 mM MgSO4, 0.1 mM CaCl2), 0.4% Glucose, 20 mM NH4Cl, 0.5% (w/v) casamino acids, 10 μg/ml FeSO4, vitamins (0.001 mg/ml of each biotin, thiamine, riboflavin, pyridoxine, niacinamide). For induction 0.1 to 0.4 mM IPTG were used.
10.3 Recombinant Protein Expression
Standard Expression Protocol: Fusion proteins, which are substrates (hereinafter “fusion proteins” or “substrates”) for caspases, caspase-2 variants, cp caspases-2 with (hereinafter “P1′ tolerable cp caspases-2”) and without mutations (hereinafter “wild-type cp caspases-2”) for increased tolerability for the amino acid in P1′ position, as described in Example 2, Section 2.2 were expressed in TY medium in shaking flasks. A 20 ml preculture was prepared by inoculation with a single colony and incubated shaking at 220 rpm, 37° C. overnight. The next day the culture was diluted 1:50 in 1000 ml fresh TY medium and incubated shaking at 220 rpm, 37° C. until induction at OD600 1.0 with 1 mM IPTG, at 37° C., 220 rpm, for 4 h.
Expression protocol for wild-type cp caspases-2 and P1′ tolerable cp caspases-2: The cp caspases-2 were expressed in TB medium in shaking flasks. A 20 ml preculture was prepared by inoculation with a single colony and incubated shaking at 220 rpm, 37° C. overnight. The next day the culture was diluted 1:50 in 1000 ml fresh TB medium and incubated shaking at 220 rpm, 37° C. until induction at OD600 1.2 with 0.4 mM IPTG, 25° C., 220 rpm, for 4 h.
10.4 Cell Lysis and Protein Purification
Fusion proteins, wild-type cp caspases-2 and P1′ tolerable cp-caspase-2 variants were purified using Immobilized Metal Affinity Purification (IMAC).
The harvested cell pellets were suspended in Tris-Buffer (50 mM Tris, 50 mM NaCl, pH 7.5), disrupted with a French press and the clarified supernatant applied to an IMAC column (HisTrap FF Crude, 1 ml, GE Healthcare). Washing was executed for five column volumes with running buffer (50 mM Tris/HCl, pH 7.4, 300 mM NaCl, 20 mM Imidazole), the fifth wash fraction had an increased imidazole concentration (40 mM). Elution was conducted for five column volumes with buffer containing 250 mM imidazole.
After affinity-chromatography imidazole and excess NaCl were exchanged to Tris-buffer with a sepharose column (HiTrap Desalting, 5 ml, GE Healthcare). All elution fractions were pooled, the concentration determined with a BCA assay, and the proteins stored in Tris-Buffer with 2 mM DTT at −80° C.
10.5 Testing of Wild-Type cp Caspases-2 and P1′ Tolerable cp Caspases-2 (Hereinafter Together “cp Caspases-2”)—In Vitro Cleavage Assay
The activity of purified caspases was assessed with an in vitro cleavage assay. The samples were analyzed with SDS-PAGE to separate cleaved and unprocessed substrate. The band intensities were measured with ImageQuant TL 1D software, version 8.1 (GE Healthcare) and used for statistical analysis and calculation of cleavage efficiency. To standardize the process samples with about 50% of cleaved fusion protein were used for calculations.
Standard conditions where defined as: enzyme to fusion protein mass ratio of 1:100 (1 mg/ml substrate and 0.01 mg/ml caspase, molar ratio 1:170) in caspase assay buffer (20 mM PIPES, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mm EDTA, 10 mM DTT, pH 7.2) and incubation at 25° C. For slowly proceeding reactions the caspase concentration was increased to 0.1 mg/ml (enzyme to fusion protein mass ratio 1:10). Fusion proteins are described in Example 2, section 2.2.
cp caspase-2 (0.01 mg/ml) (SEQ ID No. 6) cleaved 50% of the substrate VDVAD-E2 with a P1′ glycine (1 mg/ml) (SEQ ID No. 33) at 25° C., in caspase assay buffer within 1 min (
By N-terminal Edman sequencing of the processed fusion protein, it was proven, that it was only cleaved between the VDVAD recognition site and the P1′ glycine.
For in vitro cleavages that compared the activity to commercially available caspase-2 about 0.005 mg/ml wt caspase-2 (Caspase-2 (human), recombinant, active, Enzo Life Sciences Inc.; Farmingdale (N.Y.), USA) were used to cleave 1 mg/ml VDVAD-E2 with a P1′ glycine (mass ratio 1:200, molar ratio 1:340).
11.1 Cloning of Constructs
To create specific changes like deletions, insertions of linkers or tags, substitutions, site mutations of single bases or the like in initial proteins, site directed mutagenesis was performed.
The specific primers containing the desired mutations were designed with 5′ ends annealing back-to-back and were used for an exponential amplification of the whole plasmid with a high-fidelity DNA polymerase in a polymerase chain reaction (PCR).
After amplification a KLD (kinase ligase Dpnl) reaction was performed. In this treatment the PCR product was incubated with a Kinase, a Ligase and Dpnl restriction enzyme, so that the PCR fragments were phosphorylated and ligated to a circular plasmid and the bacterial derived, methylated template DNA was digested. NovaBlue heat shock cells were transformed with the constructs and a fraction of the cell suspension was plated on TY agar containing the appropriate antibiotic. Successful cloning was verified by sequencing of single colonies.
All fusion proteins and cp caspases-2 as described here were expressed and purified as described in Example 10, sections 10.3 and 10.4 unless otherwise stated.
Protein and nucleotide sequences of all constructs are listed in
11.2 Fusion Proteins as Substrates for Caspases, Caspase-2 Variants and Cp Caspases-2
Human ubiquitin-conjugating enzyme E2 L3 (E2; UniProt ID P6803612) as fusion protein was used as standard caspase substrate. A fusion protein (VDVAD-E2) with N-terminal His tag, short GSG-linker and VDVAD caspase-2 recognition site was designed. The first amino acid after the cleavage site (P1′) was a glycine (VDVAD-E2, SEQ ID No. 33). The whole protein has a size of 21.3 kDa, whereas when the tag is cleaved off, the E2 protein itself has 19.5 kDa. This difference is big enough to visualize the cleavage activity on an SDS-PAGE.
As the P1′ site is known to influence cleavage activity, E2 was expressed and purified with all twenty possible residues after the VDVAD cleavage site. E2 was also cloned with cleavage sites differing from VDVAD. All tested tag sequences fused to E2-protein are listed in Table 1.
β-galactosidase was chosen as a model protein, because due to its large size (116 kDa) it is vulnerable to unspecific cleavage. An N-terminal His tag as well as a GSG linker and the caspase-2 cleavage site VDVAD were added (SEQ ID No. 34). The first amino acid after the cleavage site (P1′=the N-terminal amino acid of the β-galactosidase) is a methionine (M, Met).
Superoxide Dismutase, SOD, was used as a model fusion protein with an N-terminal 6 His Tag, a GSG linker and the recognition site, VDVAD, fused to the N-terminus of SOD (SEQ ID No. 193). The first amino acid after the cleavage site (P1′=the N-terminal amino acid of Superoxide Dismutase) is glycine (g, Gly).
11.3 Designed Wild-Type cp Caspases-2 and P1′ Tolerable Cp-Caspase-2 Variants
Circularly permuted caspase-2: Circularly permuted caspase-2 variants (cp caspases-2) were designed. based on the sequence of human caspase-2 (UniProtKB14 ID P42575, SEQ ID No. 11); the N-terminal CARD was removed and the order of large (LS) and small subunit (SS) exchanged to create a constitutively active caspase. The SS was linked to the N-terminus of the LS via a GS-linker. Optionally the SS pro-peptide was linked to the N-terminus of the SS. In this case to ensure expression as a single chain protein, an aspartate (Asp343 in the wild-type sequence of caspase-2, Asp21 in the cp caspase-2) was mutated to alanine, to avoid cleavage of the small subunit from a p14 to a p12 chain. This resulted in the cp caspases-2 SEQ ID No. 9, SEQ ID No. 6 and, SEQ ID No. 76, both of the latter having additionally an N-terminal 6 His tag. The basic structures of these variants are shown in
The protein sequence was codon optimized for E. coli with the GeneArt™ online tool (Thermo Fisher Scientific). Between the small and the large subunit, a glycine-serine linker was added which also forms a BamHI restriction site. This enables the separate cloning of the subunits and facilitates the creation of chimera consisting of subunits from different caspases. The N-terminal His tag enabled IMAC-purification.
All cp-caspase-2 variants described under this chapter 2.3 were constructed based on SEQ ID No. 6, except otherwise described. The amino acid positions of the mutations indicated correspond to SEQ ID No. 6, unless explicitly stated otherwise. All variants have 6 His Tag, except otherwise described.
cp caspase-2 Stop and cp caspase-2 D285E: To test the influence of the propeptide annotated in UniProtKB14 (ID P42575) within the C-terminus of the large subunit, a truncated version was produced by deleting amino acids 286-292 in the cp caspase-2 of SEQ ID No. 6, thereby creating the cp caspase-2 Stop variant (SEQ ID No. 14), and an uncleavable variant (cp caspase-2 D285E) (SEQ ID No. 13) was created.
cp caspase-2 with C-terminal Strep tag: Strep tags were fused C-terminal to create cp caspase-2 Strep and cp caspase-2 D285E Strep variants (SEQ ID No. 15 and SEQ ID No. 16, respectively).
In SEQ ID No. 15, a Strep tag was fused to the C-terminus of the cp caspase 2 (SEQ ID No. 6), which was mutated to VDQQS (the substitution: D292S), as experiments had shown that VDQQE is recognized as a cleavage site. Despite the VDQQS mutation, the Strep tag was partially cleaved from the caspase. The cleavage product had the same size as the Stop variant (31.9 kDa), indicating that it had been cleaved at the DETD-R (between Asp285 and Arg286) and not at the VDQQS site.
Therefore, a Strep tag was added to the C-terminus of cp caspase-2 with the D285E and the E292S mutations. This variant (SEQ ID No. 16) was expressed as a single chain with 33.9 kDa. Proving that the mutation of Asp285 to Glu prevents cleavage. The C-terminal Strep-tag did not influence the cleavage activity of this variant.
cp caspase-2 D282T and cp caspase-2H185A D282T: Two cp caspases-2 were generated, the first with a D282T mutation and the second with an additional H185A mutation in cp caspase-2 (SEQ ID No. 6) comprising SEQ ID No. 17 and SEQ ID No. 18, respectively.
cp caspase-2 G171D, cp caspase-2 V225G, and cp caspase-2 D282E: cp caspase-2 (SEQ ID No. 6) was mutated at positions 171, 225, or 282 respectively resulting in amino acid exchanges G171D, V225G, or D282E resulting in the variants having SEQ ID No. 190, 192 and 191, respectively.
cp caspase-2 with different linkers between small and large subunit: The GS linker between small and large subunit of cp caspase-2 (SEQ ID No. 6) was mutated. Resulting variants contained no linker (cp caspase-2 A Linker, SEQ ID No. 73), a GGSGG linker (cp caspase-2 5 aa Linker, SEQ ID No. 74), and a GSAGSAAGSG linker (cp caspase-2 10 aa Linker, SEQ ID No. 75).
cp caspase-2 with partial and without small subunit propeptide: The propeptide of the small subunit of cp caspase-2 (SEQ ID No. 6) was mutated by site directed mutagenesis. Deletion of residues 8-22 produced a variant without propeptide (cp caspase-2 Δ SS Prop, SEQ ID No. 76, see also
cp caspase-2 with shifted circular permutation: cp caspase-2 Δ SS Prop (SEQ ID No. 76) was used to generate variants with shifted circular permutation. At the N-terminus of the small subunit three amino acids were deleted and added to the C-terminus of the large subunit. Because of possible auto-cleavage, detected when adding a Strep-tag to the C-terminal end of cp caspase-2, additionally the mutations D267E and D274S according to SEQ ID No. 76 were inserted. The resulting variant cp caspase-2 C-term +3 (SEQ ID No. 82) was expressed, purified and tested as described above in Example 10, sections 10.3, 10.4 and 10.5.
In parallel, a variant was generated by deletion of the 3 C-terminal residues of the large subunit and insertion of those residues to the N-terminus of the small subunit of cp caspase-2 Δ SS Prop (SEQ ID No. 76). The resulting variant cp caspase-2 N-term +3 (SEQ ID No. 83) was expressed, purified and tested as described in the standard protocol in Example 10.
Both variants were expressed with an N-terminal 6 His-tag.
cp caspase-2 C203S: The variant was created by insertion of the C203S mutation in cp caspase-2 (SEQ ID No. 6) resulting in SEQ ID No. 198.
cp caspase-2 S9 C203S: The substitution C203S was inserted in cp caspase-2 S9 (SEQ ID No. 51), resulting in SEQ ID No. 199.
cp caspase-2 N85C and cp caspase-2 A86C: The variants were created by insertion of the mutations N85C (SEQ ID No. 80) and A86C (SEQ ID No. 88) in cp caspase-2 (SEQ ID No. 6).
Homologous cp Caspases-2:
The cp caspases-2 from different species were constructed analogue to the cp caspase-2 of human origin (SEQ ID No. 6).
Based on the sequence of Tasmanian devil caspase-2 (Sarcophilus harrisii, UniProtKB14 ID G3VQP7, SEQ ID No. 95) and Ghost shark caspase-2 (Callorhinchus milii, UniProtKB14 ID V9KZT1, SEQ ID No. 113) the N-terminal CARD was removed and the order of large and small subunit exchanged to create a constitutively active caspase. The SS was linked to the N-terminus of the LS via a GS-linker. The SS pro-peptide was linked to the N-terminus of the SS. To ensure expression as a single chain protein, an aspartate (corresponding to Asp343 in the wild-type sequence of human caspase-2, Asp21 in the cp protein) was mutated to alanine, to avoid cleavage of the small subunit propeptide.
The protein sequence was codon optimized for E. coli with the GeneArt™ online tool (Thermo Fisher Scientific). Between the small and the large subunit, a glycine-serine linker was added which also forms a BamHI restriction site. This enables the separate cloning of the subunits and facilitates the creation of chimera consisting of subunits from different caspases. The N-terminal His tag enabled IMAC-purification.
Resulting variants are Sarcophilus cp caspase-2 (SEQ ID No. 64) and Callorhinchus cp caspase-2 (SEQ ID No. 68).
Mutations at positions corresponding to (at positions functionally equivalent to) residues Glu105 and Glu172 in cp caspase-2 (SEQ ID No. 6) were inserted in Sarcophilus cp caspase-2, generating variant Sarcophilus cp caspase-2 E105V E172V (SEQ ID No. 78).
Mutations at positions corresponding to Glu105 and Gly171 in cp caspase-2 (SEQ ID No. 6) were inserted in Callorhinchus cp caspase-2, generating variant Callorhinchus cp caspase-2 E105V G171D (SEQ ID No. 79).
Additionally, the variants were cloned containing an N-terminal T7AC-6 His tag (SEQ ID No. 84, 85, 86, 87).
Functionally equivalent positions are listed in Table 2.
UniProt IDs: Human (P42575), Mouse (P29594), Sheep (W5Q8H6), Tasmanian Devil (G3VQP7), Chicken (Q98943), Anolis (H9GC58), Alligator (A0A1U8D1 G6), Xenopus (F6RDY9), Danio (Q0PKX3), Ghost Shark (V9KZT1), Sea squirt (A0A1W2WKB0)
The homologous cp caspases-2 described above were fermented in benchtop fermentations.
For the benchtop fed-batch cultivations, a DASGIP® parallel bioreactor system (Eppendorf AG, Germany) enabling four parallel cultivations was used. The total vessel volume was 2.1 L with a maximum working volume of 1.8 L The bioreactors were equipped with a pH probe (Hamilton Bonaduz AG, Switzerland), an optical DO probe (Hamilton Bonaduz AG), and a DASGIP® GA4X-module (Eppendorf AG) for online off-gas monitoring. Pre-cultivation and batch phase were identical to the standardized fermentations as described in Example 18 (section 18.1.2.2) unless stated otherwise. The fed-batch phases were performed at 30° C. For biomass production the fed-batch phase was performed with an exponential feed (μ=of 0.05 h−1) for 2.74 generations resulting in a total feed time of 39 h. The calculated CDM was 34 g/L.
Induction started with fed-batch phase by adding feed medium including IPTG (so called “over feed” induction) to achieve a final IPTG concentration of 0.5 μmol IPTG/g theoretical CDM at the end of the fermentation and a protein production for 4 generations.
The production of two different cp caspase-2 homologous and variants thereof, T7AC-6H-cpCasp2_cal (SEQ ID No. 68, also called “cpCasp2-cal”), T7AC-6H-cpCasp2_sar (SEQ ID No. 64, also called “cpCasp2_sar”), T7AC-6H-cpCasp2_cal_E105V, G171D (SEQ ID No. 79, also called: “T7AC-6H-cpCasp2_cal_mut” or cpCasp2_cal_mut”) and T7AC-6H-cpCasp2_sar_E105V, E172V (SEQ ID No. 78, also called: “T7AC-6H-cpCasp2_sar_mut” or cpCasp2_sar_mut”), were investigated in benchtop fermentations with a μ=0.05 h−1 and an IPTG concentration of 0.9 μmol IPTG/g CDM during induction. The cell growth kinetics of all production clones were comparable (
Purification of the homologous cp caspases-2 was performed as described in section 18.2.2.
Michaelis Menten kinetic was determined as described under section: kinetic and P1′ tolerability were tested as described in 18.3.3.
Michaelis Menten kinetic was determined for the homologous cp caspases-2 for the following substrates: VDVADFA, VDVADGA, VDVADQA and VDVADVA, where the P1′ amino acid is indicated by bold and underlined font.
The FRET results in Table 25 show a drastic difference in catalytic efficiency between the two proteases. Adding the mutations E105V and E172V into the cpGasp2 of S. harrisii, greatly increases the catalytic efficiency kcat/KM by a factor of 2 to 4. This increase is mostly driven by an increase in the turnover number kcat, while the Michaelis constant KM remains mostly unchanged.
As can be seen in Table 26, cpCasp2_cal_mut with the two mutations E105V and G171 D shows a higher activity for both G and F at the P1′ site compared to cpCasp2_cal. In detail, the v0 for the VDVADFA substrate was three times higher for cpCasp2_cal_mut. The v0 for the VDVADGA substrate was two times higher for cpCasp2_cal_mut. This corresponds to an increase in P1′ tolerability of 173%.
Selection system to detect variants with improved P1′ tolerance A selection system was used for the improvement of cp caspase-2. It is based on a circularly permuted ATCase (aspartate transcarbamoylase) catalytic subunit and a pyrimidine auxotroph strain. The pyrBI operon (encoding regulatory pyrl and catalytic pyrB subunits of ATCase) was deleted in E. coli BL21 (DE3), so this knock-out strain can only survive in media containing pyrimidines or when the cells are complemented with a vector encoding ATCase. A cp catalytic subunit of ATCase (cp-pyrB), which harbors its new N-terminus in the interior of the protein, is used to detect specific proteases via the growth of E. coli, because fusion of any stretch of amino acids to its N-terminus renders the enzyme inactive as it can no longer fold properly due to space limitations in the interior of the protein. However, if a protease is provided that can exactly cleave off this additional stretch of amino acids, the enzyme gets reactivated.
12.1 Design of Constructs and cp Caspase-2 Mutant Libraries
Selection medium: Optimized M9 medium (see Example 10, section 10.2) Strain: E. coli BL21(DE3) with pyrBI operon exchanged to kanamycin resistance (id est: pyrBI is deleted)
Vectors: expressions of the ATCase subunits, cp-pyrB and pyrl from pETDuet™-1 vector using T7 promoters and the ampicillin-resistance as selection marker; expressions of the diverse caspase variants from pACYCDuet™-1 vector using a T7 promoter and the chloramphenicol resistance marker. Selection protocol was performed with respective cotransformations with simultaneous use of ampicillin, kanamycin and chloramphenicol in the above selection medium.
VDVAD-cpATCase
The used pETDuet-1 plasmid (substrate plasmid), contained a pyrl gene in MCSI (SEQ ID No. 20) and cp-pyrB gene in MCSII (SEQ ID No. 21). In pyrl the potential caspase cleavage site DQVD was changed to DQVE by mutation of Asp73. A 6 His tag followed by a GSG linker and a caspase recognition site were fused to the N-terminus of cp pyrBc227 [25]. This hinders the correct folding of the enzyme and makes it inactive, but proteolytic cleavage of this tag can restore its function. The first Met of cp pyrB was deleted. The amino acid after Met is Thr. The cpATCase is still active when this residue is substituted. Only mutations to His, Lys, Phe, Tyr, and Trp render it inactive. This enables the selection for caspases with improved or altered recognition site specificity and/or improved P1′ tolerance. CpATCase constructs with 6 His-GSG-VDVAD-ΔM-X-pyrB (SEQ ID No. 22) were used for in vivo selection of altered P1′ tolerance.
Construction of cp Caspase-2 Mutant Library—ep PCR and oe PCR
Mutant gene libraries of different cp caspases-2 were generated by error prone (ep) PCR and overlap extension (oe) PCR of vector and the mutated caspase gene. The linear DNA fragments were ligated using T4 DNA ligase. The amount of mutations can be modified by changing the Mg (II) and Mn (II) ion concentrations in the PCR buffer. The used concentrations caused in average one to three amino acid exchanges in the caspase. The cp caspases-2, of which mutant libraries were made of, are indicated in Table 5 in the column “Mutated Caspase”.
12.2 Selection of cp Caspase-2 Mutant Libraries
The caspase mutant libraries were transformed into E. coli BL21(DE3) ΔpyrBI electro competent cells that already contained the cpATCase plasmid with the desired protease cleavage site and P1′ residue. Selection was executed either in optimized M9 medium or on M9 agar plates at 30° C. for 24-48 h. Liquid cultures were used to enrich mutants with improved growth. IPTG concentrations in liquid culture and in agar plates between 0.025 and 1 mM were used.
Mutant libraries in E. coli BL21(DE3) ΔpyrBI cells were selected with VDVAD-cpATCase with different P1′ residues. Selections were executed with Pro, Met, Thr, and Val. Selections with P1′ Met were executed with cp ATCase without deletion of the native methionine, all other selections were executed with constructs comprising SEQ ID No. 22. Selection with Met, Thr, and Val as P1′ lead to hundreds of positive variants, thus only the largest colonies were analyzed.
All together 77 clones with a total of 263 mutations were analyzed from all selections combined. Some mutations were found several times in independent experiments. The mutations of resulting variants in comparison to SEQ ID No. 6 are shown in Table 5 below. P1′ amino acids used for selection are indicated under “P1′ cpATCase”.
Mutations of variants were analyzed and several were selected for expression (clones harboring those variants were cultivated and variants expressed and purified as described in Example 10, Sections 10.3 and 10.4) and characterization by in vitro cleavages (as described in Example 10, Section 10.5). Variants were chosen when they had been enriched in liquid culture or contained mutations that were found several times independently. Description of those variants can be found in Example 13.
cp caspase-2 S9 D285E and S9 D285: Selection of a cp caspase-2 D285E (SEQ ID No. 13) library, containing about 5,500 variants, was performed, with VDVAD-cpATCase that contained a methionine as P1′ and with an induction strength of 0.025 mM IPTG. The E105V mutation was found repeatedly among 16 analyzed clones. One selected variant with this mutation (cp caspase-2 S9 D285E, SEQ ID No. 1) was expressed, purified and tested as described in Example 10.
The selected cp caspase-2 S9 D285E was mutated to generate the cp caspase-2 S9 D285 variant (SEQ ID No. 51). The variant was expressed, purified and tested as described above (Example 10).
cp caspase-2 mS9 Pro D285E and cp caspase-2 mS9 Pro D285: The cp caspase-2 S9 D285E (SEQ ID No. 1) variant was used for a further round of mutation because of its improved P1′ tolerance. The new mutant library contained about 10,000 variants and was selected with VDVAD-ΔM-Pro-cpATCase. Selection in liquid culture enriched a variant (mS9 Pro D285E, SEQ ID No. 70) with the mutations E105V, G171 D, V225G, D282E and D285E. The caspase was expressed and purified as described above.
The selected cp caspase-2 mS9 Pro D285E (SEQ ID No. 70) was mutated to generate the cp caspase-2 mS9 Pro D285 variant (SEQ ID No. 52). The variant was expressed, purified and tested as described above.
cp caspase-2 mS9 Thr 0.8: The variant with K83E, E105V, E172V, V255M, and D285Y mutations was selected from mutated cp caspase-2 S9 D285 (SEQ ID No. 51). The new variant (SEQ ID No. 53 and SEQ ID No. 54) was enriched in liquid culture in a selection with VDVAD-Thr-cpATCase and 0.8 mM IPTG. It was expressed, purified and tested as described in Example 10.
cp caspase-2 S17: Variant with E105V, C132R, E141G, H200R, and D285E mutations that was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-cpATCase with Met as P1′ and 0.1 mM IPTG. The variant was never purified and tested in vitro, mutations at positions 105, 132 and 105 were found repeatedly in different experiments.
cp caspase-2 S20: The variant with C203Y and D285E mutations (SEQ ID No. 26) was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-cpATCase with Met as P1′ and 0.1 mM IPTG.
cp caspase-2 D285E SV4: The variant with V201A and D285E mutations (SEQ ID No. 28) was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-Val-cpATCase and 0.1 mM IPTG. The mutation V201A was found several times independently.
cp caspase-2 SV19: The cp caspase-2 SV 19 (SEQ ID No. 81) was selected from variants with mutated C-terminus with VDVAD-Val-cpATCase and 0.1 mM IPTG.
The sequence equals the consensus-sequence of 13 active variants with mutated C-terminus.
cp caspase-2 D285E SV30: The variant with E174G and D285E mutations (SEQ ID No. 30) was selected from mutated cp caspase-2 D285E (SEQ ID No. 13) with VDVAD-Val-cpATCase and 0.1 mM IPTG. The variant was enriched in liquid culture.
14.1 β-Galactosidase (as Described in Example 11, Section 11.2)
The model substrate β-galactosidase contains four DXXD and one DXXE sites, three of which are on the surface and could be accessible to the caspase.
After incubating 1 mg/ml β-galactosidase fusion protein (with N-terminal tag including the recognition site VDVAD with 0.1 mg/ml cp caspase-2 (SEQ ID No. 6) for 24 hours, no unspecific cleavage was observed (
14.2 SOD (as Described in Example 11, Section 11.2)
14.3 VDVAD-Gly-E2 (as described in Example 11, Section 11.2) cleavage Values of all Tested cp Caspases-2 (of Examples 10-13 and 16)
cp caspase-2 (0.01 mg/ml) (SEQ ID No. 6) cleaved 50% of the substrate VDVAD-E2 with a P1′ glycine (1 mg/ml) at 25° C., in caspase assay buffer within 1 min.
These conditions were defined as standard activity to which all other reactions were compared (
The activity of all cp caspases-2 with the fusion protein (substrate), VDVAD-E2 with P1′ glycine, was tested as described in Example 10, section 10.5 to compare their standard proteolytic activities. Not all tested variants cleaved this standard substrate to 50% in 1 min. A list of the activities of all cp caspases-2 is given in Table 28.
14.4 P1′ Tolerance
Cleavage site specificity and P1′ tolerance of caspases have been studied using peptide substrates, degradome analysis, and phage libraries. Peptides are not ideal for this purpose, as structure influences the cleavage activity. Degradome studies, on the other hand, are influenced by the sequences occurring in the analyzed cells. To our knowledge, so far no study has systematically tested caspase specificity and P1′ tolerance with protein substrates. Therefore, we permuted the P1′ residue after the cleavage site in the fusion protein VDVAD-E2 (Example 11, section 11.2) to evaluate the cleavage efficiency of cp caspase-2 in dependency of the P1′ residue.
Glycine was highly preferred in the P1′ position, cleavage before all other residues was at least five-times less efficient. The group of amino acids that was reasonably well tolerated comprised small, basic, and aromatic residues, as well as Asn and Met.
Table 29 (Table 29.1 and Table 29.2) shows cleavage of E2 substrates with VDVAD recognition site and different P1′ residues (Example 11, section 11.2) by cp caspases-2 (of Examples 10-13 and 16). Cleavage was carried out as described in 10.5. Activity is given in percent of activity for cleavage of VDVAD-E2 with a P1′ glycine for each cp-caspase-2 variant. Thus Table 29 (29.1 and 29.2) shows the P1′ tolerance of the respective cp caspase-2 variant. All values (means±standard deviation) were determined with at least three independent experiments, executed with 1 mg/ml E2. For Asp-E2, Glu-E2, Ile-E2, Pro-E2 and Val-E2 cp caspase-2 concentration was 0.1 mg/ml, for all others 0.01 mg/ml. The given values already consider these concentration differences.
Table 30 (Table 30.1 and Table 30.2) further below shows the cleavage activity of all cpcaspase-2 variants for all P1′ amino acids related to the cleavage activity of the standard op caspase-2 (SEQ ID No. 6) in %. Thus Table 30 shows the extent of increase (or decrease) of P1′ tolerance.
Taken together, these data show that variants of a cp caspase-2, comprising amino acid substitutions at any one or more of positions 83, 105, 171, 172, 185, 225, 255, 282, 285 of SEQ ID No. 6, display significantly improved P1′ tolerance for at least one amino acid. In most cases, these variants comprise significantly improved P1′ tolerance for multiple amino acids.
Furthermore, these data show that even though amino acid substitutions at positions 85, 86, 132, 141, 174, 200, 201, 203 of SEQ ID No. 6 do not improve P1′ tolerance, they do not hamper caspase activity significantly. Table 6, for example, shows that variants comprising amino acid substitutions at positions 85, 86, 132, 141, 174, 200, 201, or 203 of SEQ ID No. 6 still cleave about 50% of the substrate VDVAD-E2 within 2 or 3 minutes. These represent examples for functionally active variants of cp-caspases-2 of the present invention. Furthermore, all variants selected using the selection system as described in Example 12 and as shown in Table 27 are further examples of functionally active variants of cp-caspases-2, since they all have catalytic activity for the cleavage of the VDVAD P1′ motiv (a caspase-2 cleavage site). Otherwise the colonies/clones would not have grown.
15.1 System for in vivo selection of alternative caspase recognition sites The selection system described in Section 12.1 of Example 12 is used for the selection of recognition sites different to VDVAD that are accepted by cp caspase-2 mS9 Pro with a 6 His-tag (SEQ ID No. 70).
A gene library of 6 His-GSG-XDXXD-ΔM-Thr-pyrB (SEQ ID No. 22) cpATCase constructs was cloned with degenerate primers to insert random mutations in the caspase recognition sequence at the positions P5, P3, and P2 (Forward primer sequence: nnnnnnGATACCCGCGTGCAAAAAG, reverse primer sequence: ATCnnnGCCGCTGCCATGATGATG). The primers were designed with their 5′ ends back-to-back for a PCR with a high-fidelity DNA polymerase which generates a linear DNA fragment of the whole vector (described in Section 11.1, Example 11). After KLD reaction NovaBlue heat shock cells were transformed with the gene library and diluted in an overnight culture, which was used for a DNA preparation. Sequencing of the pooled gene library was used to control the quality of the DNA preparation before selection to ensure a diverse mutant library.
E. coli BL21(DE3) ΔpyrBI cells were generated that contain the cp caspase-2 mS9 Pro construct (SEQ ID No. 70) in a pACYCDuet-1 vector.
The cells were transformed with the XDXXD-cpATCase library and after recovery in SOC medium the cells were either diluted in optimized M9 minimal medium or plated on optimized M9 agar plates containing 0.1 mM IPTG and incubated at 30° C. for 24-48 h.
15.2 Selected and Identified Alternative Caspase-2 Recognition Sites for Cleavage
cp caspases-2: 79 single colonies were sequenced and the nucleotide sequence of the cpATCase was analyzed detecting alternative recognition sites tolerated by cp caspase-2 mS9 Pro.
The list of all found cleavage sites, as described in Table 31, Section 15.2, Example 15, was used to generate a sequence logo for the consensus sequence.
In a similar experiment the influence of the P1′ residue on the recognition site selection was tested.
E. coli BL21(DE3) ΔpyrBI cells were generated that contain the cp caspase-2 construct (SEQ ID No. 6) in a pACYCDuet-1 vector.
A cpATCase substrate library with an XDXXD recognition site and Pro as P1′ was generated as described above. After transformation of the cells with the gene library, the selection was executed in an optimized M9 liquid culture to enrich an optimal recognition site. After plating of the incubated culture 22 single colonies were sequenced. Four sequences could not be analyzed because of contaminations, one YDVPD site was found, all 17 other sequences showed a VDSAD recognition site.
15.3 Cleavage of Fusion Proteins (Substrates) with Alternative Caspase-2 Recognition Sites for Cleavage
cp caspases-2 Human fibroblast growth factor-2 was expressed with a modified tag. The tag T7AC-6H-GSG-VDSAD was attached on the N-terminus of the POI, resulting in the fusion protein T7AC-6H-GSG-VDSAD-hFGF2. The protein was expressed in a shaker culture as described in Example 10, section 10.3. The purification of the fusion protein was performed as described in Example 19, section 19.3. After IMAC capture and buffer exchange into PBS using UF/DF, the fusion protein was stored in aliquots at −80° C. until further use.
A Michaelis-Menten type enzyme kinetic was performed as described in Example 20 and the results were compared to the cleavage of substrate T7AC-6H-GSG-VDVAD-hFGF2 (Example 20). The experiment was performed with T7AC-6H-mS9ProD as the enzyme. As shown in
A further recommended recognition site for cp-caspases-2 resp caspases-2 was tested: VDTTD (Kitevska, T., Roberts, S. J., Pantaki-eimany, D., Boyd, S. E., Scott, F. L., and Hawkins, C. J. Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate Bioscience Reports. Bioscience Reports 34 (2014)): It had been reported that a fluorogenic substrate with VDTTD was cleaved four times more efficiently than the VDVAD substrate by wild type caspase-2.
The cleavage of 6 His-GSG-VDTTD-E2 was tested. This experiment also shows that the residues at positions P2 and P3 have a minor influence on activity (
16.1 Design of Constructs and Selection
Saturation mutagenesis with degenerate primers, designed to create all possible 19 amino acid substitutions at one site in the protein, was performed with cp caspase-2 E105V G171 D (SEQ ID No. 200) in a pACYCDuet-1 vector as a template.
For the site-specific random mutation, a PCR reaction was performed with a high-fidelity DNA polymerase, as described in Section 11.1, Example 11. The primers were designed with their 5′ ends back-to-back to create a linear DNA fragment of the whole vector. Mutations at positions Val105 and Asp171 were inserted sequentially in two separate PCR reactions.
Primers were designed with the degenerate codon NNS at the site of mutation which generates all 20 amino acids with 32 codons and reduces codon redundancy.
For the PCR different annealing temperatures between 58 and 62° C. were used to ensure optimal binding of all codon combinations. Depending on the random codon the temperature can vary up to 4° C. For the mutation at position Val105 the forward primer TCGTTGTAAAnnsATGAGCGAGTATTG (SEQ ID NO: 282) and the reverse primer TGAAATTCTGTACCCGGTG (SEQ ID NO: 283) were used. To ligate the linear fragments a KLD reaction was performed as described in Section 11.1 of Example 11. The ligated product was purified and used as a template for the following mutagenesis to insert mutations at position Asp171. The forward primer CATTTTACCnnsGAAAAAGAACTG (SEQ ID NO: 284) and the reverse primer AACATTGCTCAGAACCAG (SEQ ID NO: 285) were used. Sequencing of the pooled gene library was used to control the quality of the DNA preparation. A clear preference for the nucleotide G was observed at the degenerate position which only produced a reduced sequence space. An additional set of primers was used to exclude all codons with G nucleotides already found in the previous PCR reaction to introduce mutations for amino acids that were not found with the NNS codon. The forward primers CATTTTACChhcGAAAAAGAACTG (SEQ ID NO: 286) and CATTTTACChhgGAAAAAGAACTG (SEQ ID NO: 287) and for both the same reverse primer AACATTGCTCAGAACCAG (SEQ ID NO: 288) were used.
Following this, a KLD reaction was performed. NovaBlue heat shock cells were transformed with the ligated product and the cells were diluted into an overnight culture which was used for a DNA preparation. Sequencing of the pooled gene library from primers with NNS, HHC and HHG codons was used to control the quality of the library before selection. All nucleotides were represented in the first two degenerate positions to theoretically produce all 400 possible variants.
E. coli BL21(DE3) ΔpyrBI cells that contained the VDVAD-cpATCase substrate with P1′ Thr and Pro (SEQ ID No. 22) were transformed with the gene library. The selection, as described in Example 3, was executed either in optimized M9 medium or on optimized M9 agar plates at 30° C. for 24-48 h.
16.2 Selected and Identified cp Caspases-2 Having Mutations in Positions Glu105 and Gly171
The DNA of 161 single colonies was analyzed, detecting combinations of mutations in active variants, as shown in Table 33, Section 16.2 in Example 16.
16.3 Characterization of cp Caspases-2 Having Mutations in Positions Glu105 and Gly171
Three combinatorial mutants were expressed, purified and tested as described in Sections 10.3, 10.4 and 10.5 of Example 10.
Variant cp caspase-2 E105V G171 D (SEQ ID No. 253) was chosen for further tests because the mutations E105V and G171 D showed the highest influence on the cp caspase-2 properties when tested separately. The combination of both mutations was also found repeatedly during selections of the combinatorial library with P1′ Pro and the variant was enriched in liquid culture. The specific activity of cp caspase-2 E105V G171D was the same as for the other caspase variants, 50% of 1 mg/ml VDVAD-E2 were cleaved in 1 min by 0.01 mg/ml cp caspase, as shown in Table 28, Example 14. The P1′ tolerance was even increased compared to cp caspase-2 mS9 ProD, the highest tolerance was observed for proline in the P1′ position, as shown in Tables 29.1, 29.2, 30.1 and 30.2, Example 14.
Variant cp caspase-2 E105V G171V (SEQ ID No. 254) was found repeatedly in selections with P1′ Thr and Pro and was also enriched in liquid culture. The specific activity of the variant was the same as for the other caspase variants, 50% of 1 mg/ml VDVAD-E2 cleaved in 1 min by 0.01 mg/ml caspase, as shown in Table 28, Example 14. The values for P1′ cleavage activities are higher than for the variant with the single mutation E105V (cp caspase-2 S9, SEQ ID No. 51), the tolerance for P1′ Pro was even increased to the level of variant mS9 ProD, as shown in Tables 29.1, 29.2, 30.1 and 30.2, Example 5.
The variant cp caspase-2 E105H G171V (SEQ ID No. 256) was suggested by the molecular modelling group and was never found in a selection. It was cloned as described in Section 11.1, Example 11 and expressed, purified and tested. Its specific activity was slightly decreased, 1 mg/ml VDVAD-E2 were cleaved by 0.01 mg/ml caspase in about 3 min, as shown in Table 28, Example 14. Though the variant's P1′ tolerance was lower compared to cp caspase-2 E105V G171V, it was increased compared to cp caspase-2. The highest increase was observed for P1′ Pro, as shown in Tables 29.1, 29.2, 30.1 and 30.2.
DEVD-E2 (SEQ Id No. 57)
DEVD is the preferred cleavage site of caspases-3 and -7. DEVD-E2 (Example 11, section 11.2) was used to evaluate the influence of the P5 residue, because the influence of the amino acids in the P2 and P3 positions on caspase-2 activity are considered insignificant. The substrate was processed 140 times slower than VDVAD-E2 (SEQ ID No. 33; Example 11, section 11.2) by cp caspase-2 (SEQ ID No. 6) showing that the recognition of the P5 residue is very important for caspase-2 and cp caspase-2.
This is in accordance with results from fluorescent peptides [26, 24], and proves the initial assumption of this study that caspase-2 was more specific than other caspases, because of its pentapeptidic recognition site. This seems to be even more pronounced in the circularly permuted variant, as the literature only describes a 35-fold increase in activity with VDVAD over DEVD [26].
17.1 Comparison of Specificity with Wild-Type Caspase-2
The specificity of cp caspase-2 (SEQ ID No. 6) was compared with commercially available wild-type caspase-2 (human, recombinant, active Caspase-2, Enzo Life Sciences, Farmingdale, N.Y., USA). 72 U/ml of the wild-type caspase-2 were used for cleavage reactions, according to the specifications, this equals about 0.005 mg/ml enzyme, half the concentration used in standard reactions with cp caspase-2. But the wild-type caspase was even six times less active than cp caspase-2 under the same conditions (1 mg/ml VDVAD-E2 was processed to 50% in 6 min).
While the absolute activities of the enzymes might be difficult to compare, because of different purity and concentration, a clear discrepancy could be found between their specificities. Wild-type caspase-2 cleaved DEVD-E2 only 44 times slower than VDVAD-E2, while cp caspase-2 has a 140-fold preference for VDVAD over DEVD. Thus, the cp caspase-2 is three times more specific than the wild-type enzyme (
17.2: Production and Characterization of a Wild Type Caspase-2
For comparison of wild-type caspase-2 with cp-caspase-2 variants a human caspase-2 was produced.
Production of Wt Caspase-2:
Production of wt caspase-2 was performed in a 30 L (23 L net volume, 5 L batch volume) computer-controlled bioreactor (Bioengineering; Wald, Switzerland) equipped with standard control units (Siemens PS7, Intellution iFIX). The pH was maintained at a set-point of 7.0±0.05 by addition of 25% ammonia solution (w/w), the temperature was set to 37° C.±0.5° C. in the batch phase and 30° C.±0.5° C. in the fed-batch phase. To avoid oxygen limitation the DO level was held above 30% saturation by adjusting the stirrer speed and the aeration rate of the process air. The maximum overpressure in the head space was 1.1 bar.
Pre-cultures for inoculation were grown in synthetic media calculated to produce 3 g/L. For incubation 1 mL of a deep frozen MCB was aseptically transferred to 400 mL medium and cultivated in two 2000 mL shaking flasks at 37° C. and 180 rpm until an OD of approx. 4 was reached.
For cultivation, minimal media calculated to produce 64 g cell dry mass (CDM) in the batch phase and 890 g CDM during feed phase were used. The batch medium was prepared volumetrically; the components were dissolved in 8 L RO—H2O. The fed-batch medium was prepared gravimetrically; the final weight was 8.45 kg. All components for the fed-batch medium were weighed in and dissolved in RO—H2O separately. All components (obtained from MERCK), were added in relation to the theoretical grams of cell dry mass to be produced: The composition of the batch and the fed-batch medium is as follows: 94.1 mg/g KH2PO4, 31.8 mg/g H3PO4 (85%), 41.2 mg/g C6H5Na3O7*2 H2O, 45.3 mg/g NH4SO4, 46.0 mg/g MgCl2*2 H2O, 20.2 mg/g CaCl2*2 H2O, 50 μL trace element solution, and 3.3 g/g C6H12O6*H2O. The trace element solution was prepared in 5 N HCl and included 40 g/L FeSO4.*7H2O, 10 g/L MnSO4.*H2O, 10 g/L AlCl3.*6 H2O, 4 g/L COCl2, 2 g/L ZnSO4.*7H2O, 2 g/L Na2MoO2.*2 H2O, 1 g/L CuCl2.* 2H2O, and 0.5 g/L H3BO3. To accelerate initial growth of the population, the complex component yeast extract (150 mg/g calculated CDM) was added to the batch medium. Nitrogen level was maintained by adding 25% ammonium hydroxide solution (w/w) for pH control. Antifoam (PPG 2000) 0.5 mL/L total volume was added at the beginning.
The fed-batch phase (29 h) was performed at 30° C. with an exponential feeding strategy with a consistent growth rate of μ=0.1 h−1. The substrate feed was controlled by increasing pump speed according to the exponential growth algorithm, X=X0·eμt, with superimposed feedback control of weight loss in the substrate tank. Induction started with fed-batch phase by adding 0.5 μmol IPTG/g CDM directly to the feed-media to achieve a protein production for 4 generations. IPTG concentration was calculated with the theoretical final CDM.
In addition to standard online monitoring (pH, stirrer speed, temperature and pO2) the concentration of pO2 and O2 in the outlet air was measured with a BlueSens gas analyzer. Sampling of the standard offline process parameters started after one generation in fed-batch mode. The first sample was withdrawn from the bioreactor prior to induction. Optical density (OD600) was measured with a spectrophotometer at wavelength λ=600 nm. Samples were diluted in PBS to ensure a measurement at a linear range from 0.1 to 0.8. Cell dry mass (CDM) was determined by centrifugation of 10 mL of cell suspension for 8 min at 8500 rpm. The supernatant was discarded and cells were resuspended with RO—H2O and centrifuged. Water was discarded and cell were resuspended again with RO—H2O. Cell suspension was transferred into a beaker, which was weighted before. Beakers were dried for at least 24 h at 105° C. and weighted again. The difference in weight account for the CDM.
For the determination of the content of wt caspase-2, aliquots of approximately 1.0 mg CDM of the samples were centrifuged (10 min. at 13200 rpm); the supernatants were discarded, the insides of the tubes were carefully blotted dry and the samples were stored at −20° C.
Comparison of wt caspase-2 production with cp caspase-2 production in fermentations with a μ=0.1 h−1 and an IPTG concentration of 0.5 μmol IPTG/g CDM during induction (standard fermentations as described in section 18.1.2.2). Whereas overexpression of cp caspase-2 was possible in E. coli, the expression of soluble wt caspase-2-6H was generally low and only detectable with western blot (
For recovery the E. coli cell mass was harvested by centrifugation at 18,590 rcf for 15 minutes and the supernatant was discarded. The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0). The cells were re suspended at a concentration of 400 g wet cell mass per L. Cell lysis was performed through high pressure homogenization at 1400 bar/140 bar with two passages with an in-line counter current chiller set to 10° C. The homogenate was centrifuged at 18,590 rcf for 2.5 hours at 4° C. The pellet was discarded and the supernatant used. Before chromatography the supernatant was filtered through a 0.22 μm membrane.
The wt caspase-2 carrying a poly-his-tag was captured using immobilized metal affinity chromatography (IMAC). The following buffers were used: equilibration buffer: 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0. Elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0.
Imidazole was added to the clarified supernatant before IMAC, to a final concentration of 20 mM imidazole. 57 CV clarified supernatant were loaded to an equilibrated Ni-Sepharose 6 Fast Flow column (50×18 mm, 35 mL). A residence time of 7 minutes was used during loading and 3 minutes for subsequent steps. After loading was completed the column was washed for 10 CV with equilibration buffer. The bound wt caspase 2 was eluted using a step gradient to 100% elution buffer for 10 CV.
The elution fractions were analyzed using SDS-PAGE and all fractions containing wt caspase-2 were used for the next purification step.
The capture eluate of wt caspase-2 was buffer exchanged before the polishing chromatography step. Tangential flow ultra-/diafiltration with a 5 kDa cut off membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. In total 5 volumes were exchanged.
The capture step used cation exchange chromatography on SP Sepharose HP (5×24 mm, 0.5 mL) using the following buffers: equilibration buffer A: 50 mM sodium citrate, pH 5.0. Elution buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0.
Buffer exchanged capture eluate was loaded on the equilibrated polishing column. The residence time was held constant at 5 minutes. The column was loaded with 37 CV of buffer exchanged capture eluate. Wt caspase-2 was eluted in a linear gradient from 0-100% B in 10 CV. The elution fractions were analyzed using Western blot and SDS PAGE and the fractions positive for the small sub unit of wt caspase-2 were combined and stored at −80° C. Before performing enzyme kinetic measurements, oxidation induced activity losses were reversed by incubating wt caspase-2 with 100 mM DTT for 15 minutes.
Characterization of Wt Caspase-2
FRET Assay (as Described in Example 9, Section 9.3.3)
Michaelis Menten kinetic was determined for wt caspase-2 and cp caspase-2 for the following substrates: VDVADFA, VDVADGA, VDVADQA and VDVADVA, where the P1′ amino acid is indicated by bold and underlined font.
The FRET results in Table 34 show significant differences between the two proteases. cp caspase-2 exhibits catalytic efficiencies approximately one order of magnitude higher than wt caspase-2. While the Michaelis constant KM appears mostly unaffected by circular permutation, the turnover number kcat is the cause for the stark differences in catalytic efficiency kcat/KM between wt caspase-2 and cp caspase-2. The produced wt caspase-2 seems to exhibit slightly better P1′ tolerance compared to cp caspase-2 (both not comprising the amino acid substitutions for improved P1′ tolerance described herein), e.g. F as P1′ is cleaved with 2.5% catalytic efficiency in cp caspase 2 compared to 3.2% in wt caspase-2. This slight increase in P1′ (1.3 to 2.3-fold increase) is overshadowed by the, on average eleven times lower catalytic efficiency and eight times lower turnover number of wt caspase-2.
Tolerance for Elevated Temperatures
Cleavage of a heat stable model fusion tag protein, 6H-GSG-VDVAD-GFPmut3.1 (GFP with P1′=M), was used to quantify the tolerance of caspase-2 towards elevated temperatures.
The GFP cleavage results in Table 35 show comparable heat tolerance between the two proteases. The cleavage reaction with cp caspase-2 is 1.6-fold faster, than with wt caspase-2 at 25° C. This difference increases to 2.3-fold at 50° C., showcasing the increased stability of cp caspase-2 at elevated temperatures. In general, the cleavage reaction at 50° C. is 1.9 times faster for wt caspase-2 and 2.8 times faster for cp caspase 2. This is a clear benefit if a heat stable target protein has to be processed.
Tolerance to Chaotropic Conditions
Cleavage of a model fusion tag protein stable in 4 M urea, namely 6H-GSG-VDVAD-hFGF-2 (P1′=A), was used to quantify the tolerance of caspase-2 towards chaotropic conditions.
The FGF2 cleavage results in Table 36 show comparable tolerance for chaotropic conditions between the two proteases. In order to quantify the cleavage product in the linear range, the reaction had to be stopped at differing time points. Both proteases show almost identical behavior in the presence of 4 M urea, were the reaction rate is reduced to 1.2% and 1.3% for wt caspase-2 and cp caspase-2 respectively. For this particular model protein, cp caspase-2 exhibited a 3.2-fold increased reaction rate relative to wt caspase-2.
Manufacturability
Perhaps the biggest observable difference between the two proteases, is in their ease of manufacture. In order to express the difference in manufacturability between wt caspase-2 and cp caspase-2, we calculated the amount of dry cell mass required to produce one milligram of purified enzyme. This takes into account the differences in specific protein content of the E. coli fermentation and the differences in downstream processing yields. It does not take into account differences in biomass yield between fermentations. In order to produce 1 mg of wt caspase-2, 70 g of cell dry mass (CDM) were required. For the production of cp caspase-2, only 34 mg of CDM were needed per milligram pure enzyme. This corresponds to a difference in manufacturability of a factor of 2033.
Conclusion
FRET assay results with 4 different P1′ amino acids showed a general trend of tenfold higher catalytic efficiencies of the cp caspase-2 compared to wt caspase-2. The cleavage of non peptide substrates, showed two to three-fold faster cleavage reaction depending on the protein substrate. The circular permutation of caspase-2 has apparently lead to an increase in heat tolerance, showcased by the larger increase in turnover rate at 50° C. The tolerance to chaotropic conditions also appears slightly higher
The largest differentiating factor between wt and cp enzymes is their manufacturability. While the expression level of wt caspase-2 is very low (under the limit of quantification), cp caspase-2 reaches expression levels of 80 mg specific protein content per g CDM. This also results in much lower losses during DSP, where a process yield of about 35% can be achieved for cp caspase-2.
18.1 Upstream Processing of cp Caspase-2 and Variants
For the production of cp caspase-2 and variants with and without solubility tag lab-scale fermentations were performed as described below. Different expression clones were compared regarding cell growth and soluble recombinant protein production. For further process optimization, a series of cultivation runs was conducted.
18.1.1 Bacterial Strain, Plasmid and Wild-Type cp Caspases-2 and P1′ Tolerable cp Caspases-2
The E. coli strain BL21(DE3) [F−, fhuA2, Ion, ompT, gal, dcm, ΔhsdS λ DE3 [λ sBamHIo, ΔEcoRI-B int::(lacl::PlacUV5::T7 gene1) i21 Δnin5], purchased from Novagen, was transformed with a pET30a vector carrying the gene for the respective cp caspase-2 variant with and without solubility tag under the T7 promoter/operator system. The expression clones cultivated in lab-scale bioreactors are listed in Table 37.
18.1.2 Lab-Scale Fermentation of cp Caspase-2 and Variants.
18.1.2.1 Fermentation Media
For high cell density (HCD) cultivation experiments minimal media calculated to produce 80 g cell dry mass (CDM) in the batch phase and 1450 g CDM during feed phase were used. The batch medium was prepared volumetrically; the components were dissolved in 10 L RO—H2O. The fed-batch medium was prepared gravimetrically; the final weight was 10.1 kg. All components for the fed-batch medium were weighed in and dissolved in RO—H2O separately. All components (obtained from MERCK), were added in relation to the theoretical grams of cell dry mass to be produced: The composition of the batch and the fed-batch medium is as follows: 94.1 mg/g KH2PO4, 31.8 mg/g H3PO4 (85%), 41.2 mg/g C6H5Na3O7*2 H2O, 45.3 mg/g NH4SO4, 46.0 mg/g MgCl2*2 H2O, 20.2 mg/g CaCl2*2 H2O, 50 μL trace element solution, and 3.3 g/g C6H12O6*H2O. The trace element solution was prepared in 5 N HCl and included 40 g/L FeSO4.*7H2O, 10 g/L MnSO4.*H2O, 10 g/L AlCl3.*6 H2O, 4 g/L COCl2, 2 g/L ZnSO4.*7H2O, 2 g/L Na2MoO2.*2 H2O, 1 g/L CuCl2.*2 H2O, and 0.5 g/L H3BO3. To accelerate initial growth of the population, the complex component yeast extract (150 mg/g calculated CDM) was added to the batch medium. Nitrogen level was maintained by adding 25% ammonium hydroxide solution (w/w) for pH control. Antifoam (PPG 2000) 0.5 mL/L total volume was added at the beginning. Pre-cultures for inoculation were grown in synthetic media calculated to produce 3 g/L).
18.1.2.2 Cultivation and Induction Conditions for (Standard) Lab-Scale Fermentations of Wild-Type cp Caspases-2 and P1′ Tolerable cp Caspases-2.
All high cell densities (HCD) fermentations were performed in a 30 L (23 L net volume, 5 L batch volume) computer-controlled bioreactor (Bioengineering; Wald, Switzerland) equipped with standard control units (Siemens PS7, Intellution iFIX). The pH was maintained at a set-point of 7.0±0.05 by addition of 25% ammonia solution (w/w), the temperature was set to 37° C.±0.5° C. in the batch phase and 30° C.±0.5° C. in the fed-batch phase. To avoid oxygen limitation the DO level was held above 30% saturation by adjusting the stirrer speed and the aeration rate of the process air. The maximum overpressure in the head space was 1.1 bar. Foaming was suppressed by addition of 0.5 mL/L antifoam (PPG 2000 Sigma Aldrich) to the batch medium and by pulsed addition of antifoam during the fed-batch phase. The cultivation was inoculated with an overnight pre-culture. The pre-culture was set-up by inoculating 200 mL LB media with 1 mL of a deep frozen WCB in 2000 mL shake flasks. Cells were grown on an orbital shaker at 180 rpm and at 37° C. until the OD600 reached a value of approx. 4. Thereafter, batch was inoculated with the pre-culture to an initial OD600 of 0.10 and cultivated at 37° C. At the end of the batch phase as soon as cells entered the stationary growth phase, an exponential substrate feed was started. The fed-batch phase (29 h, unless otherwise stated in table 40) was performed at 30° C., unless otherwise stated in table 40, with an exponential feeding strategy with a consistent growth rate of μ=0.1 h−1 unless otherwise stated in table 40. The substrate feed was controlled by increasing pump speed according to the exponential growth algorithm, X=X0·eμt, with superimposed feedback control of weight loss in the substrate tank. Induction was as follows, unless otherwise stated in table 40: Induction started with fed-batch phase by adding feed medium including IPTG (so called “over feed” induction, table 40) to achieve a final IPTG concentration of 0.5 μmol IPTG/g theoretical CDM at the end of the fermentation and a protein production for 4 generations.
18.1.2.3 Further Cultivation and Induction Conditions:
Pre-cultivation and batch phase were identical to the previously described standardized fermentations. The fed-batch phases were performed at 30° C. For biomass production the first fed-batch phase was performed with an exponential feed (μ=of 0.17 h−1) for 1.72 generations. As previously described, the substrate feed was controlled by increasing pump speed according to the exponential growth algorithm, X=X0*eμt, with superimposed feedback control of weight loss in the substrate tank. In a second feed-phase a lower growth rate (0.03, 0.05 and 0.07 h−1) was adjusted resulting in a total feed time of 60.5 h, 39 h and 30 h. The calculated CDM was 70 g/L. To ensure sufficient adaption to the low growth conditions, the cells grew for 0.25 generations without induction. Then induction was performed with three different IPTG concentrations (0.5, 0.9 and 1.3 μmol/g actual CDM) for two generations. IPTG corresponding to the CDM at induction time, as mentioned before, was injected into the reactor and then IPTG calculated to the actual CDM was fed into the fermenter within the feed medium. To that end the needed IPTG was transferred into the feed bottle calculated to the IPTG needed until the theoretical CDM at the end of fermentation. Thus, the IPTG concentration related to the theoretical CDM was constant throughout the whole fermentation. 9 fermentations were performed. The results are described in 18.1.2.7
All fermentation results for the fermentations as described in table 40 can be seen in Table 41.
18.1.2.4 Fermentation Monitoring
In addition to standard online monitoring (pH, stirrer speed, temperature and pO2) the concentration of pO2 and O2 in the outlet air was measured with a BlueSens gas analyzer. Sampling of the standard offline process parameters started after one generation in fed-batch mode. The first sample was withdrawn from the bioreactor prior to induction. Optical density (OD600) was measured with a spectrophotometer at wavelength λ=600 nm. Samples were diluted in PBS to ensure a measurement at a linear range from 0.1 to 0.8. Cell dry mass (CDM) was determined by centrifugation of 10 mL of cell suspension for 8 min at 8500 rpm. The supernatant was discarded and cells were resuspended with RO—H2O and centrifuged. Water was discarded and cell were resuspended again with RO—H2O. Cell suspension was transferred into a beaker, which was weighted before. Beakers were dried for at least 24 h at 105° C. and weighted again. The difference in weight account for the CDM.
For the determination of the content of cp caspase-2 and variants, aliquots of approximately 1.0 mg CDM of the samples were centrifuged (10 min. at 13200 rpm); the supernatants were discarded, the insides of the tubes were carefully blotted dry and the samples were stored at −20° C.
18.1.2.5 Determination of Titer and Specific Titer of cp Caspase-2 Variants and Fusion Proteins in Fermentation Samples
Cell disintegration, fractionation of soluble and insoluble recombinant protein and IB dissolving: Cell disintegration was performed from fermentation samples containing approximately 1.0 mg CDM. 200 μL of cell integration buffer was added to the cell pellet and vortexed until the pellet was completely resuspended. For cell disruption 50 μL Lysozyme and 50 μL Benzonase were added and incubated while shaking at room temperature. 100 μL Triton X-100 was added and samples were incubated again while shaking. Then, samples were centrifuged at 4° C. and 13000 rpm to separate soluble proteins and inclusion bodies (IB). The supernatant was transferred into a new reaction tube for direct analysis (SDS-PAGE) or stored at −20° C.
The remaining pellet (IBs and cell debris) was washed two times by resuspending with 1 mL Tris/HCL (100 mM). After resuspending the pellet was centrifuged at 4° C. and 13000 rpm for 10 min. The supernatant was discarded. Afterwards, 400 μL IB solvent buffer was added and incubated at room temperature for 30 min. while shaking. Finally, the sample was centrifuged again and the supernatant containing dissolved IBs was used for analysis (SDS-PAGE) or stored at −20° C.
SDS-PAGE:
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate and analyze the recombinant proteins. Electrophoresis was performed by using precast gels with an acrylamide gradient (NuPAGE 4-12% BisTris, Thermo Fisher Scientific, Waltham, Mass., USA) and NuPAGE® MES SDS Running buffer. Loading samples were prepared by mixing 13 μL of the supernatant (soluble fraction) or IB supernatant (insoluble fraction) with 5 μL LDS sample buffer (4×) and 2 μL NuPAGE® reducing agent (10×) and incubating the mixture in a thermos mixer at 70° C. for 10 minutes. A ready-to-use molecular weight marker (Mark12™, Unstained Standard, Invitrogen) was directly loaded as size marker. For quantification, purified T7AC_6H_cpCasp2 standards (75, 50 and 25 μg/mL) listed in Table 9, produced as described in Example 9 (see sections 9.1, and 9.2), were used. For fusion proteins BSA (bovine serum albumin) served as a standard. Electrophoresis settings were 200 V and 400 mA for 40 to 50 minutes in a XCell SureLock™ Electrophoresis Cell chamber (Thermo Fisher Scientific). After electrophoresis the SDS Gels were fixed in fixing solution (40% ethanol; 50% dH2O; 10% acetic acid) for 30 minutes and stained afterwards with Coomassie brilliant blue R250 staining solution for 30 minutes. Finally, the gel was decolorized in a destaining solution (25% acetic acid; 8% ethanol; 67% dH2O) for at least two hours. Gels were transferred in water and scanned with a desktop scanner, converted to grey-scale and analysed using the software ImageQuant TL (7.0). The concentration of cp caspase-2 and variants was quantified via a linear regression curve.
18.1.2.6 Comparison of production of wild-type cp caspases-2 and P1′ tolerable cp caspases-2 with and without solubility tag in fermentations with a μ=0.1 h−1 and an IPTG concentration of 0.5 μmol IPTG/g CDM during induction (standard fermentations as described in section 9.1.2.2).
While overexpression of cp caspase-2 was possible in E. coli, the expression rate of soluble cp caspase-2 was generally low. In order to increase the fermentation titer, a solubility tag was added to the enzyme. The tag T7A3 (SEQ ID No. 37) is based on a highly negatively charged peptide from the T7 bacteriophage. When used on the cp caspases-2 we noticed autocatalytic cleavage of the tag and subsequently modified the tag, using a cleavage site prediction algorithm. The altered solubility tag was coined T7AC (SEQ ID No. 43)
For evaluation of the production of cp caspase-2 and variants with and without solubility tag (T7AC), standardized lab-scale fermentations (section 18.1.2.2) were performed. Expression clones were compared regarding cell growth and soluble and insoluble recombinant protein production.
Comparing the production of 6H-cpCasp2D and T7AC-6H-cpCasp2D in lab-scale fermentations, we observed that the production of 6H-cpCasp2D without solubility tag lead predominantly to inclusion body formation (
Comparing the production of three cp caspases-2 (cp caspase-2, mS9Pro E285 and mS9 Pro D285) with and without T7AC solubility tag, it turned out that the variant itself has no influence on the performance, no significant differences in cell growth and soluble cp caspase-2 expression. By means of the T7AC solubility tag the soluble expression of all three variants was significantly improved.
Cell growth kinetics off all cultivations were almost the same (VC<4%). Only at the end of the fermentations slight deviations were observed (
Furthermore, a T7A3 tag could increase the soluble titer of cp-caspase-2D as can be seen in Table 40 and 41.
18.1.2.7 Further Fermentation Processes for Wild-Type Like Cpcaspase-2 Variants and P1′ Tolerable cp Caspases-2.
For testing further process conditions, a series of cultivation runs were conducted according to 18.1.2.3. The production clone BL21(DE3)(pET30a-T7AC_6H_cpCasp2D) was used. The influence of different growth rates (μ=0.03, 0.05 and 0.07 h−1) and induction strengths (0.5, 0.9 and 1.3 μmol IPTG/g CDM) were investigated regarding cell growth and soluble and insoluble recombinant protein production. The results are shown in Table 45.
It was observed that the specific yield of soluble cp caspase-2 was higher at low growth rates and IB formation decreased. The calculated CDM was not reached at the end of fermentation with μ=0.03 h−1 due to too high expression levels (
Nevertheless, the highest volumetric soluble yield was reached with μ=0.03 h−1 and 0.9 or 0.5 μmol IPTG/g CDM (
Surprisingly the combination of using a T7AC or a T7A3 tag and low specific growth rates during induction (expression phase) and dosed IPTG concentration for tuning the expression rate, led to a titer of >5 g/L for cp-caspases-2
18.1.2.8: Application of Fermentation Processes for Production of Cp-Caspase-2 Variants
Direct comparison between fermentations of T7AC-6H-cpCasp2D and T7AC-6H-mS9 ProD is shown with two different 2-phase fed-batch cultivations, μ=0.17 followed by 0.03 h−1 for production and μ=0.17 followed by 0.05 h−1 for production with constant 0.9 μmol IPTG/g CDM as described in 18.1.2.3 and tables 40 and 41, Experiment Numbers F42 and F44, in
The processes as outlined in 18.1.2.2 to 18.1.2.8 can be applied to all cp caspases-2 irrespective if it includes or not mutations at positions that increase the P1′ tolerance.
18.1.2.9: Another preferred fermentation process for the production of a cp-caspase2, cp-caspase-2D as described in table 40 and 41 (Experiment Number F34) resulted in surprisingly high titer of soluble cp caspase 2D of 5.28 g/L as can be seen in
18.2 Downstream Processing of Wild-Type cp Caspases-2 and P1′ Tolerable Cp Caspases-2
18.2.1 Downstream Processing without Solubility Tag
The E. coli cell mass from fermentations as described under 18.1 or shake flask as described under section 10.3 was harvested by centrifugation at 18,590 rcf for 15 minutes and the supernatant was discarded. The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0). The cells were resuspended at a concentration of 150 g wet cell mass per L. Cell lysis was performed through high pressure homogenization at 700 bar/70 bar with two passages. The homogenate was centrifuged at 18,590 rcf for 2 hours. The pellet was discarded and the supernatant used. Before chromatography the supernatant was filtered through a 0.22 μm membrane.
The cp caspase-2 carrying a poly-his-tag was captured using immobilized metal affinity chromatography. The following buffers were used: equilibration buffer: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0. Wash buffer: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 30% iso-propanol, pH 7.0. Elution buffer: 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.0.
Clarified supernatant was loaded to an equilibrated Ni-Sepharose 6 Fast Flow column to a capacity of ˜40 mg/mL. A residence time of 3-5 minutes was used. After loading was completed the column was washed for 5 column volumes (5 CV) with equilibration buffer, 10 CV with wash buffer and 5 CV of equilibration buffer. The bound cp caspase-2 was eluted using a linear gradient from 0-100% elution buffer in 10 CV, with a 10 CV hold step to fully elute all protein.
The elution fractions were analyzed using SDS-PAGE and all fractions containing cp caspase-2 were used for the next purification step.
The capture eluate of cp caspase-2 was buffer exchanged before the polishing chromatography step. Tangential flow ultra-/diafiltration with a 5 kDa cut off membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. In total 5 volumes were exchanged.
The capture step used cation exchange chromatography on SOURCE 30S using the following buffers: equilibration buffer A: 50 mM sodium citrate, pH 5.0. Elution buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0.
Buffer exchanged capture eluate was loaded on the equilibrated polishing. The residence time was held constant at 5 minutes. The column was loaded to a capacity of ˜100 mg/ml. cp caspase-2 was eluted in a linear gradient from 0-100% B in 20 CV. The elution fractions were analyzed using RP-HPLC as described under 9.3.1 and the fractions showing a purity of ˜99% were combined and stored at −80° C.
18.2.2 Downstream Processing with Solubility Tag
The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 500 or 300 mM NaCl, pH 7.0 or 8.0, see Table 46). The cells were re suspended at a concentration of 300 g wet cell mass per L. Cell lysis was performed through high pressure homogenization at 1400 bar/140 bar with two passages with an in line counter current chiller set to 10° C. The homogenate was centrifuged at 18,590 rcf for 2 hours at 4° C. The pellet was discarded and the supernatant used. Before chromatography the supernatant was filtered through a 0.22 μm membrane.
The T7AC_6H-tagged cp caspase-2 and cp caspases-2 were captured using immobilized metal affinity chromatography (IMAC). The following buffers were used: equilibration buffer A1: 50 mM sodium phosphate, 500 or 300 mM NaCl, 20 mM imidazole, pH 7.0 or 8.0. Wash buffer A2: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0, 30% isopropanol. Elution buffer: 50 mM sodium phosphate, 500 or 300 mM NaCl, 500 mM imidazole, pH 7.0 or 8. See Table 27 for details on concentrations.
Imidazole was added to the clarified supernatant before IMAC, to a final concentration of 20 mM imidazole. The clarified supernatant was loaded to an equilibrated Ni-Sepharose 6 Fast Flow column. A residence time of 7 minutes was used during loading and 3 minutes for subsequent steps. After loading was completed the column was washed as shown in Table 47. The bound cp caspase 2 or cp caspase 2 variant was eluted using a step gradient to 100% elution buffer for 10 CV or a linear gradient from 0-100% B over 5 CV with a 5 CV hold step.
The elution fractions were analyzed using SODS-PAGE and all fractions containing cp caspase 2 or cp caspase 2 variants were used for the next purification step, a cation exchange chromatography (CEX) polishing step. Only for T7AC_6H-cpCasp2_cal and T7AC_6H-cpCasp2_cal_mut the capture eluate was buffer exchanged to phosphate buffered saline (PBS) using UF/DF, omitting the CEX polishing step, due to the low yield in the capture step. UF/DF for cpCasp2_cal and cpCasp2_cal_mut was performed in Amicon centrifugal filter vials with a 10 kDa nominal membrane cut off. In total 5 volumes were exchanged.
The capture eluate of cp caspase 2 or cp caspase 2 variants were buffer exchanged before the polishing chromatography step. Tangential flow ultra-/diafiltration with a 5 kDa cut off PIES membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. In total 5 volumes were exchanged.
The polishing step was CEX on SP Sepharose HP (10×85 mm, 6.7 mL) using the following buffers: equilibration buffer A: 50 mM sodium citrate, pH 5.0. Elution buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0.
Buffer exchanged capture eluate was loaded on the equilibrated polishing column. The residence time was constant at 1.5 minutes. The column was loaded with buffer exchanged capture eluate. T7AC_6H-cpCasp2D, T7AC_6H-mS9ProD and T7AC_6H-mS9ProE were eluted in a 5 CV step gradient at 45% B. The elution pools were analyzed using RP-HPLC as described under 9.3.1 and showed a purity of ˜99%. T7AC_6H_cpCasp2D_sar and T7AC_6H_cpCasp2D_sar_mut were eluted in a linear gradient from 0-100% B in 10 CV. The elution fractions were analyzed using SDS PAGE and the fractions positive for cpCasp2 were combined and stored at −80° C.
18.3 Characterization of Wild-Type cp Caspases-2 and P1′ Tolerable Cp Caspases-2
18.3.1 Purity Determination (HPLC) for cp Caspases-2 Purified as Described in Section 18.2.1 and 18.2.2
Experiments were performed on a Tosoh TSKgel Protein C4-300, L×I.D. 5 cm×4.6 mm, 3 μm column with a guard column on a Waters e2695 HPLC. Mobile phase A was water with 0.15% trifluoroacetic acid (TFA) and mobile phase B was acetonitrile with 0.15% TFA. The flowrate was 1 ml/min. Temperature of the column oven was 40° C., temperature of the autosampler 10° C. The following gradient was used as shown in Table 48.
200 μL of purified cp caspase-2 (or variant) sample (˜4 g/L) was diluted with 100 μL PBS and 100 μL 2 M Dithiothreitol (DTT). 10 μl of 0.22 μm filtered sample were injected. The outlet was monitored at 214 nm and 280 nm. The HCP peaks eluted between retention times 3.8 and 9 minutes. The cp caspase-2 peaks eluted between 9.2 and 12.4 minutes. The peak areas in the 214 nm signal were used to calculate the purity of the protein of interest.
18.3.2 Quantification of Released Fusion Tag with RP-HPLC
The calibration curve was generated mixing the substrate protein, e.g. human fibroblast growth factor 2 (hFGF-2: T7AC-6H-GSG-VDVAD-hFGF-2), and cp caspase-2 (T7AC-6H-cp-caspase2D) in a ratio 10:1 (in triplicates) and incubated for 4 hours at 25° C. while shaking. The reaction was stopped by adding formic acid to a final concentration of 0.3% or by adding cystamine to a final concentration of 10 mM. Each triplet was diluted with PBS buffer to get six different concentrations (100 μM, 46 μM, 21 μM, 10 μM, 4 μM, 2 μM).
10 μL of 0.22 μm filtered sample were injected to a reversed phase high pressure liquid chromatography (RP-HPLC) using a method outlined below. The outlet was monitored at 214 nm. The fusion tag peaks eluted between retention times 3.9 and 5.6 minutes. The peak areas in the 214 nm signal were used to calculate the quantity of the fusion tag using a linear calibration function.
Experiments were performed on a Tosoh TSKgel Protein C4-300, L×I.D. 5 cm×4.6 mm, 3 μm column with a guard column on a Waters e2695 HPLC. Mobile phase A was water with 0.15% trifluoroacetic acid (TFA) and mobile phase B was acetonitrile with 0.15% TFA. The flowrate was 1 mL/min. Temperature of the column oven was 40° C., temperature of the autosampler 10° C. The following gradient was used (Table 49):
18.3.3 Determination of enzymatic activity of wild-type like cp-caspases-2 and P1′ tolerable cp caspases-2 (prepared as described in Example 18, section 18.1 and 18.2) with FRET assay
A Förster resonance energy transfer (FRET) assay for the determination of the Michaelis-Menten enzymatic activity parameters was performed in the following way.
The substrates were obtained from Bachem AG and were of the general structure of Abz-VDVAD-XA-Dap (Dnp), where all 20 amino acids were substituted for X (the P1′ position). All substrates were dissolved in 10 mM HEPES, pH 7.5 to a concentration of 750 μM.
The buffer for the assay was 50 mM HEPES, 150 mM NaCl, pH 7.2.
The calibration curve was generated by incubating varying amounts of substrate (20 μM, 6.9 μM, 2.4 μM, 0.8 μM, 0.3 μM, 0.1 μM) with 72 μM cpCasp2 in PBS and incubated at room temperature for up to 24 hours. 100% conversion was assumed. Fluorescence was measured in black 96 well plates on a Tecan Infinite M200 Pro plate reader. Excitation wavelength was 320 nm, emission wavelength 420 nm.
Michaelis-Menten kinetics were measured by varying substrate concentrations (200 μM, 100 μM, 50 μM, 20 μM, 10 μM) at constant enzyme concentration ([E]=1 μM). The initial slope was measured by measuring the fluorescence for 3-15 minutes (or 3 to 20 hours for proline as P1′) and calculating the slope of the initial measurement in μM product generated per second. Fluorescence was measured in black 96 well plates on a Tecan Infinite M200 Pro plate reader. Excitation wavelength was 320 nm, emission wavelength 420 nm. In the FRET assay all substrates, except for proline as P1′ showed excellent linearity for at least a few minutes.
Evaluation of the data was performed by fitting the data in the TableCurve 2D v5 software to a Michaelis-Menten kinetic:
v=V
max*[S]/KM+[S]
Where v is the initial slope, Vmax is the maximum rate, KM is the Michaelis constant and [S] is the substrate concentration. The parameters Vmax and KM were fitted. kcat was calculated by dividing Vmax by the enzyme concentration [E].
An example kinetic curve can be seen in
The results of all measurements are shown in Table 50 and 51 and
Fusion proteins comprising a tag and the protein of interest (Pol) and hFGF-2 without a tag were cloned as described in Example 11, section 11.1. In table 52 all fusion proteins for cleavage reactions in Examples 11 to 13 are described.
19.2 Fermentation of Fusion Proteins
The E. coli strain BL21 (DE3) [F−, fhuA2, Ion, ompT, gal, dcm, ΔhsdS λ DE3 [λ sBamHIo, ΔEcoRI-B int::(lacl::PlacUV5::T7 gene1) i21 Δnin5], purchased from Novagen, was transformed with a pET30a vector carrying the gene for the respective fusion proteins resp fusion protein construct of table 52, under the T7 promoter/operator system. The expression clones cultivated in lab-scale bioreactors are listed in Table 52. Fermentation of fusion proteins (substrates) was performed analogous to fermentation of cp caspases-2 as described in section 18.1.2 if not stated otherwise. Fermentation media was prepared using the same components and methods listed in section 18.1.2.1 but was calculated according to the CDM and volumes given in Table 53. Fermentations were carried out as described in section 18.1.2.2. In case of TNFα (TNFalpha), the fermentations were carried out in a 15 L computer-controlled bioreactor (MBR, Zürich, Switzerland) using the same standard control units as specified in section 18.1.2.2. For rhGH, PTH and GCSF fermentations the computer-controlled fermentation system DASGIP® Bioblock with a working volume of 2 L (Eppendorf, Hamburg, Germany) was employed. This system was controlled using the DASware® control software and the standard control boxes DASGIP® TC4SC4, DASGIP® PH4PO4L and DASGIP® MX4/4 from Eppendorf. Precultures for fusion protein substrates were carried out according to the methods described in section 18.1.2.2 using semisynthetic medium (SSM), which has the same composition as the batch medium and was prepared for a CDM of 3 g/L. In case of rhGH, PTH and GCSF, inoculation was performed using 25 OD units (at a wavelength of 600 nm) of preculture. Additionally, for the cultivations in the DASGIP® Bioblock, the pH was maintained with 12.5% ammonia solution (w/w). All other fermentation parameters and procedures if not stated otherwise in this chapter or in table 53 were carried out as described in section 18.1.2.2. Fermentation monitoring was performed as described in section 18.1.2.4 with the exception of determination of CDM of rhGH, PTH and GCSF fermentations, which was performed using the same methods as described but with 1 mL of cell suspension.
Determination of specific and volumetric fusion protein titer was performed as described in section 18.1.2.5:
In addition to 6H-GSG-VDVAD-BIWA4 in Table 53 also T7AC-6H-GSG-VDVAD-BIWA4 was fermented under the same conditions as 6H-GSG-VDVAD-BIWA4. The T7AC-6H-GSG-VDVAD-BIWA4 fusion protein was also expressed as unsoluble Inclusion Bodies (IB), but with a 3-fold titer (
hFGF-2—all variants in table 53 means all hFGF-2 variants described in Table 41 except: T7AC_6H_GSG_VDSAD-hFGF2, T7AC-6H-VDVAD-hFGF2 and T7AC_6H_GSGSGSG_VDVAD-hFGF2, which were produced as described Example 10
The course of biomass and fusion protein formation for all fusion protein fermentations of table 53 can be seen in
The titer of the His tagged 6H-hFGF-2 is significantly lower than the untagged hFGF-2. Surprisingly the additional GSG and/or VDVAD sequence in the 6H-GSG-VDVAD-hFGF-2 increases the titer of the His tagged hFGF-2 by a factor of 2.5-fold, since the recognition site is not known to act as an expression enhancer. The further addition of a T7AC or aT7A3 tag further increases the titer for hFGF-2 resp. the fusion protein, which is surprising since the titer for the untagged hFGF-2 could not only be restored, but was increased about 2-fold. All this can be seen in
19.3 Purification of Fusion Proteins (of Sections 19.1 and 19.2) Before Cleavage with Wild-Type cp Caspases-2 and P1′ Tolerable cp Caspases-2
The E. coli cell harvest was solubilized using homogenization buffer: 50 mM sodium phosphate—buffer and NaCl and pH as indicated in Table 54.
The cells were re-suspended at a concentration of 150 g wet cell mass per L. Cell lysis was performed through high pressure homogenization at 1000 bar/100 bar with two passages with an in line counter current chiller set to 10° C. The homogenate was centrifuged at 18,590 rcf for 2 hours at 4° C. The pellet was discarded and the supernatant used. Before chromatography the supernatant was filtered through a 0.22 μm membrane.
The 6H_GSG_VDVAD- or T7AC_6H_GSG_VDVAD-tagged Pols (fusion proteins) were captured using immobilized metal affinity chromatography (IMAC). The following buffers were used: equilibration buffer A1: 50 mM sodium phosphate, 150 or 500 mM NaCl (see Table 54), 20 mM imidazole, pH 7.0 or pH 7.4 (see Table 54. Wash buffer A2: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0, 30% isopropanol. Elution buffer: 50 mM sodium phosphate, 150 or 500 mM NaCl (see Table 54), 500 mM imidazole, pH 7.0 or pH 7.4 (see Table 54).
Imidazole was added to the clarified supernatant before IMAC, to a final concentration of 20 mM imidazole. The clarified supernatant was loaded to an equilibrated Ni-Sepharose 6 Fast Flow column. A residence time of 7 minutes was used during loading and 3 minutes for subsequent steps. After loading was completed the column was washed with equilibration buffer for 5, 10 or 15 CV (see Table 54). The bound fusion protein was eluted using a step gradient to 100% elution buffer for 10 CV or a linear gradient from 0-100% B over 5 CV with a 5 CV hold step, see Table 54.
The elution fractions were analyzed using SOS-PAGE and all fractions containing POI were pooled. The product pool was buffer exchanged to phosphate buffered saline (PBS) using UF/DF. UF/DF for was performed in Amicon centrifugal filter vials with a 10 kDa nominal membrane cut off (3 kDa cut-off for T7AC_6H_GSG_VDVAD-PTH). In total 5 volumes were exchanged.
Purification of 6H GSG VDVAD-TNFα and T7AC 6H GSG VDVAD-TNFα was performed by IMAC capture. Different volumes of cell lysis supernatant were loaded based on the specific protein content. Apart from the differing loading times, the chromatograms of both POIs looked similar (see
Fusion proteins as produced in Example 19, sections 19.2 and 19.3, were cleaved with several cp caspases-2 at different substrate (fusion protein) concentrations with the same amount of the respective cp caspase-2 variant.
To determine the Michaelis Menten kinetic different concentrations of the fusion protein were incubated with a certain amount of different wild-type cp caspases-2 and P1′ tolerable cp-caspase-2 variants. The buffer is PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and the digests are incubated at room temperature for a certain time. The reaction was stopped by addition of 20 mM cystamine to a final concentration of 2 mM. The samples were then analyzed by RP-HPLC as described in section 18.3.2. The initial rate (v0) in μM/s of each concentration was calculated using the cleaved fusion tag peak area from HPLC at the time point of the initial slope. These data were transferred to TableCurve 2D to fit a Michaelis Menten kinetic in order to calculate values for Vmax and KM.
A Michaelis Menten kinetic was measured with 6H_GSG_VDVAD-hFGF2 and T7AG_6H_GSG_VDVAD-hFGF2 with the enzyme 6H_cpCasp2D. No significant difference in cleavage kinetics was observed (
A Michaelis Menten kinetic was measured with 6H_GSG_VDVAD-hFGF2 and T7AG_6H_GSG_VDVAD-hFGF2 with the enzyme T7AC_6H_cpCasp2D. No significant difference in cleavage kinetics was observed (
A Michaelis Menten kinetic was measured with 6H_GSG_VDVAD-hFGF2 and T7AC_6H_GSG_VDVAD-hFGF2 with the enzyme T7AC_6H_mS9ProD. No significant difference in cleavage kinetics was observed (
A Michaelis Menten kinetic was measured with 6H GSG VDVAD-hFGF2 and T7AC_6H_GSG_VDVAD-hFGF2 with the enzyme T7AC_6H_mS9ProE. No significant difference in cleavage kinetics was observed (
A Michaelis Menten kinetic was measured with T7AC_6H_VDVAD-hFGF2, T7AC 6H GSG VDVAD-hFGF2 and T7AC 6H GSGSGSG VDVAD-hFGF2 with the enzyme T7AC_6H_mS9ProD. Cleavage of hFGF2 without a linker between 6H and VDVAD was significantly less efficient (
Surprisingly with a linker between the 6H tag and the recognition site, VDVAD, the kkat is dramatically increased, which further improves the production technology for a POI, which is expressed as a fusion protein, and wherein the fusion tag is cleaved by a wild-type cp caspase-2 or a P1′ tolerable cp caspase-2.
A Michaelis Menten kinetic was measured with the fusion proteins, 6H-GSG-VDVAD-hFGF-2, 6H-GSG-VDVAD-TNFalpha, 6H-GSG-VDVAD-BIWA4. The measured values are shown in Table 61.
A fusion protein (tagged POI) at 1 g/L was incubated with a cp-caspase-2 variant in a dilution of 50:1 or 100:1 (M/M) fusion protein to cp caspase-2 variant. The digest is incubated at room temperature in PBS for 1 or 2 hours. The reaction was stopped by addition of 20 mM cysteamine to a final concentration of 2 mM. The samples were then analyzed by SDS-PAGE.
The tag cleavage of five fusion proteins with the T7AC_6H_GSG_VDVAD-tag were tested with the enzymes T7AC_6H-cpCasp2D, T7AC_6H-mS9ProD, T7AC_6H-mS9ProE.
When cleaving T7AC_6H_GSG_VDVAD-hFGF2, T7AC_6H-mS9ProD and T7AC_6H-mS9ProE have a higher yield than T7AC_6H-cpCasp2D (
When cleaving T7AC_6H_GSG_VDVAD-TNFα, T7AC_6H-mS9ProD and T7AC_6H-mS9ProE have a higher yield than T7AC_6H-cpCasp2D (
When cleaving T7AC_6H_GSG_VDVAD-rhGH, T7AC_6H-mS9ProD has a higher yield than T7AC_6H-cpCasp2D and T7AC_6H-mS9ProE (
When cleaving T7AC_6H_GSG_VDVAD-GCSF, T7AC_6H-mS9ProD and T7AC_6H-mS9ProE have a higher yield than T7AC_6H-cpCasp2D (
When cleaving T7AC_6H_GSG_VDVAD-GCSF, T7AC_6H-mS9ProD and T7AC_6H-mS9ProE have a higher yield than T7AC_6H-cpCasp2D (
When cleaving T7AC_6H_GSG_VDVAD-PTH, T7AC_6H-mS9ProD and T7AC_6H-mS9ProE have a higher yield than T7AC_6H-cpCasp2D (
Enzyme immobilization was performed through amine coupling. The primary amino groups of the lysine residues on the enzyme were coupled to activated NHS-groups, placed on spacer arms in the resin. The coupling forms a stable amide bond. cp caspase-2 was immobilized at the following concentrations 1 μM, 10 μM, 50 μM and 100 μM. The enzyme was diluted in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) to reach the desired concentration. For a 500 μl column, around 1.5-2 ml of resin slurry in 100% isopropanol was transferred to a 15 ml centrifuge tube. The first step was to wash the resin for removal of the isopropanol. This was done with 10 to 15 resin volumes of cold 1 mM HCl. Immediately after the washing step, the resin and the coupling buffer with enzyme were mixed using a vortex. The sample was left at 4° C. overnight for the coupling reaction. After the coupling the samples were mixed with blocking buffer (0.1 M Tris-HCl, pH 8.5) and kept in the buffer for 2 to 4 hours to block all non-reacted NHS groups in the resin. The samples were then washed alternating two buffers with high (0.1 M Tris-HCl, pH 8.5) respectively low (0.1 M HAc, 0.5 M NaCl, pH 4.7) pH using 3 medium volumes each time and repeating the procedure for 3 to 6 times. In each step, the buffer was added, the sample vortexed, thereafter centrifuged (1.000×g, 1 min, 4° C.) and the supernatant was discarded. The immobilized resin was then stored at 4° C. in either 20% EtOH or 0.01% NaN3 in 1×PBS to prevent microbial contamination before packed in columns.
To determine the kinetics and activity of the immobilized cp caspase-2, the columns were tested with different concentrations of the model protein, hFGF-2 at varying residence times in the column. The flow through from the sample application and first column wash was collected in fractions in 96 deep well plates containing 1/1000 formic acid to deactivate any leaked enzyme and to stop the reaction. The amount of product was quantified using the RP-HPLC method outlined in section 18.3.2.
The amount of cleavage varied with residence time (See
Production of the protein of interest, hFGF-2 The protein was produced in a fermentation as described in Example 19, section 19.2 as a fusion protein, 6H_GSG_VDVAD_hFGF-2.
Cell harvest of the fusion protein, cell disintegration and clarification The cell mass was harvested by centrifugation at 18,590 rcf for 15 minutes. The pellet was stored at −80° C. until further use and the supernatant was discarded. The E. coli harvest was solubilized using homogenization buffer: 50 mM NaPO4, 500 mM NaCl, pH 7.0. The cells were re suspended at a concentration of 30 g cell dry mass per L. Cell lysis was performed through high pressure homogenization (Panda PLUS 2000, Gea, Dusseldorf, Germany) with two passages at 1000 bar for 6H_GSG_VDVAD_hFGF-2. The homogenate was centrifuged (Beckman Coulter GmbH, Vienna, Austria) at 18,590 rcf for 2 hours. The supernatant was filtered through a 0.2 μm membrane (Kleenpak™ Capsule with Fluorodyne® EX Grade EDF Membrane, Pall, New York, USA).
Chromatographic Purification Steps
Preparative chromatography runs were performed on an Äkta Pure 25 system, equipped with a S9 sample pump (GE Healthcare, Uppsala, Sweden).
6H_GSG_VDVAD_hFGF-2 was captured on a Ni-Sepharose 6 Fast Flow column equilibrated with 50 mM NaPO4, 500 mM NaCl, 5 mM imidazole, pH 7.0. The imidazole concentration of the clarified supernatant was adjusted to 5 mM using a solution of 8 M imidazole. The residence time throughout the capture step was 2 minutes. After sample application, the column was washed for 15 CV with equilibration buffer and 6H_GSG_VDVAD_hFGF-2 was eluted using a linear gradient for 5 CV with elution buffer, 50 mM NaPO4, 500 mM NaCl, 500 mM imidazole, pH 7.0, followed by a 5 CV hold step. The tagged POI (fusion protein) eluted in the linear portion of the gradient. A 30-minute CIP cycle using 0.1 M NaOH was used after elution.
The eluate was buffer exchanged using UF/DF using a membrane with a nominal 5 kDa cut-off in Amicon Ultra spin vials (Merck). 5 volumes were exchanged in a discontinuous fashion, until the imidazole concentration was reduced to 5 mM. The buffer exchanged product pool was digested using a 1:100 (w/w/) dilution of T7AC-&H cp caspase-2D (produced as described in Example 18, sections 18.1 and 18.2) per fusion protein at room temperature for 4 hours, to ensure full cleavage of the tag.
This solution (the enzymatic tag removal pool) was loaded on the subtractive IMAC step, using the same column and buffers as before. The hFGF-2 product (which was cleaved by the cp-caspase-2 variant in the step before from the fusion protein) eluted in the flow-through of the chromatographic run. After a wash step with equilibration buffer for 5 CV, the remaining impurities and tagged enzyme were eluted using a 5 CV step gradient with elution buffer. The flow-through was pooled and analyzed:
HCP ELISA, dsDNA Quantification and Endotoxin Assay
The analytical assays for HCP determination via ELISA, dsDNA quantification via PicoGreen assay and Endotoxin quantification via recombinant Factor C assay were performed as previously described by Sauer et al. 2019. A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance. Protein Expr Purif, 153, 70-82. doi:10.1016/j.pep.2018.08.009.
Mass Spectrometric Analysis
For intact mass analysis, the proteins were directly injected to a LC-ESI-MS system (LC: Dionex Ultimate 3000 LC, Thermo, Sunnyvale, Calif., USA). A gradient from 10 to 80% acetonitrile in 0.05% trifluoroacetic acid (using a Thermo ProSwift™ RP-4H column (0.2×250 mm)) at a flow rate of 8 μL/min was applied (30-minute gradient time). Detection was performed with a Q-TOF instrument (Bruker maXis 4G, Billerica, Mass., USA) equipped with the standard ESI source in positive ion, MS mode (range: 400-3000 Da). Instrument calibration was performed using ESI calibration mixture (Agilent, Santa Clara, Calif., USA). Data was processed using Data Analysis 4.0 (Bruker) and the spectrum was deconvoluted by MaxEnt.
6H_GSG_VDVAD_hFGF-2 was purified using a downstream process consisting of IMAC capture step, buffer exchange, enzymatic tag removal and hFGF-2 was then purified by a subtractive IMAC step. The capture IMAC chromatogram can be seen in
The cleavage of 6H_GSG_VDVAD_FGF2 with 6H-cpCasp2 yielded a 99% stoichiometric yield, which equals a 91% mass yield, due to the loss of the tag (Table 62 and lane ETR in
The second, subtractive IMAC step can be used to bind all previously co-purified metal binding host cell proteins, the cleaved tag and eventually residual uncleaved fusion protein as well as the 6H tagged cp-caspase-2, since the Pol, hFGF-2 does not have a his-tag after tag cleavage, shown in
This process sequence is generic and can be applied to any fusion protein comprising an appropriate tag as described in Example 19, sections 19.1-19.3 (this is a tag comprising 6H and the VDVAD recocnition site for a cp caspase-2 variant) and any Pol.
Table 62 shows yield and purity data for FGF2.
†= the stochiometric yield of the cleavage reaction was 99%, but the mass yield was 91 % due to the loss of the tag.
The final fraction was analyzed using LG-MS to confirm the correct mass and N-terminus. The theoretical mass of native hFGF2 is 17,090.5 Da. The major ion detected in MS had a mass of 17,090.2 Da and matches the expected value very well (
| Number | Date | Country | Kind |
|---|---|---|---|
| 19191767.3 | Aug 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/072934 | 8/14/2020 | WO |
