Patent Application
20200318120
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 18, 2019, is named 319573_ST25.txt and is 57,522 bytes in size.
The present invention relates to recombinant proteins, and more particularly to recombinant proteins expressed in gram-negative bacteria such as Escherichia coli (E. coli). The invention also relates to nucleic acids, vectors and gram-negative bacteria for expression of recombinant proteins as well as to separation matrices with covalently linked recombinant protein ligands and to methods of separating immunoglobulins on such matrices.
Expression of heterologous proteins in E. coli (E. coli) is commonly used for recombinant proteins in laboratory and commercial scale. Expression in E. coli using secretion generally means transport of the produced protein over the inner membrane separating the cytoplasm and the periplasm. By secretion to the periplasm, the protein many times also leaks out to the extracellular medium (Mergulhão et al., Biotech Adv 23, 177-202, 2005). Secretion has many advantages compared to cytoplasmic expression such as facilitating correct protein folding, correct N-terminal processing, simplification of down-stream processing and prevention of aggregation into inclusion bodies. However far from all proteins have been successfully expressed in soluble form in periplasm. Some of the problems that might arise is poor secretion and no, or incorrect, processing of the signal peptide. A particular example of proteins having issues with inadequate secretion is in the expression of immunoglobulin binders based on the native or mutated Fc-binding domains B or C of Staphylococcus aureus Protein A (L Abrahmsén et al. EMBO J 4(13B), 3901-3906, 1985). Such immunoglobulin binders are commonly used as ligands in affinity chromatography separation of monoclonal antibodies, a major category of modern pharmaceuticals.
Protein expression is depending on a promoter sequence that starts the transcription of the messenger ribonucleotide acid (mRNA) followed by a ribosome-binding site (RBS) that attracts the translation machinery followed by a signal peptide sequence that is facilitating the transport of the protein to the periplasm. The mature protein is often cloned after the signal peptide and the mature protein is cleaved off from the signal peptide by a signal peptidase when passing the membrane. However, an issue when cloning constructs after a signal peptide is that the restriction enzymes often needs a specific sequence to cleave the DNA, and this leaves a cloning scar after the signal peptide sequence.
Accordingly, there is a need for improvements in the expression of heterologous proteins in E. coli and other gram-negative bacteria, in particular for immunoglobulin-binding proteins derived from the B and C domains of Staphylococcus aureus Protein A.
One aspect of the invention is to provide a functional protein which is easily expressed and secreted in gram-negative bacteria like E. coli. This is achieved with a recombinant protein comprising a functional polypeptide and, linked to the N-terminus of said functional polypeptide, an N-terminal spacer having a length such that the distance between a signal peptide cleaving site and an N-terminus proximal structural unit of the functional polypeptide is 14-24 amino acid residues.
One advantage is that the expression level is improved by the introduction of the N-terminal spacer. A further advantage is that the selectivity of the signal peptide cleavage is improved.
A second aspect of the invention is to provide a nucleic acid molecule encoding for the recombinant protein. This is achieved with a nucleic acid molecule comprising the following elements in the 5′ to 3′ direction, said elements being operatively linked:
a) an inducible or constitutive promoter DNA sequence;
b) a DNA sequence encoding a signal peptide;
c) a DNA sequence encoding an N-terminal spacer; and
d) a DNA sequence encoding a functional or immunoglobulin-binding polypeptide.
A third aspect of the invention is to provide a cloning vector which expresses and secretes the recombinant protein of any preceding claim into the bacterial periplasm of a gram-negative cell. This is achieved with a cloning vector comprising the above nucleic acid molecule.
A fourth aspect of the invention is to provide a gram-negative bacterium transformed by the cloning vector.
A fifth aspect of the invention is to provide a method of expressing and secreting the recombinant protein in a gram-negative bacterium. This is achieved by a method comprising the steps of providing the gram-negative bacterium and culturing the gram-negative bacterium.
A sixth aspect of the invention is to provide a separation matrix comprising the recombinant protein covalently linked to a support.
A seventh aspect of the invention is to provide a method of separating an immunoglobulin, comprising the steps of:
i) providing the above separation matrix wherein the recombinant protein comprises an immunoglobulin-binding polypeptide;
ii) contacting the separation matrix with a liquid sample containing an immunoglobulin, to bind the immunoglobulin;
iii) optionally washing the separation matrix with a washing liquid;
iv) contacting the separation matrix with an elution liquid, to elute the immunoglobulin.
Further suitable embodiments of the invention are described in the dependent claims.
The terms “antibody” and “immunoglobulin” are used interchangeably herein, and are understood to include also fragments of antibodies, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments.
The terms an “Fc-binding polypeptide” and “Fc-binding protein” mean a polypeptide or protein respectively, capable of binding to the crystallisable part (Fc) of an antibody and includes e.g. Staphylococcus aureus Protein A and Streptococcus Protein G, or any fragment or fusion protein thereof that has maintained said binding property.
The terms an “Fab-binding polypeptide” and “Fab-binding protein” mean a polypeptide or protein respectively, capable of binding to the antigen-binding part (Fab) of an antibody and includes e.g. Peptostreptococcus magnus Protein L, Streptococcus Protein G, native Staphylococcus aureus Protein A or any fragment or fusion protein thereof that has maintained said binding property.
The term “linker” herein means an element linking two polypeptide units, monomers or domains to each other in a multimer.
The term “% identity” with respect to comparisons of amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST™) described in Altschul et al. (1990) J. Mol. Biol., 215: 403-410. A web-based software for this is freely available from the US National Library of Medicine at http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. Here, the algorithm “blastp (protein-protein BLAST)” is used for alignment of a query sequence with a subject sequence and determining i.a. the % identity.
The abbreviation “DsbA” herein means E. coli, Thiol:disulfide interchange protein, UniProt P0AEG4.
The abbreviation “OmpA” herein means E. coli, Outer membrane protein A, UniProt P0A910.
The abbreviation “PrA” herein means Staphylococcus aureus protein A, UniProt P38507.
The abbreviation “GIII” herein means Gene 3 from Bacteriophage M13, UniProt P69168.
The term “signal peptide” herein means a short (usually 16-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. It may also be referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide. The signal peptide is normally cleaved off from the protein by a signal peptidase enzyme.
The term “signal peptide cleavage site” herein means a dipeptide between which the signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases the dipeptide is Ala-Ala. The signal peptide cleavage site can be calculated with algorithms such as SignalP 4.1, available on-line at http://www.cbs.dtu.dk/services/SignalP/ (Center for Biological Sequence Analysis, Technical University of Denmark).
The term “heterologous expression” herein means the expression of a gene or part of a gene in a host organism, which does not naturally have this gene or gene fragment. “Secreted” refers to across the inner membrane of gram negative bacteria, such as E. coli.
The cytoplasm (cytoplasmic), is the space inside of the inner cell membrane in gram negative bacteria containing the genetic material. The term “cytoplasmic expression” herein means protein expression within the cytoplasm.
The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in gram-negative bacteria.
The term “promoter” herein means a region of DNA that initiates transcription (writing to mRNA) of a particular gene. Promoters are normally located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5′ region of the sense strand). A promoter can be inducible, meaning that the expression of genes operably linked to the promoter can be turned on by the presence of an inducer substance. Alternatively, the promoter may be constitutive, i.e. that it is not regulated by any inducer substance.
The abbreviation “RBS” herein means a ribosome-binding site, or ribosomal binding site. This is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation.
The abbreviation “RhaBAD” herein means the E. coli rhamnose operon promoter (also called rhamnose promoter) of genes RhaB, RhaA and RhaD. This is a promoter widely used in molecular biology.
The abbreviation “T5” herein means Bacteriophage T5 promoter for E. coli RNA polymerase, with an embedded lac operator. An operator is a segment of DNA to which a transcription factor binds to regulate gene expression by repressing it.
The abbreviation “pD861-SR” herein refers to a plasmid for E. coli protein expression with rhamnose promoter (RhaBAD) and a strong ribosome-binding site (SR).
The abbreviation “pJ401” herein refers to a plasmid for E. coli protein expression, with bacteriophage T5 promoter and dual embedded lac operator mirrored on each side of the promoter.
The abbreviation “OptEc” herein means optimized for E. coli expression, i.e. codon triplets are chosen to fit E. coli translation machinery.
The abbreviation “FspI” herein means a DNA restriction enzyme from Fischerella species (ATCC 29114). It cleaves blunt at the sequence TGCGCA.
The abbreviation “KpnI” herein means a DNA restriction enzyme from Klebsiella pneumoniae OK8 (ATCC 49790). It cleaves with overhang at the sequence GGTACC.
The abbreviation “SRP” herein means the signal recognition particle pathway, a universally conserved pathway for targeting polypeptides for secretion via the cotranslational pathway.
The abbreviation “Sec” herein means secretion or Type II secretory pathway, a system which is responsible for the secretion of proteins through the cell membrane.
The term “E. coli K12-017” herein means an E. coli expression strain, as described by Olsson M. O. and Isaksson L. A. in Molec. Gen. Genet. 169, 251-257 (1979).
The term “functional polypeptide” herein means a polypeptide with a technically useful property. Examples of such properties are: a) highly specific binding to a target species (for use as affinity binders, in particular as ligands for affinity chromatography), b) therapeutic properties (for use as a medicament), c) enzymatic properties (for use as biocatalysts) and d) signal-emitting properties (for use as reporter proteins, such as fluorescent reporter proteins).
As used herein, the terms “comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
In one aspect, illustrated by
The functional polypeptide can be an immunoglobulin-binding polypeptide. Such a polypeptide may e.g. comprise one or more immunoglobulin-binding domains derived from a bacterial protein selected from the group consisting of Staphylococcus aureus Protein A, Peptostreptococcus magnus Protein L and Streptococcus Protein G, such as from the group consisting of Staphylococcus aureus Protein A and Peptostreptococcus magnus Protein L. The immunoglobulin-binding domain(s) can e.g. have at least 80%, such as at least 90 or 95%, sequence identity with Domain E, D, A, B or C of Staphylococcus aureus Protein A, with Protein Z (a variant of Domain B of Staphylococcus aureus Protein A), Zvar or Zvar2 (alkali-stabilized mutants of Protein Z) or with Domain 1, 2, 3, 4 or 5 of Peptostreptococcus magnus Protein L. In this context, the immunoglobulin-containing domain(s) can be defined by, or have at least 80%, such as at least 90 or 95% sequence identity with, an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11. SEQ ID NO. 1-7 (Staphylococcus aureus Protein A domains E, D, A, B and C, Protein Z and Zvar) are listed in
SEQ ID NO. 1-11 can all be characterized as Fc-binding domains derived from Staphylococcus aureus Protein A. Such domains may further be alkali-stabilized by mutations of the native domains, as has been done in Zvar and Zvar2. Further examples of such alkali-stabilized domains can be SEQ ID NO. 48-93 (listed under Example 6) and other examples are given e.g. in U.S. Pat. Nos. 8,329,860, 8,754,196, 9,040,661, 9,403,883, JP 2006304633A, U.S. Pat. No. 8,674,073, US 2010/0221844, US 2012/0208234, U.S. Pat. No. 9,051,375, US 2014/0031522, US 2014/0107315, US 2013/0096276, US 2013/0274451, US 2005/0143566, US 2016/0159855, US 2016/0168209, US 2016/0237124, WO 2014/146350, WO 2016/079033, WO 2016/079034, WO 2016/152946, PCT EP2017/061162, PCT EP2017/061164, PCT EP2017/061160, PCT EP2017/061158, PCT EP2017/061159, U.S. Ser. No. 14/961,164, U.S. Ser. No. 15/348,699 and U.S. Ser. No. 15/282,367, all of which are hereby incorporated by reference in their entireties. Specifically, the alkali-stabilized Fc-binding domains may have at least 80%, such as at least 90% or at least 95%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 7-11, 48-64 and 74-93.
The immunoglobulin-binding polypeptide can suitably be a multimer of immunoglobulin-binding domains, e.g. Fc-binding domains, as discussed above. The multimer can e.g. be a dimer, trimer, tetramer, pentamer, hexamer or a heptamer, such as a dimer, tetramer or hexamer. Suitably, the multimer may comprise at least four domains. The domains can be directly linked to each other (e.g. in the case of SEQ ID NO. 1-8, 48-64 and 74-93), but they can also be linked to each other via linkers, typically comprising 1-25 (such as 3-20) amino acid residues (e.g. in the case of SEQ ID NO 9-11). Examples of suitable linkers include APKADNKFNKE, APKVDAKFDKE, APK, APKVDA, AKFDKE, APKVFDKE, APAKFDKE, VDAKFDKE, APKKFDKE, APKYEDGKQYTVDAKFDKE and APKYEDGVDAKFDKE. Some specific examples of multimers include SEQ ID NO. 12 (tetramer), SEQ ID NO. 13 (hexamer) and SEQ ID NO. 65-73 (dimers).
The recombinant protein may comprise a coupling moiety at or proximal to the C-terminus or the N-terminus, such as at the C-terminus. This coupling moiety can be used for specific coupling to a support as discussed below and may comprise a cysteine, allowing for coupling by thioether bonds. Alternatively, or additionally, the coupling moiety may comprise one or more lysines, such as a cluster of 2-8 lysines.
The functional or immunoglobulin-binding polypeptide may have secondary and tertiary structures and may suitably comprise one or more structural units, as exemplified by alpha helices, beta sheets and/or beta barrels. In particular, the polypeptide may comprise a plurality of alpha helices, such as at least three alpha helices. The Fc-binding domains derived from Staphylococcus aureus Protein A (e.g. SEQ ID NO. 1-11, 48-64 and 74-93) each comprise three alpha helices, so the number of alpha helices in a multimer as discussed above may be three times the number of domains in the multimer. The first alpha helix in the domains as exemplified by SEQ ID NO. 1-11, 48-64 and 74-93 starts at position 9 (using the position nomenclature of
The N-terminal spacer suitably has a length such that the number of amino acid residues between the signal peptide cleaving site and an N-terminus proximal structural unit of the functional or immunoglobulin-binding polypeptide is 14-24. As discussed above, this structural unit can suitably be an alpha helix (or alternatively a beta sheet or beta barrel). The number of amino acid residues between the N-terminus of the functional or immunoglobulin-binding polypeptide and the N-terminus proximal structural unit can vary, e.g. between 0 (SEQ ID NO. 9-11) and 11 (SEQ ID NO. 2), and accordingly, the length of the N-terminal spacer may vary, e.g. between 3-24 amino acid residues, such as between 8-24 amino acid residues or 14-24 amino acid residues.
For example, if the N-terminal spacer is linked to an Fc-binding domain having at least 90% identity to SEQ ID NO. 1-8, 48-64 or 74-93, the N-terminal spacer may e.g. have a length of 8-12 amino acid residues and if the N-terminal spacer is linked to an Fc-binding domain having at least 90% identity to SEQ ID NO. 9-11, the N-terminal spacer may e.g. have a length of 16-20 amino acid residues. The N-terminal spacer may e.g. consist of amino acid residues selected from the group consisting of alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine and valine. For improved alkali stability, it may be advantageous to exclude asparagines. In this case, the N-terminal spacer may e.g. consist of amino acid residues selected from the group consisting of alanine, aspartic acid, glutamine, glutamic acid, glycine, histidine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine and valine. It can be advantageous if the N-terminal spacer does not comprise any clusters of arginines or lysines, and/or if it comprises at most two amino acid residues selected from the group consisting of lysine and arginine. In some embodiments, the two N-terminal amino acid residues of the N-terminal spacer may be AQ (alanine followed by glutamine).
In particular, the N-terminal spacer may comprise, comprise essentially or have, an amino acid sequence having at least 80% sequence identity to, or being defined by, an amino acid sequence selected from the group consisting of SEQ ID NO. 16-18, 29-30, 33-40, 43-45 and 47. The N-terminal spacer may further comprise, comprise essentially or have an amino acid sequence having at least 80% sequence identity to, or being defined by, an amino acid sequence selected from the group consisting of AQYEDGKQYTADNKFNKE, AQYEDGKQYTVDAKFDKE, AQKDQTWYTGVDAKFDKE, AQHDEAQQEAVDAKFDKE, AQGGGSGGGSVDAKFDKE, AQYEDGKQYGTVDAKFDKE, AQYEDGKQGTVDAKFDKE, AQYEDGKQYTTLEKGTVDAKFDKE, AQYEDGKQYTTLEKPVAGGTVDAKFDKE, AQYEDGKQYTVDAKFDKE, AQYEDGKQYTETVDAKFDKE, AQYEDGKQYTDTVDAKFDKE, AQYEDGKQYTATVDAKFDKE, AQYEDGKQYEDTVDAKFDKE, AQHHHHHHHHGTVDAKFDKE, AQHHHHHHGTVDAKFDKE and AQHDEAQQEAGTVDAKFDKE. The latter sequences are particularly advantageous in combination with immunoglobulin-binding polypeptides derived from SEQ ID NO. 9-11.
In terms of alkali stability it can further be advantageous to use an N-terminal spacer comprising, comprising essentially or having, an amino acid sequence having at least 80% sequence identity to, or being defined by, an amino acid sequence selected from the group consisting of SEQ ID NO. 35, 37, 38, 40, 43, 44 and 47. The N-terminal spacer may also for alkali stability comprise, comprise essentially or have an amino acid sequence having at least 80% sequence identity to, or being defined by, an amino acid sequence selected from the group consisting of AQYEDGKQYTVDAKFDKE, AQYEDGKQYTETVDAKFDKE, AQYEDGKQYTDTVDAKFDKE, AQYEDGKQYEDTVDAKFDKE, AQIIIIIIIIIIIIIIIIGTVDAKFDKE, AQIIIIIIIIIIIIGTVDAKFDKE and AQHDEAQQEAGTVDAKFDKE. The latter sequences are particularly advantageous in combination with immunoglobulin-binding polypeptides derived from SEQ ID NO. 9-11.
The alkali stability of the recombinant protein can be assessed by coupling it to an SPR chip, e.g. to Biacore CMS sensor chip as described in the examples of WO2016079033, using e.g. NHS- or maleimide coupling chemistries, and measuring the immunoglobulin-binding capacity of the chip, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22+/−2° C. The incubation can e.g. be performed in 0.5 M NaOH for a number of 10 min cycles, such as 100, 200 or 300 cycles. The IgG capacity of the matrix after 100 10 min incubation cycles in 0.5 M NaOH at 22+1-2° C. can be at least 55, such as at least 60, at least 80 or at least 90% of the IgG capacity before the incubation. Alternatively, the remaining IgG capacity after 100 cycles for a particular mutant measured as above can be compared with the remaining IgG capacity for a parental recombinant protein. In this case, the remaining IgG capacity for the mutant may be at least 105%, such as at least 110%, at least 125%, at least 150% or at least 200% of the parental recombinant protein.
In a second aspect, the present invention discloses a nucleic acid molecule encoding for the recombinant protein as disclosed above. The nucleic acid molecule comprises the following operatively linked elements in the 5′ to 3′ direction:
a) an inducible or constitutive promoter DNA sequence, e.g. the RhaBAD or T5 promoter sequence. RhaBAD is inducible with rhamnose and T5 with Isopropyl β-D-1-thiogalactopyranoside (IPTG). An example of a constitutive promoter is the spa promoter (the promoter of the naturally occurring Staphylococcus aureus Protein A);
b) a DNA sequence encoding a signal peptide, e.g. the OmpA (SEQ ID NO. 14) or DsbA (SEQ ID NO. 15) signal peptides, or a signal peptide having at least 80% sequence identity with either of these;
c) a DNA sequence encoding an N-terminal spacer as discussed above; and
d) a DNA sequence encoding a functional or immunoglobulin-binding polypeptide as discussed above.
The nucleic acid molecule may further comprise a ribosome-binding site (RBS) and an origin of replication. It can suitably also comprise an antibiotic resistance marker.
In a third aspect, the present invention discloses a cloning vector, e.g. a plasmid, which expresses and secretes the recombinant protein as disclosed above into the bacterial periplasm of a gram-negative cell, e.g. E. coli. The cloning vector comprises the nucleic acid molecule as discussed above.
In a fourth aspect, the present invention discloses a gram-negative bacterium transformed by the cloning vector disclosed above. The bacterium may e.g. be identified as Escherichia coli, in particular E. coli of the K12 strain, such as E. coli K12-017. Other examples of gram-negative bacteria include e.g. the genus Pseudomonas, such as e.g. Pseudomonas fluorescens. The transformation may be achieved e.g. by the heat shock method, although other methods such as electroporation are also possible.
In a fifth aspect, the present invention discloses a method of expressing and secreting the recombinant protein discussed above in a gram-negative bacterium. This method comprises the steps of:
i) providing the gram-negative bacterium disclosed above; and
ii) culturing the gram-negative bacterium.
If the nucleic acid molecule as discussed above comprises an inducible promoter, the method may further comprise a step of inducing recombinant protein expression in the gram-negative bacterium.
In a sixth aspect, the present invention discloses a separation matrix comprising the recombinant protein disclosed above, covalently linked to a support.
As the skilled person will understand, the expressed recombinant protein should be purified to an appropriate extent before being immobilized to a support. Such purification methods are well known in the field, and the immobilization of protein-based ligands to supports is easily carried out using standard methods. Suitable methods and supports will be discussed below in more detail.
The alkali stability of the matrix can be assessed by measuring the immunoglobulin-binding capacity, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22+/−2° C. The incubation can e.g. be performed in 0.5 M NaOH for a number of 15 min cycles, such as 100, 200 or 300 cycles, corresponding to a total incubation time of 25, 50 or 75 h. The IgG capacity of the matrix after 96-100 15 min incubation cycles or a total incubation time of 24 or 25 h in 0.5 M NaOH at 22+/−2° C. can be at least 80, such as at least 85, at least 90 or at least 95% of the IgG capacity before the incubation.
The solid support of the matrix according to the invention can be of any suitable well-known kind. A conventional affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy (—COOH), carboxamido (—CONH2, possibly in N-substituted forms), amino (—NH2, possibly in substituted form), oligo- or polyethylenoxy groups on their external and, if present, also on internal surfaces. The solid support can suitably be porous. The porosity can be expressed as a Kay or Kd value (the fraction of the pore volume available to a probe molecule of a particular size) measured by inverse size exclusion chromatography, e.g. according to the methods described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13. By definition, both Kd and Kay values always lie within the range 0-1. The Kay value can advantageously be 0.6-0.95, e.g. 0.7-0.90 or 0.6-0.8, as measured with dextran of Mw 110 kDa as a probe molecule. An advantage of this is that the support has a large fraction of pores able to accommodate both the recombinant protein of the invention and immunoglobulins binding to the recombinant protein and to provide mass transport of the immunoglobulins to and from the binding sites.
The recombinant protein may be attached to the support via conventional coupling techniques utilising e.g. thiol, amino and/or carboxy groups present in the ligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents. Between the support and the recombinant protein, a molecule known as a spacer can be introduced, which improves the availability of the recombinant protein and facilitates the chemical coupling of the recombinant protein to the support. Depending on the nature of the recombinant protein and the coupling conditions, the coupling may be a multipoint coupling (e.g. via a plurality of lysines) or a single point coupling (e.g. via a single cysteine). Alternatively, the recombinant protein may be attached to the support by non-covalent bonding, such as physical adsorption or biospecific adsorption.
In some embodiments, the matrix comprises 5-25, such as 5-20 mg/ml, 5-15 mg/ml, 5-11 mg/ml or 6-11 mg/ml of the recombinant protein coupled to the support. The amount of coupled protein can be controlled by the concentration of protein used in the coupling process, by the activation and coupling conditions used and/or by the pore structure of the support used. As a general rule, the absolute binding capacity of the matrix increases with the amount of coupled protein, at least up to a point where the pores become significantly constricted by the coupled protein. The relative binding capacity per mg coupled protein will decrease at high coupling levels, resulting in a cost-benefit optimum within the ranges specified above.
In certain embodiments, the protein is coupled to the support via thioether bonds. Methods for performing such coupling are well-known in this field and easily performed by the skilled person in this field using standard techniques and equipment. Thioether bonds are flexible and stable and generally suited for use in affinity chromatography. In particular, when the thioether bond is via a terminal or near-terminal cysteine residue on the recombinant protein, the mobility of the coupled recombinant protein is enhanced which provides improved binding capacity and binding kinetics. In some embodiments, the recombinant protein is coupled via a C-terminal cysteine provided on the protein as described above. This allows for efficient coupling of the cysteine thiol to electrophilic groups, e.g. epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling. Alternatively, the recombinant protein may be covalently linked to the support via one or more amide bonds. This can be achieved e.g. through reaction between one or more lysines in the protein and one or more activated carboxyl groups on the support. The activation can e.g. be made by the commonly known N-hydroxysuccinimide (NHS) reagent. Yet another alternative is that the protein is linked via one or more secondary amine links. Such links can be formed from lysines in the protein and either hydroxyl groups on the support, which have been activated using e.g. tresyl chloride or tosyl chloride chemistry, or by reductive amination reaction between the lysines and aldehydes on the support. The aldehydes can e.g. be formed from vicinal diols on the support through periodate oxidation.
In certain embodiments, the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include e.g. dextran, starch, cellulose, pullulan, agar, agarose etc. Polysaccharides are inherently hydrophilic with low degrees of nonspecific interactions, they provide a high content of reactive (activatable) hydroxyl groups and they are generally stable towards alkaline cleaning solutions used in bioprocessing.
In some embodiments, the support comprises agar or agarose. The supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (S Hjertén: Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the base matrices are commercially available products, such as crosslinked agarose beads sold under the name of SEPHAROSE™ FF (GE Healthcare). In an embodiment, which is especially advantageous for large-scale separations, the support has been adapted to increase its rigidity using the methods described in U.S. Pat. Nos. 6,602,990 or 7,396,467, which are hereby incorporated by reference in their entirety, and hence renders the matrix more suitable for high flow rates.
In certain embodiments, the support, such as a polysaccharide or agarose support, is crosslinked, such as with hydroxyalkyl ether crosslinks. Crosslinker reagents producing such crosslinks can be e.g. epihalohydrins like epichlorohydrin, diepoxides like butanediol diglycidyl ether, allylating reagents like allyl halides or allyl glycidyl ether. Crosslinking is beneficial for the rigidity of the support and improves the chemical stability. Hydroxyalkyl ether crosslinks are alkali stable and do not cause significant nonspecific adsorption.
Alternatively, the solid support is based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides etc. In case of hydrophobic polymers, such as matrices based on divinyl and monovinyl-substituted benzenes, the surface of the matrix is often hydrophilised to expose hydrophilic groups as defined above to a surrounding aqueous liquid. Such polymers are easily produced according to standard methods, see e.g. “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Alternatively, a commercially available product, such as SOURCE™ (GE Healthcare) is used. In another alternative, the solid support according to the invention comprises a support of inorganic nature, e.g. silica, zirconium oxide etc.
In certain embodiments, the solid support is in another form such as a surface, a chip, capillaries, or a filter (e.g. a membrane or a depth filter matrix).
In a seventh aspect, the present invention discloses a method of separating an immunoglobulin, comprising the steps of:
i) providing the separation matrix as discussed above, wherein the recombinant protein comprises an immunoglobulin-binding polypeptide, e.g. comprising one or more Fc-binding domains derived from Staphylococcus aureus Protein A;
ii) contacting the separation matrix with a liquid sample containing an immunoglobulin, to bind the immunoglobulin;
iii) optionally washing the separation matrix with a washing liquid;
iv) contacting the separation matrix with an elution liquid, to elute the immunoglobulin;
v) optionally cleaning the separation matrix with a cleaning liquid. Alkali cleaning liquids are commonly used in bioprocessing and, provided that the recombinant protein is alkali-stable, the cleaning liquid may comprise at least 0.1 M NaOH or KOH, such as at least 0.5 M NaOH or KOH, or 0.5-2.5 M NaOH or KOH.
First, two different signal peptides were tested together with four different N-terminal spacers to find the best cleavage and protein expression. This experiment (Example 1) was performed with Zvar2 monomer using the RhaBAD promoter system. In Example 2, the most promising signal peptide and N-terminal start was cloned in to Zvar2 hexamer. This construct was tested in fed-batch fermentations with and without the N-terminal start sequence. This study was performed using the T5 promoter expression system. In Example 3, all the native domains of Staphylococcus aureus protein A, Zvar monomer (Zvar1), Zvar2 monomer (Zvar21) and Zvar tetramer (Zvar4) were tested with the chosen signal peptide and N-terminal start to see if the start sequence also had an effect on closely related domains. Also Example 3 was performed with the T5 expression system. Example 4 was performed with a set of different N-terminal spacers, Example 5 was a scale-up of fermentations using a couple of selected spacers and Example 6 was an investigation using the AQYEDGKQYTGT N-terminal spacer in combination with different mutants of an immunoglobulin-binding protein.
Genes as described in Table 1 were synthesized by a contract manufacturer of synthetic genes (ATUM, CA, USA). Double stranded dideoxy ribonucleic acid (dsDNA) was synthesized based on the amino acid (AA) sequence and was optimized for expression in E. coli by a manufacturer proprietary algorithm. The dsDNA was inserted into expression vector pD861-SR or pJ401 with a signal peptide either from DsbA or OmpA (Table 2).
All plasmids were transformed into Emil K12-017 using chemically competent cells. To modify the vectors, a short spacer sequence of 8 AA was inserted between the FspI and KpnI cleave sites. The 8 AA sequences were taken from the first AA in the mature protein of OmpA (AQKDQTWYTGGT, SEQ ID NO 16), DsbA (AQYEDGKQYTGT, SEQ ID NO 17), SpA (AQHDEAQQEAGT, SEQ ID NO 18) or the flexible linker structure from M13 phage GIII AA 236-243 (AQGGGSGGGSGT, SEQ ID NO 19), with modifications in OmpA to fit the restriction site and in OmpA and SpA to mutate asparagines to glutamines (
Plasmids were cleaved with restriction enzymes FspI and KpnI (New England Biolabs (NEB), MA, USA), 6 μl NEB buffer 2.1 was mixed with 6 μg plasmid and 2 μl FspI in total 58 μl. The solution was incubated in 37° C. for 1 hour before KpnI was added. The incubation was continued 2 h, followed by addition of 1 μl Calf intestine phosphatase (CIP) followed by 30 min further incubation. Excised bases were removed from the digested plasmids using QIAquick PCR Purification kit (Qiagen, Hilden, Germany).
Oligonucleotides were ordered from Integrated DNA technologies (IDT, IA, USA). All oligonucleotides were modified with a five prime phosphate group by manufacturer. Two complementary oligonucleotide pairs were mixed in ligation buffer and heated to 95° C., 4 min, followed by cooling down to room temperature. The hybridized fragment was ligated into the FspI and KpnI cleaved plasmids by use of T4 DNA ligase (NEB, MA, USA). A complete list of oligonucleotides used is found in Table 3.
Ligated plasmids were transformed into chemically competent E. coli K12-017 cells. The cells were thawed on ice for 30 min, 100 μl competent cells were added to 10 μl ligation reaction. Cells were incubated on ice for 20 min followed by heat shock at 42° C. for 1 min, followed by incubation on ice 5 min. Followed by addition of 900 μl SOC-medium (NEB, MA, USA). Transformation reaction was incubated in 37° C. in a rotary shake incubator for 60 min, 100 μl of each reaction was spread on Luria agar plates containing appropriate selective antibiotics. Positive clones were screened with Polymerase chain reaction (PCR), and clones with correct insert were selected. Selected clones were grown in 10 ml Luria broth (LB) over-night (o/n) containing appropriate antibiotics, followed by plasmid preparations using Qiagen Plasmid Miniprep kit (Qiagen, Hilden, Germany). Plasmids were sent to GATC Biotech for sequence verification (GATC Biotech, Cologne, Germany).
Escherichia coli strain K12-017 transformed with the recombinant plasmids pGE0138-145 were cultured in 30° C. for 4 h in 100 ml Terrific Broth (TB) medium (containing 12 g tryptone, 24 g yeast extract, 5 g glycerol (85%), 2.31 g KH2PO4, 12.54 g K2HPO4 and 50 mg of kanamycin sulphate per liter). Cultures were induced when OD600 nm reached 1-2, using 4 mM L-rhamnose (Sigma Aldrich, MO, USA). Cultures were further incubated in 30° C. 17-20 h. Culture solution was subjected to low-speed centrifugation (at 4,000 rpm) for 20 min in a swing out rotor to collect wet cell pellet. The bacterial cell pellets were suspended in 20 ml of a 25 mM phosphate buffer solution (pH 7.4), and the cells were lysed by heat treatment at 85° C. for 10 min in a heat block. Followed by a high-speed centrifugation (at 13 000 rpm) for 10 min to separate the supernatant. Then the supernatants were filtered through 0.2 μm syringe filter to remove any residual particles before it was applied to an IgG Sepharose 6FF (GE Healthcare Bio-Sciences, Uppsala, Sweden) affinity chromatography column. The column was equilibrated with loading buffer (25 mM phosphate pH7, 250 mM NaCl) followed by loading of sample. After loading, the column was washed with 5 column volumes (CV) of loading buffer and one CV of low salt wash buffer (50 mM acetate pH6), followed by elution with 50 mM acetic acid, pH2.8. Absorbance at 237 nm was measured in-line using an ÄKTA explorer 100 chromatography system (GE Healthcare Bio-Sciences, Uppsala, Sweden) and peak integration of the eluted peak was performed in the system software (Unicorn 5.1).
In the first work of optimization of the expression system, two different signal peptides were tested, utilizing different secretion pathways in the cell. DsbA utilizes the signal recognition particle (SRP) pathway and OmpA the Sec pathway. Results from protein expression using DsbA and OmpA signal peptides, showed that the OmpA signal peptide had higher protein expression; 2-5 times higher compared to DsbA signal peptide. Moreover, the first N-terminal AA after the cleave site of the signal peptide had a significant impact on the expression levels. In this study, the DsbA start AA had the highest expression level together with both signal peptides. The lowest expression level was seen for the Flex8AA start sequence using both signal peptides.
Eluates from IgG Sepharose 6FF were analysed using LC/MS (Waters, PA, USA). Results showed that the eluate from the reference construct pGE0120, with AQGT as N-terminal start sequence had a range of different signal peptide cleave sites (
Six, 1 L working volume fermenters, GRETA (Belach Bioteknik, Skogås, Sweden) were used. Starting volume was set to 750 ml, and end volume was approximately 1 L. Aeration was set to 1 L/min, temperature, pH and antifoam were controlled automatically. pH was kept at set value by the addition of 25% ammonia and 2 M phosphoric acid. Antifoam control was automatically adding Breox FMT 30 (BASF, Ludwigshafen, Germany), when high foaming occurred. pH and dissolved oxygen (DO) were controlled with probes from Broadley James (CA, USA). DO was kept constant at 30% by increasing stirrer speed from 300 rpm up to 1500 rpm. When the stirrer reached max speed, DO second set point was 20%, and was held constant by addition of mixing pure oxygen into the airflow. Terrific Broth (TB) supplemented with 50 mg/L kanamycin or neomycin was used in the shake flask pre-cultivations and was started by addition of 100 μl cell suspension to 100 mL TB supplemented with 50 μg/mL kanamycin or neomycin and incubated in 37° C. for 17 h. Main fermentation media was inoculated with 10 ml pre-cultivation. Glucose feed with 60% (w/v) (VWR, PA, USA) was fed into the fermenter according to a pre-set profile after initial batch glucose was consumed. Total length of the main fermentation was 26 h.
Fermentation samples were heat treated at 85° C. for 5 min in 1.5 ml tubes. Followed by high speed centrifugation (at 13500 rpm, in benchtop centrifuge) for 5 min to separate the supernatant. Supernatants were filtered through 0.2 μm syringe filter to remove any residual particles before they were applied to an IgG Sepharose 6FF HiTrap™ column (GE Healthcare Bio-Sciences, Uppsala Sweden). The column was equilibrated with loading buffer (Phosphate buffered Saline (Medicago, Uppsala, Sweden)) followed by loading of 50 μl sample. After loading, the column was washed with 5 CV of loading buffer, followed by elution with 200 mM phosphate buffer, pH2.9. Absorbance at 237 nm was measured in-line and peak integration of the eluted peak was performed in the chromatography system software. Protein concentration of the samples were determined by a standard curve using a purified protein of known concentration.
Plasmid pGE0002 containing Zvar2 hexamer (Zvar26) was cleaved with FspI and KpnI restriction enzymes. The N-terminal start from DsbA was ligated into the cleaved vector, and the resulting plasmid was denoted pGE0180. These two vectors were transformed into E. coli K12-017 and the resulting constructs were expressed in fed-batch fermentations to see differences in protein expression and signal peptide cleavage patterns. Protein concentration analysis using standard curve showed that the protein expression increased from 3.5 g/L to 16.8 g/L, i.e. a more than 4 times increase in protein expression (
Eluates from IgG Sepharose were analysed using LC/MS (Waters, PA, USA). The results showed that the eluate from the reference construct pGE0002 showed a range of different signal peptide sites including cleave sites with extra 6 AA and 9 AA, previously seen in experiment (
Protein expression in fed-batch fermenter and concentration analysis using standard curve performed as in Example 2.
Filtered samples (See above) were applied to an IgG Sepharose 6FF Tricorn 10 column with a CV of 10.5 ml (GE Healthcare Bio-Sciences, Uppsala, Sweden). The column was equilibrated with high salt loading buffer (50 mM phosphate, pH7.0, 500 mm NaCl) followed by loading of 0.5-5 mL sample based on concentration calculations (See above). After loading, the column was washed with 5 CV of loading buffer, followed by elution with 100 mM acetic acid. Eluted samples were collected and concentrated to approximately 1 mg/ml using Vivaspin 5, 3000 Da cut-off (GE Healthcare Bio-Sciences, Uppsala, Sweden). The amount of protein was estimated by absorbance measurements at 280 nm using specific absorbance coefficients, calculated based on Pace et al. Protein Science 4, 2411-2423, (1995) and Beer Lamberts law. Sample concentration was back-calculated using total protein mass in elution pool and volume of injected sample. The concentration was confirmed by amino acid analysis. The homogeneity and the molecular weights were analysed using mass spectrometry on a Waters Q-Tof (PA, USA).
To determine the effects of N-terminal start sequence on other domains from Staphylococcus aureus protein A and alkali stabilized domain Zvar monomer (Zvar1) and tetramer (Zvar4), new plasmids were constructed with the additional N-terminal and expressed in fed-batch fermentations using plasmid pJ401. The results showed that E-domain had no measurable expression without insert. However, with 8AA DsbA start, protein expression was measured to 2.9 g/L. Both the D- and A-domain showed a low expression that was not improved by adding N-terminal start. Both C- and B-domain (which have the same AA sequence in the start) showed very similar expression, with a clear increase in expression with 8AA. DsbA start. However, the largest increase of expression was seen on Zvar- and Zvar2 monomer (Zvar1 and Zvar21) with more than 4 times and 10 times protein expression respectively. Also, Zvar tetramer (Zvar4) showed a significant increase in protein expression with 8AA DsbA start, even though the increase was not as dramatic as for the monomer.
In the first experiment with N-terminal spacer, two different signal peptides from two secretion pathways were tested, and it was shown that OmpA signal peptide, utilizing Sec dependent pathway had the highest expression and signal peptide cleavage in this experiment. Other signal peptides using this pathway are for example PhoA (E. coli alkaline phosphatase), MalE (E. coli maltose binding protein) and PelB (Erwinia carotovora Pectate lyase B). On the contrary, the reference construct (with AQGT start) had higher protein expression and signal peptide cleavage using DsbA signal peptide, indicating that it is a combination of signal peptide and N-terminus. Furthermore, four different N-terminals were tested and all worked better compared to the reference construct using OmpA signal peptide. Surprisingly, when the signal peptide cleavage worked optimally, also protein expression was increased, indicating that signal peptidase cleavage might be the rate limiting enzyme in the transport of proteins over the inner membrane. Without being bound by theory, a hypothesis is that protein “gets stuck” in the pores of the inner membrane and halts further expression when secretion is not working optimally. In cultivations of high protein expression, more than 50% of the protein was found outside of the cells in fermentation broth indicating periplasmic leakage to extracellular space. When other domains from Staphylococcus aureus protein A were tested, it was seen that the E-domain had no measurable protein expression, which was unexpected given that it is the first domain after the signal peptide in native Staphylococcus aureus protein A. However, one important difference is that the first AA after cleave site have been changed from AQHDEA to AQGTVDEA for cloning purposes, and it might be this substitution/insert that affects the signal peptide function negatively. Another difference is that the signal peptide has been substituted from native SpA to OmpA, this in combination with N-terminal start might not be optimal. When adding 8AA DsbA N-terminus to the E-domain in combination with the OmpA signal peptide, protein transport and expression function properly. Moreover, the A and D domains that are very closely related in the N-terminal proximal structural unit express poorly and are not improved by additional AA, whereas the B and C-domains, identical in this unit, express well with the AQGT start, and expression increases additionally when 8AA DsbA are added in the N-terminus. Also, Zvar as monomer and tetramer increase expression with additional AA. However, the most dramatical increase is seen in Zvar2 monomer where protein expression goes from 0.68 g/L to 7.28 g/L.
Without being bound by theory, a hypothesis of the difference between Zvar and Zvar2 is that Zvar2 has two mutations in the first alpha helix in position Q9 and N11, which is estimated to increase alpha-helix structure in the start of the protein, as calculated with algorithms such as GORIV, available on-line at https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html
(PRABI, Rhône-Alpes Bioinformatics Center), thus increasing steric hindrance of the signal peptidase to cleave the signal peptide of the nascent protein.
The N-terminal inserts were each designed to investigate the effect of the insert sequence on one or more of the following parameters: protein yield, signal peptide cleavage and alkaline stability. The designs can be divided into four separate categories as follows:
The aim of category 1 was mainly to investigate the effect of the insert length on signal peptide cleavage and protein yield, and did not explicitly address the suspected truncation site at alkaline conditions. Categories 2 and 3 were mostly aimed at addressing the alkaline stability issue, although they also provide some more data for the other two issues. Category 4 was a more open category, providing a wider set of sequences to provide data in all three areas of interest. The nucleotide sequences were codon optimized for E. coli, but with some degeneracy added in the more repetitive sequences. Inserts belonging to category 1 and 4 were designed to be complementary to the digestion sites of FspI in the 5′ end, and KpnI in the 3′ end. Since the glycine-threonine truncation site was a part of the KpnI restriction site, inserts belonging to categories 2 and 3 were designed to instead be complementary to the adjacent AccI restriction site in the 3′ end, while retaining the 5′ complementarity to FspI. The amino acid sequences of all inserts are shown in table 6. A schematic illustration of the general construct is shown in
AQGT
AQYEDGKQYTGT
AQYEDGKQYGT
AQYEDGKQGT
AQYEDGKGT
AQYEDGGT
AQYEDGKQYTTLEKGT
AQYEDGKQYTTLEKPVAGGT
AQYEDGKQYT
AQYEDGKQ
AQYEDGKQYTET
AQYEDGKQYTDT
AQYEDGKQYTAT
AQYEDGKQYEDT
AQRRRRRRRRGT
AQKKKKKKKKGT
AQHHHHHHHHGT
AQHHHHHHGT
AQHHHHGT
AQWSHPQFEKGT
AQHDEAQQEAGT
The constructs were assembled by ligating different hybridized oligonucleotides into the plasmid of either pGE0002 or pGE0180, digested with the relevant restriction enzymes to remove the original N-terminal insert. After ligation, the plasmids were transformed into E. coli K12-017 cells. Positive colonies were screened using colony PCR and electrophoresis.
Two or three clones from each construct were chosen and sent to GATC biotech (Cologne, Germany) for sequence verification.
One sequence-verified clone for each construct was selected for protein expression in shake flask cultivation.
The protein solutions for each construct yielded from the shake flask cultivation were purified using IgG Sepharose 6FF (GE Healthcare, Uppsala, Sweden). The purified protein samples were investigated for signal peptide cleavage using LC/MS analysis (Waters, PA, USA). A protein with a correctly cleaved signal peptide only has one main mass (example Total Ion Count (TIC) shown in
In many cases, the peaks were hard to interpret conclusively due to the purity of the samples and insufficient separation during the liquid chromatography step. However, it could be seen that the following constructs had a correct signal peptide cleavage: pGE0180, DsbA7, DsbA6, DsbA8_noGT, DsbA8_DT, DsbA8_ET, DsbA8_AT, DsbA7_EDT, DsbA12, DsbA16, H8, H6 and SPA.
After the signal peptide cleavage investigation, the protein solutions of constructs with correct signal peptide cleavage were further purified using a Capto Q ImpRes anion exchange column (GE Healthcare, Uppsala, Sweden). Purified samples were subjected to treatment with 1 M sodium hydroxide (NaOH) for 0, 4 and 24 hours. Vivaspin columns (GE Healthcare, Uppsala, Sweden) were used to separate the main protein from any small peptides that might have been truncated. Both the peptide samples and the main proteins were investigated using LC/MS (Waters, PA, USA).
Peptides appeared in the mass spectra with +2 charge, that is, at a mass over charge (m/z)-value corresponding to half their molecular weight. In the cases investigated here, the peptides all had a molecular weight between about 800 Da and 1600 Da. As such, the relevant cluster of peaks appeared between 400 and 800 m/z, as can be seen for pGE0180 to the left in
If peptides were present, the MassLynx search function was used on the TIC to extract the peaks of different peptide masses as extracted ion chromatograms (XIC). The XIC peaks were integrated and the integrated areas were then used to compare the amounts of different peptides in the samples.
The second analysis with spin columns washed with isopropanol yielded mostly similar results to the initial run, although the peak areas were reduced. The notable exception is DsbA6, which was revealed to have some cleavage. This was not immediately detectable from the peptide analysis, but became apparent when investigating the retentate, where DsbA6 showed a clear truncated peak in much the same manner as pGE0180, while DsbA8_noGT and DsbA_DT showed no such peak.
Interesting constructs from the shake-flask study and subsequent analyses of the purified protein were selected for a larger scale cultivation in a multifermenter (Belach Bioteknik, Skogås, Sweden). At the end of the fermentation, the OD000 nm and protein concentration were measured (see
Since the different proteins have somewhat varying extinction coefficients, it stands to reason that they might vary in absorbance at 237 nm as well, which is where the protein concentration was estimated. Therefore, the ratio between the pGE0180 extinction coefficient (0.294) and the calculated extinction coefficient for each other protein was used to estimate a more “true” value.
The fed-batch cultures of constructs DsbA8_noGT and DsbA8_DT were heat treated to extract periplasmic protein, and centrifuged and diafiltered to remove cellular debris. The filtrate was conditioned to remove some contaminants via precipitation, and prepare the solution for purification with Capto S ImpAct (GE Healthcare, Uppsala, Sweden) using a pH gradient. The elute was reduced to remove dimers, and desalted to remove the DTT and get the correct buffer for the anion exchange purification, where a salt gradient was used. The final purified solution was concentrated via membrane ultrafiltration to reach the concentration needed for immobilization at the desired ligand densities. The end product was analysed with SEC for purity and LC/MS for molecular weight, with satisfactory results.
The purified proteins (>90% purity) were coupled to highly cross-linked agarose beads, aiming for ligand densities similar to those in a previous experiment where Zvar2 was immobilized on a base matrix of similar properties and analyzed for dynamic binding capacity. The coupled gels were packed in Tricorn 5/100 columns (GE Healthcare, Uppsala, Sweden). The packing of the gel bed was evaluated through the asymmetry of an acetone peak. The packed columns were used to measure the dynamic binding capacity of the gels using IgG. One measurement per construct was made. The results are summarized in table 7.
The N-terminal spacer AQYEDGKQYTGT and a C-terminal cysteine was introduced in a number of monomeric and dimeric further mutants of Zvar2 (as listed below), using the OmpA signal peptide. The structure of the mature protein was thus: AQYEDGKQYTGT-IgG-binding polypeptide-C.
Plasmids were transformed into chemically competent E. coli K12-017 cells. The cells were thawed on ice for 30 min, 50 μl competent cells were added to 20 ng plasmids. Cells were incubated on ice for 20 min followed by heat shock at 42° C. for 1 min, followed by incubation on ice 5 min and addition of 400 μl SOC-medium (NEB, MA, USA). Transformation reaction was incubated in 37° C. in a rotary shake incubator for 60 min, 200 μl of each reaction was spread on Luria agar plates containing appropriate selective antibiotics.
E. coli strain K12-017 transformed with the recombinant plasmids (Table 8) were cultured in 37° C. in 100 ml Terrific Broth (TB) medium (containing 12 g tryptone, 24 g yeast extract, 5 g glycerol (85%), 2.31 g KH2PO4, 12.54 g K2HPO4 and 50 mg of kanamycin sulphate per liter). Cultures were induced when OD600 nm reached 1, using 1 mM IPTG (Sigma Aldrich, MO, USA). Cultures were further incubated in 30° C. 17-20 h. Culture solution was subjected to low-speed centrifugation (at 4,000 rpm) for 20 min in a swing out rotor to collect wet cell pellet. The bacterial cell pellets were suspended in 10 ml of a 25 mM phosphate buffer solution (pH 7.4), and the cells were lysed by heat treatment at 85° C. for 15 min in a heat block. Followed by a high-speed centrifugation (at 10 000×g) for 10 min to separate the supernatant. Then supernatants were filtered through 0.2 μm syringe filter to remove any residual particles before it was applied to an XK 16-6 IgG Sepharose 6FF (GE Healthcare Bio-Sciences, Uppsala, Sweden). The column was equilibrated with loading buffer (25 mM phosphate pH7, 250 mM NaCl) followed by loading of sample. After loading the column was washed with 5 column volumes (CV) of loading buffer and one CV of low salt wash buffer (50 mM acetate pH6), followed by elution with 50 mM acetic acid, pH2.8. Absorbance at 237 nm was measured in-line using an ÄKTA explorer 100 (GE Healthcare Bio-Sciences, Uppsala, Sweden) and peak integration of the eluted peak was performed in the system software (Unicorn 5.1).
The IgG-binding fraction was eluted and collected in a pool and the volumes and protein concentrations of the pools were noted—see table 8. The amount of IgG-binding protein expressed may in several cases be higher than the amount recovered, due to loss of protein secreted into the culture medium and/or to overloading of the IgG column.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. Any patents or patent applications mentioned in the text are hereby incorporated by reference in their entireties, as if they were individually incorporated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1708277.7 | May 2017 | GB | national |
This application claims the priority benefit of PCT/EP2018/063435 filed on May 23, 2018, which claims priority benefit of Great Britain Application No. 1708277.7 filed on May 24, 2017, the entire contents of which are hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/063435 | 5/23/2018 | WO | 00 |
