Patent Grant
11168116
The present invention is filed under 35 U.S.C. § 371 as the U.S. national phase of International Application No. PCT/EP2017/059715, filed Apr. 25, 2017, which designated the U.S. and claims the right of priority of European patent application 16 166 773.8 filed with the European Patent Office on Apr. 25, 2016 purposes. The entire disclosures of the above-identified priority applications are hereby fully incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 3, 2018, is named SCH-4600-US_SeqListing.txt and is 27 kilobytes in size.
The present invention relates to novel stabilized streptavidin muteins, methods of producing such muteins by means of recombinant DNA technology as well as the use of these streptavidin muteins for the isolation, purification and determination of biological substances such as recombinant proteins under denaturing conditions. The invention thus also relates to solid phases having immobilized thereon the streptavidin muteins of the invention as well as to nucleic acid molecules encoding the muteins of the invention. The invention further relates to kits, suitable for detection, isolation or purification of proteins, containing muteins of the invention, for example, immobilized on a solid phase.
High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm. However, when a secretion vector is used, they can also form in the periplasmic space. Inclusion bodies are however, not restricted to E. coli; they can also form in yeast, mammalian, and insect cells, for example. In case of the formation of inclusion bodies, fusion proteins carrying polyhistidine affinity tags can be purified by the use of denaturing conditions such as 6 M guanidinium hydrochloride (also known as guanidine hydrochloride) or 8 M urea during the purification process (see, for example, Palmer & Wingfield “Preparation and Extraction of Insoluble Inclusion-Body Proteins from Escherichia coli”, Curr Protoc Protein Sci. 2004 November; CHAPTER: Unit-6.3. doi:10.1002/0471140864.ps0603s38 or Bornholst & Falke “Purification of Proteins Using Polyhistidine Affinity Tags”, Methods Enzymol. 2000; 326: 245-254). Interaction of the affinity chromatography resin with the polyhistidine tag such as a hexahistidine (His6) tag does not require a specific conformation of the peptide tag, which makes effective purification with the use of denaturing conditions possible. For this reason, the His6 tag has been seen by Schmidt and Skerra as the better alternative for isolation of recombinant proteins from solubilized inclusion bodies in the presence of, for example, 6 M guanidine hydrochloride than the STREP-TAG® II (IBA GmbH) affinity tag (Schmidt & Skerra, “The strep-tag system for one-step purification and high-affinity detection or capturing of proteins.” Nature Protocols 2 (2007), 1528-1535). When using a streptavidin binding affinity tag such as the STREP-TAG® affinity tag, it has thus been recommended to fuse both such a streptavidin binding affinity tag and a His6 tag to a recombinant protein, to be able to purify the recombinant protein from inclusion bodies first under denaturing conditions by means of the His6 tag and, after refolding of the protein, follow up with a second affinity purification under physiological conditions by means of the streptaividin binding peptide. So doing should provide the ability of obtaining highly pure and homogenous protein preparations from inclusion bodies.
Apart from the limitation of not being suitable for protein purification under denaturing conditions, the STREP-TAG®II affinity tag (Schmidt & Skerra, Nature Protocols 2 (2007), supra or U.S. Pat. No. 5,506,121) that has the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, SEQ ID NO: 3) has been widely recognized as a very versatile affinity tag. The STREP-TAG®II affinity tag is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The currently most widely and most efficient streptavidin based receptor for the STREP-TAG®II affinity tag are streptavidin muteins with improved binding affinity that are named STREP-TACTIN® (IBA GmbH) (Voss & Skerra, Protein Engineering 10 (1997), 975-982; U.S. Pat. No. 6,103,493 or European Patent 0 835 934). The STREP-TAG®II affinity tag binds to the biotin binding pocket of streptavidin (and these streptavidin muteins) enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the affinity resins. The system consisting of the STREP-TAG®II affinity tag and the STREP-TACTIN® streptavidin mutein has provided powerful applications in the last over 15 years for purification, detection and assay of recombinant proteins (reviewed in Schmidt & Skerra, Nature Protocols 2, supra) and even of cells (Knabel et al., “Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer”, Nature Medicine 8 (2002), 631-637).
For this reason, it would be desirable to have at hand a tool and a system that allows purification of fusion proteins under denaturing conditions by means of affinity chromatography using streptavidin binding affinity peptides. It is therefore an object of the present invention to provide a tool and a system that allows purification of fusion proteins under denaturing conditions by means of streptavidin based affinity chromatography.
In a first aspect the invention provides a mutein, selected from muteins of streptavidin, wherein the mutein
(a) has a Cys residue at sequence position 127 with reference to the amino acid sequence of wild-type streptavidin as set forth in SEQ ID NO: 1 and
(b) comprises at least one mutation in the region of the amino acid positions 115 to 121 with reference to the amino acid sequence of wild type streptavidin as set forth in SEQ ID NO: 1.
In a second aspect, the invention provides a nucleic acid molecule, comprising a sequence coding for a streptavidin mutein of the invention, i.e. a streptavidin mutein that
(a) has a Cys residue at sequence position 127 with reference to the amino acid sequence of wild-type streptavidin as set forth in SEQ ID NO: 1 and
(b) comprises at least one mutation in the region of the amino acid positions 115 to 121 with reference to the amino acid sequence of wild type streptavidin as set forth in SEQ ID NO: 1.
The nucleic acid of this second aspect of the invention might be a vector comprising at least one copy of such nucleic acid molecule in an operatively functionally environment.
In a third aspect, the invention provides a cell that is transformed or transfected with a nucleic acid or a vector according to the second aspect.
The invention also provides a method of producing a streptavidin mutein according to the first aspect, comprising:
(a) transforming a suitable host cell with a vector which contains a nucleic acid coding for the streptavidin mutein,
(b) culturing the host cell under conditions in which an expression of the streptavidin mutein takes place,
(c) isolating the mutein.
The invention also provides a method of isolating, purifying or determining under denaturing conditions a protein that is fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 8) in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys or b) with a peptide sequence that comprises a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence -His-Pro-Baa-, where Baa is glutamine, asparagine or methionine, and wherein the other binding module has the sequence -Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- (SEQ ID NO: 9) where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg, comprising contacting a sample containing said protein with a streptavidin mutein as described herein, under suitable conditions to bind the peptide sequence to the streptavidin mutein, and separating the resulting complex from said sample.
The invention also provides a method of immobilizing under denaturing conditions a protein which is fused with a) a peptide sequence of formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 8) in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys or b) with a peptide sequence that comprises a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence -His-Pro-Baa-, where Baa is glutamine, asparagine or methionine, and wherein the other binding module has the sequence -Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa-(SEQ ID NO: 9) where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg, comprising contacting said protein to a solid phase which carries a streptavidin mutein according to the first aspect, under conditions for immobilizing said protein.
The invention also provides a solid phase having immobilized thereon comprising a streptavidin mutein of the invention. Such a solid phase may be an affinity chromatography matrix.
The invention further provides a reagent kit comprising a streptavidin mutein as described herein or a solid phase having immobilized thereon a streptavidin as described herein. Such a kit may further comprise at least one reagent selected from the group consisting of a conventional buffer, an auxiliary substance and an additive. The invention also provides a streptavidin mutein as described herein immobilized on a solid support such as a chromatography resin, an ELISA plate or a chip for surface plasmon resonance (SPR) measurements.
These aspects of the invention will be more fully understood in view of the following description, drawings and non-limiting examples.
It has been found in the present invention that the replacement of the histidine residue that is present at sequence position 127 of wild-type streptavidin provide muteins that allow the purification of fusion proteins by means of streptavidin binding peptides under denaturing conditions. This finding is in particular surprising since the replacement of His 127 by a cystein residue in wild-type streptavidin has lead to an only insignificant increase in stability of streptavidin in 7 M guanidine hydrochloride (Reznik et al., “Streptavidins with intersubunit crosslinks have enhanced stability”, Nat Biotechnol. 1996 August; 14(8):1007-11.). It could have thus not been expected that this amino acid exchange will enable streptavidin muteins that reversibly bind streptavidin binding peptides to be used for protein purification by affinity chromatography under denaturing conditions. This holds in particular true, since in accordance with the recommendation of Schmidt & Skerra, Nature Protocols 2, supra to use His6 tags/immobilized metal chelate affinity chromatography (IMAC) for protein purification under denaturing conditions, it has also been found in the present invention that the streptavidin mutein “1” of U.S. Pat. No. 6,103,493 (that has the amino sequence Val44-Thr45-Ala46-Arg47 (SEQ ID NO: 10) at amino acid positions 44 to 47 of the wt-streptavidin sequence, while being significantly more stable under physiological conditions than muteins of the present invention, is not suitable for protein purification under denaturing conditions (cf. Examples 4 and 5 of the present application). It has rather been found here that streptavidin muteins which comprise at least one mutation in the region of the amino acid positions 115 to 121 with reference to the amino acid sequence of wild type streptavidin as set forth in SEQ ID NO: 1 profit from the replacement of His 127 by Cys. Such streptavidin muteins comprising at least one mutation in the region of the segment of amino acid positions 115 to 121 have been described in International patent application WO 20140/76277. Accordingly, the present invention has the advantage that it abolishes the need to fuse both a streptavidin binding affinity tag and a His6 tag to a recombinant protein in case the protein will form inclusion bodies during its production.
As an added advantage, the muteins of the present invention are stable under denaturing conditions such as exposure to 4 M guanidinium hydrochloride as well as to exposure to strong bases such as NaOH (see Example 4). Accordingly, the muteins of the present invention can also be advantageously used in applications such as biosensors for e.g. ForteBio's OCTET® or GE's BIACORE® family of instruments that provide label-free, real-time measurements for the analysis of protein:protein, protein:peptide, and protein:small molecule interactions or chips for high throughput analysis of a multitude of analytes bound to its surface or beads such as magnetic beads or ALPHASCREEN® (PerkinElmer Life Sciences, Inc.) beads or LUMINEX® (Luminex Corporation) beads for e.g. protein:protein interaction analysis. In all such examples, the coating of the respective solid phase with a streptavidin mutein described provides a generic platform for the simple, reproducible, mild and stable immobilization of an arbitrary protein to said solid phase. At the same time, the stability of the streptavidin muteins to high concentrations of alkali (hydroxide ions) allow regeneration of the solid phase for further use.
The streptavidin muteins of the present invention may correspond to the amino acid sequence of wt-streptavidin outside the region of the amino acid positions 115-121 and apart from the presence of the cysteine residue at sequence position 127. On the other hand, the amino acid sequence according to the invention can also be different to the wt-streptavidin sequence outside the region of the amino acids 115 to 121. Such variants of the streptavidin sequence include naturally occurring as well as artificially produced variants and the modifications are understood as substitution including those disclosed by U.S. Pat. No. 6,103,493, those containing a disulfide bond, insertions, deletions of amino acid residues as well as N- or/and C-terminal deletions or additions.
Preferred streptavidin muteins according to the invention comprise at least one mutation at the amino acid positions 117, 120 and 121 and/or comprise a deletion of amino acids 118 and 119 and substitution of at least of amino acid position 121.
It is noted in this context that deletion of amino acids in loops may not only be tolerated but may even be favorable for purification of fusion proteins carrying streptavidin binding peptides. Accordingly, streptavidin muteins containing additional deletions, substitutions or additions outside the preferred changes at the positions specified within the present invention also fall within the scope of the present invention.
Thus, the term “mutation” as used herein also includes a deletion of an amino acid residue (however, the Cys residue at position 127 of the wild-type sequence of streptavidin will always be present in a mutein of the invention). In this respect, it is however noted that a streptavidin mutein in which the entire loop of the amino acids 114 to 121 of streptavidin (TTEANAWK) or the loop region of amino acids 115 to 121 of streptavidin (TEANAWK) is deleted is not encompassed in the present invention. Rather in the muteins of the invention that contain one or more mutations within the segment of amino acids 115 to 121, at least one amino acid is present at one of the position 115 to 121. In some of these embodiments, an amino acid is present at positions 117, 118, 119, 120 and 121 while the amino acid at position 118 and/or 119 is deleted. Thus, in such muteins the segment formed by sequence positions 115 to 121 is shorted by either one or two amino acids. In line with the above disclosure that muteins in which the entire segment of amino acids 115 to 121 is deleted are not being used in the present invention, the muteins of streptavidin as described in Fletcher et al, Journal of Biotechnology 2003, are not encompassed by the present invention. This means that the following streptavidin muteins are excluded herein: 1. A mutein in which the wild type amino acid residues Thr-Thr-Glu-Asp-Asn-Ala-Trp-Lys (TTEANAWK, SEQ ID NO: 17) at sequence positions 114 to 121 are deleted. This mutein is designated SAPV in Fletcher et al. 2. The two muteins designated SAPV-Alb5 and SAPV-84 in Fletcher et al in which the deleted nine amino acid residues Thr-Thr-Glu-Asp-Asn-Ala-Trp-Lys (SEQ ID NO: 17) are replaced by the amino acid sequence HPYFYAPELLFFAK (SEQ ID NO: 18) or EGGKETLTPSELRDLV (SEQ ID NO: 19).
Preferred streptavidin muteins of the invention are derived from streptavidin variants which are shortened at the N- or/and the C-terminus. The minimal streptavidins which are N- and C-terminally shortened known from the state of the art are particularly preferred. A preferred polypeptide according to the present invention comprises outside of the mutagenized region the amino acid sequence of a minimal streptavidin which begins N-terminally in the region of the amino acid positions 10 to 16 and terminates C-terminally in the region of the amino acid positions 133 to 142. Such a streptavidin mutein polypeptide corresponds preferably to a minimal streptavidin outside of the mutation region which comprises an amino acid sequence from position Ala13 to Ser139 and optionally has an N-terminal methionine residue instead of Ala13. In this application the numbering of amino acid positions refers throughout to the numbering of mature wt-streptavidin (Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882, cf. also
Streptavidin muteins carrying one or more mutations in the region of the amino acid positions 115 to 121 according to the invention that are especially preferred may be characterized in different subclasses.
First, amino acids that are found at positions 117, 120, and 121 can be regarded separately depending on the presence or non-presence of the deletion of the two amino acids at positions 118 and 119. Amino acids that are found at positions 117, 120, and 121 in these two different cases and which, therefore, may contribute to binding of streptavidin binding peptides in each case, are summarized in
Muteins without deletion may be characterized as follows: They carry at position 117 most preferably a large hydrophobic residue like Trp, Tyr or Phe or a charged residue like Glu, Asp or Arg or a hydrophilic residue like Asn or Gln, or, less preferred, the hydrophobic residues Leu, Met or Ala, or the polar residues Thr, Ser or His, in combination with i) a small residue like Ser or Ala or, most preferably, Gly at position 120 which is then combined with a hydrophobic residue at position 121, most preferably with a bulky hydrophobic residue like Trp, Tyr or Phe or in combination with ii) a hydrophobic residue at position 120 which is Leu, Ile, Met, or Val or, more preferably, Tyr or Phe, which is then combined with a small residue like Gly, Ala, or Ser, or with Gin, or with a hydrophobic residue like Leu, Val, Ile, Trp, Tyr, Phe, or Met at position 121.
Muteins with deleted amino acid positions 118 and 119 may be characterized as follows: Position 117 may be any amino acid with bulky hydrophobic residues like Phe, Tyr or Trp being less preferred and position 120 is then most preferably a Trp and less preferably Val and position 121 is also a hydrophobic amino acid, most preferably Met, Leu, Tyr or Phe, or position 121 is a small hydrophilic residue, most preferably Ser or Thr, or position 121 is Arg. An overview of illustrative specific mutein sequences is also shown in Tables 1-5 given below.
In some embodiments, a streptavidin mutein of the invention has a first sequence motif for positions 117, 120, and 121 at the sequence 117 to 121 of the wild type sequence that comprises a Gly residue at sequence position 120 (Gly120) as most important feature. Such a motif carries preferably a Phe or a Tyr, or less preferably a Met residue at sequence position 121 and a Glu, an Asp, an Arg, a His, a Leu, a Met, an Asn, a Gln, a Thr or a Ser residue at sequence position 117, position 117 thus being more variable in this motif. Such a mutein may have the wild type streptavidin amino acid Asn118 and/or Ala119 at sequence positions 118 and 119 (see also
In a second sequence motif for positions 117 to 121 a streptavidin mutein disclosed here comprises a hydrophobic or aromatic amino acid residue at sequence position 120 (see also
In a third sequence motif for streptavidin muteins disclosed here having mutations at sequence positions 117 to 121 the residues at sequence positions 118 and 119 are deleted (see also
However, by no means should the consensus sequences 1 to 3 explained above and shown in
Tables 1 to 5: Examples of mutations of muteins of the invention at sequence positions 117 to 121 of the amino acid sequence of wt-streptavidin
Tyr
117Asn118Ala119Phe120Met121
Tyr
117Asn118Ala119Tyr120Ala121
Ala
117---118---119Trp120Tyr121
Asp
117Asn118Ala119Gly120Phe121
Arg
117Asn118Ala119Gly120Phe121
Gln
117Asn118Ala119Gly120Phe121
Phe
117Asn118Ala119Ser120Trp121
Asp
117Asn118Ala119Val120Met121
Ala
117---118---119Trp120Met121
Glu
117Asn118Ala119Gly120Phe121
Tyr
117Asn118Ala119Tyr120Ser121
Phe
117Asn118Ala119Tyr120Gly121
Tyr
117Asn118Ala119Gly120Phe121
Arg
117Asn118Ala119Tyr120Ala121
Trp
117Asn118Ala119Tyr120Gly121
Leu
117Asn118Ala119Gly120Phe121
His
117Asn118Ala119Gly120Tyr121
Met
117Asn118Ala119Gly120Phe121
Arg
117Asn118Ala119Gly120Tyr121
Glu
117Asn118Ala119Gly120Trp121
His
117Asn118Ala119Gly120Phe121
Ser
117Asn118Ala119Gly120Phe121
Thr
117Asn118Ala119Gly120Phe121
Asn
117Asn118Ala119Gly120Phe121
Glu
117Asn118Ala119Gly120Met121
Trp
117Asn118Ala119Cys120Cys121
Met
117Asn118Ala119Phe120Val121
Ala
117Asn118Ala119Asp120Trp121
Ser
117Asn118Ala119Met120Met121
Arg
117Asn118Ala119Val120Val121
Ser
117Asn118Ala119Ser120Phe121
Ala
117---118---119Trp120Asp121
Arg
117Asn118Ala119Arg120Ala121
Ser
117Asn118Ala119Ala120Phe121
Gly
117Asn118Ala119Met120Met121
Glu
117Asn118Ala119Gly120Phe121
Asp
117Asn118Ala119Gly120Tyr121
Glu
117Asn118Ala119Gly120Tyr121
Arg
117Asn118Ala119Met120Met121
Arg
117Asn118Ala119Gly120Phe121
Ala
117Asn118Ala119Pro120Ala121
Ala
117Asn118Ala119Met120Val121
Gln
117Asn118Ala119Ser120Ala121
Ala
117Asn118Ala119Gly120Phe121
Gln
117Asn118Ala119Met120Val121
Asn
117Asn118Ala119Gly120Tyr121
Ala
117Asn118Ala119Ala120Val121
Ser
117Asn118Ala119Met120Ile121
His
117Asn118Ala119Gly120Tyr121
Ser
117Asn118Ala119Met120Ala121
Gln
117Asn118Ala119Val120Ala121
Tyr
117Asn118Ala119Tyr120Met121
Leu
117Asn118Ala119Trp120Gly121
His
117Asn118Ala119Ser120Met121
Tyr
117Asn118Ala119Phe120Leu121
Phe
117Asn118Ala119Phe120Leu121
Tyr
117Asn118Ala119Leu120Trp121
Phe
117Asn118Ala119Tyr120Ile121
Tyr
117Asn118Ala119Tyr120Leu121
Tyr
117Asn118Ala119Tyr120Gln121
Phe
117Asn118Ala119Ile120Trp121
Thr
117---118---119Trp120Leu121
His
117---118---119Trp120Leu121
Ile
117---118---119Trp120Arg121
His
117---118---119Trp120Thr121
Thr
117---118---119Trp120Arg121
Ala
117---118---119Trp120Arg121
Arg
117---118---119Trp120Ser121
Asn
117---118---119Trp120Arg121
Lys
117---118---119Trp120Ser121
Ser
117---118---119Val120Phe121
Lys
117---118---119Trp120Thr121
Ala
117---118---119Trp120Tyr121
His
117---118---119Trp120Met121
His
117---118---119Trp120Tyr121
Glu
117---118---119Trp120Tyr121
Gln
117---118---119Trp120Tyr121
As disclosed by Tables 1 to 5, illustrative streptavidin muteins of the invention comprise or have one of the sequences of any of SEQ ID NOs: 21 to 97 at sequence positions 117 to 121 of the amino acid sequence of wild type streptavidin. These streptavidin muteins can either have the wildtype streptavidin sequence at any of the other sequence positions or the sequence of any known streptavidin muteins, for example, the sequence of the known muteins “1” or “2” that comprise the amino acid sequence Val44-Thr45-Ala46-Arg47 (SEQ ID NO: 10) or Ile44-Gly45-Ala46-Arg47 (SEQ ID NO: 11) at amino acid positions 44 to 47.
In some embodiments, a mutein of the invention comprises at sequence positions 117 to 121 an amino acid sequence of consisting of the group of
Also such a mutein may comprise, for example, either the wild-type streptavidin sequence, the sequence of mutein “1” or of mutein “2” at any other position (apart from the Cys residue at position 127 that is shared by all muteins of the present invention).
In other illustrative embodiments, the streptavidin muteins of the invention may, in addition to the Cys residue at sequence position 127, comprise or consist of the sequence of any of the following muteins that are shown in FIG. 4 of International Patent Application WO 2014/076277, i.e. the following muteins:
m400, m402, m4001, mutein“1”-m36, mutein“1”-m23, mutein“1”-m41, mutein“1”-m4, mutein“1”-m12, mutein“1”-m22, mutein“1”-m31, mutein“1”-m32, mutein“1”-m35, mutein“1”-m38, mutein“1”-m40, mutein“1”-m42, mutein“1”-m45, mutein“1”-m46, mutein“1”-m47, mutein“1”-m7, mutein“1”-m10, mutein“1”-m17, mutein“1”-m21, mutein“1”-m24, mutein“1”-m27, mutein“1”-m28, mutein“1”-m30, mutein“1”-m33, mutein“1”-m1, mutein“1”-m3, mutein“1”-m8, mutein“1”-m15, mutein“1”-m6, mutein“1”-m9, mutein“1”-m20, mutein“1”-m34, mutein“1”-m14, mutein“1”-m18, mutein“1”-m19, m4001-m8, m4001-m21, m4001-m9, m4001-m1, m4001-m2, m4001-m3, m4001-m5, m4001-m13, m4001-m14, m4001-m24, m4001-m4, m4001-m6, m4001-m7, m4001-m10, m4001-m15, m4001-m23, m4001-m17, m4001-m12, m4001-m20, mutein“1”-m101, mutein“1”-m106, mutein“1”-m111, mutein“1”-m100, mutein“1”-m110, mutein“1”-m104, mutein“1”-m108, mutein“1”-m207, mutein“1”-m212, mutein“1”-m202, mutein“1”-m204, mutein“1”-m206, mutein“1”-m208, mutein“1”-m203, mutein“1”-m209, mutein“1”-m200, mutein“1”-m201, mutein“1”-m211, mutein“1”-m300, mutein“1”-m301, mutein“1”-m302, mutein“1”-m303, mutein“1”-m304 and m1-9.
Turning now to the practical uses of the muteins of the invention (for example, for affinity chromatography under denaturing conditions), it may be desirable to employ a ligand which, due to a higher binding affinity or/and due to being present at higher concentrations than the streptavidin binding peptide, can detach the binding of the streptavidin binding peptide (for example, a Strep-Tag® (II) peptide or a Di-tag peptide as described herein) from the streptavidin mutein according to the invention. Usually this ligand acts as competitor of the Strep-Tag® peptide. This (competitive) ligand is usually present in free form, meaning not fused to any protein or other molecule. In this manner it is possible to release (generally by competitive elution) bound streptavidin binding peptide ligands or proteins to which a streptavidin binding peptide such as the Strep-Tag® (II) peptide or a Di-tag peptide is fused under the chosen, for example, denaturing elution conditions. This is, e.g., important for elution of the bound fusion protein from the streptavidin mutein affinity column or to reverse the binding of multimeric low affinity fusion proteins carrying a streptavidin binding peptide that are multimerized via a backbone of streptavidin mutein (multimers) of the present invention. Hence, in this aspect, the present invention concerns those streptavidin muteins whose binding affinity for peptide ligands is such that they can be competitively eluted by other streptavidin ligands like, e.g., biotin, iminobiotin, lipoic acid, thiobiotin, desthiobiotin, diaminobiotin, HABA (hydroxyazobenzene-benzoic acid) or/and dimethyl-HABA. The use of coloured substances such as HABA or dimethyl-HABA may have the advantage that the elution from a column can be checked visually. However, irrespective of this, the binding affinity of the streptavidin muteins of the present invention for peptide ligands, particularly for Strep-Tag® II is usually higher than that of wt-streptavidin or than that of the muteins “1” or “2” disclosed in U.S. Pat. No. 6,103,493. Therefore, in some embodiments higher affinity ligands like thiobiotin or biotin are preferred for sharp elution. Alternatively, also isolated peptide ligands binding to the biotin binding pocket, e.g. as described herein, may be used for competitive elution. For the sake of completeness, it is noted that the interaction/binding of a streptavidin binding peptide (that is usually fused or conjugated to a protein of interest) to a streptavidin mutein of the invention may not necessarily be disrupted by competitive elution but also by any other means that is able to disrupt this non-covalent complex. For example, if such fusion proteins are immobilized on a surface that is coated with a streptavidin mutein of the invention such as a surface plasmon resonsance chip, an ELISA plate or even a chromatography resin, the binding can be disrupted by change of the pH, for example, by addition of a base such as NaOH. Such an approach might even be preferred for the regeneration of a surface plasmon resonsance chip but also a chromatography resin that has a streptavidin mutein of the invention immobilized thereon.
It may be preferable for certain detection methods to use the streptavidin muteins of the present invention in a labeled form. Accordingly, a further subject matter of this invention is a polypeptide according to the invention which is characterized in that it carries at least one label. Suitable labeling groups are known to a person skilled in the art and comprise the usual radiolabels, fluorescent labels, luminescent labels and chromophore labels as well as substances and enzymes which generate a substance that can be determined in a chemical or enzymatic reaction. In this connection, all labels known for wt-streptavidin can also be coupled to the streptavidin muteins according to the present invention.
A further aspect of the present invention concerns a nucleic acid which comprises a sequence coding for a streptavidin mutein of the present invention. Such a nucleic acid is optionally operatively linked to a sequence coding for a signal peptide and, in a particular embodiment, the sequence coding for the signal peptide is the sequence for the OmpA signal peptide. Moreover, it is also possible to use other signal peptides and this may even be preferable especially depending on the expression system or host cell used. A large number of such signal peptides are known in the state of the art and will not be elucidated in detail here. However, cytoplasmic expression is preferred, i.e. with a start methionine instead of the signal sequence (cf. Schmidt & Skerra, J. Chromatogr. A 676 (1994), 337-345).
A further aspect of the present invention concerns a vector which contains at least one copy of an aforementioned nucleic acid in an operatively functional environment. An operatively functional environment is understood as those elements which enable, favor, facilitate or/and increase the expression, i.e. transcription or/and a subsequent processing, of the mRNA. Examples of such elements are promoters, enhancers, transcription initiation sites and termination sites, translation initiation sites, polyA-sites etc.
The vector is selected depending on the intended expression system and for this single copy plasmids, multi-copy plasmids as well as vehicles which facilitate an integration of the nucleic acid into the host genome come into consideration. A large number of suitable vectors are known from the state of the art and will not be described in detail here. They optionally contain standard elements used for vectors such as resistances, selection markers or/and elements which for example enable an amplification of the nucleic acid or the induction of expression.
A further aspect of the present invention concerns a cell which is transformed or transfected with such a vector which carries as an insert at least one copy of a nucleic acid sequence coding for a streptavidin mutein according to the invention. The selection of the cell is not particularly critical and in general it is possible to use any cells that are suitable for such purposes. Prokaryotic as well as eukaryotic cells and yeasts come into consideration. For practical reasons prokaryotic cells are generally preferred and in particular E. coli for the expression of an unglycosylated protein as in the present case.
Yet a further aspect of the present invention concerns a process for the production of a streptavidin mutein according to the invention which is characterized by the following steps:
(a) transforming a suitable host cell with a vector which contains a nucleic acid coding for the streptavidin mutein,
(b) culturing the host cell under conditions in which an expression of the streptavidin mutein takes place,
(c) isolating the polypeptide.
With respect to the production process, streptavidin muteins according to the invention may have a toxic effect due to their ability to bind to endogeneous biotin. Hence, when culturing the host cell the conditions should be selected such that the expression product that forms is either transported from the inside of the host cell used, for example, into the periplasm or into the culture medium by means of a suitable signal sequence or it aggregates inside the cell in the form of insoluble inclusion bodies. In the former case the streptavidin mutein according to the invention can be isolated from the periplasmic cell fraction or the cell supernatant whereas in the latter case, step (c) of the process according to the invention comprises the lysis of host cells, the isolation of the streptavidin mutein in the form of inclusion bodies and the renaturation of the streptavidin mutein. In this case E. coli is preferred as the host cell.
The practical applications for the streptavidin muteins or the streptavidin mutein/peptide ligand system according to the invention are essentially the same as those for conventional streptavidin/biotin or streptavidin/peptide ligand systems. However, as explained above, the muteins described here have the advantage that all these practical applications can be carried out in the presence of one or more chaotrope reagents that provide for denaturing conditions. This advantage over the conventional streptavidin/biotin system or over the system as disclosed by U.S. Pat. No. 6,103,493 applies in particular to affinity chromatography and to purification, isolation or determination methods for recombinant proteins.
Accordingly, the invention also concerns the use of a streptavidin mutein according to the invention in a method of isolating, purifying, detecting or immobilizing under denaturing conditions a protein. The protein of interest can be fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 8) in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys. Alternatively, the protein of interest can be fused b) with a peptide sequence that comprises a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence -His-Pro-Baa-, where Baa is glutamine, asparagine or methionine, and wherein the other binding module has the sequence -Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- (SEQ ID NO: 9) where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg. Affinity peptides with such a sequential arrangement of at least two streptavidin binding modules are known from International Patent Application WO 02/077018 or U.S. Pat. No. 7,981,632, for example.
In this method of isolating, purifying or detecting, a liquid containing the protein to be isolated or purified is contacted with the optionally immobilized streptavidin mutein under suitable and at the same time denaturing conditions in order to bind the peptide sequence to the streptavidin mutein. Thereafter, the resulting complex is separated from the liquid and the protein is released from the complex or detected. In some embodiments, the peptide sequence is preferably the one of the STREP-TAG®II affinity tag. In other embodiments, the peptide sequence is preferably the di-tag3 sequence (WSHPQFEKGGGSGGGSGGGSWSHPQFEK; SEQ ID NO: 5), the di-tag2 sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 6) that are described in International Patent Application WO02/077018 or U.S. Pat. No. 7,981,632 or the sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 7, also known as TWIN-STREP-TAG™ (IBA GmbH)). The peptide sequence is preferably fused to the N- or/and C-terminus of the protein. The streptavidin mutein can be bound to a solid phase or can be capable of binding to it.
An advantage of utilizing the streptavidin mutein/peptide ligand system according to the invention in an isolation or purification method is that (apart from the denaturing conditions) very mild conditions can be used to elute the fusion protein carrying the peptide ligand. Hence it is possible to incubate a solid phase coupled to the streptavidin mutein, such as for example an affinity chromatography column to which the fusion protein has been adsorbed, with an adequate concentration of a ligand selected from biotin and derivatives thereof in order to release the fusion protein from the complex again. In this connection, the use of biotin has proven to be particularly advantageous.
The streptavidin muteins according to the invention can be used in detection methods in an essentially similar manner to the corresponding methods that are known for conventional streptavidin. A further application is the qualitative or quantitative determination under denaturing conditions of a protein of interest which may be fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys. Alternatively, the protein of interest may be fused b) with a peptide sequence that comprises a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence -His-Pro-Baa-, where Baa is glutamine, asparagine or methionine, and wherein the other binding module has the sequence -Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- (SEQ ID NO: 9) where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg. In this method the protein to be determined is contacted under denaturing but otherwise suitable conditions with a labeled streptavidin mutein in order to bind the peptide sequence to the streptavidin mutein, is washed and the label is determined. Such a determination method can, for example, be carried out qualitatively to detect proteins in Western blots or quantitatively as in an ELISA. Suitable labels are all known radioactive and non-radio-active labeling groups e.g. luminescent groups, enzymes, metals, metal complexes etc. The streptavidin can be directly labeled e.g. by covalent coupling. However, indirect labels such as labeled anti-streptavidin antibodies or biotinylated enzymes, etc. can also be used.
A further advantageous aspect of the invention is the use of the streptavidin muteins according to the invention to immobilize under denaturing conditions a protein of interest which is fused with a) a peptide sequence Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys. Alternatively, the protein of interest is fused b) with a peptide sequence that comprises a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence -His-Pro-Baa-, where Baa is glutamine, asparagine or methionine, and wherein the other binding module has the sequence -Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- (SEQ ID NO: 9) where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg. In one embodiment, the peptide sequence is the sequence (WSHPQFEKGGGSGGGSGGGSWSHPQFEK; SEQ ID NO: 5). In another embodiment, the peptide sequence is the sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 7). This immobilization is preferably carried out on solid phases coated with streptavidin muteins of the invention such as microtitre plates, beads (e.g. made of agarose or other polymers like, e.g., polymethacrylate), microbeads made of organic or paramagnetic materials, nanobeads made of organic or paramagnetic materials or sensor chips such as BIACORE® (Global Life Sciences Solutions USA LLC) chips or other support as e.g. used for lateral flow assays or for making protein arrays
The terms “denaturing conditions”, “denaturation” and “denaturing” are used herein in accordance with their regular meaning in the field of protein chemistry/protein folding. Accordingly, “denaturation” is used to refer to the process in which proteins lose the quaternary structure, tertiary structure and/or secondary structure which is present in their native state, by exposure to and contact with a chaotropic agent. A chaotropic agent (chaotrope) is a compound that is able to disrupt in aqueous solution the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity). This has an effect on the stability of the native state of proteins that are present in the aqueous solution by weakening the hydrophobic effect. A chaotropic agent reduces, for example, the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation. Thus, “denaturing conditions” as used herein refer to such conditions in which a chaotropic agent is present in an amount that is able to induce at least partial loss of the folded structure of a protein of interest. Examples of chaotropic agents that can be used in methods of the invention include, but are not limited to urea, thiourea, guanidine hydrochloride, lithium perchlorate, hydroxide ions, and combinations thereof. Illustrative examples of denaturing conditions include the presence of guanidinium chloride in about 2 M to about 6 M (at high concentrations of guanidinium chloride as about 6 M proteins lose their ordered structure, and they tend to become randomly coiled), the presence of urea in about 1 to about 8 M, or the use of a concentrated solution of lithium perchlorate (for example, about 4.5 M). The presence of hydroxide ions, for example, in concentrations of about 0.05 M, or about 1 M, or about 2 M (which is equivalent to a 1N or 2 N NaOH solution) is a further example of denaturing conditions as used in methods of the present invention.
In this context, it is noted that it is also possible to use the streptavidin muteins according to the invention in a conventional streptavidin/biotin (derivative) system. Put differently, this means the use of the streptavidin muteins according to the invention to determine or isolate substances which carry a group capable of binding to streptavidin. If only a part of the wt-streptavidin is replaced by the streptavidin muteins according to the invention, particular effects can be achieved in this connection via the formation of mixed tetramers.
A further aspect of the present invention relates to a solid phase having immobilized thereon a streptavidin mutein as described herein. Illustrative examples of suitable solid phases include a cellulose membrane, a plastic membrane, a biosensor, a lateral flow device or an affinity chromatography matrix. Examples of affinity chromatography matrices include, to mention only a few, a polysaccharide gel, a polyacrylamide gel, an agarose gel, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide) silica, poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of an acrylate or an acrylamide and a diol, a co-polymer of a polysaccharide and N,N′-methylenebisacrylamide and a combination of any two or more thereof. Further examples of well-known affinity chromatography matrices (or stationary phases) that are also suitable in the present invention, include derivatized silica or a crosslinked gel. A crosslinked gel (which is typically manufactured in a bead form) may be based on a natural polymer, i.e. on a polymer class that occurs in nature. For example, a natural polymer on which a chromatography stationary phase is based is a polysaccharide. A respective polysaccharide is generally crosslinked. An example of a polysaccharide matrix is an agarose gel (for example, SUPERFLOW™ (Sterogene Bioseparations) agarose or a SEPHAROSE® (GE Healthcare) material such as SUPERFLOW™ SEPHAROSE® resin that are commercially available in different bead and pore sizes) or a gel of crosslinked dextran(s). A further illustrative example is a particulate cross-linked agarose matrix, to which dextran is covalently bonded, that is commercially available (in various bead sizes and with various pore sizes) as SEPHADEX® resin or SUPERDEX® resin, both available from GE Healthcare. Another illustrative example of such a chromatography material is SEPHACRYL® resin which is also available in different bead and pore sizes from GE Healthcare.
Yet a further aspect of the invention concerns a reagent kit that contains a streptavidin mutein according to the invention or a solid phase having immobilized thereon a streptavidin mutein as described herein. The kit may optionally contain standard buffer and auxiliary substances and additives. Such a reagent kit is in particular intended to be used in one of the isolation, purification, assay or determination methods described above. However, the kit is also suitable for other methods in which the conventional streptavidin/biotin system is used e.g. for nucleic acid hybridization assays or immunoassays. The reagent kit can contain the streptavidin mutein according to the invention as free, non-modified protein or/and in a solid phase-bound or/and labeled form.
The invention is further illustrated by the following tables, figures and examples.
General Methods
DNA manipulations were carried out by conventional genetic engineering methods (see e.g. Sambrook et al., Molecular Cloning. A Laboratory Manual (1989), Cold Spring Harbor Press), E. coli K12 TOP10 (Life Technologies) for cloning and E. coli BL21 for expression of the feline immunodeficiency virus Gag protein (FIV GAG) fused to the streptavidin peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 7) under formation of inclusion bodies. Cytosolic expression of the streptavidin muteins for subsequent protein isolation for coupling to SEPHAROSE® resin was carried out according to Schmidt & Skerra, J. Chromatogr. A 676 (1994), 337-345). Sequencings were carried out according to the standard dideoxy technique by Sequence Laboratories Gottingen GmbH. Primers and oligonucleotides were synthesized using an Applied Biosystems Expedite DNA synthesizer
The expression vectors encoding the muteins m302C and m1-9C were generated by site specific mutagenesis starting from the expression vector of the parental muteins m302 and m1-9, respectively (see International Patent Application WO 2014/076277, Example 8 for a description of the pASK75 based expression vector for the muteins m302 and m1-9). The codon for the mutation His 127->Cys was introduced via the primers that were used for amplification of the expression vector. These two primers having opposite directions for amplification were annealed to the expression vector and then the expression vector was amplified by PCR.
The streptavidin muteins “1” (SEQ ID NO: 12), m1-9 (SEQ ID NO: 15) and m302 (SEQ ID NO: 13) were produced as described, for example, in International Patent Application WO 2014/076277. The known expression system for recombinant minimal streptavidin (Schmidt and Skerra (1994), supra) was also used to produce the streptavidin muteins m1-9C (SEQ ID NO: 16) and m302C (SEQ ID NO: 14) on a preparative scale. For this the major part of the coding region was removed from the vector pSA1 which contains the coding region of wt-streptavidin and the T7 promoter by using the singular SacII and HindIII restriction sites and replaced by the corresponding regions from the mutated pASK-IBA2-SAm1 plasmids. wt-streptavidin and the streptavidin muteins were subsequently expressed in the form of cytoplasmic inclusion bodies, solubilized, renatured and purified by fractional ammonium sulphate precipitation as described by Schmidt and Skerra (1994) supra. The purity of the obtained streptavidin muteins was checked with an Agilent 2100 Bioanalyzer.
In order to determine the stability of the streptavidin muteins SDS-PAGE analysis was carried out. The samples of the muteins for the SDS-PAGE were not boiled prior to loading onto the gel in order to be able to determine the influence of the denaturing effect of the SDS (detergent) on the streptavidin muteins. The SDS-PAGE was conducted under non-reducing conditions and the gel was stained with Coomassie Brilliant Blue.
Notably, as evident from the single prominent band in the lane of mutein “1”, the tetrameric form of this mutein remains intact even in the presence of SDS, indicating that the mutein “1” is very stable under denaturing conditions. In contrast to this, the mutein m1-9 is far less stable, as evident from the band of the monomeric subunits in the SDS-PAGE extended degree of instability. The instability is even much more pronounced for the mutein m302. This mutein completely dissociates into its monomers under the denaturing conditions of the SDS page. As also evident from
The streptavidin muteins m1-9 (SEQ ID NO: 15) and m1-9C (SEQ ID NO: 16) as well as streptavidin mutein “1” (SEQ ID NO: 12) of U.S. Pat. No. 6,103,493 were prepared as described in Example 3 of the present application as well as in Example 9 of International Patent Application WO 2014/076277. Then, the streptavidin muteins were coupled to NHS-activated SEPHAROSE® 4 Fast Flow resin (GE Healthcare) according to the instructions of the manufacturer (cf. Schmidt and Skerra, 1994, supra). Sepharose gel loading (SUPERFLOW™) with the respective streptavidin mutein was determined with a PIERCE™ BCA assay (Thermo Fisher Scientific) according to the instructions of the manufacturer and as described in International Patent Application WO 2014/076277.
In order to examine the behaviour of these two streptavidin muteins and the streptavidin mutein “1” of U.S. Pat. No. 6,103,493 immobilized in this manner in the affinity purification of STREP-TAG® II affinity tag-carrying fusion proteins under denaturing conditions, the recombinant feline immunodeficiency virus Gag protein fused to the streptavidin peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 7, also known under its trademark “TWIN-STREP-TAG™”) was expressed using the expression vector pASG-103 (IBA GmbH, catalogue number 5-4103-001) in E. coli BL 21. Use of this expression vector leads to the formation of inclusion bodies of the FIV GAG protein in the cytosol of E. coli. BL21.
The recombinant protein expression and cell harvest was carried out in accordance with the following protocol. Transformed E. coli BL21 cells were cultured in LB medium containing ampicillin and induced when reaching a sufficient OD. The cell pellet was harvested by centrifugation and resuspended in Buffer W (100 mM Tris-Cl pH8, 150 mM NaCl, 1 mM EDTA). The cells were then disrupted by ultra-sonication in Buffer W followed by centrifugation. The pellet was lysed in Buffer W (at a weight of 100 mg/ml buffer) containing 8 M urea or 6 M guanidine hydrochloride by stirring for 15-60 minutes at room temperature. The sample was adjusted to concentrations of 4 M, 6 M urea, 4 M and 6 M guanidine hydrochloride by dilution with Buffer W, followed by centrifugation. The supernatant was then applied on a 1 ml column of mutein “1” (i.e. a commercially available STREP-TACTIN® column) and of mutant m1-9 and m1-9C, respectively. As mentioned above, in each case, SUPERFLOW® resin was used as affinity chromatography resin to which the respective streptavidin mutein was coupled. The columns had been equilibrated with Buffer W and the appropriate urea or guanidine hydrochloride concentrations of the sample. For experiments with a concentration of 4 M urea and 4 M guanidine, respectively, 2 ml of the sample containing the fusion protein were applied on the column. For experiments with a concentration of 6 M urea and 6 M guanidine, respectively, 3 ml of the sample containing the fusion protein were applied to provide equal amounts of protein. Columns were washed with one column volume (CV) Buffer W+urea/guanidine per wash step until the A280 nm of the flowthrough was below 0.1 or reached a constant signal. The elution was carried out by consecutively applying 0.8, 1.4 and 0.8 CV of Buffer W+50 mM biotin and urea/guanidine to the columns of the employed streptavidin mutein.
The effectiveness of the affinity purification was analysed by SDS-PAGE.
In addition to the SDS-PAGE analysis, it was tested using 1 mM HABA or 10 mM NaOH whether the resins can be appropriately regenerated. When so doing, the following observation was made.
The resin of the STREP-TACTIN® mutein “1” could be successfully regenerated using buffer R (100 mM Tris-Cl pH8, 150 mM NaCl, 1 mM EDTA, 1 mM HABA) after being subjected to purification under denaturing conditions both with urea and guanidine. The resin of the mutein m1-9 could be successfully regenerated using 10 mM NaOH after urea purification but was destroyed in the course of the guanidine purification, while the resin of the mutein m1-9C could be successfully regenerated using 10 mM NaOH after purification using either urea or guanidine as chaotrope. In addition, it was found that resins of the mutein m1-9C can be regenerated using NaOH in concentrations of up to 500 mM while resins of the mutein m1-C were destroyed with NaOH concentrations higher than 100 mM.
The conclusion of these experiments can be summarized as follows. The mutein “1” (also known as STREP-TACTIN®) is not suited for the purification of proteins under denatured conditions. While the mutein m1-9 could be used for purification with 4 M and 6M urea, this mutein is not suited for purification when the denaturing buffer contains guanidine since guanidine destroys the mutein m1-9. As guanidine is a widely used denaturant for purification of proteins from inclusion bodies, the practical use of mutein m1-9 is therefore severly limited. In contrast to this, the mutein m1-9C was successfully used for the purification of the FIV GAG fusion protein when either 4 M or 6 M urea and also 4 M guanidine chloride was used as chaotrope. In addition, the mutein m1-9C was found to be stable against exposure to guanidine and allows the regeneration and reuse of the resin material. Accordingly, in case a protein is to be purified under denatured conditions and a broad range of denaturing conditions including high concentrations of ureas and guanidine is needed, the mutein m1-9C is the molecule of choice. In addition, due to the stability of resins of the mutein m1-9C to NaOH concentrations of up to 500 mM, resins of mutein m1-9C can be advantageously used for affinity purification of proteins in commercial settings that require cleaning-in-place (CIP) protocols for removal of impurifications, as 500 mM NaOH has been found to be a very effective CIP reagent. Thus, due to its high affinity for affinity peptides containing streptavidin binding sequences such as the STREP-TAG® peptide sequence, mutant m1-9C enables now very efficient purification of commercial fusion proteins under physiological and/or denaturing conditions using efficient CIP thereby contributing to improved safety for the use of said fusion proteins.
The melting temperature was determined under physiological conditions for wild-type core streptavidin (SEQ ID NO: 2, available from IBA GmbH under catalogue number 02-20203) streptavidin muteins “1” (SEQ ID NO: 12), m1-9 (SEQ ID NO: 15) and m1-9C (SEQ ID NO: 16) in order to assess their thermal stability, using the Thermofluor based assay described by Boivin et al. Protein Expression and Purification 91 (2013) 192-206. In more detail, the measurements were carried in Buffer W (100 mM Tris-Cl pH 8, 150 mM NaCl, 1 mM EDTA) with a total sample volume of 25 μl. SYPRO™ orange was used as thermofluor, with the sample containing 2 μl SYPRO™ orange (this solution was prepared by pre-diluting 5000x SYPRO™ orange stock solution gel stain S5692-50 UL (Sigma) to a concentration of 62x to yield a final concentration of 5x SYPRO™ orange in the sample). The protein concentration was 5 μM. For the measurements, the samples were pipetted into a 96 well RT-PCR plate and the wells were sealed with transparent film. The thermocycler was then started, using a heating rate of 1 C°/min and the increase of the fluorescence over time was monitored.
Using this method, the following melting temperatures were obtained (see also
As evident from Table 6 the mutein ‘1” has the same melting temperature of 75° C. as wild-type streptavidin and is thus significantly more thermostable than the mutein m1-9. However, and in contrast to the mutein “1”, despite its significantly lower thermostability, the mutein m1-9 is suitable for being used in affinity purification of proteins under denaturing conditions (see Example 4 above). This finding is more than surprising since Reznik et al, Nat Biotechnol. 1996 supra, found a high correlation for streptavidin of biotin binding ability with thermal stability, suggesting that maintenance of the tetrameric structure is essential for streptavidin to maintain its biotin-binding ability and thus also for the ability of streptavidin muteins to maintain their binding ability for streptavidin binding peptides that competitively bind biotin. Rather, in light of the melting temperature of only 51° C., the person skilled in the art would have expected that the mutein m1-9 is not suitable at all for affinity purification of proteins under denaturing conditions. The person skilled in the art would also not have expected that the introduction of a Cys residue at position 127 of the sequence of streptavidin as in mutein m1-9C will improve the stability to the level of the initial mutant “1” or of wild-type streptavidin while maintaining the ability, for example, of the mutein m1-9 for affinity purification of proteins under denaturing conditions.
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of certain embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
List of amino acid sequences disclosed in the present application:
Tyr
117Asn118Ala119Phe120Met121
Tyr
117Asn118Ala119Tyr120Ala121
Ala
117---118---119Trp12Tyr121
Asp
117Asn118Ala119Gly120Phe121
Arg
117Asn118Ala119Gly120Phe121
Gln
117Asn118Ala119Gly120Phe121
Phe
117Asn118Ala119Ser120Trp121
Asp
117Asn118Ala119Val120Met121
Asp
117---118---119Trp120Met121
Glu
117Asn118Ala119Gly120Phe121
Tyr
117Asn118Ala119Tyr120Ser121
Phe
117Asn118Ala119Tyr120Gly121
Tyr
117Asn118Ala119Tyr120Phe121
Arg
117Asn118Ala119Tyr120Ala121
Tyr
117Asn118Ala119Tyr120Gly121
Leu
117Asn118Ala119Gly120Phe121
His
117Asn118Ala119Gly120Tyr121
Met
117Asn118Ala119Gly120Phe121
Arg
117Asn118Ala119Gly120Tyr121
Glu
117Asn118Ala119Gly120Trp121
His
117Asn118Ala119Gly120Phe121
Ser
117Asn118Ala119Gly120Phe121
Thr
117Asn118Ala119Gly120Phe121
Asn
117Asn118Ala119Gly120Phe121
Glu
117Asn118Ala119Gly120Met121
Trp
117Asn118Ala119Cys120Cys121
Met
117Asn118Ala119Phe120Val121
Ala
117Asn118Ala119Asp120Trp121
Ser
117Asn118Ala119Met120Met121
Arg
117Asn118Ala119Val120Val121
Ser
117Asn118Ala119Ser120Phe121
Ala
117---118---119Trp120Asp121
Arg
117Asn118Ala119Arg120Ala121
Ser
117Asn118Ala119Ala120Phe121
Gly
117Asn118Ala119Met120Met121
Glu
117Asn118Ala119Gly120Phe121
Asp
117Asn118Ala119Gly120Tyr121
Glu
117Asn118Ala119Gly120Tyr121
Arg
117Asn118Ala119Met120Met121
Arg
117Asn118Ala119Gly120Phe121
Ala
117Asn118Ala119Ala120Met121
Ala
117Asn118Ala119Met120Val121
Gln
117Asn118Ala119Ser120Ala121
Ala
117Asn118Ala119Gly120Phe121
Gln
117Asn118Ala119Met120Val121
Asn
117Asn118Ala119Gly120Tyr121
Ala
117Asn118Ala119Ala120Val121
Ser
117Asn118Ala119Met120Ile121
His
117Asn118Ala119Gly120Tyr121
Ser
117Asn118Ala119Met120Ala121
Gln
117Asn118Ala119Val120Ala121
Tyr
117Asn118Ala119Tyr120Met121
Leu
117Asn118Ala119Trp120Gly121
His
117Asn118Ala119Ser120Met121
Tyr
117Asn118Ala119Phe120Leu121
Phe
117Asn118Ala119Phe120Leu121
Tyr
117Asn118Ala119Leu120Trp121
Phe
117Asn118Ala119Tyr120Ile121
Tyr
117Asn118Ala119Tyr120Leu121
Tyr
117Asn118Ala119Tyr120Gln121
Phe
117Asn118Ala119Ile120Trp121
Thr
117Asn118Ala119Trp120Leu121
His
117Asn118Ala119Trp120Leu121
Ile
117---118---119Trp120Arg121
His
117Asn118Ala119Trp120Thr121
Thr
117---118---119Trp120Arg121
Ala
117---118---119Trp120Arg121
Arg
117---118---119Trp120Ser121
Asn
117---118---119Trp120Arg121
Lys
117---118---119Trp120Ser121
Ser
117---118---119Val120Phe121
Lys
117---118---119Trp120Thr121
Ala
117---118---119Trp120Tyr21
His
117---118---119Trp120Met121
His
117---118---119Trp120Tyr121
Glu
117---118---119Trp120Tyr121
Gln
117---118---119Trp120Tyr121
Xaa Asn Ala Gly Zaa,
Zaa is Leu, Ile, Met, Gln, Gly, Trp, Ser, Ala, Val
| Number | Date | Country | Kind |
|---|---|---|---|
| 16166773 | Apr 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/059715 | 4/25/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/186669 | 11/2/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5506121 | Skerra et al. | Apr 1996 | A |
| 6103493 | Skerra et al. | Aug 2000 | A |
| 7981632 | Schmidt | Jul 2011 | B2 |
| 10065996 | Schmidt | Sep 2018 | B2 |
| 20180346527 | Schmidt | Dec 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 0835934 | Apr 1998 | EP |
| 199711183 | Mar 1997 | WO |
| 02077018 | Oct 2002 | WO |
| 2013017954 | Feb 2013 | WO |
| 2014076277 | May 2014 | WO |
| Entry |
|---|
| International Search Report dated Jul. 27, 2017 in PCT/EP2017/059715 (5 pages). |
| Written Opinion dated Jul. 27, 2017 in PCT/EP2017/059715 (6 pages). |
| Reznik Go et al: “Streptavidins With Intersubunit Crosslinks Have Enhanced Stability”, Nature Biotechnology, Gale Group Inc, US, vol. 14, Aug. 1, 1998, pp. 1007-1011. |
| Aslan et al: “Engineering a novel, stable dimeric streptavidin with lower isoelectric point”, Journal of Biotechnology, Elsevier, Amsterdam, NL, vol . 128, No. 2, Jan. 13, 2007, pp. 213-225. |
| Voss S et al: “Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification”, Protein Engineering, Oxford University Press, Surrey, GB, vol. 10, No. 8, Aug. 1, 1997, pp. 975-982. |
| Sano T et al: “Intersubunit Contacts Made by Tryptophan-120 With Biotin Are Essential for Both Strong Biotin Binding and Biotin-Induced Tighter Subunit Association of Streptavidin”, Proceedings National Academy of Sciences PNAS, National Academy of Sciences, US, vol. 92, Apr. 1, 1995, pp. 3180-3184. |
| Schmidt and Skerra, “The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins”, Nature Protocols, 2007, 2(6):1528-1535. |
| Young et al., “Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications”, Biotechnol. J., 2012, 7, 620-634. |
| Argarana et al., Molecular Cloning and Nucleotide Sequence of the Streptavidin Gene. Nucleic Acids Res. Feb. 25, 1986;14(4):1871-1882. |
| Bornholst and Falke, Purification of Proteins Using Polyhistidine Affinity Tags. Methods Enzymol. 2000;326:245-254. |
| Fletcher et al., Self-assembly of proteins and their nucleic acids. J Nanobiotechnology. Jan. 28, 2003;1(1):1 (16 pages). |
| Knabel et al., Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nat Med. Jun. 2002;8(6):631-637. |
| Schmidt and Skerra, One-step affinity purification of bacterially produced proteins by means of the “Strep tag” and immobilized recombinant core streptavidin. J Chromatogr A. Aug. 5, 1994;676(2):337-345. |
| Schmidt and Skerra, The Strep-tag System for One-Step Purification and High-Affinity Detection or Capturing of Proteins. Nat Protoc. 2007;2(6):1528-1535. |
| Number | Date | Country | |
|---|---|---|---|
| 20190112344 A1 | Apr 2019 | US |
