STREPTAVIDIN MUTEINS AND METHODS OF USING THEM

Abstract

The invention concerns novel streptavidin muteins and methods of determining, immobilizing, isolating or purifying proteins under denaturing conditions. In one embodiment such a mutein has an Cys residue at sequence position 127 of the wild-type sequence of streptavidin and comprises at least one mutation in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptavidin.

Claims

1. 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) wherein the sequence Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 10) or the sequence Ile.sup.44Gly.sup.45Ala.sup.46Arg.sup.47 (SEQ ID NO: 11) is present in the region of amino acid positions 44 to 47 with reference to the amino acid sequence of wild-type streptavidin.

2. A mutein according to claim 1, wherein the mutein comprises at sequence positions 117 to 121 with reference to the amino acid sequence of wild-type streptavidin an amino acid sequence selected from the group consisting of TABLE-US-00009 (SEQ ID NO: 56) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121, (SEQ ID NO: 57) Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121, (SEQ ID NO: 58) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121, (SEQ ID NO: 22) Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 (SEQ ID NO: 26) Gln.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121, (SEQ ID NO: 44) Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 (SEQ ID NO: 66) Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121, and (SEQ ID NO: 95) His.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121.

3. A mutein according to claim 1, wherein the mutein is a mutein of a minimal streptavidin which begins N-terminally in the region of the amino acids 10 to 16 of wild type streptavidin and terminates C-terminally in the region of the amino acids 133-142 of wild type streptavidin.

4. A mutein according to claim 1, wherein the mutein comprises the sequence of the mutein m302C (SEQ ID NO: 14).

5. The mutein according to claim 4, wherein the mutein consists of the sequence of mutein m1-9C (SEQ ID NO: 16) or m302C (SEQ ID NO: 14).

6. A nucleic acid molecule, comprising a sequence coding for a streptavidin mutein as defined in claim 1.

7. 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, the method comprising contacting a sample containing the protein with a streptavidin mutein of claim 1 under suitable conditions to bind the peptide sequence to the streptavidin mutein, and separating the resulting complex from said sample.

8. The method according to claim 7, wherein the streptavidin mutein is bound to a solid phase or is capable of binding thereto.

9. The method according to claim 8, wherein in order to release the fusion protein from the complex the complex is incubated with an adequate amount of a ligand for the streptavidin mutein selected from biotin and derivatives thereof.

10. The method according to claim 7, wherein the denaturing conditions are caused by the presence of a chaotropic agent.

11. The method according to claim 10, wherein the chaotropic agent is selected from the group consisting of urea, thiourea, guanidine hydrochloride, lithium perchlorate hydroxide ions, and combinations thereof.

12. The method according to claim 8, wherein the solid phase is an affinity chromatography matrix.

13. The method according to claim 12, wherein the affinity chromatography matrix is selected from the group consisting of a cellulose membrane, a plastic membrane, 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 copolymer of a polysaccharide and N,N′-methylenebisacrylamide and a combination of any two or more thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0055] FIG. 1A shows the full length amino acid sequence of mature wild-type streptavidin (residues 1 to 159; SEQ ID NO: 1), while FIG. 1B shows the amino acid sequence of a shortened version of mature wild-type streptavidin (residues 14 to 139; SEQ ID NO: 2) that was used in the present invention for the generation of inventive muteins.

[0056] FIG. 2 shows three sequence motives of muteins of the invention having at least one mutation within the peptide segment of residues 117 to 121 of the streptavidin sequence. Preferred residues are shown in bold print, less preferred residues are shown in normal print. Amino acid residues shall be considered positionwise and each may be in principle combined with any other occurring at another position. Sequence motif 1 is characterized in that glycine is highly preferred at position 120 and may be combined with a large hydrophobic residue, preferably tyrosine or phenylalanine, or, less preferably, methionine at position 121 and a charged, preferably glutamate, aspartate, arginine or histidine, or, less preferably, a hydrophilic residue like glutamine, asparagine, serine or threonine or a hydrophobic residue like leucine or methionine at position 117. Sequence motif 2 is characterized in that a large hydrophobic residue, preferably tyrosine or phenylalanine but not tryptophane is highly preferred at position 120 instead of small glycine while leucine, isoleucine or methionine are less preferred at this position 120. Then also hydrophobic residues are preferred at positions 117 and 121, whereby aromatic tyrosine or phenylalanine are preferred for position 117 and large but non-aromatic hydrophobic residues, most preferably leucine, isoleucine and methionine, are preferred for position 121. Less preferred for position 117 are the residues arginine, tryptophane or glutamine and for position 121 the residues glutamine, glycine, tryptophane, serine, alanine or valine. Sequence motif 3 is characterized in that amino acids at positions 118 and 119 are deleted. In this case, tryptophane at original position 120 is strongly preferred and valine is less preferred and these residues may be combined with preferably tyrosine at position 121, whereby also other residues like leucine, methionine, threonine, serine, phenylalanine or arginine may occur at position 121, and with most preferably a hydrogen bond acceptor and/or donator like histidine, gltuamine or glutamate or, less preferably also other residues like threonine, arginine, asparagine, lysine, serine, alanine or isoleucine at position 117.

[0057] FIG. 3 shows the amino acid sequence of all muteins experimentally generated and characterised herein (muteins m302 (SEQ ID NO: 13), m302C (SEQ ID NO: 14), m1-9 (SEQ ID NO: 15), and m1-9C (SEQ ID NO: 16)), aligned with the amino acid sequence of amino acid positions 14 to 139 of wild type streptavidin (SEQ ID NO: 2) and of mutein “1” (SEQ ID NO: 12) described in U.S. Pat. No. 6,103,493 (that is commercially available under its trademark STREP-TACTIN®. When the muteins were produced by E. coli in the cytosol as inclusion bodies for subsequent refolding, purification and analysis, the resulting protein sequences were produced as shown with an additional methionine at the N-terminal end (position 13, not shown in FIG. 4). Deleted amino acids in the muteins m302 and m302C are indicated by a dash (-). For these two muteins, amino acid numbering has been conducted in a manner maintaining comparability at equivalent positions. It should, however, be noted that the muteins m302 and m302C in which the amino acid residues at sequence positions 118 and 119 are deleted, are with 125 amino acid residues two amino acids shorter than mutein “1” and the muteins m1-9 and m1-9C (who have a length of 127 amino acid residues). In the muteins of the invention (m302C and m1-9C) the Cys residue at sequence position 127 is underlined and depicted in bold. In addition, the mutated amino acids in the region of sequence positions 117 to 121 in the muteins m302, m302C, m1-9, and m1-9C are in bold print.

[0058] FIG. 4 shows a SDS-polyacrylamide gel electrophoresis (PAGE) analysis of the stability of the muteins m302, m302C, m1-9, and m1-9C in comparison to mutein “1”. The samples 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.

[0059] FIG. 5 shows a SDS-PAGE gel analysis of the efficiency of the purification of the feline immunodeficiency virus Gag protein (FIV GAG) to which the streptavidin peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.2-Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 110) was fused C-terminally by affinity chromatography under denaturing conditions. The SDS-PAGE analysis of FIG. 5 shows the comparison of the affinity chromatography using the inventive streptavidin mutein m1-9C with the muteins “1” and m1-9. In more detail, [0060] lane 1 shows the eluate of the purification using mutein “1” as affinity reagent in the presence of 4 M urea, [0061] lane 2 shows the eluate of the purification using mutein “1” as affinity reagent in the presence of 4 M guanidinium hydrochloride, [0062] lane 3 shows the molecular marker, [0063] lane 4 shows the eluate of the purification using mutein m1-9 as affinity reagent in the presence of 4 M urea, [0064] lane 5 shows the eluate of the purification using mutein m1-9 as affinity reagent in the presence of 6 M urea, [0065] lane 6 shows the eluate of the purification using mutein m1-9 as affinity reagent in the presence of 4 M guanidinium hydrochloride, [0066] lane 7 shows the eluate of the purification using mutein m1-9 as affinity reagent in the presence of 6 M guanidinium hydrochloride, [0067] lane 8 shows the eluate of the purification using mutein m1-9C as affinity reagent in the presence of 4 M urea, [0068] lane 9 shows the eluate of the purification using mutein m1-9C as affinity reagent in the presence of 6 M urea, [0069] lane 10 shows the eluate of the purification using mutein m1-9C as affinity reagent in the presence of 4 M guanidinium hydrochloride, [0070] lane 11 shows the eluate of the purification using mutein m1-9C as affinity reagent in the presence of 6 M guanidinium hydrochloride.

[0071] FIG. 6 shows the melting temperature curves for wild-type streptavidin (wt-strep, SEQ ID NO: 2), streptavidin mutein “1” (SEQ ID NO: 12), streptavidin mutein m1-9 (SEQ ID NO: 15) and streptavidin mutein m1-9C (SEQ ID NO: 16).

EXAMPLES

General Methods

[0072] 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).sub.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 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 Göttingen GmbH. Primers and oligonucleotides were synthesized using an Applied Biosystems Expedite DNA synthesizer.

Example 1: Mutagenesis/Generation of Expression Vectors Encoding Muteins m302C and m1-9

[0073] 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.

Example 2: Production of Streptavidin Muteins on a Preparative Scale

[0074] 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.

Example 3: Analysis of the Stability of Streptavidin Muteins under Denaturing Conditions

[0075] 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 Brillant Blue. FIG. 4 shows the result of the SDS-PAGE, comparing the stability of the muteins m302, m302C, m1-9, and m1-9C to mutein “1” under these denaturing conditions.

[0076] 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 FIG. 4, the introduction of the Cys residue at position 127 greatly increases the stability of the muteins m1-9 and m302 under these conditions. The mutein m1-9C seems to be present only in its tetrameric form. Under these conditions, the relative gain in stabilization seems to be even more pronounced for the mutein 302. The mutein m302C seems to be able to maintain its tetrameric structure to a large extent, only a small fraction of this streptavidin mutein dissociates into its monomer under these denaturing conditions.

Example 4: Production of Fusion Protein and Purification of the Fusion Protein from Inclusion Bodies via Affinity Chromatography under Denaturing Conditions

[0077] 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 (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 BCA assay (Pierce) according to the instructions of the manufacturer and as described in International Patent Application WO 2014/076277.

[0078] 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-TAGII 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).sub.2-Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (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.

[0079] 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.

[0080] The effectiveness of the affinity purification was analysed by SDS-PAGE. FIG. 5 shows the comparison of the affinity chromatography using the inventive streptavidin mutein m1-9C with the muteins “1” and m1-9. While protein purity was high in all experiments, no resin allowed the purification of the feline immunodeficiency virus Gag protein at a concentration of 6 M guanidine hydrochloride. As can be taken from FIG. 5, only small amount of protein in 4 M urea containing Buffer W was purified with mutein “1” (STREP-TACTIN®), while purification using mutein m1-9 was successful under denaturing conditions with 4 M urea and 6 M urea. It is noted here that for mutein m1-9 using 6 M urea the gel of FIG. 5 shows a band of lower protein concentration. However, later results showed that there is no difference between 4 M and 6 M urea. In addition, it was observed for mutein m1-9 that it can be used for affinity purification using 4 M guanidine but more fusion protein was eluted when urea was used as denaturant. For the mutein m1-9C it was found that its use as affinity material for the purification resulted in an eluate that showed high protein concentration, when 4 M or 6 M urea was used.

[0081] 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.

[0082] 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.

[0083] The conclusion of these experiments can be summarized as follows. The mutein “1” (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 severely 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.

Example 5: Determination of the Melting Temperature (Tm) of the Streptavidin Muteins

[0084] 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 5000× SYPRO™ orange stock solution gel stain S5692-50 UL (Sigma) to a concentration of 62× to yield a final concentration of 5× SYPRO 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 flourescence over time was monitored.

[0085] Using this method, the following melting temperatures were obtained (see also FIG. 6 that shows the melting curves of the respective protein):

TABLE-US-00007 TABLE 6 melting points of muteins Melting Temperature Mutein (Tm) (° C.) wt-streptavidin (amino 75 acids 14 to 139) mutein “1” 75 m1-9 51 m1-9C 75

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] List of amino acid sequences disclosed in the present application:

SEQ ID NO: 1 amino acid sequence of mature wild-type streptavidin (residues 1 to 159)
SEQ ID NO: 2 amino acid sequence of mature wild-type streptavidin (residues 14 to 139)
SEQ ID NO: 3 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, STREP-TAG® II affinity tag
SEQ ID NO: 4 Trp-Arg-His-Pro-Gln-Phe-Gly-Gly, STREP-TAG® affinity tag
SEQ ID NO: 5 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, di-tag3 sequence
SEQ ID NO: 6 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, di-tag2 sequence

SEQ ID NO: 7 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).SUB.2.-Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, TWIN-STREP-TAG®

[0092] SEQ ID NO: 8 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
SEQ ID NO: 9 Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- 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
SEQ ID NO: 10 Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47
SEQ ID NO: 11 Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47

SEQ ID NO: 12: Mutein “1”

[0093] SEQ ID NO: 13: Mutein m302
SEQ ID NO: 14: Mutein m302C
SEQ ID NO: 15: Mutein m1-9
SEQ ID NO: 16: Mutein m1-9C

SEQ ID NO: 17 Thr-Thr-Glu-Asp-Asn-Ala-Trp-Lys (TTEANAWK)

SEQ ID NO: 18 HPYFYAPELLFFAK

SEQ ID NO: 19 EGGKETLTPSELRDLV

[0094] SEQ ID NO: 20 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 (wild type streptavidin)

TABLE-US-00008 SEQ ID NO: 21 m36 Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Met.sup.121 is: YNAFM SEQ ID NO: 22 m23 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 is: YNAYA SEQ ID NO: 23 m41 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: AXXWY SEQ ID NO: 24 m4 Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: DNAGF SEQ ID NO: 25 m12 Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: RNAGF SEQ ID NO: 26 m22 Gln.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: QNAGF SEQ ID NO: 27 m31 Phe.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Trp.sup.121 is: FNASW SEQ ID NO: 28 m32 Asp.sup.117Asn.sup.118Ala.sup.119Val.sup.120Met.sup.121 is: DNAVM SEQ ID NO: 29 m35 Ala.sup.117---.sup.118---.sup.119Trp.sup.120met.sup.121 is: AXXWM SEQ ID NO: 30 m38 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: ENAGF SEQ ID NO: 31 m40 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ser.sup.121 is: YNAYS SEQ ID NO: 32 m42 Phe.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gly.sup.121 is: FNAYG SEQ ID NO: 33 m45 Tyr.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: YNAGF SEQ ID NO: 34 m46 Arg.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 is: RNAYA SEQ ID NO: 35 m47 Trp.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gly.sup.121 is: WNAYG SEQ ID NO: 36 m7 Leu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: LNAGF SEQ ID NO: 37 m10 Hisn7Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: HNAGY SEQ ID NO: 38 m17 Met.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: MNAGF SEQ ID NO: 39 m21 Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: RNAGY SEQ ID NO: 40 m24 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Trp.sup.121 is: ENAGW SEQ ID NO: 41 m27 His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: HNAGF SEQ ID NO: 42 m28 Ser.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: SNAGF SEQ ID NO: 43 m30 Thr.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: TNAGF SEQ ID NO: 44 m33 Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: NNAGF SEQ ID NO: 45 m1 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Met.sup.121 is: ENAGM SEQ ID NO: 46 m3 Trp.sup.117Asn.sup.118pda.sup.119cys.sup.120Cys.sup.121 is: WNACC SEQ ID NO: 47 m8 Met.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Val.sup.121 is: MNAFV SEQ ID NO: 48 m15 Ala.sup.117Asn.sup.118Ala.sup.119Asp.sup.120Trp.sup.121 is: ANADW SEQ ID NO: 49 m6 Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 is: SNAMM SEQ ID NO: 50 m9 Arg.sup.117Asn.sup.118Ala.sup.119Val.sup.120Val.sup.121 is: RNAVV SEQ ID NO: 51 m20 Ser.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Phe.sup.121 is: SNASF SEQ ID NO: 52 m34 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Asp.sup.121 is: AXXWD SEQ ID NO: 53 m14 Arg.sup.117Asn.sup.118Ala.sup.119Arg.sup.120Ala.sup.121 is: RNARA SEQ ID NO: 54 m18 Ser.sup.117Asn.sup.118Ala.sup.119Ala.sup.120Phe.sup.121 is: SNAAF SEQ ID NO: 55 m19 Gly.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 is: GNAMM SEQ ID NO: 56 m8 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: ENAGF SEQ ID NO: 57 m21 Aspu7Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: DNAGY SEQ ID NO: 58 m9 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: ENAGY SEQ ID NO: 59 m1 Arg.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 is: RNAMM SEQ ID NO: 60 m2 Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: RNAGF SEQ ID NO: 61 m3 Ala.sup.117Asn.sup.118Ala.sup.119Pro.sup.120Ala.sup.121 is: ANAPA SEQ ID NO: 62 m5 Ala.sup.117Asn.sup.118Ala.sup.119Met.sup.120Val.sup.121 is: ANAMV SEQ ID NO: 63 m13 Gln.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Ala.sup.121 is: QNASA SEQ ID NO: 64 m14 Ala.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: ANAGF SEQ ID NO: 65 m24 Gln.sup.117Asn.sup.118Ala.sup.119Met.sup.120Val.sup.121 is: QNAMV SEQ ID NO: 66 m4 Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: NNAGY SEQ ID NO: 67 m6 Ala.sup.117Asn.sup.118Ala.sup.119Ala.sup.120Val.sup.121 is: ANAAV SEQ ID NO: 68 m7 seru7Asn.sup.118pda.sup.119Met.sup.120Ile.sup.121 is: SNAMI SEQ ID NO: 69 m10 His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: HNAGY SEQ ID NO: 70 m15 Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Ala.sup.121 is: SNAMA SEQ ID NO: 71 m23 Gln.sup.117Asn.sup.118Ala.sup.119Val.sup.120Ala.sup.121 is: QNAVA SEQ ID NO: 72 m17 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Met.sup.121 is: YNAYM SEQ ID NO: 73 m12 Leu.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Gly.sup.121 is: LNAWG SEQ ID NO: 74 m20 His.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Met.sup.121 is: HNASM SEQ ID NO: 75 m101 Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 is: YNAFL SEQ ID NO: 76 m106 Phe.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 is: FNAFL SEQ ID NO: 77 m111 Tyr.sup.117Asn.sup.118Ala.sup.119Leu.sup.120Trp.sup.121 is: YNALW SEQ ID NO: 78 m100 phen7Asn.sup.118Ala.sup.119Tyr.sup.120Ile.sup.121 is: FNAYI SEQ ID NO: 79 m110 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Leu.sup.121 is: YNAYL SEQ ID NO: 80 m104 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gln.sup.121 is: YNAYQ SEQ ID NO: 81 m108 Phe.sup.117Asn.sup.118Ala.sup.119Ile.sup.120Trp.sup.121 is: FNAIW SEQ ID NO: 82 m207 Thr.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 is: TXXWL SEQ ID NO: 83 m212 His.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 is: HXXWL SEQ ID NO: 84 m202 i1e.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: IXXWR SEQ ID NO: 85 m204 His.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 is: HXXWT SEQ ID NO: 86 m206 Thr.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: TXXWR SEQ ID NO: 87 m208 Aia.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: AXXWR SEQ ID NO: 88 m203 Arg.sup.117---.sup.118---.sup.119Trp.sup.120Ser.sup.121 is: RXXWS SEQ ID NO: 89 m209 Asn.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: NXXWR SEQ ID NO: 90 m200 Lys.sup.117---.sup.118---.sup.119Trp.sup.120Ser.sup.121 is: KXXWS SEQ ID NO: 91 m201 Ser.sup.117---.sup.118---.sup.119Val.sup.120phe.sup.121 is: SXXVF SEQ ID NO: 92 m211 Lys.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 is: KXXWT SEQ ID NO: 93 m300 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: AXXWY SEQ ID NO: 94 m301 His.sup.117---.sup.118---.sup.119Trp.sup.120Met.sup.121 is: HXXWM SEQ ID NO: 95 m302 His.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: HXXWY SEQ ID NO: 96 m303 Glu.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: EXXWY SEQ ID NO: 97 m304 Gln.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: QXXWY SEQ ID NO: 98 motif1 Xaa Asn Ala Gly Zaa, (FIG. 2) Xaa is Glu, Asp, Arg, His, Asn, Gln, Thr, Ser, Leu, Met and Zaa motif2 is Phe, Tyr, Met is XNAGX consensus Aaa.sup.117Baa.sup.120Caa.sup.121, sequence2 wherein Aaa may be Tyr, Phe, Arg, Trp or Gln, Baa may be Tyr, Phe, Leu, Ile or Met and Caa may be any amino acid, wherein Caa.sup.121 is preferably a Leu, an Ile, a Met, a Gly, a Gly, a Trp, a Ser, an Ala or a Val residue SEQ. ID NO: 99 Motif2 Xaa-Asn-Ala-Yaa-Zaa, (FIG. 2) wherein Xaa is Tyr, Phe, Arg, Trp, Gln; Yaa is Tyr, Phe, Leu, Ile, Met and Zaa is Leu, Ile, Met, Gln, Gly, Trp, Ser, Ala, Val Is XNAXX SEQ ID NO: motif3 Daa.sup.117Eaa.sup.118Faa.sup.119Gaa.sup.120Haa.sup.121, wherein Daa and Haa may 100 consensus be any amino acid and Eaa and Faa are both deleted and Gaa sequence3 may be Trp or Val, wherein Daa.sup.117 is preferably a His, a Glu, a Gln, a Thr, an Ala or an Ile residue and wherein Haa.sup.121 is preferably a Tyr, a Leu, a Met, or an Arg residue is XXXXX