Streptavidin muteins and methods of using them

Abstract

The invention concerns novel streptavidin muteins. In one embodiment such a mutein (a) contains two or more mutations in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptavidin of which amino acid residues 14 to 139 are as set forth at SEQ ID NO:212 and (b) has a higher binding affinity than each of (i) a streptavidin mutein 1 (SEQ ID NO: 16) that comprises the amino acid sequence Val44-Thr45-Ala46-Arg47 (SEQ ID NO: 98), or (ii) wild type-streptavidin of which amino acid residues 14 to 139 are shown as SEQ ID NO: 212 for peptide ligands comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 100).

Claims

1. A mutein, selected from muteins of streptavidin, wherein the mutein (a) contains two or more mutations in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptayidin of which amino acid residues 14 to 139 are as set forth at SEQ ID NO: 212 and (b) has a higher binding affinity than each of (i) a streptavidin mutein 1 (SEQ ID NO: 112) that comprises the amino add sequence Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47(SEQ ID NO: 98), or (ii) wild type-streptavidin of which amino add residues 14 to 139 are shown as SEQ HD NO: 212 for peptide ligands comprising the amino add sequence Trp-Ser-His-Pro-Gin-Phe-Glu-Lys (SEQ ID NO: 100), wherein the mutein carries as mutated residue at sequence position 117 an amino add residue selected from the group consisting of Tyr, Phe, Ile and Thr or, wherein the mutein carries as mutated residue at sequence position 120 an amino acid residue selected from the group consisting of Phe, Tyr, Val, and Leu, or wherein the mutein carries as mutated residue at sequence position 121 an amino add residue selected from the group consisting of Ala, Trp, Ser, Ile, Val, and Arg.

2. A mutein according to claim 1, wherein an amino acid residue is present at sequence position 114, wherein the amino acid residue is either the wild type threonine or any other amino acid.

3. A mutein according to claim 1, wherein the mutein has a binding affinity for peptide ligands comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 100) that is at least 1.1 times higher (expressed by the ratio of the respective Ka) than a streptavidin mutein that comprises the amino acid sequence Va1.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 98) or the sequence Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 99) at amino acid positions 44 to 47.

4. A mutein, selected from muteins of streptavidin, wherein the mutein (a) contains two or more mutations in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptavidin of which amino acid residues 14 to 139 are as set forth at SEQ ID NO: 212 and (b) has a higher binding affinity than each of (i) a streptavidin mutein 1(SEQ ID NO: 112) that comprises the amino acid sequence Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47(SEQ ID NO: 98), or (ii) wild type-streptavidin of which amino acid residues 14 to 139 are shown as SEQ ID NO: 212 for peptide ligands comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 100), wherein the mutein comprises with reference to the amino acid sequence of wild type streptavidin of which amino acid residues 14 to 139 are set forth as SEQ ID NO: 212 an amino acid sequence selected from the group consisting of Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Met.sup.121 depicted as SEQ ID NO: 21, Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 depicted as SEQ ID NO: 22, Ala.sup.117---.sup.118---.sup.119TRP.sup.120Tyr.sup.121 depicted as SEQ ID NO: 23, Tyr.sup.117Asn.sup.118Ala.sup.119Met.sup.120 depicted as SEQ ID NO: 33, Gly.sup.117Asn.sup.118Met.sup.120Met.sup.121 depicted as SEQ ID NO: 55, Tyr.sup.117Asn.sup.118Ala.sup.110Phe.sup.120Leu.sup.121 depicted as SEQ ID NO: 75, Phe.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 depicted as SEQ ID NO: 76, Tyr.sup.117Asn.sup.118Ala.sup.119Leu.sup.120Leu.sup.121 depicted as SEQ ID NO: 77, Thr.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 depicted as SEQ ID NO: 82 His.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 depicted as SEQ ID NO: 83 Ile.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 depicted as SEQ ID NO: 84 His.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 depicted as SEQ ID NO: 85 Thr.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 depicted as SEQ ID NO: 86 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 depicted as SEQ ID NO: 87 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 depicted as SEQ ID NO: 93 Glu.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 depicted as SEQ ID NO: 96, and Gln.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 depicted as SEQ ID NO: 97 at sequence positions 117 to 121.

5. A mutein, selected from muteins of streptavidin, wherein the mutein (a) contains two or more mutations in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptavidin of which amino acid residues 14 to 139 are as set forth at SEQ ID NO: 212 and (b) has a higher binding affinity than each of (i) a streptavidin mutein 1 (SEQ ID NO: 112) that comprises the amino acid sequence Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47(SEQ ID NO: 98), or (ii) wild type-streptavidin of which amino acid residues 14 to 139 are shown as SEQ ID NO: 212 for peptide ligands comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 100), wherein the mutein comprises the sequence of any of the following muteins: mutein1-m36 (SEQ ID NO: 117), mutein1-m23 (SEQ ID NO: 118), mutein1-m41 (SEQ ID NO: 119), mutein1-m4 (SEQ ID NO: 120), mutein1-m12 (SEQ ID NO: 121), mutein1-m22 (SEQ ID NO: 122), mutein1-m31 (SEQ ID NO: 123), mutein1-m32 (SEQ ID NO: 124), mutein1-m35 (SEQ ID NO: 125), mutein1-m38 (SEQ ID NO: 126), mutein1-m40 (SEQ ID NO: 127), mutein1-m42 (SEQ ID NO: 128), mutein1-m45 (SEQ ID NO: 129), mutein1-m46 (SEQ ID NO: 130), mutein1-m47 (SEQ ID NO: 131), mutein1-m7 (SEQ ID NO: 132), mutein1-m10 (SEQ ID NO: 133), mutein1-m17 (SEQ ID NO: 134), mutein1-m27 (SEQ ID NO: 137), mutein1-m28 (SEQ ID NO: 138), mutein1-m30 (SEQ ID NO: 139), mutein1-m33 (SEQ ID NO: 140), mutein1-m3 (SEQ ID NO: 142), mutein1-m6 (SEQ ID NO: 145), mutein1-m20 (SEQ ID NO: 147), mutein1-m34 (SEQ ID NO: 148),), mutein1-m18 (SEQ ID NO: 150), mutein1-m19 (SEQ ID NO: 151), mutein1-m101 (SEQ ID NO: 171), mutein1-m106 (SEQ ID NO; 172), mutein1-m111 (SEQ ID NO: 173), mutein1-m100 (SEQ ID NO: 174), mutein1-m110 (SEQ ID NO: 175), mutein1-m104 (SEQ ID NO: 176), mutein1-m108 (SEQ ID NO: 177), mutein1-m207 (SEQ ID NO: 178), mutein1-m212 (SEQ ID NO: 179), mutein1-m202 (SEQ ID NO: 180), mutein1-m204 (SEQ ID NO: 181), mutein1-m206 (SEQ ID NO: 182), mutein1-m208 (SEQ ID NO: 183), mutein1-m203 (SEQ ID NO: 184), mutein1-m209 (SEQ ID NO: 185), mutein1-m200 (SEQ ID NO: 186), mutein1-m201 (SEQ ID NO: 187), mutein1-m211 (SEQ ID NO: 188), mutein1-m300 (SEQ ID NO: 189),), mutein1-m303 (SEQ ID NO: 192), mutein1-m304, and (SEQ ID NO; 193).

6. A mutein according to claim 4, wherein the binding affinity for the peptide ligand is such that a competitive elution can take place by streptavidin ligands selected from biotin, thiobiotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA or/and dimethyl-HAB A.

7. A method of producing of a streptavidin mutein according to claim 1, 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.

8. A method of isolating, purifying or determining a protein that is fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 101) 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: 108) 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 the protein with a streptavidin mutein of claim 1, under suitable conditions to bind the peptide sequence to the streptavidin mutein, and separating resulting complex from said sample.

9. A mutein according to claim 4, in which the amino acid residue at at least one of positions 118 and 119 is deleted.

10. A mutein according to claim 4, 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.

11. A mutein according to claim 4, wherein with reference to the amino acid sequence of wild type streptavidin of which amino acid residues 14 to 139 are as set forth at SEQ ID NO: 212 Glu is replaced by a hydrophobic aliphatic amino acid at sequence position 44, an arbitrary amino acid is present at sequence position 45, a hydrophobic aliphatic amino acid is present at sequence position 46 or/and Val is replaced by a basic amino acid at sequence position 47.

12. A mutein according to claim 11, wherein the sequence Val -Thr.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 98) is present in the region of amino acid positions 44 to 47.

13. A mutein according to claim 11, wherein the Ile.sup.44Gly.sup.45Ala.sup.46Arg.sup.47(SEQ ID NO: 99) is present in the region of amino acid positions 44 to 47.

14. A nucleic acid molecule, comprising a nucleic acid sequence coding for a streptavidin mutein according to claim 4.

15. A nucleic acid molecule, comprising a nucleic acid sequence coding for a streptavidin mutein according to claim 4.

16. A method of isolating, purifying or determining a protein that is fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 101) 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: 108) 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 the protein with a streptavidin mutein of claim 4, under suitable conditions to bind the peptide sequence to the streptavidin mutein, and separating resulting complex from said sample.

17. A nucleic acid molecule, comprising a nucleic acid sequence coding for a streptavidin mutein according to claim 5.

18. A method of isolating, purifying or determining a protein that is fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 101) 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: 108) 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 the protein with a streptavidin mutein of claim 5, under suitable conditions to bind the peptide sequence to the streptavidin mutein, and separating resulting complex from said sample.

19. The mutein of claim 4, further comprising one or more mutations in the region of the amino acid positions 44 to 53 with reference to the amino acid sequence of wild type streptavidin.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A and 1B show graphs showing the binding affinity of recombinant streptavidin mutein 1 disclosed in U.S. Pat. No. 6,103,493 compared to two selected streptavidin muteins according to the present invention in an ELISA;

(2) FIG. 1A shows the improved affinity of the disulfide containing streptavidin mutein m4001 of the present invention shown in Table 2 for the peptide ligand Strep-tagII in an ELISA as compared to streptavidin mutein 1 disclosed by U.S. Pat. No. 6,103,493. For this rows of an ELISA plate were each coated with equivalent concentrations of the respective recombinant streptavidin mutein 1 (rhombus) and m4001 (triangles). After saturating with BSA and washing, the wells were incubated with a purified fusion protein consisting of E. coli alkaline phosphatase Strep-tagII fusion protein (BAP-StrepII)-expressed by pASK75-phoA, the sequence of which is disclosed by SEQ ID NO. 1, and purified by Strep-Tactin affinity chromatography - at the concentrations shown in the graph. After washing to remove unbound protein, the activity of bound BAP-StrepII fusion protein was measured in the presence of p-nitrophenyl phosphate. The data were fitted by non-linear regression by the least squared error method. The following K.sub.D values were obtained: 0.11 M for streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and 0.02 M for m4001. The fitted values for each concentration are shown as well for m4001 (+) and streptavidin mutein 1 of U.S. Pat. No. 6,103,493 (x) demonstrating that experimental data and fit are in good agreement.

(3) FIG. 1B shows the improved affinity of the streptavidin mutein m4 of the present invention shown in Table 3 for the peptide ligand Strep-tagII in an ELISA when compared to streptavidin mutein 1 disclosed by U.S. Pat. No. 6,103,493. For this rows of an ELISA plate were each coated with equivalent concentrations of the respective recombinant streptavidin mutein 1 (rhombus) and m4 (triangles). After saturating with BSA and washing, the wells were incubated with a purified fusion protein consisting of E. coli alkaline phosphatase Strep-tagII fusion protein (BAP-StrepII)-expressed by pASK75-phoA, the sequence of which is disclosed by SEQ ID NO. 1, and purified by Strep-Tactin affinity chromatography - at the concentrations shown in the graph. After washing to remove unbound protein, the activity of bound BAP-StrepII fusion protein was measured in the presence of p-nitrophenyl phosphate. The data were fitted by non-linear regression by the least squared error method. The following K.sub.D values were obtained: 0.11 M for mutein 1 of U.S. Pat. No. 6,103,493 and 0.009 M for m4. The fitted values for each concentration are shown as well for m4 (+) and streptavidin mutein 1 of U.S. Pat. No. 6,103,493 (x) demonstrating that experimental data and fit are in good agreement.

(4) FIG. 2A shows a non limiting overview about the mutations at sequence positions 117 to 121 found in streptavidin muteins of the invention in which amino acid positions were subjected to mutagenesis. FIG. 2A shows the mutations found in muteins in which amino acid residues 118 and 119 were present and also found in muteins in which amino acid residues 118 and 119 were deleted.

(5) FIG. 2B shows the 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, glutamine or glutamate or, less preferably also other residues like threonine, arginine, asparagine, lysine, serine, alanine or isoleucine at position 117.

(6) FIG. 3 shows the amino acid sequence of sequence positions 14 to 139 of wild-type streptavidin (SEQ ID NO: 15).

(7) FIG. 4 shows the amino acid sequence of all muteins experimentally generated and characterised herein aligned with the amino acid sequence of amino acid positions 14 to 139 of wild type streptavidin and of the muteins 1 and 2 described in U.S. Pat. No. 6,103,493. When the muteins were secreted by E. coli to the periplasm, as done during screening within the filter sandwich assay, the resulting protein sequences were produced with an additional alanine at the N-terminal end (position 13). 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). Deleted amino acids are indicated by a dash (-). Amino acid numbering has been conducted in case of deletions in a manner maintaining comparability at equivalent positions. It has, however, to be noted that the molecules containing deletions are decreased in length by the number of deletions which is 2 for the corresponding muteins of FIG. 4. This means that the muteins of FIG. 4 without deletions contain a total of 127 amino acids while those with deletions contain a total of 125 amino acids. The amino acid residues resulting from randomized codons in the different libraries are shown in bold. The sequence identifiers corresponding to the amino acid sequences depicted in FIG. 4 are as follows:

(8) SEQ ID NO: 212 wildtype (Wt) streptavidin,

(9) SEQ ID NO: 112 mutein1,

(10) SEQ ID NO: 113 mutein2,

(11) SEQ ID NO: 114 mutein m400,

(12) SEQ ID NO: 115 mutein m402,

(13) SEQ ID NO: 116 mutein m4001,

(14) SEQ ID NO: 117 mutein1-m36,

(15) SEQ ID NO: 118 mutein1-m23,

(16) SEQ ID NO: 119 mutein1-m41,

(17) SEQ ID NO: 120 mutein1-m4,

(18) SEQ ID NO: 121 mutein1-m12,

(19) SEQ ID NO: 122 mutein1-m22,

(20) SEQ ID NO: 123 mutein1-m31,

(21) SEQ ID NO: 124 mutein1-m32,

(22) SEQ ID NO: 125 mutein1-m35,

(23) SEQ ID NO: 126 mutein1-m38,

(24) SEQ ID NO: 127 mutein1-m40,

(25) SEQ ID NO: 128 mutein1-m42,

(26) SEQ ID NO: 129 mutein1-m45,

(27) SEQ ID NO: 130 mutein1-m46,

(28) SEQ ID NO: 131 mutein1-m47,

(29) SEQ ID NO: 132 mutein1-m7,

(30) SEQ ID NO: 133 mutein1-m10,

(31) SEQ ID NO: 134 mutein1-m17,

(32) SEQ ID NO: 135 mutein1-m21,

(33) SEQ ID NO: 136 mutein1-m24,

(34) SEQ ID NO: 137 mutein1-m27,

(35) SEQ ID NO: 138 mutein1-m28,

(36) SEQ ID NO: 139 mutein1-m30,

(37) SEQ ID NO: 140 mutein1-m33,

(38) SEQ ID NO: 141 mutein1-m1,

(39) SEQ ID NO: 142 mutein1-m3,

(40) SEQ ID NO: 143 mutein1-m8,

(41) SEQ ID NO: 144 mutein1-m15,

(42) SEQ ID NO: 145 mutein1-m6,

(43) SEQ ID NO: 146 mutein1-m9,

(44) SEQ ID NO: 147 mutein1-m20,

(45) SEQ ID NO: 148 mutein1-m34,

(46) SEQ ID NO: 149 mutein1-m14,

(47) SEQ ID NO: 150 mutein1-m18,

(48) SEQ ID NO: 151 mutein1-m19,

(49) SEQ ID NO: 152 m4001-m8,

(50) SEQ ID NO: 153 m4001-m21,

(51) SEQ ID NO: 154 m4001-m9,

(52) SEQ ID NO: 155 m4001-m1,

(53) SEQ ID NO: 156 m4001-m2,

(54) SEQ ID NO: 157 m4001-m3,

(55) SEQ ID NO: 158 m4001-m5,

(56) SEQ ID NO: 159 m4001-m13,

(57) SEQ ID NO: 160 m4001-m14,

(58) SEQ ID NO: 161 m4001-m24,

(59) SEQ ID NO: 162 m4001-m4,

(60) SEQ ID NO: 163 m4001-m6,

(61) SEQ ID NO: 164 m4001-m7,

(62) SEQ ID NO: 165 m4001-m10,

(63) SEQ ID NO: 166 m4001-m15,

(64) SEQ ID NO: 167 m4001-m23,

(65) SEQ ID NO: 168 m4001-m17,

(66) SEQ ID NO: 169 m4001-m12,

(67) SEQ ID NO: 170 m4001-m20,

(68) SEQ ID NO: 171 mutein1-m101,

(69) SEQ ID NO: 172 mutein1-m106,

(70) SEQ ID NO: 173 mutein1-m111,

(71) SEQ ID NO: 174 mutein1-m100,

(72) SEQ ID NO: 175 mutein1-m110,

(73) SEQ ID NO: 176 mutein1-m104,

(74) SEQ ID NO: 177 mutein1-m108,

(75) SEQ ID NO: 178 mutein1-m207,

(76) SEQ ID NO: 179 mutein1-m212,

(77) SEQ ID NO: 180 mutein1-m202,

(78) SEQ ID NO: 181 mutein1-m204,

(79) SEQ ID NO: 182 mutein1-m206,

(80) SEQ ID NO: 183 mutein1-m208,

(81) SEQ ID NO: 184 mutein1-m203,

(82) SEQ ID NO: 185 mutein1-m209,

(83) SEQ ID NO: 186 mutein1-m200,

(84) SEQ ID NO: 187 mutein1-m201,

(85) SEQ ID NO: 188 mutein1-m211,

(86) SEQ ID NO: 189 mutein1-m300,

(87) SEQ ID NO: 190 mutein1-m301,

(88) SEQ ID NO: 191 mutein1-m302,

(89) SEQ ID NO: 192 mutein1-m303,

(90) SEQ ID NO: 193 mutein1-m304, and

(91) SEQ ID NO: 194 mutein m1-9.

EXAMPLES

(92) General Methods

(93) 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 TG1 (Stratagene) was used for library expression of secreted streptavidin muteins, E. coli K12 TOP10 (Life Technologies) for cloning and library expression, and E. coli K12 JM83 (Yanisch-Peron et al., (1985), Gene 33, 103-119) for periplasmic expression of E. coli cytochromeb562 and alkaline phosphatase, both fused to Strep-tagII. Cytosolic expression of the muteins for subsequent protein isolation for coupling to Sepharose or for coating microtitre plates was carried out according to Schmidt and Skerra (1994), supra. Plasmid sequencings were carried out according to the standard dideoxy technique by Sequence Laboratories Gttingen GmbH. The primers and oligonucleotides were synthesized using an Applied Biosystems Expedite DNA synthesizer.

Example 1: Preparation of Library1

(94) A plasmid bank with DNA sequences which code for streptavidin derivatives mutagenized in the region of amino acid positions 44 to 52 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm1 using pfu-polymerase (Fermentas) and the following primers P1 and P2:

(95) P1: 5-TCG TGA CCG CGG GTG CAG ACG GAG CTC TGA CCG GTA CCT ACN N(C/G)N N(G/T)G CGC GTG GCA ACG CCG AGN N(C/G)C GCT ACG TCC TGA CCG GTC GTT (SEQ ID NO. 5) where the 3 terminal T was linked via a phosphorothioate bond and

(96) P2: 5-AGT AGC GGT AAA CGG CAG A (SEQ ID NO. 6).

(97) DNA sequences were generated in this manner which contained 32-fold degenerated codons at each of the positions 44, 45 and 52 of streptavidin mutein 1 encoding all of the 20 amino acids or a stop codon. The resulting PCR product was purified by gel electrophoresis, cleaved with SacII and HindIII and ligated into the correspondingly cleaved vector fragment of pASK-IBA2-SAm1.

(98) E. coli TOP10 cells were transformed with the vector mixture using the calcium chloride method (Sambrook et al., 1989).

Example 2: Preparation of Library2

(99) A plasmid bank with DNA sequences which code for streptavidin derivatives mutagenized in the region of amino acid positions 44 to 52 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm1 using pfu-polymerase (Fermentas) and the following primers P2 and P3:

(100) P3: 5-CTG ACC GGT ACC TAC G(G/C)T TGC NN(G/C) NN(G/T) GGC AAC GCC GAG TGC CGC TAC GTC CTG A (SEQ ID NO. 7) where the 3 terminal A was linked via a phosphorothioate bond and

(101) P2: 5-AGT AGC GGT AAA CGG CAG A (SEQ ID NO. 6).

(102) DNA sequences were generated in this manner which contained fixed mutations Thr45-->Cys and Ser52-->Cys, a 2-fold degenerated codon at position 44 encoding Gly or Ala and 32-fold degenerated codons encoding all of the 20 amino acids or a stop codon at each of the positions 46 and 47 of streptavidin mutein 1. The resulting PCR product was purified by gel electrophoresis, cleaved with KpnI and HindIII and ligated into the correspondingly cleaved vector fragment of pASK-IBA2-SAm1.

(103) E. coli TOP10 cells were transformed with the vector mixture using the calcium chloride method (Sambrook et al., 1989).

Example 3: Preparation of Library3

(104) A plasmid bank with DNA sequences which code for streptavidin mutein 1 derivatives mutagenized in the region of amino acid positions 115 to 121 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm1 using PfuUltra polymerase (Stratagene) and the following primers P4 and P5:

(105) P4: 5-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA (SEQ ID NO. 8) which was phosphorylated at the 5 end and

(106) P5: 5-GTT (A/C)NN CTC GGT GGT GCC GGA GGT (SEQ ID NO. 9) equally phosphorylated at the 5 end.

(107) Linear DNA sequences of the whole vector were generated in this manner which contained streptavidin mutein 1 gene variants with 32-fold degenerated codons at each of the positions 117, 120 and 121 encoding all of the 20 amino acids or a stop codon. The resulting PCR product was purified by gel electrophoresis and ligated. This strategy of amplifying the whole vector with a blunt end generating proof-reading polymerase using phosphorylated primers has the advantage that no restriction enzymes have to be used and, moreover, that a one fragment ligation can be performed which, being a monomolecular reaction, is concentration independent and more efficient than a two fragment ligation as used for the generation of libraries 1 and 2.

(108) E. coli TOP10 or/and TG1 cells were transformed with the ligated vector mixture using electroporation with a Bio-Rad MicroPulser using the manufacturers standard program Ec2 (0.2 cm cuvettes; 2.5 kV).

Example 4: Preparation of Library4

(109) A plasmid bank with DNA sequences which code for streptavidin mutein m4001 derivatives mutagenized in the region of amino acid positions 115 to 121 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm4001 using PfuUltra polymerase (Stratagene) and the following primers P4 and P5:

(110) P4: 5-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA (SEQ ID NO. 8) which was phosphorylated at the 5 end and

(111) P5: 5-GTT (A/C)NN CTC GGT GGT GCC GGA GGT (SEQ ID NO. 9) equally phosphorylated at the 5 end.

(112) Linear DNA sequences of the whole vector were generated in this manner which contained streptavidin mutein m4001 gene variants with 32-fold degenerated codons at each of the positions 117, 120 and 121 encoding all of the 20 amino acids or a stop codon. The resulting PCR product was purified by gel electrophoresis and ligated.

(113) E. coli TOP10 or/and TG1 cells were transformed with the ligated vector mixture using electroporation with a Bio-Rad MicroPulser using the manufacturers standard program Ec2 (0.2 cm cuvettes; 2.5 kV).

Example 5: Preparation of Library5

(114) A plasmid bank with DNA sequences which code for streptavidin mutein 1 derivatives mutagenized in the region of amino acid positions 115 to 121 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm1 using PfuUltra polymerase (Stratagene) and the following primers P4 and P6:

(115) P4: 5-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA (SEQ ID NO. 8) which was phosphorylated at the 5 end and

(116) P6: 5-GTT A(A/T)A CTC GGT GGT GCC GGA GGT (SEQ ID NO. 10) equally phosphorylated at the 5 end.

(117) Linear DNA sequences of the whole vector were generated in this manner which contained streptavidin mutein 1 gene variants with a 2-fold degenerated codon at position 117 encoding Phe or Tyr and 32-fold degenerated codons at each of the positions 120 and 121 encoding all of the 20 amino acids or a stop codon. The resulting PCR product was purified by gel electrophoresis and ligated.

(118) E. coli TOP10 or/and TG1 cells were transformed with the ligated vector mixture using electroporation with a Bio-Rad MicroPulser using the manufacturers standard program Ec2 (0.2 cm cuvettes; 2.5 kV).

Example 6: Preparation of Library6

(119) A plasmid bank with DNA sequences which code for streptavidin mutein 1 derivatives mutagenized in the region of amino acid positions 115 to 121 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm1 using PfuUltra polymerase (Stratagene) and the following primers P7 and P8:

(120) P7: 5-N(G/C)N N(G/T)T CCA CGC TGG TCG GCC AC (SEQ ID NO. 11) which was phosphorylated at the 5 end and

(121) P8: 5-N(A/C)N NCT CGG TGG TGC CGG AGG T (SEQ ID NO. 12) equally phosphorylated at the 5 end.

(122) Linear DNA sequences of the whole vector were generated in this manner which contained streptavidin mutein 1 gene variants with deleted amino acid positions 118 and 119 and 32-fold degenerated codons at each of the positions 117, 120 and 121 encoding all of the 20 amino acids or a stop codon. The resulting PCR product was purified by gel electrophoresis and ligated.

(123) E. coli TOP10 or/and TG1 cells were transformed with the ligated vector mixture using electroporation with a Bio-Rad MicroPulser using the manufacturers standard program Ec2 (0.2 cm cuvettes; 2.5 kV).

Example 7: Preparation of Library7

(124) A plasmid bank with DNA sequences which code for streptavidin mutein 1 derivatives mutagenized in the region of amino acid positions 115 to 121 (with reference to wt-streptavidin) was prepared by PCR amplification of pASK-IBA2-SAm1 using PfuUltra polymerase (Stratagene) and the following primers P9 and P10:

(125) P9: 5-GGN N(G/T)T CCA CGC TGG TCG GCC AC (SEQ ID NO. 13) which was phosphorylated at the 5 end and

(126) P10: 5-A(C/A)N NCT CGG TGG TGC CGG AGG T (SEQ ID NO. 14) equally phosphorylated at the 5 end.

(127) Linear DNA sequences of the whole vector were generated in this manner which contained streptavidin mutein 1 gene variants with deleted amino acid positions 118 and 119, a fixed Trp at position 120 and 32-fold degenerated codons at each of the positions 117 and 121 encoding all of the 20 amino acids or a stop codon. The resulting PCR product was purified by gel electrophoresis and ligated.

(128) E. coli TOP10 or/and TG1 cells were transformed with the ligated vector mixture using electroporation with a Bio-Rad MicroPulser using the manufacturers standard program Ec2 (0.2 cm cuvettes; 2.5 kV).

Example 8: Identification of Streptavidin Muteins with an Increased Binding Affinity for Peptide Ligands in a Filter Sandwich Assay (Cf. U.S. Pat. No. 6,103,493)

(129) In order to identify streptavidin muteins with an increased binding affinity for peptide ligands, a fusion protein was prepared comprising the alkaline phosphatase of E. coli (BAP) and the Strep-tagII peptide (WSHPQFEK) which was attached to its C-terminus as encoded by pASK75-phoA (SEQ ID NO. 1). For this pASK75-phoA was expressed with JM83 and the recombinant protein was purified as described in U.S. Pat. No. 6,103,493 with the sole difference that Strep-Tactin instead of streptavidin affinity chromatography and using desthiobiotin instead of diaminobiotin as the eluting agent according to the procedure of Schmidt and Skerra (2007), supra, was used. Desthiobiotin was removed by dialysis prior to using the BAP-Strep-tagII fusion protein (also denoted BAP-StrepII) in further assays.

(130) E. coli cells (TG1 or TOP10) transformed with the plasmid banks obtained in example 1-7 were plated out on nitrocellulose acetate membranes (type OE66, 110 mm diameter, Whatman) which had been placed on an agar plate containing LB medium which contained 100 g/ml ampicillin. The membrane was incubated for 24 hours at 30 C. until colonies became visible.

(131) During this incubation, a second membrane was prepared. An Immobilon-P membrane (Millipore) of similar size was coated at room temperature for ca. 6 hours with a total volume of 10 ml of rabbit anti-streptavidin immunoglobulin (Sigma) diluted 1:200 with PBS (4 mM KH2PO4, 16 mM Na2HPO4, 115 mM NaCl) and afterwards was blocked for ca. 2 hours in 3% w/v bovine serum albumin (BSA), 0.5% v/v Tween in PBS.

(132) This second membrane was washed with PBS and placed on an agar plate containing LB medium which contained 100 g/ml ampicillin and 0.2 g/ml anhydrotetracyclin. Subsequently the nitrocellulose membrane with the colonies on the upper side was placed on the second membrane and the relative positions of the two membranes were marked. After incubation overnight at room temperature, the upper membrane with the colonies was removed and stored on a fresh LB ampicillin agar plate at 4 C. The second membrane was also removed from the agar plate and washed three times for 30 minutes while shaking in PBS/Tween (0.1% v/v Tween20 in PBS). Subsequently the membrane was admixed with 10 ml fresh PBS/Tween solution containing the purified BAP-Strep-tagII fusion protein (2 g/ml). After incubating for one hour at room temperature, the membrane was washed again twice in PBS/Tween and twice in PBS buffer. The signal generation took place for 1 to 2 hours in the presence of 10 ml AP buffer (100 mM Tris-Cl pH 8.8, 100 mM NaCl, 5 mM MgCl.sub.2) with addition of 30 l bromo-chloro-indolylphosphate (BCIP) (50 mg/ml in dimethylformamide) and 5 l nitroblue tetrazolium (NBT) (75 mg/ml in 70% v/v dimethylformamide). The color spots which formed in this process were assigned to corresponding colonies on the first membrane. After isolation and culture of several signal generating clones, the corresponding plasmid DNA was isolated, sequenced and the deduced amino acid sequence at the randomized positions is shown in Tables 1-7 together with the relative signal intensity obtained in the filter assay described above. Signal intensities from different libraries cannot be compared as they arose from different non parallel experiments. Surprisingly, sequencing of signal positive clones from library3 unexpectedly revealed in some cases the deletion of amino acids at positions 118 and 119. These deletions can be explained by the presence of defective primers in the P4 and P5 primer preparations, each shortened at the 5 end by 3 bases.

Example 9: Production of Streptavidin Muteins on a Preparative Scale

(133) The known expression system for recombinant minimal streptavidin (Schmidt and Skerra (1994), supra) was used to produce streptavidin muteins 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. Each streptavidin mutein described in the present application was obtained at >90% purity. Disulfide formation of the cysteines at positions 45 and 52 in purified streptavidin mutein m4001 was determined to be 98.6% by probing a 234 M solution (determined by using the theoretic molar extinction coefficient 280=42060 cm.sup.1M.sup.1 for the monomer) in comparison to a serial dilution of a reduced 1,4-dithio-D-threitol (DTT) standard with Ellman's reagent (5,5-dithiobis-(2-nitrobenzoic acid)) and measuring absorbance at 412 nm.

Example 10: ELISA

(134) An ELISA was carried out to determine the binding affinity of the streptavidin muteins for the peptide ligand Strep-tagII.

(135) The wells of a 96-well microtitre plate (Costar) were coated overnight at 4 C. with 100 l of a solution of recombinant streptavidin muteins of the invention (m400 from library 1; m4001 from library 2; m4, m23, m36 m41, m45 from library 3; m101, m111 from library5; m207, m212 from library 6; and m301, m302 from library 7) as well as streptavidin mutein 1 of U.S. Pat. No. 6,103,493 at a concentration in each case of 15 g/ml in 10 mM NaBO.sub.3, pH 8.5. Further proceeding was at room temperature (23 C.). The wells were blocked for 2 hours with each 200 l 3% w/v BSA, 0.5% v/v Tween20 in TBS (100 mM Tris-Cl pH8, 115 mM NaCl). After washing three times with TBS/Tween (TBS containing 0.05% v/v Tween20), 50 l of the same buffer was added to each well. 50 l 0.3 M BAP-StrepII fusion protein in TBS/Tween (prepared by 67 fold dilution with TBS/T of a 20 M purified and dialysed BAP-StrepII stock solution in PBS) was added to the first well of each row and mixed. A dilution series was set up in the other wells of a row by pipetting 50 l (from a total of 100 II) out of the first well and mixing it with the contents (50 l) of the next well in the same row etc. In this manner concentrations of the fusion protein between 150 nM in the first well of each row and 0.146484 nM in the eleventh well were obtained.

(136) After incubating for one hour the solutions were removed and the wells were each washed twice with TBS/Tween and twice with TBS. Subsequently 100 l of a solution of 0.5 mg/ml p-nitrophenyl phosphate in 1 mM ZnSO.sub.4, 5 mM MgCl.sub.2, 1 M Tris-Cl pH8 was pipetted into each well. Data of each well were raised by measuring absorbance at 405 nm subtracted by absorbance at 595 nm using a BioTek microplate reader e1808. The activity of the bound BAP-StrepII fusion protein in each well was measured as difference value between the data obtained prior and after a 20 min incubation at 23 C. under shaking and is given in milli optical density units (mOD) for (A.sub.405A.sub.595) in FIG. 1.

(137) The data were evaluated assuming a single binding equilibrium between streptavidin mutein monomers (P) and the BAP-StrepII fusion protein (L) which yielded a dissociation constant K.sub.D=[P]*[L]/[P*L]. Under the assumption that [P].sub.tot=[P]+[P*L] and that [L] is very much larger than [P*L] so that [L].sub.tot is approximately the same as [L], the amount of bound fusion enzyme BAP-StrepII is determined as [P*L]=[L].sub.tot*[P].sub.tot/(K.sub.D+[L].sub.tot). This equation was used for fitting the measured data for [P*L](in terms of enzyme activity, (A.sub.405A.sub.595)/t) against [L].sub.tot (the concentration of the applied BAP-StrepII fusion enzyme) by non-linear least squares regression, with K.sub.D and [P].sub.tot (corresponding to the asymptotic activity value, ((A.sub.405A.sub.595)/t)max) as the parameters. FIG. 1 shows the graph of experimental results obtained from 2 selected streptavidin muteins of the invention in comparison to streptavidin mutein 1 of U.S. Pat. No. 6,103,493. ((A.sub.405A.sub.595) for the control where no streptavidin (mutein) was immobilized and where the wells were blocked only was <1 for each of the BAP-StrepII concentrations during the 20 min incubation (not shown in the graph)). Table 8 gives the evaluated results for K.sub.D and ((A.sub.405A.sub.595)/t)max for a larger selection of streptavidin muteins of the invention in comparison to streptavidin mutein 1 of U.S. Pat. No. 6,103,493. It can be seen from these data that the streptavidin muteins of the invention have a significantly higher affinity for Strep-tagII fused to BAP than streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and, therefore, are more suitable for efficient immobilization of Strep-tag(II) fusion proteins to a solid phase, as shown in this ELISA.

Example 11: Affinity Chromatography

(138) The streptavidin muteins (m400 from library 1; m4001 from library 2; m4, m23, m36 m41, m45 from library 3; m111 from library5; m207, m212 from library 6; and m301, m302 from library 7) were prepared as described in example 9 as well as streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and wt-streptavidin. Then, the proteins 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 with the respective streptavidin mutein was determined with a BCA assay (Pierce) according to the instructions of the manufacturer. Briefly, 50 l of a 10% v/v Sepharose gel suspension in buffer (100 mM Tris-Cl pH8) were mixed with the freshly prepared BCA reagent and incubated for 30 min at 37 C. At each measurement a reference curve was determined in parallel with standards of streptavidin mutein 1 at different concentrations dissolved in the same buffer. The determined Sepharose gel loading with the different streptavidin muteins with respect to the fitted (2nd order polynomial) reference curve is shown in Table 9 and 10 as result of the mean value of 3 independent measurements. As validity control for this BCA assay based determination method for solid phase bound streptavidin muteins, a reference Sepharose gel of known loading with streptavidin mutein 1 was measured in parallel. The resulting BCA derived value deviated by less than 1% from the reference measurement value thereby proving the BCA assay to provide reliable data for determination of Sepharose bound streptavidin muteins.

(139) In order to examine the behaviour of the streptavidin muteins including streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and wt-streptavidin immobilized in this manner in the affinity purification of Strep-tagII-carrying fusion proteins, the recombinant cytochromeb562 (Schmidt and Skerra 1994, supra) fused to the Strep-tagII (also denoted cytb562-StrepII) was expressed via the tet promoter/operator controlled plasmid pASK-IBA2-cytochromeb562 (SEQ ID NO. 2), essentially as described in Schmidt & Skerra (2007), supra. Briefly, E. coli JM83 was transformed with pASK-IBA2-cytochromeb562 and cultivated at 37 C. in LB medium containing 100 g/ml ampicillin. Expression was induced at an OD.sub.550=0.5 with 0.2 g/ml anhydrotetracycline and continued for 3 h at 37 C. Cells were then harvested via centrifugation and resuspended in a hundredth volume (with respect to the culture volume) pre-chilled buffer W (100 mM Tris-Cl pH8, 150 mM NaCl, 1 mM EDTA), e.g. in 10 ml when derived from a culture volume of 1 liter. Cells are lysed via sonication and cell debris were removed by centrifugation (30000 g, 15 min, 4 C.). The cleared supernatant was then subjected to Strep-Tactin affinity chromatography to purify the recombinant cytochromeb562-Strep-tagII fusion protein. After purification, desthiobiotin was removed by dialysis against buffer W and such prepared cytochromeb562-Strep-tagII fusion protein was then used for the following affinity chromatography experiments to characterize the streptavidin muteins of the invention in comparison to streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and in comparison to wt streptavidin.

(140) In a first affinity chromatography experiment, 450 l Sepharose gel (derived from 4.5 ml of a 10% suspension) with each of the different streptavidin muteins of the invention as well as streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and wt streptavidin was filled into a 2 ml column (Pierce, Cat. no. 89896) between 2 polyethylene filter discs. Then, 3 ml of 1 mg/ml purified cytochromeb562-Strep-tagII fusion protein in buffer W was applied at gravity flow to each column. In this way, each column was largely overloaded and cytochromeb562 Strep-tagII fusion protein emerges in the eluate. Then, each column was washed 2 times with 2.5 ml buffer W. Retained cytochrome was eluted with 10 mM biotin in buffer W and quantified spectrophotometrically by measuring absorbance of the eluate at 280 nm using the molar extinction coefficient E280=8250 M.sup.1 cm.sup.1. Results are given in Table 9. All muteins of the invention retained significantly more cytochromeb562 Strep-tagII fusion protein in comparison to mutein 1 of U.S. Pat. No. 6,103,493 (up to 3 times more) and by far more in comparison to wt streptavidin (up to 28 times more). Results were normalized to the amount of immobilized streptavidin (mutein). Thus, with the muteins of the invention, affinity columns of significantly improved Strep-tagII fusion protein binding capacity can be prepared.

(141) In a second affinity chromatography experiment, Sepharose gel with an aggregate of 1 mg streptavidin mutein of the invention, of 1 mg streptavidin mutein 1 of U.S. Pat. No. 6,103,493 and of 1 mg wt streptavidin (derived from the corresponding amount of a 10% suspension) was filled into a 2 ml column (Pierce, Cat. no. 89896) between 2 polyethylene filter discs. Then, 10 times 5 ml of 10 g/ml purified cytochromeb562-Strep-tagII fusion protein (500 g in total) in buffer W was applied at gravity flow to each column. The flow rate was in all cases between 0.6 and 0.8 ml per min. Each column was washed with 1 column volume (CV) buffer W. Captured cytochromeb562-Strep-tagII fusion protein was eluted by the addition of 10 mM biotin in buffer W and quantified spectrophotometrically by measuring absorbance of the eluate at 280 nm using the molar extinction coefficient E280=8250 M.sup.1 cm-1. Results are given in Table 10. All muteins of the invention captured significantly more cytochromeb562 Strep-tagII fusion protein in comparison to mutein 1 of U.S. Pat. No. 6,103,493 (up to more than 3 times more) per immobilized streptavidin (mutein). Thus, with the muteins of the invention, affinity columns can be prepared providing significantly improved yields of a Strep-tagII fusion protein applied in comparatively diluted form as it is, e.g., the case for recombinant proteins secreted by mammalian cells to the cell culture medium. Recoveries of up to nearly 70% of the applied Strep-tagII fusion protein were obtained with the muteins of the invention while using an affinity material amount providing only a theoretic 2 fold excess of immobilized Strep-tagII binding sites over the applied Strep-tagII ligand at the fusion protein (cytochromeb562 in this case), thereby demonstrating the efficiency of affinity capture of Strep-tagII fusion proteins using streptavidin muteins of the invention immobilized to a resin.

(142) It has further to be noted that, in contrast to streptavidin mutein 1 of U.S. Pat. No. 6,103,493, the use of desthiobiotin did not lead to efficient elution in the case of most of the streptavidin muteins of the invention. When biotin was used instead, sharp elution was also achieved in the case of the muteins.

Example 12: Affinity Increase Due to Mutations in the Region of Amino Acids 115-121 are Independent from the Context of Amino Acid Region 43-52

(143) Mutein m4001-m9 (FIG. 4, comprising the amino acid sequence of SEQ ID NO: 58 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 at positions 117 to 121 of the streptavidin amino acid sequence) was one of the top results of screening a library of streptavidin muteins based on streptavidin mutein m4001 carrying the amino acids Ala, Cys, Gly, Cys at positions 44, 45, 46, 52, respectively (FIG. 4), where amino acid positions 117, 120 and 121 were randomized. The selection criterion/quality determining parameter was signal intensity produced in the filter sandwich assay (Example 8). Based on these results, the identified amino acids Glu, Gly and Tyr at positions 117, 120 and 121 were transferred from the context of mutein m4001 into the context of mutein 1 to obtain streptavidin mutein m1-9 (see FIG. 4 for the complete amino acid sequence of mutein m1-9). Thus, a result (the improved binding affinity towards streptavidin binding peptides) obtained for the amino acid region 115-121 in the context of a certain amino acid sequence for the loop formed by amino acid residues 43-52, i.e. the sequence of m4001, was combined with another amino acid sequence for region 43-52, i.e. the sequence of mutein 1 in this case. Affinities were measured via BiaCore for the interaction of this new combined streptavidin mutein m1-9 with 2 different Strep-tagII (mono-tag) or di-tag3 fusion proteins and compared to the affinities of the fusion proteins to the streptavidin mutein 1 (Table 11). The mutein m1-9 provides an affinity increase of a factor of around 10 for GFP-StrepII (Green Fluorescent Protein) and of around 30 for Cytb562-StrepII. This is in the same range as the affinity increases measured for a selection of streptavidin muteins of the present invention raised directly from the random libraries for their interaction with BAP-Strep-tagII (mono-tag) fusion protein (Table 8) and confirms the advantage that the muteins of the invention provide over the known mutein 1.

(144) The mutein (combination product) m1-9 was also tested in similar affinity chromatography experiments as described for a selection of streptavidin muteins raised directly from the random libraries in Example 10. Also in this practical application related setting, the mutein m1-9 emerged to be significantly superior over mutein 1, in a similar degree as compared to the streptavidin muteins that were selected directly from the random libraries,

(145) These results obtained with the mutein m1-9 demonstrate that the affinity increases generated by replacing amino acids in the region of amino acid positions 115-121 for a certain amino acid sequence context in the region of amino acid positions 43-52 can be combined with another amino acid sequence context in the region of amino acid positions 43-52 while preserving the beneficial properties of these mutations. Thus, this confirms that the results obtained for the mutations in region 115-121 are context independent and may, if wanted, be combined with other beneficial amino acid sequences in other regions of streptavidin. Thus, the current invention does not only provide novel advantageous streptavidin muteins but provides the additional benefit that the mutations identified here can also improve the properties of known streptavidin muteins.

Example 13: Repeated Affinity Chromatography Cycles with Crude Lysates (E. Coli)

(146) The streptavidin mutein m1-9 was immobilized on agarose (Superflow) essentially as described in Example 11. The resulting resin had a biotin binding capacity of 233 nmol/ml (corresponding to a loading density of 3.1 mg mutein m1-9 per ml resin, assuming an activity of 100%). A column was filled with 0.5 ml of the resin and used for the repeated purification of GFP-StrepII from a crude E. coli extract under gravity flow to test its suitability for repeated purification cycles. The yield and purity of GFP-StrepII were determined after each purification cycle.

(147) For this purpose, a cleared lysate of the total soluble content of E. coli cells after cytosolic expression of GFP-StrepII was prepared using Buffer W (100 mM Tris-Cl pH8, 150 mM NaCl, 1 mM EDTA) according to standard procedures described in the manuals of IBA GmbH (e.g. Manual (Twin) Strep-tag (version PR02-0025) available as PDF file at http://www.iba-lifesciences.com/technical-support.html). The lysate contained approximately 1.2 mg GFP-StrepII per ml. The column with 0.5 ml resin with mutein m1-9 was loaded in a first step with 0.25 ml cleared lysate. The column was washed 5 times with 1 column volume (CV) corresponding to 0.5 ml Buffer W and then eluted with 10 mM biotin in Buffer W. The column was regenerated (released from biotin) by washing it 2 times with 5 CV 10 mM NaOH. Then, a second amount of 0.25 ml cleared lysate was applied and again contained GFP-StrepII was isolated and the column was regenerated as described above. Then, in a third purification attempt on the same 0.5-ml column, the triple amount (0.75 ml) of cleared lysate was applied and the same process for purification of contained GFP-StrepII and column regeneration as described above was used. Then, in a fourth purification attempt on the same 0.5-ml column, again 0.75 ml cleared lysate were applied and the same process for purification of contained GFP-StrepII and column regeneration as described above was used. The results of this sequential purification experiment using the same column at each step are summarized in Table 12. As can be seen from Table 12, in each step the essentially same amount as applied of the Strep-Tag II fusion protein could be purified with a purity of more than 90%. Thus, this experiment demonstrates that affinity columns with streptavidin muteins of the invention can be reliably used for repeated purifications of a recombinant protein with constant high yields and high purity. Alternatively to using 10 CV 10 mM NaOH, the column can also be regenerated by washing with larger volumes of 10 mM HABA in Buffer W. This may be advisable if biotin has to be removed under milder conditions if, e.g., the resin or other support coated with a streptavidin mutein of the invention is sensitive against alkaline pH.

Example 14: Interaction of Mutein m1-9 with 2 Sequentially Arranged Streptavidin Binding Peptide Moieties (Di-Tag3)

(148) The present invention provides streptavidin muteins with significantly increased affinities for Strep-tagII fusion proteins than known streptavidin muteins. However, limitations may remain when higher affinities are required in a given application. This may be the case in purification scenarios where at least one of the binding partners-Strep-tagII fusion protein or the respective streptavidin mutein-is present or applied at very low concentration. Such examples are poor expression of the Strep-tagII fusion protein and/or using large buffer volumes for cell lysis or secreting the Strep-tagII fusion protein to the cell culture medium. In all these cases a large sample volume containing the target protein at low concentration is applied to the affinity column. On the other hand, also the dilution of the streptavidin mutein reaction partner leads to suboptimal performance which is e. g., the case for batch purification in contrast to column purification. However, also other applications downstream purification may take advantage from a higher affinity. Illustrative examples are the directed, mild and stable immobilization of dedicated target proteins (or other molecules chemically fused with a Strep-tagII) to be analyzed on a solid phase coated with a streptavidin mutein for assay development. Examples for solid phases and corresponding assays are microplates for ELISA, Biosensors for e.g. ForteBio's Octet or GE's BiaCore family of instruments providing label-free, real-time measurements for the analysis of protein:protein, protein:peptide, and protein:small molecule interactions, chips for high throughput analysis of a multitude of analytes bound to its surface or beads, like magnetic beads or Alphascreen beads or Luminex beads for e.g. protein:protein interaction analysis. In all such examples, the coating of the respective solid phase with a streptavidin mutein should provide a generic platform for the simple, reproducible, mild and stable immobilization of an arbitrary protein to said solid phase. This will be extremely helpful as otherwise, a specific immobilization procedure has to be developed separately for each protein to be immobilized and analyzed.

(149) Consequently, there may be still room for improved affinities to address these applications beyond affinity purification in a better way. Therefore, the binding characteristics of the streptavidin muteins of the invention was also tested for a tandem arrangement of two Strep-tagII binding sequences connected by a short linker (WSHPQFEKGGGSGGGSGGGSWSHPQFEK; SEQ ID NO: 103) that is named di-tag3 and that has been described in U.S. Pat. No. 7,981,632. U.S. Pat. No. 7,981,632 also describes for the Strep-tag affinity system the advantages of such a tandem arrangement-leading to simultaneous binding of both Strep-tagII sequences to a tetrameric streptavidin mutein thereby providing higher binding stability under maintenance of efficient competitive elution. While in the present example, the data were generated using di-tag3 fusion proteins, the benefits are, however, not limited to using this particular di-tag3 streptavidin binding mutein. Rather, the avidity effect will be also achieved with any other sequentially arranged streptavidin binding modules described in U.S. Pat. No. 7,981,632, thereby leading to other affinity characteristics being still enhanced.

(150) Consequently, the affinity increase of the interaction between the streptavidin muteins of the present invention, exemplified by mutein m1-9, and two sequentially arranged Strep-tagII binding moieties, exemplified by di-tag3, in comparison to its binding affinity for the monovalent Strep-tagII was analyzed via real-time interaction analysis on a BiaCore T100 instrument and compared with the affinity increase of the same di-tag3 versus Strep-tagII in case of mutein 1 (Table 11). The di-tag3 and Strep-tagII were presented at the C-terminus of two different recombinant proteins, namely GFP and cytochrome b562. The result was very surprising. While the di-tag3 led to an affinity increase in case of mutein 1 of a factor of merely 10 in case of GFP and merely 200-400 in case of cytochrome b562, the respective increase in case of the mutein m1-9 of the present invention was 300-600 and 10,000-40,000. Thus, the mutein m1-9 of the present invention provides an avidity effect comparable to IgG antibodies (Roitt et al., third ed., Mosby, St Louis, pages 6.3-6.4 1993) which is much more pronounced (increased by a factor of 50-100) than the avidity effect provided by the state of the art mutein 1.

(151) The off-rate (0.000015 s.sup.1) for avidic binding of di-tag3 to mutein m1-9 specifies an interaction with a half live of T.sub.1/2=ln 2/koff=46,209 sec=770 min=>12 h. In case of a monovalent interaction such a slow off-rate cannot be efficiently disrupted by competitive elution as it would need 2 days to release approximately 95% of the bound molecules under the assumption that no rebinding can occur. Thus, such a monovalent interaction would not be suitable for affinity chromatography using competitive elution which is preferable because it can be accomplished under mild physiological conditions and as it provides high purities as non-specific binding contaminants are minimally released from the resin during elution. However, the off-rate of an avidic binding interaction resembles under competitive conditions the off-rate of the single interacting moiety with the slowest off-rate. Therefore, a di-tag3 fusion protein can still be eluted rather efficiently from affinity resins carrying (immobilized thereon) streptavidin muteins of the present invention. This ability to elute these fusion proteins is consistent with the observations made in U.S. Pat. No. 7,981,632 for the streptavidin binding peptide di-tag3 interacting with mutein 1. The elution behavior from a column having immobilised thereon the mutein m1-9 was in fact similar for GFP-StrepII and GFP-di-tag3 illustrating that the theoretical considerations made above are of practical relevance (data not shown). The off-rate of GFP-StrepII specifies a T.sub.1/2=161 sec so that roughly 11 min are needed to displace 95% of the bound molecules. This is no obstacle for efficient elution during affinity chromatography. To obtain the target protein as concentrated as possible, the column may be in a first step submersed with 1 column volume (CV) of elution buffer containing a competitor. Then, the flow can be stopped for 10-20 min to allow for displacement of the bound target protein prior to eluting it by re-starting flow with elution buffer again. Alternatively, the elution may be performed at very slow flow rates to provide the target protein at higher concentrations.

(152) On the other hand, the very stable interaction between di-tag3 and the mutein m1-9 under non-competitive conditions (T.sub.1/2=>12 h) makes this interaction very attractive to be generically used for the directed, mild and stable immobilization of any given target protein that is fused (chemically or recombinantly) to di-tag3 (or any other sequentially arranged streptavidin mutein binding moieties as, e.g., described in U.S. Pat. No. 7,981,632) on a solid phase during the development of assays in analytical settings as described above (ELISA, Alphascreen, Luminex, BiaCore, Octet, to name a few). Moreover, reversibilty of the interaction in the presence of a competitor enables mild regeneration of the device or sample coated with a streptavidin mutein of the present invention to remove the bound di-tag3 fusion protein after analysis of a certain analyte and to couple another di-tag3 fusion protein for analysis of another analyte binding to the di-tag3 fusion protein. Mild regeneration conserves the device or sample and the coupled proteinaceous receptor being a streptavidin mutein of the present invention in this example.

(153) Another illustrative example where this property can be exploited is affinity determination via a surface plasmon resonance technology such as BiaCore. A suitable chip (e.g. CM5 in the case of BiaCore) is coated with a streptavidin mutein of the present invention. A di-tag3 fusion protein for which the affinity for a ligand is aimed to be determined is stably bound to the chip via its fused di-tag3 stably binding to the immobilized streptavidin mutein of the present invention. Then, after determining the binding kinetics of the ligand applied at a certain concentration, the chip has to be regenerated from the ligand which has to be applied at another concentration. If this is not possible without damaging the di-tag3 fusion protein, the same chip can be regenerated from the whole complex, i.e. di-tag3 fusion protein with bound ligand, by adding a competitor, e.g. biotin, and washing off the competitor, e.g. by using another competitor of reduced affinity (e.g. HABA (=2-(4-hydroxyphenylazo)benzoic acid)), in further steps and finally washing the chip with buffer alone. Finally, the chip is regenerated for binding a new amount of the di-tag3 fusion protein for analysis of ligand binding applied at another concentration without the need of using a new chip.

(154) The high affinity of di-tag3 fusion proteins for a mutein such as mutein m1-9 should lead to a very efficient exploitation of the binding sites of immobilized streptavidin mutein m1-9 during affinity chromatography. To test this assumption, a 0.6 ml column was packed with immobilized mutein m1-9 described above (233 nmol biotin binding capacity per m1). The column provides thus an amount of 140 nmol biotin binding sites. The column was overloaded with GFP-di-tag3 fusion protein by addition of 2.6 mg of the GFP fusion protein. The column was then washed with 5 CV buffer W and remaining GFP-di-tag3 fusion protein was eluted with 10 mM biotin in buffer W. Eluted GFP-di-tag3 was quantified to be 2.13 mg by measuring absorbance at 280 nm and using the theoretical extinction coefficient of E0.1%=1.053. This amount of 2.13 mg corresponds to 71 nmol GFP-di-tag3 fusion protein. Thus, the stochiometry is 2:1. It can be deduced from this stochiometry that each binding peptide di-tag3 occupies 2 binding sites on the mutein m1-9 and that each binding site is in complex with a Strep-tagII moiety, thereby corresponding to a capacity exploitation of near to 100%. From this result and from the strong avidity effect shown by the BiaCore data, irrespective whether the chip was loaded at low or at high density with mutein m1-9 (see Table 11), it can be deduced that one di-tag3 binding peptide occupies very efficiently the 2 binding sites that are located close to each other on one tetramer (in fact, each tetramer has 4 binding sites which are located pair wise in close proximity; Weber et al., 1989, Science 243, 85-88) so that there is no or only little competition by different di-tag3 sequences for such binding sites located in close proximity at the concentrations used in this experiment. This further means that it should be possible to dimerize in a well defined manner di-tag3 (or other sequentially arranged streptavidin binding epitopes as, e.g., disclosed in U.S. Pat. No. 7,981,632) fusion proteins or proteins conjugated to di-tag3 on a streptavidin mutein tetramer of the invention. Due to the very slow off-rate (T.sub.1/2=>12h) there is no considerable exchange of the complex forming partners within the time frame of standard analytical assays which are accomplished usually in the 1 h range. Thus, when using 2 differently labeled streptavidin mutein m1-9 preparations (the label may be a fluorescent label, a chromophoric label, an enzymatic label, a magnetic label (magnetic bead), or other beads as used in the alphascreen or luminex assay platforms, or simply an agarose bead of a certain size or any other addressable property) each labeled variant is complexed by a different di-tag3 fusion protein. In this case both complex preparations may be mixed without that significant interchange between labeled streptavidin muteins and di-tag3 fusion proteins occurs at standard assay durations being <1 h. To provide a clarifying example: Preparation 1 is composed of mutein m1-9-label1 complexed with di-tag3-fusion-protein X and preparation 2 is composed of mutein m1-9-label2 complexed with di-tag3-fusion-protein Y. Then both preparations may be mixed without formation of significant populations of mutein m1-9-label1 complexed with di-tag3-fusion-protein Y and mutein m1-9-label2 complexed with di-tag3-fusion-protein X. This property enables multiplex assays where e.g. in a sample or in a specimen (e.g. in immunocytochemistry) or on a cell or on any other entity, different targets may be simultaneously addressed by a dedicated label bound via a streptavidin mutein of the invention to a ligand specific for a certain target fused or conjugated to di-tag3 without getting artifacts from interchanged detection complexes. But as the di-tag3:streptavidin mutein complex can still be efficiently reversed by the addition of a competitor like biotin, this methodology optionally additionally allows the efficient removal of the label from the target. This may be important for re-using the sample or specimen or cell or any other biological entity, which may be a precious unique specimen, in further assays. As ligands are multimerized by this strategy (dimerized on one labelled streptavidin mutein tetramer or multimerized on labeled streptavidin mutein multimers multimerized e.g. via chemical crosslinking), also low affinity ligands may be used in this methodology for multiplexed labeling assays. In this case, also the ligand can be easily removed from the sample or specimen or cell or any other entity after monomerization by competitive disruption of the complex between di-tag3 and streptavidin mutein of the invention and subsequent washing (cf. Streptamer technology as e.g. described in Stemberger et al., 2012, PLoSONE, Volume 7| Issue 4|e35798). This is a further advantage of the streptavidin muteins of the present invention, illustrating the superior properties of these muteins over the known streptavidin muteins used for interacting with streptavidin binding peptides.

(155) The invention is further elucidated by the electronically filed sequence protocol, in which inter alia:

(156) SEQ ID NO 1: shows the nucleotide sequence of the expression vector pASK75-phoA which contains a sequence coding for the PhoA signal peptide (bold) followed by the sequence coding for E. coli alkaline phosphatase (BAP, underlined, continuous line) followed by the sequence coding for a linker (underlined, dotted line), followed by the sequence coding for the Strep-tagII (underlined, dashed line). The gene is operatively linked to the tetracyclin promoter/operator (tetP/O) for transcription regulation. The vector is suitable for periplasmic expression of a BAP-Strep-tagII fusion protein. General use of this tet-promoter based expression system is described in U.S. Pat. No. 5,849,576.

(157) SEQ ID NO 2: shows the nucleotide sequence of the expression vector pASK-IBA2-cytochromeb562 which contains a sequence coding for the OmpA signal peptide (bold) followed by the sequence coding for E. coli cytochromeb562 (Cytb562, underlined, continuous line) followed by the sequence coding for a linker (underlined, dotted line), followed by the sequence coding for the Strep-tagII (underlined, dashed line). The gene for the cytochromeb562 Strep-tagII fusion protein (also denoted cytb562-StrepII) is operatively linked to the tetracyclin promoter/operator (tetP/O) for transcription regulation. The vector is suitable for periplasmic expression of a cytb562-Strep-tagII fusion protein. General use of this tet-promoter based expression system is described in U.S. Pat. No. 5,849,576.

(158) SEQ ID NO 3: shows the nucleotide sequence of the expression vector pASK-IBA2-SAm1 which contains a sequence coding for the OmpA signal peptide (bold) followed by the sequence coding for streptavidin mutein 1 disclosed by U.S. Pat. No. 6,103,493 (Ala13-Ser139, underlined, continuous line). The gene is operatively linked to the tetracyclin promoter/operator (tetP/O) for transcription regulation. The vector is suitable for periplasmic expression of streptavidin mutein 1. General use of this tet-promoter based expression system is described in U.S. Pat. No. 5,849,576.

(159) SEQ ID NO 4: shows the nucleotide sequence of the expression vector pASK-IBA2-SAm4001 which contains a sequence coding for the OmpA signal peptide (bold) followed by the sequence coding for streptavidin mutein m4001 of the present invention (Ala13-Ser139, underlined, continuous line). The gene is operatively linked to the tetracyclin promoter/operator (tetP/O) for transcription regulation. The vector is suitable for periplasmic expression of streptavidin mutein m4001. General use of this tet-promoter based expression system is described in U.S. Pat. No. 5,849,576.

(160) SEQ ID NO 5: shows the nucleotide sequence of the oligonucleotide primer P1,

(161) SEQ ID NO 6: shows the nucleotide sequence of the oligonucleotide primer P2,

(162) SEQ ID NO 7: shows the nucleotide sequence of the oligonucleotide primer P3,

(163) SEQ ID NO 8: shows the nucleotide sequence of the oligonucleotide primer P4,

(164) SEQ ID NO 9: shows the nucleotide sequence of the oligonucleotide primer P5,

(165) SEQ ID NO 10: shows the nucleotide sequence of the oligonucleotide primer P6,

(166) SEQ ID NO 11: shows the nucleotide sequence of the oligonucleotide primer P7,

(167) SEQ ID NO 12: shows the nucleotide sequence of the oligonucleotide primer P8,

(168) SEQ ID NO 13: shows the nucleotide sequence of the oligonucleotide primer P9,

(169) SEQ ID NO 14: shows the nucleotide sequence of the oligonucleotide primer P10.

(170) 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.

(171) 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.

(172) 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.

(173) 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.

(174) TABLE-US-00001 TABLE1 Library1 custom character Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51 relative (SEQIDNO:203) signal Wt Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 (SEQIDNO:15) mutein1 Val.sup.44Thr.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 + (SEQIDNO:16) m400 custom character Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 ++ (SEQIDNO:18) m402 Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51 ++ (SEQIDNO:19)

(175) TABLE-US-00002 TABLE2 Library2 custom character Cys.sup.45 Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 relative (SEQIDNO:204) signal Wt Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 (SEQIDNO:205) mutein1 Val.sup.44Thr.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 + (SEQIDNO:16) m4001 custom character Cys.sup.45 Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 +++ (SEQIDNO:20)

(176) TABLE-US-00003 TABLE3 Library3(SEQIDNO:206) custom character Asn.sup.118Ala.sup.119 relativesignal Wt(SEQIDNO:207) Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 mutein1 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 m36(SEQIDNO:21) Asn.sup.118Ala.sup.119 +++++++ m23(SEQIDNO:22) Asn.sup.118Ala.sup.119 ++++++ m41(SEQIDNO:23) custom character ++++++ m4(SEQIDNO:24) Asn.sup.118Ala.sup.119 +++++ m12(SEQIDNO:25) Asn.sup.118Ala.sup.119 +++++ m22(SEQIDNO:26) Asn.sup.118Ala.sup.119 +++++ m31(SEQIDNO:27) Asn.sup.118Ala.sup.119 +++++ m32(SEQIDNO:28) Asn.sup.118Ala.sup.119 +++++ m35(SEQIDNO:29) +++++ m38(SEQIDNO:30) Asn.sup.118Ala.sup.119 custom character +++++ m40(SEQIDNO:31) Asn.sup.118Ala.sup.119 +++++ m42(SEQIDNO:32) Asn.sup.118Ala.sup.119 +++++ m45(SEQIDNO:33) Asn.sup.118Ala.sup.119 +++++ m46(SEQIDNO:34) Asn.sup.118Ala.sup.119 +++++ m47(SEQIDNO:35) Asn.sup.118Ala.sup.119 +++++ m7(SEQIDNO:36) Asn.sup.118Ala.sup.119 ++++ m10(SEQIDNO:37) custom character Asn.sup.118Ala.sup.119 ++++ m17(SEQIDNO:38) Asn.sup.118Ala.sup.119 ++++ m21(SEQIDNO:39) Asn.sup.118Ala.sup.119 ++++ m24(SEQIDNO:40) Asn.sup.118Ala.sup.119 ++++ m27(SEQIDNO:41) Asn.sup.118Ala.sup.119 ++++ m28(SEQIDNO:42) Asn.sup.118Ala.sup.119 ++++ m30(SEQIDNO:43) Asn.sup.118Ala.sup.119 custom character ++++ m33(SEQIDNO:44) Asn.sup.118Ala.sup.119 ++++ Library3(SEQIDNO:206) Asn.sup.118Ala.sup.119 relativesignal m1(SEQIDNO:45) Asn.sup.118Ala.sup.119 +++ m3(SEQIDNO:46) Asn.sup.118Ala.sup.119 +++ m8(SEQIDNO:47) Asn.sup.118Ala.sup.119 +++ m15(SEQIDNO:48) Asn.sup.118Ala.sup.119 +++ m6(SEQIDNO:49) custom character Asn.sup.118Ala.sup.119 ++ m9(SEQIDNO:50) Asn.sup.118Ala.sup.119 ++ m20(SEQIDNO:51) Asn.sup.118Ala.sup.119 ++ m34(SEQIDNO:52) ++ m14(SEQIDNO:53) Asn.sup.118Ala.sup.119 + m18(SEQIDNO:54) Asn.sup.118Ala.sup.119 + m19(SEQIDNO:55) Asn.sup.118Ala.sup.119 +

(177) TABLE-US-00004 TABLE4 Library4(SEQIDNO:208) custom character Asn.sup.118Ala.sup.119 relativesignal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 m4001 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 m8(SEQIDNO:56) Asn.sup.118Ala.sup.119 ++++++ m21(SEQIDNO:57) Asn.sup.118Ala.sup.119 ++++++ m9(SEQIDNO:58) Asn.sup.118Ala.sup.119 custom character +++++ m1(SEQIDNO:59) Asn.sup.118Ala.sup.119 ++++ m2(SEQIDNO:60) Asn.sup.118Ala.sup.119 ++++ m3(SEQIDNO:61) Asn.sup.118Ala.sup.119 ++++ m5(SEQIDNO:62) Asn.sup.118Ala.sup.119 ++++ m13(SEQIDNO:63) Asn.sup.118Ala.sup.119 ++++ m14(SEQIDNO:64) Asn.sup.118Ala.sup.119 ++++ m24(SEQIDNO:65) custom character Asn.sup.118Ala.sup.119 ++++ m4(SEQIDNO:66) Asn.sup.118Ala.sup.119 +++ m6(SEQIDNO:67) Asn.sup.118Ala.sup.119 +++ m7(SEQIDNO:68) Asn.sup.118Ala.sup.119 +++ m10(SEQIDNO:69) Asn.sup.118Ala.sup.119 +++ m15(SEQIDNO:70) Asn.sup.118Ala.sup.119 +++ m23(SEQIDNO:71) Asn.sup.118Ala.sup.119 custom character +++ m17(SEQIDNO:72) Asn.sup.118Ala.sup.119 ++ m12(SEQIDNO:73) Asn.sup.118Ala.sup.119 + m20(SEQIDNO:74) Asn.sup.118Ala.sup.119 +

(178) TABLE-US-00005 TABLE5 Library5(SEQIDNO:209) custom character Asn.sup.118Ala.sup.119 relativesignal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lsy.sup.121 mutein1 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lsy.sup.121 m101(SEQIDNO:75) Asn.sup.118Ala.sup.119 ++++++ m106(SEQIDNO:76) Asn.sup.118Ala.sup.119 ++++++ m111(SEQIDNO:77) Asn.sup.118Ala.sup.119 custom character ++++++ m100(SEQIDNO:78) Asn.sup.118Ala.sup.119 +++++ m110(SEQIDNO:79) Asn.sup.118Ala.sup.119 +++++ m104(SEQIDNO:80) Asn.sup.118Ala.sup.119 ++++ m108(SEQIDNO:81) Asn.sup.118Ala.sup.119 ++++

(179) TABLE-US-00006 TABLE6 Library6(SEQIDNO:210) custom character relativesignal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 mutein1 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 m207(SEQIDNO:82) ++++++ m212(SEQIDNO:83) ++++++ m202(SEQIDNO:84) +++++ m204(SEQIDNO:85) +++++ m206(SEQIDNO:86) +++++ m208(SEQIDNO:87) +++++ m203(SEQIDNO:88) custom character ++++ m209(SEQIDNO:89) ++++ m200(SEQIDNO:90) +++ m201(SEQIDNO:91) +++ m211(SEQIDNO:92) +++

(180) TABLE-US-00007 TABLE7 Library7(SEQIDNO:209) custom character relativesignal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 mutein1 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 m300(SEQIDNO:93) +++++++ m301(SEQIDNO:94) +++++++ m302(SEQIDNO:95) +++++++ m303(SEQIDNO:96) +++++++ m304(SEQIDNO:97) +++++++

(181) TABLE-US-00008 TABLE 8 K.sub.D (mutein1)/ ((A.sub.405 Mutein K.sub.D [nM] K.sub.Dmutein A.sub.595)/t).sub.max mutein 1 (SEQ ID NO: 110 1.0 703 16) m4 (SEQ ID NO: 24) 9 12.2 716 m23 (SEQ ID NO: 22) 12 9.2 968 m36 (SEQ ID NO: 21) 12 9.2 945 m41 (SEQ ID NO: 23) 13 8.5 613 m45 (SEQ ID NO: 33) 18 6.1 566 m101 (SEQ ID NO: 75) 14 7.9 589 m111 (SEQ ID NO: 77) 13 8.5 492 m207 (SEQ ID NO: 82) 27 4.1 493 m212 (SEQ ID NO: 83) 17 6.5 607 m301 (SEQ ID NO: 94) 10 11.0 725 m302 (SEQ ID NO: 95) 5 22.0 726 m402 (SEQ ID NO: 19) 92 1.2 880 m4001 (SEQ ID NO: 20) 20 5.5 986 m1-9 (SEQ ID NO: 194) 25 1696 m4001 (SEQ ID NO: 20) 27 1049 m4 (SEQ ID NO: 24) 12 518 m23 (SEQ ID NO: 22) 13 644

(182) TABLE-US-00009 TABLE 9 Retained Retained Cytb.sub.562-StrepII Sepharose immobilization Cytb.sub.562- per gel loading degree Retained StrepII per immobilized (100% gel relative to Cytb.sub.562- immobilized mutein relative suspension) mutein 1 StrepII mutein to mutein 1 Variant [g/ml] [%] [g] [g/g] [%] mutein 1 2747 100 156 0.13 100 streptavidin 2034 74 12 0.01 11 wt m4 3557 129 564 0.35 280 m23 3452 126 479 0.31 245 m36 2985 109 429 0.32 254 m41 3284 120 289 0.20 155 m45 2711 99 472 0.39 308 m111 2277 83 318 0.31 246 m207 2767 101 358 0.29 229 m212 3489 127 390 0.25 197 m301 3149 115 400 0.28 225 m302 2804 102 350 0.28 221 m402 1776 51 111 0.14 111 m4001 2429 65 175 0.16 127 Streptavidin muteins correspond to those listed in Table 8

(183) TABLE-US-00010 TABLE 10 captured Cytb.sub.562-StrepII Sepharose column size per yield of gel loading for 1 mg captured immobilized Cytb.sub.562- (100% gel immobilized Cytb.sub.562- mutein relative StrepII suspension) mutein StrepII to mutein 1 applied Variant [g/ml] [l] [g] [%] [%] mutein 1 2747 364 98 100 20 streptavidin 2034 492 4 4 1 wt m4 3557 281 321 327 64 m23 3452 290 301 308 60 m36 2985 335 307 314 61 m41 3284 305 235 240 47 m45 2711 369 335 342 67 m111 2277 439 337 344 67 m207 2767 361 274 279 55 m212 3489 287 233 238 47 m301 3149 318 285 291 57 m302 2804 357 313 320 63 m402 1776 563 153 156 31 m4001 2429 412 168 171 34 Streptavidin muteins correspond to those listed in Table 8

(184) TABLE-US-00011 TABLE 11 Relative streptavidin Streptavidin mutein density on k.sub.on 10.sup.5 [M.sup.1 k.sub.off 10.sup.4 Fusion protein mutein chip [RU] s.sup.1] [s.sup.1] K.sub.D [pM] GFP-StrepII mutein 1 325 1.00 300.00 300000 5567 1.90 250.00 130000 m1-9 170 1.50 47.00 32000 4350 1.70 38.00 23000 GFP-di-tag3 mutein 1 325 0.80 43.00 57000 5567 1.50 17.00 11000 m1-9 170 2.80 0.15 53 4350 2.20 0.17 79 Cytb.sub.562-StrepII mutein 1 325 0.17 2900.00 16700000 5567 0.14 2400.00 16700000 m1-9 170 0.70 410.00 586000 4350 0.52 250.00 485000 Cytb.sub.562-di-tag3 mutein 1 325 0.60 58.00 97000 5567 1.20 53.00 44000 m1-9 170 10.00 0.15 14 4350 4.40 0.19 43

(185) TABLE-US-00012 TABLE 12 0.5 ml column with 3.1 mg/ml streptavidin mutein m1-9 Purification Purification Purification Purification 1 2 3 4 Applied 0.25 0.25 0.75 0.75 volume of cleared lysate [ml] Yield of GFP- 0.57 0.54 1.80 1.82 StrepII [mg] Purity [%] 91.6 93.5 98.7 94.5

(186) TABLE-US-00013 TABLE13 SEQIDNO Name Sequence SEQIDNO.1 E.colialkalinephosphataseStrep-tagIIfusionprotein(BAP-StrepII)- expressedbypASK75-phoA SEQIDNO.2 plasmidpASK-IBA2-cytochromeb562 SEQIDNO3 nucleotidesequenceoftheexpressionvectorpASK-IBA2-SAm1whichcontainsa sequencecodingfortheOmpAsignalpeptidefollowedbythesequencecodingfor streptavidinmutein1 disclosedbyU.S.Pat.No.6,103,493 SEQIDNO4 nucleotidesequenceoftheexpressionvectorpASK-IBA2-SAm4001whichcontainsa sequencecodingfortheOmpAsignalpeptidefollowedbythesequencecodingfor thestreptavidinmuteinm4001ofthepresentinvention SEQIDNO.5 Primer1(P1) 5-TCGTGACCGCGGGTGCAGACGGAGCTCTGA CCGGTACCTACNN(C/G)NN(G/T)GCGCGTG GCAACGCCGAGNN(C/G)CGCTACGTCCTGA CCGGTCGTT Is: TCGTGACCGCGGGTGCAGACGGAGCTCTGA CCGGTACCTACNNSNNKGCGCGTGGCAACG CCGAGNNSCGCTACGTCCTGACCGGTCGTT SEQIDNO.6 Primer2(P2) 5-AGTAGCGGTAAACGGCAGA SEQIDNO.7 Primer3(P3) 5-CTGACCGGTACCTACG(G/C)TTGCNN(G/C) NN(G/T)GGCAACGCCGAGTGCCGCTACGTC CTGA Is: CTGACCGGTACCTACGSTTGCNNSNNKGGC AACGCCGAGTGCCGCTACGTCCTGA SEQIDNO.8 Primer4(P4) 5-GCCNN(G/C)NN(G/T)TCCACGCTGGTCGGCCA Is: GCCNNSNNKTCCACGCTGGTCGGCCA SEQIDNO.9 Primer5(P5) 5-GTT(A/C)NNCTCGGTGGTGCCGGAGGT Is: GTTMNNCTCGGTGGTGCCGGAGGT SEQIDNO.10 Primer6(P6) 5-GTTA(A/T)ACTCGGTGGTGCCGGAGGT Is: GTTAMACTCGGTGGTGCCGGAGGT SEQIDNO.11 Primer7(P7) 5-N(G/C)NN(G/T)TCCACGCTGGTCGGCCAC Is: NSNNKTCCACGCTGGTCGGCCAC SEQIDNO.12 Primer8(P8) 5-N(A/C)NNCTCGGTGGTGCCGGAGGT Is: NMNNCTCGGTGGTGCCGGAGGT SEQIDNO.13 Primer9(P9) 5-GGNN(G/T)TCCACGCTGGTCGGCCAC Is: GGNNKTCCACGCTGGTCGGCCAC SEQIDNO.14 Primer10(P10) 5-A(C/A)NNCTCGGTGGTGCCGGAGGT Is: AMNNCTCGGTGGTGCCGGAGGT SEQIDNO:15 wildtypestreptavidin Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 is:ESAVGNAES SEQIDNO:16 mutein1 Val.sup.44Thr.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 is:TARGNAES SEQIDNO:17 mutein2(44-47) IGAR SEQIDNO:18 m400 Gly.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 is:GCARGNAEC SEQIDNO:19 m402 Ala.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 is:ACARGNAEC SEQIDNO:20 m4001 custom character Cys.sup.45 Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 is:ACGRGNAEC SEQIDNO:21 m36 Asn.sup.118Ala.sup.119 is:YNAFM SEQIDNO:22 m23 Asn.sup.118Ala.sup.119 is:YNAYA SEQIDNO:23 m41 is:AXXWY SEQIDNO:24 m4 Asn.sup.118Ala.sup.119 is:DNAGF SEQIDNO:25 m12 Asn.sup.118 is:RNAGF SEQIDNO:26 m22 custom character Asn.sup.118Ala.sup.119 is:QNAGF SEQIDNO:27 m31 Asn.sup.118Ala.sup.119 is:FNASW SEQIDNO:28 m32 Asn.sup.118Ala.sup.119 is:DNAVM SEQIDNO:29 m35 is:AXXWM SEQIDNO:30 m38 Asn.sup.118Ala.sup.119 is:ENAGF SEQIDNO:31 m40 Asn.sup.118Ala.sup.119 is:YNAYS SEQIDNO:32 m42 Asn.sup.118Ala.sup.119 custom character is:FNAYG SEQIDNO:33 m45 Asn.sup.118Ala.sup.119 is:YNAGF SEQIDNO:34 m46 Asn.sup.118Ala.sup.119 is:RNAYA SEQIDNO:35 m47 Asn.sup.118Ala.sup.119 is:WNAYG SEQIDNO:36 m7 Asn.sup.118Ala.sup.119 is:LNAGF SEQIDNO:37 m10 Asn.sup.118Ala.sup.119 is:HNAGY SEQIDNO:38 m17 Asn.sup.118Ala.sup.119 custom character is:MNAGF SEQIDNO:39 m21 Asn.sup.118Ala.sup.119 is:RNAGY SEQIDNO:40 m24 Asn.sup.118Ala.sup.119 is:ENAGW SEQIDNO:41 m27 Asn.sup.118Ala.sup.119 is:HNAGF SEQIDNO:42 m28 Asn.sup.118Ala.sup.119 is:SNAGF SEQIDNO:43 m30 Asn.sup.118Ala.sup.119 is:TNAGF SEQIDNO:44 m33 Asn.sup.118Ala.sup.119 custom character is:NNAGF SEQIDNO:45 m1 Asn.sup.118Ala.sup.119 is:ENAGM SEQIDNO:46 m3 Asn.sup.118Ala.sup.119 is:WNACC SEQIDNO:47 m8 Asn.sup.118Ala.sup.119 is:MNAFV SEQIDNO:48 m15 Asn.sup.118Ala.sup.119 is:ANADW SEQIDNO:49 m6 Asn.sup.118Ala.sup.119 is:SNAMM SEQIDNO:50 m9 Asn.sup.118Ala.sup.119 is:RNAVV SEQIDNO:51 m20 custom character Asn.sup.118Ala.sup.119 is:SNASF SEQIDNO:52 m34 is:AXXWD SEQIDNO:53 m14 Asn.sup.118Ala.sup.119 is:RNARA SEQIDNO:54 m18 Asn.sup.118Ala.sup.119 is:SNAAF SEQIDNO:55 m19 Asn.sup.118Ala.sup.119 is:GNAMM SEQIDNO:56 m8 Asn.sup.118Ala.sup.119 is:ENAGF SEQIDNO:57 m21 Asn.sup.118Ala.sup.119 custom character is:DNAGY SEQIDNO:58 m9 Asn.sup.118Ala.sup.119 is:ENAGY SEQIDNO:59 m1 Asn.sup.118Ala.sup.119 is:RNAMM SEQIDNO:60 m2 Asn.sup.118Ala.sup.119 is:RNAGF SEQIDNO:61 m3 Asn.sup.118Ala.sup.119 is:ANAPA SEQIDNO:62 m5 Asn.sup.118Ala.sup.119 is:ANAMV SEQIDNO:63 m13 Asn.sup.118Ala.sup.119 is:QNASA SEQIDNO:64 m14 custom character Asn.sup.118Ala.sup.119 is:ANAGF SEQIDNO:65 m24 Asn.sup.118Ala.sup.119 is:QNAMV SEQIDNO:66 m4 Asn.sup.118Ala.sup.119 is:NNAGY SEQIDNO:67 m6 Asn.sup.118Ala.sup.119 is:ANAAV SEQIDNO:68 m7 Asn.sup.118Ala.sup.119 is:SNAMI SEQIDNO:69 m10 Asn.sup.118Ala.sup.119 is:HNAGY SEQIDNO:70 m15 custom character Asn.sup.118Ala.sup.119 is:SNAMA SEQIDNO:71 m23 Asn.sup.118Ala.sup.119 is:QNAVA SEQIDNO:72 m17 Asn.sup.118Ala.sup.119 is:YNAYM SEQIDNO:73 m12 Asn.sup.118Ala.sup.119 is:LNAWG SEQIDNO:74 m20 Asn.sup.118Ala.sup.119 is:HNASM SEQIDNO:75 m101 Asn.sup.118Ala.sup.119 is:YNAFL SEQIDNO:76 m106 custom character Asn.sup.118Ala.sup.119 is:FNAFL SEQIDNO:77 m111 Asn.sup.118Ala.sup.119 is:YNALW SEQIDNO:78 m100 Asn.sup.118Ala.sup.119 is:FNAYI SEQIDNO:79 m110 Asn.sup.118Ala.sup.119 is:YNAYL SEQIDNO:80 m104 Asn.sup.118Ala.sup.119 is:YNAYQ SEQIDNO:81 m108 Asn.sup.118Ala.sup.119 is:FNAIW SEQIDNO:82 m207 custom character is:TXXWL SEQIDNO:83 m212 is:HXXWL SEQIDNO:84 m202 is:IXXWR SEQIDNO:85 m204 is:HXXWT SEQIDNO:86 m206 is:TXXWR SEQIDNO:87 m208 is:AXXWR SEQIDNO:88 m203 is:RXXWS SEQIDNO:89 m209 is:NXXWR SEQIDNO:90 m200 is:KXXWS SEQIDNO:91 m201 is:SXXVF SEQIDNO:92 m211 is:KXXWT SEQIDNO:93 m300 custom character is:AXXWY SEQIDNO:94 m301 is:HXXWM SEQIDNO:95 m302 is:HXXWY SEQIDNO:96 m303 is:EXXWY SEQIDNO:97 m304 is:QXXWY SEQIDNO:98 streptavidinmutein Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (44-47= VTAR) is:VTAR SEQIDNO:99 streptavidinmutein Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 is:IGAR SEQIDNO:100 Strep-tagIIaffinity Trp-Ser-His-Pro-Gln-Phe-Glu-Lys peptideligand is:WSHPQFEK SEQIDNO:101 peptidesequence Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa inwhichXaarepresentsanarbitraryaminoacid andYaaandZaaeitherbothdenoteGlyorYaa denotesGluandZaadenotesArgorLys isWXHPQFXX SEQIDNO:102 Strep-tagstreptavidin Trp-Arg-His-Pro-Gln-Phe-Gly-Gly(WRHPQFGG) bindingpeptide SEQIDNO:103 Peptidesequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.3- Trp-Ser-His-Pro-Gln-Phe-Glu-Lys IsWSHPQFEKGGGSGGGSGGGSWSHPQFEK SEQIDNO:104 GFP-StrepII SEQIDNO:105 GFP-di-tag3 SEQIDNO:106 Cytb562-StrepII SEQIDNO:107 Cytb562-di-tag3 SEQIDNO:108 peptidesequence Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa whereOaaisTrp,LysorArg,Xaaisanyamino acidandwhereeitherYaaandZaaarebothGlyor YaaisGluandZaaisLysorArgisXXHPQFXX SEQIDNO:109 di-tag2 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.2- Trp-Ser-His-Pro-Gln-Phe-Glu-Lys IsWSHPQFEKGGGSGGGSWSHPQFEK SEQIDNO:110 Peptidesequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK SEQIDNO:111 Wildtype FIG.4 SEQIDNO:112 mutein1 FIG.4 SEQIDNO:113 mutein2 FIG.4 SEQIDNO:114 m400 FIG.4 SEQIDNO:115 m402 FIG.4 SEQIDNO:116 m4001 FIG.4 SEQIDNO:117 mutein1-m36 FIG.4 SEQIDNO:118 mutein1-m23 FIG.4 SEQIDNO:119 mutein1-m41 FIG.4 SEQIDNO:120 mutein1-m4 FIG.4 SEQIDNO:121 mutein1-m12 FIG.4 SEQIDNO:122 mutein1-m22 FIG.4 SEQIDNO:123 mutein1-m31 FIG.4 SEQIDNO:124 mutein1-m32 FIG.4 SEQIDNO:125 mutein1-m35 FIG.4 SEQIDNO:126 mutein1-m38 FIG.4 SEQIDNO:127 mutein1-m40 FIG.4 SEQIDNO:128 mutein1-m42 FIG.4 SEQIDNO:129 mutein1-m45 FIG.4 SEQIDNO:130 mutein1-m46 FIG.4 SEQIDNO:131 mutein1-m47 FIG.4 SEQIDNO:132 mutein1-m7 FIG.4 SEQIDNO:133 mutein1-m10 FIG.4 SEQIDNO:134 mutein1-m17 FIG.4 SEQIDNO:135 mutein1-m21 FIG.4 SEQIDNO:136 mutein1-m24 FIG.4 SEQIDNO:137 mutein1-m27 FIG.4 SEQIDNO:138 mutein1-m28 FIG.4 SEQIDNO:139 mutein1-m30 FIG.4 SEQIDNO:140 mutein1-m33 FIG.4 SEQIDNO:141 mutein1-m1 FIG.4 SEQIDNO:142 mutein1-m3 FIG.4 SEQIDNO:143 mutein1-m8 FIG.4 SEQIDNO:144 mutein1-m15 FIG.4 SEQIDNO:145 mutein1-m6 FIG.4 SEQIDNO:146 mutein1-m9 FIG.4 SEQIDNO:147 mutein1-m20 FIG.4 SEQIDNO:148 mutein1-m34 FIG.4 SEQIDNO:149 mutein1-m14 FIG.4 SEQIDNO:150 mutein1-m18 FIG.4 SEQIDNO:151 mutein1-m19 FIG.4 SEQIDNO:152 m4001-m8 FIG.4 SEQIDNO:153 m4001-m21 FIG.4 SEQIDNO:154 m4001-m9 FIG.4 SEQIDNO:155 m4001-m1 FIG.4 SEQIDNO:156 m4001-m2 FIG.4 SEQIDNO:157 m4001-m3 FIG.4 SEQIDNO:158 m4001-m5 FIG.4 SEQIDNO:159 m4001-m13 FIG.4 SEQIDNO:160 m4001-m14 FIG.4 SEQIDNO:161 m4001-m24 FIG.4 SEQIDNO:162 m4001-m4 FIG.4 SEQIDNO:163 m4001-m6 FIG.4 SEQIDNO:164 m4001-m7 FIG.4 SEQIDNO:165 m4001-m10 FIG.4 SEQIDNO:166 m4001-m15 FIG.4 SEQIDNO:167 m4001-m23 FIG.4 SEQIDNO:168 m4001-m17 FIG.4 SEQIDNO:169 m4001-m12 FIG.4 SEQIDNO:170 m4001-m20 FIG.4 SEQIDNO:171 mutein1-m101 FIG.4 SEQIDNO:172 mutein1-m106 FIG.4 SEQIDNO:173 mutein1-m111 FIG.4 SEQIDNO:174 mutein1-m100 FIG.4 SEQIDNO:175 mutein1-m110 FIG.4 SEQIDNO:176 mutein1-m104 FIG.4 SEQIDNO:177 mutein1-m108 FIG.4 SEQIDNO:178 mutein1-m207 FIG.4 SEQIDNO:179 mutein1-m212 FIG.4 SEQIDNO:180 mutein1-m202 FIG.4 SEQIDNO:181 mutein1-m204 FIG.4 SEQIDNO:182 mutein1-m206 FIG.4 SEQIDNO:183 mutein1-m208 FIG.4 SEQIDNO:184 mutein1-m203 FIG.4 SEQIDNO:185 mutein1-m209 FIG.4 SEQIDNO:186 mutein1-m200 FIG.4 SEQIDNO:187 mutein1-m201 FIG.4 SEQIDNO:188 mutein1-m211 FIG.4 SEQIDNO:189 mutein1-m300 FIG.4 SEQIDNO:190 mutein1-m301 FIG.4 SEQIDNO:191 mutein1-m302 FIG.4 SEQIDNO:192 mutein1-m303 FIG.4 SEQIDNO:193 mutein1-m304 FIG.4 SEQIDNO:194 Muteinm1-9 FIG.4 SEQIDNO:195 loopoftheaminoacids TTEANAWK 114to121of streptavidin SEQIDNO:196 loopregionofamino TEANAWK acids115to121of streptavidin SEQIDNO:197 mutein HPYFYAPELLFFAK SEQIDNO:198 mutein EGGKETLTPSELRDLV motif1 Xaa.sup.117Gly.sup.120Yaa.sup.121, consensussequence1 whereinXaamaybeanyaminoacidandYaamay bePheorTyrorMet isXGX-sonotmorethan4aminoacids SEQIDNO:199 motif1 XaaAsnAlaGlyZaa, (FIG.2B) XaaisGlu,Asp,Arg,His,Asn,Gln,Thr,Ser,Leu, MetandZaaisPhe,Tyr,MetisXNAGX motif2 Aaa.sup.117Baa.sup.120Caa.sup.121, consensussequence2 whereinAaamaybeTyr,Phe,Arg,TrporGln,Baa maybeTyr,Phe,Leu,IleorMetandCaamaybe anyaminoacid,whereinCaa121ispreferablya Leu,anIle,aMet,aGly,aGly,aTrp,aSer, anAlaoraValresidue SEQIDNO:200 Motif2 Xaa-Asn-Ala-Yaa-Zaa, (FIG.2B) whereinXaaisTyr,Phe,Arg,Trp,Gln; YaaisTyr,Phe,Leu,Ile,Metand ZaaisLeu,Ile,Met,Gln,Gly,Trp,Ser,Ala,Val IsXNAXX SEQIDNO:201 motif3 Daa.sup.117Eaa.sup.118Faa.sup.119Gaa.sup.120Haa.sup.121,whereinDaaand consensussequence3 HaamaybeanyaminoacidandEaaandFaaare bothdeletedandGaamaybeTrporVal, whereinDaa.sup.117ispreferablyaHis,aGlu,aGln,a Thr,anAlaoranIleresidueandwhereinHaa.sup.121is preferablyaTyr,aLeu,aMet,oranArgresidue isXXXXX SEQIDNO:202 Motif2 Xaa------YaaZaa, (FIG.2B) whereinXaaisHis,Glu,Gln,Thr,Ala,Ile,Arg, Asn,Lys,Ser; YaaisTrp,Valand ZaaisTyr,Leu,Met,Arg,Thr,Ser,Phe IsXXXXX SEQIDNO:203 Library1 custom character Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51 isXXARGNAEX SEQIDNO:204 Library2 (Ala/Gly)44Cys45Xaa46Xaa47Gly48Asn49Ala50Glu51 Cys52isXCXXGNAEC SEQIDNO:205 Wt(Library2) Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 isESAVGNAES SEQIDNO:206 Library3 custom character Asn.sup.118Ala.sup.119 IsXNAXX SEQIDNO:207 Wt(Library2)mutein1 Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 (Library2)Wt(Library4) IsANAWK m4001(Library4) Wt(Library5) mutein1 (Library5) Wt(Library6) mutein1 (Library6) Wt(Library7) mutein1 (Library7) SEQIDNO:208 Library4 custom character Asn.sup.118Ala.sup.119 SEQIDNO:209 Library5 Asn.sup.118Ala.sup.119 SEQIDNO:210 Library6 SEQIDNO:211 Library7 SEQIDNO:212 Wtstreptavidinamino EAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAP acidsequence(residues ATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTT 14to139) EANAWKSTLVGHDTFTKVKPSAAS

Streptavidin muteins and methods of using them

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Cpc classification

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C07K1/22

CHEMISTRY; METALLURGY

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G01N2333/36

PHYSICS

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G01N33/68

PHYSICS

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C12N15/62

CHEMISTRY; METALLURGY

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C07K2319/22

CHEMISTRY; METALLURGY

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C07K14/36

CHEMISTRY; METALLURGY

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C07K2319/70

CHEMISTRY; METALLURGY

International classification

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C07K14/36

CHEMISTRY; METALLURGY

Classification Explorer

G01N33/68

PHYSICS

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C07K1/22

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/62

CHEMISTRY; METALLURGY

Abstract

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