Streptavidin muteins and methods of using them

Abstract

The invention concerns novel streptavidin muteins. In one embodiment such a the mutein (a) contains at least two cysteine residues in the region of the amino acid positions 44 to 53 with reference to the amino acid sequence of wild type streptavidin as set forth at SEQ ID NO: 212 and (b) has a higher binding affinity than (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).

Claims

1. A mutein, selected from muteins of streptavidin, wherein the mutein (a) contains at least two cysteine residues in the region of the amino acid positions 44 to 53 with reference to the amino acid sequence of wild type streptavidin as set forth at SEQ ID NO: 212 and (b) has a higher binding affinity than (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).

2. A mutein according to claim 1, wherein the two cysteine residues are present at opposing positions in the loop formed by the residues of amino acid positions 44 to 53.

3. A mutein according to claim 1, wherein the two cysteine residues are present at amino acid positions 45 and 52 of wild type streptavidin.

4. A mutein according to claim 1, wherein the two cysteine residues are present at positions 44 and 53 of wild type streptavidin.

5. A mutein according to claim 1, wherein the two cysteine residues are present at positions 46 and 51 of wild type streptavidin.

6. A mutein according to claim 1, wherein the two cysteine residues are present at positions 47 and 50 of wild type streptavidin.

7. A mutein according to claim 1, wherein the two cysteine residues are present at positions 48 and 49 of wild type streptavidin.

8. A mutein according to claim 1, wherein a Gly or an Ala residue is present at amino acid position 44 of wild type streptavidin.

9. A mutein according to claim 1, wherein the amino acid residue at sequence position 46 is glycine.

10. A mutein according to claim 9, wherein the amino acid residue at sequence position 46 is glycine and the amino acid residue at sequence position 47 is arginine.

11. A mutein according to claim 1, wherein the mutein comprises an amino acid sequence at sequence positions 44 to 52 with reference to the amino acid sequence of wild type streptavidin as set forth at SEQ ID NO: 212 selected from the group consisting of TABLE-US-00028 (SEQ ID NO: 18) Gly.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 (SEQ ID NO: 19) Ala.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52, and (SEQ ID NO: 20) Ala.sup.44Cys.sup.45Gly.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52.

12. A mutein according to claim 1, wherein the mutein comprises the sequence of a mutein selected from the group consisting of mutein m400 (SEQ ID NO: 114), mutein m402 (SEQ ID NO: 115) and mutein m4001 (SEQ ID NO: 116).

13. A streptavidin mutein according to claim 1, wherein the mutein contains at least one mutation in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptavidin of which amino acid residues 14 to 139 are as set forth at SEQ ID NO: 212.

14. A mutein according to claim 13, wherein the mutein 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, wherein the mutein carries as mutated residue at sequence position 117 an amino acid residue selected from the group consisting of Glu, Asp, His, Gln and Arg or, wherein the mutein carries either the wild-type Trp at sequence position 120 or carries as mutated residue at sequence position 120 an amino acid residue selected from the group consisting of Ser, Gly, Met, and Pro, or wherein the mutein carries as mutated residue at sequence position 121 an amino acid residue selected from the group consisting of Leu, Tyr, Phe, and Met.

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

16. A mutein according to claim 1, wherein the binding affinity for the peptide ligand is such that a competitive elution can take place by streptavidin ligands selected from the group consisting of biotin, thiobiotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA and dimethyl-HABA.

17. A nucleic acid molecule, comprising a nucleic acid sequence coding for a streptavidin mutein according to claim 1.

18. 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.

19. 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.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1B show two graphs each 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-Tag®II in an ELISA as compared to streptavidin mutein “1” disclosed by U.S. Pat. No. 6,103,493.

(3) 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-Tag®II 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 μM for streptavidin mutein “1” of U.S. Pat. No. 6,103,493 and 0.02 μM 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.

(4) FIG. 1B shows the improved affinity of the streptavidin mutein m4 of the present invention shown in Table 3 for the peptide ligand Strep-Tag®II 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-Tag®II 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 μM for mutein “1” of U.S. Pat. No. 6,103,493 and 0.009 μM 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.

(5) 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.

(6) 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.

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

(8) 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:

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

(10) SEQ ID NO: 112 mutein“1”,

(11) SEQ ID NO: 113 mutein“2”,

(12) SEQ ID NO: 114 mutein m400,

(13) SEQ ID NO: 115 mutein m402,

(14) SEQ ID NO: 116 mutein m4001,

(15) SEQ ID NO: 117 mutein“1”-m36,

(16) SEQ ID NO: 118 mutein“1”-m23,

(17) SEQ ID NO: 119 mutein“1”-m41,

(18) SEQ ID NO: 120 mutein“1”-m4,

(19) SEQ ID NO: 121 mutein“1”-m12,

(20) SEQ ID NO: 122 mutein“1”-m22,

(21) SEQ ID NO: 123 mutein“1”-m31,

(22) SEQ ID NO: 124 mutein“1”-m32,

(23) SEQ ID NO: 125 mutein“1”-m35,

(24) SEQ ID NO: 126 mutein“1”-m38,

(25) SEQ ID NO: 127 mutein“1”-m40,

(26) SEQ ID NO: 128 mutein“1”-m42,

(27) SEQ ID NO: 129 mutein“1”-m45,

(28) SEQ ID NO: 130 mutein“1”-m46,

(29) SEQ ID NO: 131 mutein“1”-m47,

(30) SEQ ID NO: 132 mutein“1”-m7,

(31) SEQ ID NO: 133 mutein“1”-m10,

(32) SEQ ID NO: 134 mutein“1”-m17,

(33) SEQ ID NO: 135 mutein“1”-m21,

(34) SEQ ID NO: 136 mutein“1”-m24,

(35) SEQ ID NO: 137 mutein“1”-m27,

(36) SEQ ID NO: 138 mutein“1”-m28,

(37) SEQ ID NO: 139 mutein“1”-m30,

(38) SEQ ID NO: 140 mutein“1”-m33,

(39) SEQ ID NO: 141 mutein“1”-m1,

(40) SEQ ID NO: 142 mutein“1”-m3,

(41) SEQ ID NO: 143 mutein“1”-m8,

(42) SEQ ID NO: 144 mutein“1”-m15,

(43) SEQ ID NO: 145 mutein“1”-m6,

(44) SEQ ID NO: 146 mutein“1”-m9,

(45) SEQ ID NO: 147 mutein“1”-m20,

(46) SEQ ID NO: 148 mutein“1”-m34,

(47) SEQ ID NO: 149 mutein“1”-m14,

(48) SEQ ID NO: 150 mutein“1”-m18,

(49) SEQ ID NO: 151 mutein“1”-m19,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(69) SEQ ID NO: 171 mutein“1”-m101,

(70) SEQ ID NO: 172 mutein“1”-m106,

(71) SEQ ID NO: 173 mutein“1”-m111,

(72) SEQ ID NO: 174 mutein“1”-m100,

(73) SEQ ID NO: 175 mutein“1”-m110,

(74) SEQ ID NO: 176 mutein“1”-m104,

(75) SEQ ID NO: 177 mutein“1”-m108,

(76) SEQ ID NO: 178 mutein“1”-m207,

(77) SEQ ID NO: 179 mutein“1”-m212,

(78) SEQ ID NO: 180 mutein“1”-m202,

(79) SEQ ID NO: 181 mutein“1”-m204,

(80) SEQ ID NO: 182 mutein“1”-m206,

(81) SEQ ID NO: 183 mutein“1”-m208,

(82) SEQ ID NO: 184 mutein“1”-m203,

(83) SEQ ID NO: 185 mutein“1”-m209,

(84) SEQ ID NO: 186 mutein“1”-m200,

(85) SEQ ID NO: 187 mutein“1”-m201,

(86) SEQ ID NO: 188 mutein“1”-m211,

(87) SEQ ID NO: 189 mutein“1”-m300,

(88) SEQ ID NO: 190 mutein“1”-m301,

(89) SEQ ID NO: 191 mutein“1”-m302,

(90) SEQ ID NO: 192 mutein“1”-m303,

(91) SEQ ID NO: 193 mutein“1”-m304, and

(92) SEQ ID NO: 194 mutein m1-9.”

EXAMPLES

(93) General Methods

(94) 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 Göttingen GmbH. The primers and oligonucleotides were synthesized using an Applied Biosystems Expedite DNA synthesizer.

Example 1: Preparation of Library1

(95) 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:

(96) TABLE-US-00001 P1: (SEQ ID NO. 5) 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
where the 3′ terminal T was linked via a phosphorothioate bond and

(97) TABLE-US-00002 P2: (SEQ ID NO. 6) 5′-AGT AGC GGT AAA CGG CAG A

(98) 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.

(99) E. coli TOP0 cells were transformed with the vector mixture using the calcium chloride method (Sambrook et al., 1989).

Example 2: Preparation of Library2

(100) 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:

(101) TABLE-US-00003 P3: (SEQ ID NO. 7) 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
where the 3′ terminal A was linked via a phosphorothioate bond and

(102) TABLE-US-00004 P2: (SEQ ID NO. 6) 5′-AGT AGC GGT AAA CGG CAG A.

(103) DNA sequences were generated in this manner which contained fixed mutations Thr45.fwdarw.Cys and Ser52.fwdarw.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.

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

Example 3: Preparation of Library3

(105) 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:

(106) TABLE-US-00005 P4: (SEQ ID NO. 8) 5′-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA
which was phosphorylated at the 5′ end and

(107) TABLE-US-00006 P5: (SEQ ID NO. 9) 5′-GTT (A/C)NN CTC GGT GGT GCC GGA GGT
equally phosphorylated at the 5′ end.

(108) 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.

(109) E. coli TOP0 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

(110) 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:

(111) TABLE-US-00007 P4: (SEQ ID NO. 8) 5′-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA
which was phosphorylated at the 5′ end and

(112) TABLE-US-00008 P5: (SEQ ID NO. 9) 5′-GTT (A/C)NN CTC GGT GGT GCC GGA GGT
equally phosphorylated at the 5′ end.

(113) 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.

(114) 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

(115) 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:

(116) TABLE-US-00009 P4: (SEQ ID NO. 8) 5′-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA
which was phosphorylated at the 5′ end and

(117) TABLE-US-00010 P6: (SEQ ID NO. 10) 5′-GTT A(A/T)A CTC GGT GGT GCC GGA GGT
equally phosphorylated at the 5′ end.

(118) 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.

(119) 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

(120) 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:

(121) TABLE-US-00011 P7: (SEQ ID NO. 11) 5′-N(G/C)N N(G/T)T CCA CGC TGG TCG GCC AC
which was phosphorylated at the 5′ end and

(122) TABLE-US-00012 P8: (SEQ ID NO. 12) 5′-N(A/C)N NCT CGG TGG TGC CGG AGG T
equally phosphorylated at the 5′ end.

(123) 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.

(124) 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

(125) 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:

(126) TABLE-US-00013 P9: (SEQ ID NO. 13) 5′-GGN N(G/T)T CCA CGC TGG TCG GCC AC
which was phosphorylated at the 5′ end and

(127) TABLE-US-00014 P10: (SEQ ID NO. 14) 5′-A(C/A)N NCT CGG TGG TGC CGG AGG T
equally phosphorylated at the 5′ end.

(128) 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.

(129) 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)

(130) 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-Tag®II 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-Tag®II fusion protein (also denoted BAP-StrepII) in further assays.

(131) 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 μg/ml ampicillin. The membrane was incubated for 24 hours at 30° C. until colonies became visible.

(132) 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.

(133) This second membrane was washed with PBS and placed on an agar plate containing LB medium which contained 100 μg/ml ampicillin and 0.2 μg/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 μg/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-CI pH 8.8, 100 mM NaCl, 5 mM MgCl.sub.2) with addition of 30 μl bromo-chloro-indolylphosphate (BCIP) (50 mg/ml in dimethylformamide) and 5 μl 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

(134) 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 μM solution (determined by using the theoretic molar extinction coefficient ε.sub.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

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

(136) The wells of a 96-well microtitre plate (Costar) were coated overnight at 4° C. with 100 μl 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 μg/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 μl 3% w/v BSA, 0.5% v/v Tween20 in TBS (100 mM Tris-CI pH8, 115 mM NaCl). After washing three times with TBS/Tween (TBS containing 0.05% v/v Tween20), 50 μl of the same buffer was added to each well. 50 μl 0.3 μM BAP-StrepII fusion protein in TBS/Tween (prepared by 67fold dilution with TBS/T of a 20 μM 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 μl (from a total of 100 μl) out of the first well and mixing it with the contents (50 μl) 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.

(137) 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 μl 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-CI pH8 was pipetted into each well. Data of each well were raised by measuring absorbance at 405 nm substracted by absorbance at 595 nm using a BioTek microplate reader el808. 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 Δ(A.sub.405−A.sub.595) in FIG. 1.

(138) 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].Math.[L]/[P.Math.L]. Under the assumption that [P].sub.tot=[P]+[P.Math.L] and that [L] is very much larger than [P.Math.L] so that [L].sub.tot is approximately the same as [L], the amount of bound fusion enzyme BAP-StrepII is determined as [P.Math.L]=[L].sub.tot[P].sub.tot/(K.sub.D+[L].sub.tot). This equation was used for fitting the measured data for [P.Math.L](in terms of enzyme activity, Δ(A.sub.405−A.sub.595)/Δt) 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, (Δ(A.sub.405−A.sub.595)/Δt)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. (Δ(A.sub.405−A.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 (Δ(A.sub.405−A.sub.595)/Δt)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

(139) 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 μl of a 10% v/v Sepharose gel suspension in buffer (100 mM Tris-CI 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.

(140) 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 μg/ml ampicillin. Expression was induced at an OD.sub.550=0.5 with 0.2 μg/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-CI 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.

(141) In a first affinity chromatography experiment, 450 μl 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 ε.sub.280=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-Tag®II fusion protein binding capacity can be prepared.

(142) 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 μg/ml purified cytochromeb562-Strep-Tag®II fusion protein (500 μg 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 ε.sub.280=8250 M.sup.−1 cm.sup.−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-Tag®II 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-Tag®II fusion protein were obtained with the muteins of the invention while using an affinity material amount providing only a theoretic 2fold excess of immobilized Strep-Tag®II binding sites over the applied Strep-Tag®II ligand at the fusion protein (cytochromeb562 in this case), thereby demonstrating the efficiency of affinity capture of Strep-Tag®II fusion proteins using streptavidin muteins of the invention immobilized to a resin.

(143) 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

(144) 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”.

(145) 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,

(146) 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)

(147) 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.

(148) 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-CI 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)

(149) 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-Tag®II fusion protein or the respective streptavidin mutein—is present or applied at very low concentration. Such examples are poor expression of the Strep-Tag®II fusion protein and/or using large buffer volumes for cell lysis or secreting the Strep-Tag®II 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.

(150) 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-Tag®II 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-Tag®II 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.

(151) 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”.

(152) 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/k.sub.off=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 T2=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.

(153) 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, reversibility 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.

(154) 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.

(155) 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 ml). 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=>12 h) 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.

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

(157) 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-Tag®II (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-Tag®II 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 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-Tag®II (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.

(159) 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.

(160) 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.

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

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

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

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

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

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

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

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

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

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

(171) 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.

(172) 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.

(173) 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.

(174) 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.

(175) TABLE-US-00015 TABLE 1 Library 1  relative (SEQ ID NO: 203) Xaa.sup.44Xaa.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Xaa.sup.52 signal Wt (SEQ ID NO: 15) Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 − mutein “1” Val.sup.44Thr.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 + (SEQ ID NO: 16) m400 (SEQ ID NO: 18) Gly.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 ++ m402 (SEQ ID NO: 19) Ala.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 ++

(176) TABLE-US-00016 TABLE 2 Library 2 (Ala/Gly).sup.44Cys.sup.45Xaa.sup.46Xaa.sup.47Gly.sup.48Asn.sup.49Ala.sup.50 relative (SEQ ID NO: 204) Glu.sup.51Cys.sup.52 signal Wt  Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 − (SEQ ID NO: 205) mutein “1” Val.sup.44Thr.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 + (SEQ ID NO: 16) m4001 Ala.sup.44Cys.sup.45Gly.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 +++ (SEQ ID NO: 20)

(177) TABLE-US-00017 TABLE 3 Library 3 (SEQ ID NO: 206) Xaa.sup.117Asn.sup.118Ala.sup.119Xaa.sup.120Xaa.sup.121 relative signal Wt (SEQ ID NO: 207) Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − mutein “1” Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − m36 (SEQ ID NO: 21) Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Met.sup.121 m23 (SEQ ID NO: 22) Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 ++++++ m41 (SEQ ID NO: 23) Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 ++++++ m4 (SEQ ID NO: 24) Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 +++++ m12 (SEQ ID NO: 25) Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 +++++ m22 (SEQ ID NO: 26) Gln.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 +++++ m31 (SEQ ID NO: 27) Phe.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Trp.sup.121 +++++ m32 (SEQ ID NO: 28) Asp.sup.117Asn.sup.118Ala.sup.119Val.sup.120Met.sup.121 +++++ m35 (SEQ ID NO: 29) Ala.sup.117---.sup.118---.sup.119Trp.sup.120Met.sup.121 +++++ m38 (SEQ ID NO: 30) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 +++++ m40 (SEQ ID NO: 31) Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ser.sup.121 +++++ m42 (SEQ ID NO: 32) Phe.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gly.sup.121 +++++ m45 (SEQ ID NO: 33) Tyr.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 +++++ m46 (SEQ ID NO: 34) Arg.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 +++++ m47 (SEQ ID NO: 35) Trp.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gly.sup.121 +++++ m7 (SEQ ID NO: 36) Leu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m10 (SEQ ID NO: 37) His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 ++++ m17 (SEQ ID NO: 38) Met.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m21 (SEQ ID NO: 39) Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 ++++ m24 (SEQ ID NO: 40) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Trp.sup.121 ++++ m27 (SEQ ID NO: 41) His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m28 (SEQ ID NO: 42) Ser.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m30 (SEQ ID NO: 43) Thr.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m33 (SEQ ID NO: 44) Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m1 (SEQ ID NO: 45) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Met.sup.121 +++ m3 (SEQ ID NO: 46) Trp.sup.117Asn.sup.118Ala.sup.119Cys.sup.120Cys.sup.121 +++ m8 (SEQ ID NO: 47) Met.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Val.sup.121 +++ m15 (SEQ ID NO: 48) Ala.sup.117Asn.sup.118Ala.sup.129Asp.sup.120Trp.sup.121 +++ m6 (SEQ ID NO: 49) Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 ++ m9 (SEQ ID NO: 50) Arg.sup.117Asn.sup.118Ala.sup.119Val.sup.120Val.sup.121 ++ m20 (SEQ ID NO: 51) Ser.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Phe.sup.121 ++ m34 (SEQ ID NO: 52) Ala.sup.117---.sup.118---.sup.119Trp.sup.120Asp.sup.121 ++ m14 (SEQ ID NO: 53) Arg.sup.117Asn.sup.118Ala.sup.119Arg.sup.120Ala.sup.121 + m18 (SEQ ID NO: 54) Ser.sup.117Asn.sup.118Ala.sup.119Ala.sup.120Phe.sup.121 + m19 (SEQ ID NO: 55) Gly.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 +

(178) TABLE-US-00018 TABLE 4 Library 4 (SEQ ID NO: 208) Xaa.sup.117Asn.sup.118Ala.sup.119Xaa.sup.120Xaa.sup.121 relative signal 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 (SEQ ID NO: 56) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++++ m21 (SEQ ID NO: 57) Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 ++++++ m9 (SEQ ID NO: 58) Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 +++++ m1 (SEQ ID NO: 59) Arg.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 ++++ m2 (SEQ ID NO: 60) Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m3 (SEQ ID NO: 61) Ala.sup.117Asn.sup.118Ala.sup.119Pro.sup.120Ala.sup.121 ++++ m5 (SEQ ID NO: 62) Ala.sup.117Asn.sup.118Ala.sup.119Met.sup.120Val.sup.121 ++++ m13 (SEQ ID NO: 63) Gln.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Ala.sup.121 ++++ m14 (SEQ ID NO: 64) Ala.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 ++++ m24 (SEQ ID NO: 65) Gln.sup.117Asn.sup.118Ala.sup.119Met.sup.120Val.sup.121 ++++ m4 (SEQ ID NO: 66) Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 +++ m6 (SEQ ID NO: 67) Ala.sup.117Asn.sup.118Ala.sup.119Ala.sup.120Val.sup.121 +++ m7 (SEQ ID NO: 68) Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Ile.sup.121 +++ m10 (SEQ ID NO: 69) His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 +++ m15 (SEQ ID NO: 70) Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Ala.sup.121 +++ m23 (SEQ ID NO: 71) Gln.sup.117Asn.sup.118Ala.sup.119Val.sup.120Ala.sup.121 +++ m17 (SEQ ID NO: 72) Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Met.sup.121 ++ m12 (SEQ ID NO: 73) Leu.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Gly.sup.121 + m20 (SEQ ID NO: 74) His.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Met.sup.121 +

(179) TABLE-US-00019 TABLE 5 Library 5 (Phe/Tyr).sup.117Asn.sup.118Ala.sup.119Xaa.sup.120 (SEQ ID NO: 209) Xaa.sup.121 relative signal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − mutein “1” Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − m101 (SEQ ID NO: 75) Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 ++++++ m106 (SEQ ID NO: 76) Phe.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 ++++++ m111 (SEQ ID NO: 77) Tyr.sup.117Asn.sup.118Ala.sup.119Leu.sup.120Trp.sup.121 ++++++ m100 (SEQ ID NO: 78) Phe.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ile.sup.121 +++++ m110 (SEQ ID NO: 79) Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Leu.sup.121 +++++ m104 (SEQ ID NO: 80) Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gln.sup.121 ++++ m108 (SEQ ID NO: 81) Phe.sup.117Asn.sup.118Ala.sup.119Ile.sup.120Trp.sup.121 ++++

(180) TABLE-US-00020 TABLE 6 Library 6 (SEQ ID NO: 210) Xaa.sup.117---.sup.118---.sup.119Xaa.sup.120Xaa.sup.121 relative signal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − mutein “1” Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − m207 (SEQ ID NO: 82) Thr.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 ++++++ m212 (SEQ ID NO: 83) His.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 ++++++ m202 (SEQ ID NO: 84) Ile.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 +++++ m204 (SEQ ID NO: 85) His.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 +++++ m206 (SEQ ID NO: 86) Thr.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 +++++ m208 (SEQ ID NO: 87) Ala.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 +++++ m203 (SEQ ID NO: 88) Arg.sup.117---.sup.118---.sup.119Trp.sup.120Ser.sup.121 ++++ m209 (SEQ ID NO: 89) Asn.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 ++++ m200 (SEQ ID NO: 90) Lys.sup.117---.sup.118---.sup.119Trp.sup.120Ser.sup.121 +++ m201 (SEQ ID NO: 91) Ser.sup.117---.sup.118---.sup.119Val.sup.120Phe.sup.121 +++ m211 (SEQ ID NO: 92) Lys.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 +++

(181) TABLE-US-00021 TABLE 7 Library 7 (SEQ ID NO: 209) Xaa.sup.117---.sup.118---.sup.119Trp.sup.120Xaa.sup.121 relative signal Wt Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − mutein “1” Ala.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 − m300 (SEQ ID NO: 93) Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 +++++++ m301 (SEQ ID NO: 94) His.sup.117---.sup.118---.sup.119Trp.sup.120Met.sup.121 +++++++ m302 (SEQ ID NO: 95) His.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 +++++++ m303 (SEQ ID NO: 96) Glu.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 +++++++ m304 (SEQ ID NO: 97) Gln.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 +++++++

(182) TABLE-US-00022 TABLE 8 K.sub.D K.sub.D(mutein“1”)/ Mutein [nM] K.sub.Dmutein (Δ(A.sub.405-A.sub.595)/Δt).sub.max mutein ,,1” 110 1.0 703 (SEQ ID NO: 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

(183) TABLE-US-00023 TABLE 9 Retained Sepharose immobilization Retained Cytb.sub.562-Strepll gel loading degree Retained Cytb.sub.562-Strepll per immobilized (100% gel relative to Cytb.sub.562- per immobilized mutein relative suspension) mutein “1” Strepll mutein to mutein “1” Variant [μg/ml] [%] [μg] [μg/μg] [%] 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

(184) TABLE-US-00024 TABLE 10 captured Cytb.sub.562- Strepll per yield Sepharose column size immobilized of gel loading for 1 mg captured mutein Cytb.sub.562- (100% gel immobilized Cytb.sub.562- relative to Strepll suspension) mutein Strepll mutein “1” applied Variant [μg/ml] [μl] [μg] [%] [%] 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

(185) TABLE-US-00025 TABLE 11 Relative streptavidin mutein density Streptavidin on chip K.sub.on × 10.sup.5 k.sub.off × 10.sup.−4 K.sub.D Fusion protein mutein [RU] [M.sup.−1s.sup.−1] [s.sup.−1] [pM] GFP-Strepll 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-Strepll 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

(186) TABLE-US-00026 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 Strepll [mg] Purity [%] 91.6 93.5 98.7 94.5

(187) TABLE-US-00027 TABLE 13 SEQ ID NO Name Sequence SEQ ID NO. 1 E. coli alkaline phosphatase Strep-tag II fusion protein (BAP-StrepII) -  expressed by pASK75-phoA SEQ ID NO. 2 plasmid pASK-IBA2-cytochromeb562 SEQ ID NO 3 nucleotide sequence of the expression vector pASK-IBA2-SAm1 which contains a sequence coding for the OmpA signal peptide followed by the sequence coding for streptavidin mutein “1” disclosed by U.S. Pat. No. 6,103,493 SEQ ID NO 4 nucleotide sequence of the expression vector pASK-IBA2-SAm4001 which contains a sequence coding for the OmpA signal peptide followed by the sequence coding for streptavidin mutein m4001 of the present invention SEQ ID NO. 5 Primer 1 (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 Is: TCG TGA CCG CGG GTG CAG ACG GAG CTC TGA CCG GTA CCT ACN NSN NKG CGC GTG GCA ACG CCG AGN NSC GCT ACG TCC TGA CCG GTC GTT SEQ ID NO. 6 Primer 2 (P2) 5′-AGT AGC GGT AAA CGG CAG A SEQ ID NO. 7 Primer 3 (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 Is: CTG ACC GGT ACC TAC GST TGC NNS NNK GGC AAC GCC GAG TGC CGC TAC GTC CTG A SEQ ID NO. 8 Primer 4 (P4) 5′-GCC NN(G/C) NN(G/T) TCC ACG CTG GTC GGC CA Is: GCC NNS NNK TCC ACG CTG GTC GGC CA SEQ ID NO. 9 Primer 5 (P5) 5′-GTT (A/C)NN CTC GGT GGT GCC GGA GGT Is: GTT MNN CTC GGT GGT GCC GGA GGT SEQ ID NO. 10 Primer 6 (P6) 5′-GTT A(A/T)A CTC GGT GGT GCC GGA GGT Is: GTT AMA CTC GGT GGT GCC GGA GGT SEQ ID NO. 11 Primer 7 (P7) 5′-N(G/C)N N(G/T)T CCA CGC TGG TCG GCC AC Is: NSN NKT CCA CGC TGG TCG GCC AC EQ ID NO. 12 Primer 8 (P8) 5′-N(A/C)N NCT CGG TGG TGC CGG AGG T Is: NMN NCT CGG TGG TGC CGG AGG T SEQ ID NO. 13 Primer 9 (P9) 5′-GGN N(G/T)T CCA CGC TGG TCG GCC AC Is: GGN NKT CCA CGC TGG TCG GCC AC SEQ ID NO. 14 Primer 10 (P10) 5′-A(C/A)N NCT CGG TGG TGC CGG AGG T Is: AMN NCT CGG TGG TGC CGG AGG T SEQ ID NO: 15 wild type streptavidin Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 is: ESAVGNAES SEQ ID NO: 16 mutein 1 Val.sup.44Thr.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 is: VTARGNAES SEQ ID NO: 17 mutein 2 (44-47) IGAR SEQ ID NO: 18 m400 Gly.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 is: GCARGNAEC SEQ ID NO: 19 m402 Ala.sup.44Cys.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 is: ACARGNAEC SEQ ID NO: 20 m4001 Ala.sup.44Cys.sup.45Gly.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Cys.sup.52 is: ACGRGNAEC SEQ ID NO: 21 m36 Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Met.sup.121 is: YNAFM SEQ ID NO: 22 m23 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 is: YNAYA SEQ ID NO: 23 m41 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: AXXWY SEQ ID NO: 24 m4 Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: DNAGF SEQ ID NO: 25 m12 Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: RNAGF SEQ ID NO: 26 m22 Gln.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: QNAGF SEQ ID NO: 27 m31 Phe.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Trp.sup.121 is: FNASW SEQ ID NO: 28 m32 Asp.sup.117Asn.sup.118Ala.sup.119Val.sup.120Met.sup.121 is: DNAVM SEQ ID NO: 29 m35 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Met.sup.121 is: AXXWM SEQ ID NO: 30 m38 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: ENAGF SEQ ID NO: 31 m40 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ser.sup.121 is: YNAYS SEQ ID NO: 32 m42 Phe.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gly.sup.121 is: FNAYG SEQ ID NO: 33 m45 Tyr.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: YNAGF SEQ ID NO: 34 m46 Arg.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ala.sup.121 is: RNAYA SEQ ID NO: 35 m47 Trp.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gly.sup.121 is: WNAYG SEQ ID NO: 36 m7 Leu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: LNAGF SEQ ID NO: 37 m10 His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: HNAGY SEQ ID NO: 38 m17 Met.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: MNAGF SEQ ID NO: 39 m21 Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: RNAGY SEQ ID NO: 40 m24 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Trp.sup.121 is: ENAGW SEQ ID NO: 41 m27 His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: HNAGF SEQ ID NO: 42 m28 Ser.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: SNAGF SEQ ID NO: 43 m30 Thr.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: TNAGF SEQ ID NO: 44 m33 Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: NNAGF SEQ ID NO: 45 m1 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Met.sup.121 is: ENAGM SEQ ID NO: 46 m3 Trp.sup.117Asn.sup.118Ala.sup.119Cys.sup.120Cys.sup.121 is: WNACC SEQ ID NO: 47 m8 Met.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Val.sup.121 is: MNAFV SEQ ID NO: 48 m15 Ala.sup.117Asn.sup.118Ala.sup.119Asp.sup.120Trp.sup.121 is: ANADW SEQ ID NO: 49 m6 Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 is: SNAMM SEQ ID NO: 50 m9 Arg.sup.117Asn.sup.118Ala.sup.119Val.sup.120Val.sup.121 is: RNAVV SEQ ID NO: 51 m20 Ser.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Phe.sup.121 is: SNASF SEQ ID NO: 52 m34 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Asp.sup.121 is: AXXWD SEQ ID NO: 53 m14 Arg.sup.117Asn.sup.118Ala.sup.119Arg.sup.120Ala.sup.121 is: RNARA SEQ ID NO: 54 m18 Ser.sup.117Asn.sup.118Ala.sup.119Ala.sup.120Phe.sup.121 is: SNAAF SEQ ID NO: 55 m19 Gly.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 is: GNAMM SEQ ID NO: 56 m8 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: ENAGF SEQ ID NO: 57 m21 Asp.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: DNAGY SEQ ID NO: 58 m9 Glu.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: ENAGY SEQ ID NO: 59 m1 Arg.sup.117Asn.sup.118Ala.sup.119Met.sup.120Met.sup.121 is: RNAMM SEQ ID NO: 60 m2 Arg.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: RNAGF SEQ ID NO: 61 m3 Ala.sup.117Asn.sup.118Ala.sup.119Pro.sup.120Ala.sup.121 is: ANAPA SEQ ID NO: 62 m5 Ala.sup.117Asn.sup.118Ala.sup.119Met.sup.120Val.sup.121 is: ANAMV SEQ ID NO: 63 m13 Gln.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Ala.sup.121 is: QNASA SEQ ID NO: 64 m14 Ala.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Phe.sup.121 is: ANAGF SEQ ID NO: 65 m24 Gln.sup.117Asn.sup.118Ala.sup.119Met.sup.120Val.sup.121 is: QNAMV SEQ ID NO: 66 m4 Asn.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: NNAGY SEQ ID NO: 67 m6 Ala.sup.117Asn.sup.118Ala.sup.119Ala.sup.120Val.sup.121 is: ANAAV SEQ ID NO: 68 m7 Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Ile.sup.121 is: SNAMI SEQ ID NO: 69 m10 His.sup.117Asn.sup.118Ala.sup.119Gly.sup.120Tyr.sup.121 is: HNAGY SEQ ID NO: 70 m15 Ser.sup.117Asn.sup.118Ala.sup.119Met.sup.120Ala.sup.121 is: SNAMA SEQ ID NO: 71 m23 Gln.sup.117Asn.sup.118Ala.sup.119Val.sup.120Ala.sup.121 is: QNAVA SEQ ID NO: 72 m17 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Met.sup.121 is: YNAYM SEQ ID NO: 73 m12 Leu.sup.117Asn.sup.118Ala.sup.119Trp.sup.120Gly.sup.121 is: LNAWG SEQ ID NO: 74 m20 His.sup.117Asn.sup.118Ala.sup.119Ser.sup.120Met.sup.121 is: HNASM SEQ ID NO: 75 m101 Tyr.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 is: YNAFL SEQ ID NO: 76 m106 Phe.sup.117Asn.sup.118Ala.sup.119Phe.sup.120Leu.sup.121 is: FNAFL SEQ ID NO: 77 m111 Tyr.sup.117Asn.sup.118Ala.sup.119Leu.sup.120Trp.sup.121 is: YNALW SEQ ID NO: 78 m100 Phe.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Ile.sup.121 is: FNAYI SEQ ID NO: 79 m110 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Leu.sup.121 is: YNAYL SEQ ID NO: 80 m104 Tyr.sup.117Asn.sup.118Ala.sup.119Tyr.sup.120Gln.sup.121 is: YNAYQ SEQ ID NO: 81 m108 Phe.sup.117Asn.sup.118Ala.sup.119Ile.sup.120Trp.sup.121 is: FNAIW SEQ ID NO: 82 m207 Thr.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 is: TXXWL SEQ ID NO: 83 m212 His.sup.117---.sup.118---.sup.119Trp.sup.120Leu.sup.121 is: HXXWL SEQ ID NO: 84 m202 Ile.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: IXXWR SEQ ID NO: 85 m204 His.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 is: HXXWT SEQ ID NO: 86 m206 Thr.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: TXXWR SEQ ID NO: 87 m208 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: AXXWR SEQ ID NO: 88 m203 Arg.sup.117---.sup.118---.sup.119Trp.sup.120Ser.sup.121 is: RXXWS SEQ ID NO: 89 m209 Asn.sup.117---.sup.118---.sup.119Trp.sup.120Arg.sup.121 is: NXXWR SEQ ID NO: 90 m200 Lys.sup.117---.sup.118---.sup.119Trp.sup.120Ser.sup.121 is: KXXWS SEQ ID NO: 91 m201 Ser.sup.117---.sup.118---.sup.119Val.sup.120Phe.sup.121 is: SXXVF SEQ ID NO: 92 m211 Lys.sup.117---.sup.118---.sup.119Trp.sup.120Thr.sup.121 is: KXXWT SEQ ID NO: 93 m300 Ala.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: AXXWY SEQ ID NO: 94 m301 His.sup.117---.sup.118---.sup.119Trp.sup.120Met.sup.121 is: HXXWM SEQ ID NO: 95 m302 His.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: HXXWY SEQ ID NO: 96 m303 Glu.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: EXXWY SEQ ID NO: 97 m304 Gln.sup.117---.sup.118---.sup.119Trp.sup.120Tyr.sup.121 is: QXXWY SEQ ID NO: 98 streptavidin mutein Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (44-47 = VTAR) is: VTAR SEQ ID NO: 99 streptavidin mutein Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 is: IGAR SEQ ID NO: 100 Strep-tag II affinity peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ligand is: WSHPQFEK SEQ ID NO: 101 peptide sequence Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys is WXHPQFXX SEQ ID NO: 102 Strep-tag streptavidin Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (WRHPQFGG) binding peptide SEQ ID NO: 103 Peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.3-Trp- Ser-His-Pro-Gln-Phe-Glu-Lys Is WSHPQFEKGGGSGGGSGGGSWSHPQFEK SEQ ID NO: 104 GFP-StrepII SEQ ID NO: 105 GFP-di-tag3 SEQ ID NO: 106 Cytb562-StrepII SEQ ID NO: 107 Cytb562-di-tag3 SEQ ID NO: 108 peptide sequence Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg is XXHPQFXX SEQ ID NO: 109 di-tag2 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.2-Trp- Ser-His-Pro-Gln-Phe-Glu-Lys Is WSHPQFEKGGGSGGGSWSHPQFEK SEQ ID NO: 110 Peptide sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK SEQ ID NO: 111 Wildtype FIG. 4 SEQ ID NO: 112 mutein″1 FIG. 4 SEQ ID NO: 113 mutein″2 FIG. 4 SEQ ID NO: 114 m400 FIG. 4 SEQ ID NO: 115 m402 FIG. 4 SEQ ID NO: 116 m4001 FIG. 4 SEQ ID NO: 117 mutein“1”-m36 FIG. 4 SEQ ID NO: 118 mutein“1”-m23 FIG. 4 SEQ ID NO: 119 mutein“1”-m41 FIG. 4 SEQ ID NO: 120 mutein“1”-m4 FIG. 4 SEQ ID NO: 121 mutein“1”-m12 FIG. 4 SEQ ID NO: 122 mutein“1”-m22 FIG. 4 SEQ ID NO: 123 mutein“1”-m31 FIG. 4 SEQ ID NO: 124 mutein“1”-m32 FIG. 4 SEQ ID NO: 125 mutein“1”-m35 FIG. 4 SEQ ID NO: 126 mutein“1”-m38 FIG. 4 SEQ ID NO: 127 mutein“1”-m40 FIG. 4 SEQ ID NO: 128 mutein“1”-m42 FIG. 4 SEQ ID NO: 129 mutein“1”-m45 FIG. 4 SEQ ID NO: 130 mutein“1”-m46 FIG. 4 SEQ ID NO: 131 mutein“1”-m47 FIG. 4 SEQ ID NO: 132 mutein“1”-m7 FIG. 4 SEQ ID NO: 133 mutein“1”-m10 FIG. 4 SEQ ID NO: 134 mutein“1”-m17 FIG. 4 SEQ ID NO: 135 mutein“1”-m21 FIG. 4 SEQ ID NO: 136 mutein“1”-m24 FIG. 4 SEQ ID NO: 137 mutein“1”-m27 FIG. 4 SEQ ID NO: 138 mutein“1”-m28 FIG. 4 SEQ ID NO: 139 mutein“1”-m30 FIG. 4 SEQ ID NO: 140 mutein“1”-m33 FIG. 4 SEQ ID NO: 141 mutein“1”-ml FIG. 4 SEQ ID NO: 142 mutein“1”-m3 FIG. 4 SEQ ID NO: 143 mutein“1”-m8 FIG. 4 SEQ ID NO: 144 mutein“1”-m15 FIG. 4 SEQ ID NO: 145 mutein“1”-m6 FIG. 4 SEQ ID NO: 146 mutein“1”-m9 FIG. 4 SEQ ID NO: 147 mutein“1”-m20 FIG. 4 SEQ ID NO: 148 mutein“1”-m34 FIG. 4 SEQ ID NO: 149 mutein“1”-m14 FIG. 4 SEQ ID NO: 150 mutein“1”-m18 FIG. 4 SEQ ID NO: 151 mutein“1”-m19 FIG. 4 SEQ ID NO: 152 m4001-m8 FIG. 4 SEQ ID NO: 153 m4001-m21 FIG. 4 SEQ ID NO: 154 m4001-m9 FIG. 4 SEQ ID NO: 155 m4001-ml FIG. 4 SEQ ID NO: 156 m4001-m2 FIG. 4 SEQ ID NO: 157 m4001-m3 FIG. 4 SEQ ID NO: 158 m4001-m5 FIG. 4 SEQ ID NO: 159 m4001-m13 FIG. 4 SEQ ID NO: 160 m4001-m14 FIG. 4 SEQ ID NO: 161 m4001-m24 FIG. 4 SEQ ID NO: 162 m4001-m4 FIG. 4 SEQ ID NO: 163 m4001-m6 FIG. 4 SEQ ID NO: 164 m4001-m7 FIG. 4 SEQ ID NO: 165 m4001-m10 FIG. 4 SEQ ID NO: 166 m4001-m15 FIG. 4 SEQ ID NO: 167 m4001-m23 FIG. 4 SEQ ID NO: 168 m4001-m17 FIG. 4 SEQ ID NO: 169 m4001-m12 FIG. 4 SEQ ID NO: 170 m4001-m20 FIG. 4 SEQ ID NO: 171 mutein“1”-m101 FIG. 4 SEQ ID NO: 172 mutein“1”-m106 FIG. 4 SEQ ID NO: 173 mutein“1”-m111 FIG. 4 SEQ ID NO: 174 mutein“1”-m100 FIG. 4 SEQ ID NO: 175 mutein“1”-m110 FIG. 4 SEQ ID NO: 176 mutein“1”-m104 FIG. 4 SEQ ID NO: 177 mutein“1”-m108 FIG. 4 SEQ ID NO: 178 mutein“1”-m207 FIG. 4 SEQ ID NO: 179 mutein“1”-m212 FIG. 4 SEQ ID NO: 180 mutein“1”-m202 FIG. 4 SEQ ID NO: 181 mutein“1”-m204 FIG. 4 SEQ ID NO: 182 mutein“1”-m206 FIG. 4 SEQ ID NO: 183 mutein“1”-m208 FIG. 4 SEQ ID NO: 184 mutein“1”-m203 FIG. 4 SEQ ID NO: 185 mutein“1”-m209 FIG. 4 SEQ ID NO: 186 mutein“1”-m200 FIG. 4 SEQ ID NO: 187 mutein“1”-m201 FIG. 4 SEQ ID NO: 188 mutein“1”-m211 FIG. 4 SEQ ID NO: 189 mutein“1”-m300 FIG. 4 SEQ ID NO: 190 mutein“1”-m301 FIG. 4 SEQ ID NO: 191 mutein“1”-m302 FIG. 4 SEQ ID NO: 192 mutein“1”-m303 FIG. 4 SEQ ID NO: 193 mutein“1”-m304 FIG. 4 SEQ ID NO: 194 Mutein m1-9 FIG. 4 SEQ ID NO: 195 loop of the amino acids 114 TTEANAWK to 121 of streptavidin SEQ ID NO: 196 loop region of amino acids TEANAWK 115 to 121 of streptavidin SEQ ID NO: 197 mutein HPYFYAPELLFFAK SEQ ID NO: 198 mutein EGGKETLTPSELRDLV motif1 Xaa.sup.117Gly.sup.120Yaa.sup.121, consensus sequence1 wherein Xaa may be any amino acid and Yaa may be Phe or Tyr or Met is XGX- so not more than 4 amino acids SEQ ID NO: 199 motif1 Xaa Asn Ala Gly Zaa, (FIG. 2B) Xaa is Glu, Asp, Arg, His, Asn, Gln, Thr, Ser, Leu, Met and Zaa is Phe, Tyr, Met is XNAGX motif2 Aaa.sup.112Baa.sup.120Caa.sup.121, consensus sequence2 wherein Aaa may be Tyr, Phe, Arg, Trp or Gln, Baa may be Tyr, Phe, Leu, Ile or Met and Caa may be any amino acid, wherein Caa121 is preferably a Leu, an Ile, a Met, a Gly, a Gly, a Trp, a Ser, an Ala or a Val residue SEQ ID NO: 200 Motif2 Xaa-Asn-Ala-Yaa-Zaa, (FIG. 2B) wherein Xaa is Tyr, Phe, Arg, Trp, Gln; Yaa is Tyr, Phe, Leu, Ile, Met and Zaa is Leu, Ile, Met, Gln, Gly, Trp, Ser, Ala, Val Is XNAXX SEQ ID NO: 201 motif3 Daa.sup.117Eaa.sup.118Faa.sup.119Gaa.sup.120Haa.sup.121, wherein Daa and Haa consensus sequence3 may be any amino acid and Eaa and Faa are both deleted and Gaa may be Trp or Val, wherein Daa.sup.117 is preferably a His, a Glu, a Gln, a Thr, an Ala or an Ile residue and wherein Haa.sup.121 is preferably a Tyr, a Leu, a Met, or an Arg residue is XXXXX SEQ ID NO: 202 Motif2 Xaa --- --- Yaa Zaa, (FIG. 2B) wherein Xaa is His, Glu, Gln, Thr, Ala, Ile, Arg, Asn,  Lys, Ser; Yaa is Trp, Val and Zaa is Tyr, Leu, Met, Arg, Thr, Ser, Phe Is XXXXX SEQ ID NO: 203 Library 1 Xaa.sup.44Xaa.sup.45Ala.sup.46Arg.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Xaa.sup.52 is XXARGNAEX SEQ ID NO: 204 Library 2 (Ala/Gly)44Cys45Xaa46Xaa47Gly48Asn49Ala50Glu51 Cys52 is XCXXGNAEC SEQ ID NO: 205 Wt (Library 2) Glu.sup.44Ser.sup.45Ala.sup.46Val.sup.47Gly.sup.48Asn.sup.49Ala.sup.50Glu.sup.51Ser.sup.52 is ESAVGNAES SEQ ID NO: 206 Library 3 Xaa.sup.117Asn.sup.118Ala.sup.119Xaa.sup.120Xaa.sup.121 Is XNAXX SEQ ID NO: 207 Wt (Library 2) mutein “1” Ala.sup.171Asn.sup.118Ala.sup.119Trp.sup.120Lys.sup.121 (Library 2) Wt (Library 4) Is ANAWK m4001 (Library 4) Wt (Library 5) mutein “1” (Library 5) Wt (Library 6) mutein “1” (Library 6) Wt (Library 7) mutein “1” (Library 7) SEQ ID NO: 208 Library 4 Xaa.sup.117Asn.sup.118Ala.sup.119Xaa.sup.120Xaa.sup.121 is XNAXX SEQ ID NO: 209 Library 5 (Phe/Tyr).sup.117Asn.sup.118Ala.sup.119Xaa.sup.120Xaa.sup.121 is XNAXX SEQ ID NO: 210 Library 6 Xaa.sup.117---.sup.118---.sup.119Xaa.sup.120Xaa.sup.121 is XXXXX SEQ ID NO: 211 Library 7 Xaa.sup.117---.sup.118---.sup.119Trp.sup.120Xaa.sup.121 is XXXWX SEQ ID NO: 212 Wt streptavidin amino acid EAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRY sequence (residues 14 to VLTGRYDSAPATDG 139) SGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINT QWLLTSGTTEANAWKST LVGHDTFTKVKPSAAS