Streptavidin muteins and methods of using them

Abstract

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

What is claimed is: 
     
         1 . A mutein, selected from muteins of streptavidin, wherein the mutein
 (a) has a Cys residue at sequence position 127 with reference to the amino acid sequence of wild-type streptavidin as set forth in SEQ ID NO: 1, and   (b) wherein the sequence Val 44 -Thr 45 -Ala 46 -Arg 47  (SEQ ID NO: 10) or the sequence Ile 44 Gly 45 Ala 46 Arg 47  (SEQ ID NO: 11) is present in the region of amino acid positions 44 to 47 with reference to the amino acid sequence of wild-type streptavidin.   
     
     
         2 . A mutein according to  claim 1 , wherein the mutein comprises at sequence positions 117 to 121 with reference to the amino acid sequence of wild-type streptavidin an amino acid sequence selected from the group consisting of 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 56) 
                 
                     
                   Glu 117 Asn 118 Ala 119 Gly 120 Phe 121 , 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 57) 
                 
                     
                   Asp 117 Asn 118 Ala 119 Gly 120 Tyr 121 , 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 58) 
                 
                     
                   Glu 117 Asn 118 Ala 119 Gly 120 Tyr 121 , 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 22) 
                 
                     
                   Asp 117 Asn 118 Ala 119 Gly 120 Phe 121   
                 
                     
                     
                 
                     
                   (SEQ ID NO: 26) 
                 
                     
                   Gln 117 Asn 118 Ala 119 Gly 120 Phe 121 , 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 44) 
                 
                     
                   Asn 117 Asn 118 Ala 119 Gly 120 Phe 121   
                 
                     
                     
                 
                     
                   (SEQ ID NO: 66) 
                 
                     
                   Asn 117 Asn 118 Ala 119 Gly 120 Tyr 121 , and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 95) 
                 
                     
                   His 117 --- 118 --- 119 Trp 120 Tyr 121 . 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         3 . A mutein according to  claim 1 , wherein the mutein is a mutein of a minimal streptavidin which begins N-terminally in the region of the amino acids 10 to 16 of wild type streptavidin and terminates C-terminally in the region of the amino acids 133-142 of wild type streptavidin. 
     
     
         4 . A mutein according to  claim 1 , wherein the mutein comprises the sequence of the mutein m302C (SEQ ID NO: 14). 
     
     
         5 . The mutein according to  claim 4 , wherein the mutein consists of the sequence of mutein m1-9C (SEQ ID NO: 16) or m302C (SEQ ID NO: 14). 
     
     
         6 . A nucleic acid molecule, comprising a sequence coding for a streptavidin mutein as defined in  claim 1 . 
     
     
         7 . A method of isolating, purifying or determining under denaturing conditions a protein that is fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 8) in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys, or b) with a peptide sequence that comprises a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence -His-Pro-Baa-, where Baa is glutamine, asparagine or methionine, and wherein the other binding module has the sequence -Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- (SEQ ID NO: 9) where Oaa is Trp, Lys or Arg, Xaa is any amino acid and where either Yaa and Zaa are both Gly or Yaa is Glu and Zaa is Lys or Arg, the method comprising contacting a sample containing the protein with a streptavidin mutein of  claim 1  under suitable conditions to bind the peptide sequence to the streptavidin mutein, and separating the resulting complex from said sample. 
     
     
         8 . The method according to  claim 7 , wherein the streptavidin mutein is bound to a solid phase or is capable of binding thereto. 
     
     
         9 . The method according to  claim 8 , wherein in order to release the fusion protein from the complex the complex is incubated with an adequate amount of a ligand for the streptavidin mutein selected from biotin and derivatives thereof. 
     
     
         10 . The method according to  claim 7 , wherein the denaturing conditions are caused by the presence of a chaotropic agent. 
     
     
         11 . The method according to  claim 10 , wherein the chaotropic agent is selected from the group consisting of urea, thiourea, guanidine hydrochloride, lithium perchlorate hydroxide ions, and combinations thereof. 
     
     
         12 . The method according to  claim 8 , wherein the solid phase is an affinity chromatography matrix. 
     
     
         13 . The method according to  claim 12 , wherein the affinity chromatography matrix is selected from the group consisting of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide) silica, poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of an acrylate or an acrylamide and a diol, a copolymer of a polysaccharide and N,N′-methylenebisacrylamide and a combination of any two or more thereof.