Abstract: The invention concerns a polypeptide selected from muteins of streptavidin which is characterized in that it (a) contains at least one mutation in the region of the amino acid positions 44 to 53 with reference to wild type-(wt)-streptavidin and (b) has a higher binding affinity than wt-streptavidin for peptide ligands comprising the amino acid sequence Trp-X-His-Pro-Gln-Phe-Y-Z in which X represents an arbitrary amino acid and Y and Z either both denote Gly or Y denotes Glu and Z denotes Arg or Lys. In addition nucleic acids coding for the polypeptide, a vector containing this nucleic acid, a cell transfected with the vector as well as the use of a polypeptide in a method for the isolation, purification or determination of proteins are disclosed. Yet a further subject matter is a reagent kit containing the polypeptide.