MUTAGENESIS OF A FLEXIBLE LOOP IN STREPTAVIDIN LEADS TO HIGHER AFFINITY FOR THE STREP-TAG-II PEPTIDE AND IMPROVED PERFORMANCE IN RECOMBINANT PROTEIN-PURIFICATION

The Strep-tag, an artificial peptide ligand of streptavidin, has gaine d use as an affinity handle for the purification and detection of reco mbinant fusion proteins, In an attempt to achieve tighter complexation of the peptide, streptavidin was engineered and the amino acid residu es 44-47 in the flexible loop from 44 to 53, which is close to the bin ding site, were subjected to random mutagenesis. A fusion between alka line phosphatase and the Strep-tag II sequence, an improved version of the Strep-tag, was constructed as a molecular probe for peptide bindi ng, By means of a filter-sandwich assay, two streptavidin mutants with significantly stronger binding activity for the Strep-tag II. were th us identified from a library of Escherichia coli colonies, Both in an ELISA with the alkaline phosphatase fusion and in a fluorescence titra tion experiment with the synthetic Strep-tag II peptide, which carried an anthraniloyl group as chromophore, their affinities were found to be higher by more than one order of magnitude compared with wild-type streptavidin, The nature of the amino acid exchanges and an enhanced e lectrophoretic mobility of the streptavidin tetramers suggest an alter ed loop conformation to be part of the optimized binding mechanism, Wh en one of the streptavidin mutants was immobilized on a chromatographi c column it exhibited clearly improved performance in the purification of Strep-tag II fusion proteins, and desthiobiotin turned out to be a suitable reagent for mild competitive elution.