Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification

Selma Voss, Arne Skerra

Research output: Contribution to journalArticlepeer-review

241 Scopus citations

Abstract

The Strep-tag, an artificial peptide ligand of streptavidin, has gained use as an affinity handle for the purification and detection of recombinant fusion proteins. In an attempt to achieve tighter complexation of the peptide, streptavidin was engineered and the amino acid residues 44-47 in the flexible loop from 44 to 53, which is close to the binding site, were subjected to random mutagenesis. A fusion between alkaline phosphatase and the Strep-tag II sequence, an improved version of the Strep-tag, was constructed as a molecular probe for peptide binding. By means of a filter-sandwich assay, two streptavidin mutants with significantly stronger binding activity for the Strep-tag II were thus identified from a library of Escherichia coli colonies. Both in an ELISA with the alkaline phosphatase fusion and in a fluorescence titration 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 electrophoretic mobility of the streptavidin tetramers suggest an altered loop conformation to be part of the optimized binding mechanism. When one of the streptavidin mutants was immobilized on a chromatographic 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.

Original languageEnglish
Pages (from-to)975-982
Number of pages8
JournalProtein Engineering
Volume10
Issue number8
DOIs
StatePublished - 1997
Externally publishedYes

Keywords

  • Alkaline phosphatase
  • Aminobenzoic acid
  • Escherichia coli secretion
  • Filter-sandwich assay
  • Tetracycline promoter

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