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Abstract

The facile detection and purification of a recombinant protein without detailed knowledge about its individual biochemical properties constitutes a problem of general interest in protein engineering. The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study. Because of its widespread use as part of a variety of conjugates and other affinity reagents, streptavidin constitutes the binding partner of choice both for detection and purification purposes. The streptavidin-affinity tag was engineered at the C-terminus of the V_H domain as part of the D1.3 Fv fragment which was functionally expressed in Escherichia coli. Irrespective of whether it was displayed by the V_H or the V_L domain, the optimized version of the affinity peptide termed 'Strep-tag' allowed the detection of the Fv fragment both on Western blots and in ELISAs by a streptavidin-alkaline phosphatase conjugate. In addition, the one-step purification of the intact Fv fragment carrying a single Strep-tag at the C-terminus of only one of its domains was achieved by affinity chromatography with streptavidin-agarose using very mild elution conditions.