ONE-STEP AFFINITY PURIFICATION OF BACTERIALLY PRODUCED PROTEINS BY MEANS OF THE STREP TAG AND IMMOBILIZED RECOMBINANT CORE STREPTAVIDIN

The ''Strep tag'' is a nine amino acid peptide with intrinsic streptav idin-binding activity. If this sequence is genetically fused to the C- terminus of a polypeptide the recombinant protein can be directly puri fied by affinity chromatography from the host cell extract on immobili zed streptavidin. However, variations were observed in the suitability of different commercial streptavidin-agarose preparations for this pu rpose. Therefore, the influence of the source of streptavidin, the cou pling chemistry, and the nature of the affinity chromatography resin w as investigated. A procedure was developed for the production of recom binant core streptavidin in Escherichia coli, followed by its efficien t refolding and purification with an overall yield of up to 140 mg fun ctional protein per 1 1 bacterial culture. When coupled to activated C H-Sepharose 4B this truncated form of streptavidin showed a performanc e in the affinity chromatography of Strep tag fusion proteins that was superior to all other combinations tested. In contrast to its convent ional preparation from Streptomyces strains the recombinant core strep tavidin was produced without a proteolytic processing step. Thus, dele terious effects during the streptavidin affinity purification of prote ins due to residual proteolytic activity in the immobilized streptavid in were avoided. The versatility of the optimized purification system was demonstrated with five different Strep tag fusion proteins.