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 streptavidin-binding activity. If this sequence is genetically fused to the C-terminus of a polypeptide the recombinant protein can be directly purified by affinity chromatography from the host cell extract on immobilized streptavidin. However, variations were observed in the suitability of different commercial streptavidin-agarose preparations for this purpose. Therefore, the influence of the source of streptavidin, the coupling chemistry, and the nature of the affinity chromatography resin was investigated. A procedure was developed for the production of recombinant core streptavidin in Escherichia coli, followed by its efficient refolding and purification with an overall yield of up to 140 mg functional protein per 11 bacterial culture. When coupled to activated CH-Sepharose 4B this truncated form of streptavidin showed a performance in the affinity chromatography of Strep tag fusion proteins that was superior to all other combinations tested. In contrast to its conventional preparation from Streptomyces strains the recombinant core streptavidin was produced without a proteolytic processing step. Thus, deleterious effects during the streptavidin affinity purification of proteins due to residual proteolytic activity in the immobilized streptavidin were avoided. The versatility of the optimized purification system was demonstrated with five different Strep tag fusion proteins.
Schmidt
T G
TG
Abteilung Molekulare Membranbiologie, Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Skerra
A
A
D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D019809:Streptavidin; D013302:Streptomyces / Q000737:chemistry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019809:Streptavidin