Strep-tag

Abstract: We describe a general purification method for recombinant proteins based upon the selective interaction between a poly-histidine peptide, which is fused to the protein of interest, and a novel metal chelate adsorbent. The principle of the technique is illustrated with mouse dihydrofolate reductase. DNA elements coding for adjacent histidines were fused to the mouse dihydrofolate reductase gene. Subsequent expression in E. coli resulted in the production of hybrid proteins that could be purified by immobilized metal ion affinity chromatography, followed by removal of the histidine affinity peptide with carboxypeptidase A.

Abstract: Publisher Summary This chapter discusses the fundamental molecular properties of avidin and streptavidin. Carbohydrate-free avidin can be obtained by use of deglycosylating enzymes and it has been shown to be present in significant amounts in some commercial preparations from which it may be separated by use of lectin columns. Improvement on the original use of biotinyl cellulose came with the introduction of iminobiotinyl derivatives of Sepharose that utilized the pH dependence of the binding 24 to achieve efficient elution. In iminobiotin, the ureido group becomes a guanidinium group; the form in which this is uncharged is strongly bound. The gene for streptavidin has been cloned and sequenced with the ultimate objective of using it in general expression systems for detecting and isolating fusion proteins. The stability is greatly enhanced by biotin binding, because the total free energy of binding is about 330 kJ/mol of tetramer. The dissociation constant for biotin is so low that it can be estimated only from the ratio of the rate constants for binding and exchange. The binding is accompanied by a red shift of the tryptophan spectrum and by a decrease in fluorescence, either of which can be used as the basis for quantitative assays.

Abstract: The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.

Abstract: The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.