Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent.
Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope.
Witte, C.P., Noel, L., Gielbert, J., Parker, J. Single-step Strep-tag ® purification for the isolation and identification of protein complexes from mammalian cells. Junttila, M.R., Saarinen, S., Schmidt, T., Kast, J. Exploiting features of adenovirus replication to support mammalian kinase production. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics. Comparison of affinity tags for protein purification.
Lichty, J.J., Malecki, J.L., Agnew, H.D., Michelson-Horowitz, D.J. Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. A short polypeptide marker sequence useful for recombinant protein identification and purification. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase. A single step purification for recombinant proteins. Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins. The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification. Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site. Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. One-step affinity purification of bacterially produced proteins by means of the “Strep tag” and immobilized recombinant core streptavidin. Genetically engineered avidins and streptavidins. Laitinen, O.H., Hytonen, V.P., Nordlund, H.R. Characterization and crystallization of core streptavidin. Pähler, A., Hendrickson, W.A., Kolks, M.A., Argaraña, C.E. The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Use of the Strep-tag and streptavidin for detection and purification of recombinant proteins. 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. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody ( StrepMAB-Classic). 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.
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. 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.
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