The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins

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.

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

“…A molecular mass of 23,746.5 Da was calculated based on the amino acid sequence of the expressed protein (49,50). PA2602 expression was induced in BL21(DE3)pLysS cells (Novagen) and purified using Strep-tag affinity chromatography according to the manufacturer's protocol (IBA) (51,52). We employed the following modifications to the standard protein expression/purification procedures: (i) cells were incubated overnight at 18°C during protein expression; (ii) cell lysis buffer contained 100 mM Tris (pH 8.0), 1.15 M NaCl, 1 mM EDTA, 5 mM Tween 20 (1.5%, v/v), and a Roche complete protease inhibitor; and (iii) cells were lysed using a French pressure cell (model FA-078, SLM Aminco).…”

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

“…To facilitate purification of the ClpP/R core, as well as its individual rings, we generated two different transgenic plant lines expressing tagged ClpR4 or ClpP3 subunits by complementing homozygous CLPR4 and CLPP3 null mutants (clpr4-1 and clpp3-1) with, respectively, a 13 35S-driven CLPR4 or CLPP3 cDNA fused to C-terminal StrepII tags (Figure 1). The eightresidue StrepII tag (Schmidt and Skerra, 2007) was attached to the C terminus rather than the N terminus to prevent interference with the N-terminal chloroplast-targeting peptide. A C-terminal tag also likely minimizes interference with the Clp core function because the N-terminal domains of ClpP directly modulate chaperone interactions for substrate delivery in the E. coli system (Gribun et al, 2005).…”

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

“…Therefore, we incorporated a second affinity tag to allow double affinity purification of the reporters, adding a StrepTagII affinity tag to the C terminus of the reporter (Fig. 1B) [33]. The reporters were first purified by Ni-NTA affinity chromatography via the N-terminal His6 tag and then subjected to StrepTactin affinity chromatography, yielding high-purity BoTest ™ reporters (Fig.…”

“…Chapman (University of Wisconsin-Madison) [32]. The constructs were engineered to encode the 8-amino-acid StrepTagII sequence (WSHPQFEK) C terminal of the YFP moiety using standard polymerase chain reaction (PCR) techniques [33]. The resulting constructs, pTrcHisA-CFP-SNAP 141-206 -YFP-StrepTagII and pTrcHisA-CFP-Syb2 33-94 -YFP-StrepTagII, were further modified to incorporate Venus mutations into YFP using standard site-directed mutagenesis and molecular biology techniques [34].…”