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T G Schmidt

Publications and source records attributed to T G Schmidt.

6 recordsLinked to original sources
PubMed1999

Applications of a peptide ligand for streptavidin: the Strep-tag.

The Strep-tag constitutes a nine amino acid-peptide that binds specifically to streptavidin and occupies the same pocket where biotin is normally complexed. Since the Strep-tag participates in a reversible interaction it can be applied for the efficient purification of corresponding fusion proteins on affinity columns with immobilized streptavidin. Elution of the bound recombinant protein can be effected under mild buffer conditions by competition with biotin or a suitable derivative. In addition, Strep-tag fusion proteins can be easily detected in immunochemical assays, like Western blots or ELISAs, by means of commercially available streptavidin-enzyme conjugates. The Strep-tag/streptavidin system has been systematically optimized over the past years, including the engineering of streptavidin itself. Structural insight into the molecular mimicry between the peptide and biotin was furthermore gained from X-ray crystallographic analysis. As a result the system provides a reliable and versatile tool in recombinant protein chemistry. Exemplary applications of the Strep-tag are discussed in this review.

Affinity Labels↗
PubMed1996

Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin.

The Strep-tag is a selected nine-amino acid peptide (AWRHPQFGG) that displays intrinsic binding affinity towards streptavidin and has been used as an affinity tag for recombinant proteins. In order to elucidate the molecular mechanism underlying this type of artificial protein-peptide recognition, X-ray crystallographic analyses and binding measurements were carried out. The crystal structure of the complex between recombinant core streptavidin and the synthesized peptide was solved and refined at 1.7 A resolution (space group I4(1)22; unit cell dimensions a = b = 58.3 A, c = 176.9 A). The Strep-tag was bound at the same surface pocket where biotin, the natural ligand of streptavidin, gets complexed. The peptide backbone exhibited 3(10)-helical conformation, with eight of the residues involved in protein contacts. The C-terminal Gly-Gly moiety of the Strep-tag participated in a salt bridge to Arg84 of streptavidin with its free carboxylate group. This finding explained why the use of the Strep-tag in fusions with recombinant proteins was restricted to their carboxyl end. Employing a synthetic peptide spot assay, the variant Strep-tag II was screened, which did not have this limitation. The isomorphous crystal structure of its complex with streptavidin revealed that a glutamate side-chain provided the salt bridge in this case, with an otherwise almost unchanged mode of binding. Affinity constants between the peptides and streptavidin were measured by isothermal titration calorimetry. A value of 2.7 x 10(4) M-1 was determined for the Strep-tag peptide, and slightly tighter binding was seen when the Strep-tag was applied as part of a bacterially produced fusion protein. This affinity is significantly higher, compared with values previously reported for shorter streptavidin-binding peptides, and agrees well with the remarkable selectivity observed in recombinant protein purification applications.

Amino Acid Sequence↗
PubMed1994

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.

Bacterial Proteins↗
PubMed1993

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment.

The facile detection and purification of a recombinant protein without detailed knowledge about its individual biochemical properties constitutes a problem of general interest in protein engineering. The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study. Because of its widespread use as part of a variety of conjugates and other affinity reagents, streptavidin constitutes the binding partner of choice both for detection and purification purposes. The streptavidin-affinity tag was engineered at the C-terminus of the VH domain as part of the D1.3 Fv fragment which was functionally expressed in Escherichia coli. Irrespective of whether it was displayed by the VH or the VL domain, the optimized version of the affinity peptide termed 'Strep-tag' allowed the detection of the Fv fragment both on Western blots and in ELISAs by a streptavidin-alkaline phosphatase conjugate. In addition, the one-step purification of the intact Fv fragment carrying a single Strep-tag at the C-terminus of only one of its domains was achieved by affinity chromatography with streptavidin-agarose using very mild elution conditions.

Affinity Labels↗