Journal of Molecular Biology
Regular articleMolecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin
Abstract
TheStrep-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 Å resolution (space groupI4122; unit cell dimensionsa=b= 58.3 Å,c=176.9 Å). TheStrep-tag was bound at the same surface pocket where biotin, the natural ligand of streptavidin, gets complexed. The peptide backbone exhibited 310-helical conformation, with eight of the residues involved in protein contacts. TheC-terminal Gly-Gly moiety of theStrep-tag participated in a salt bridge to Arg84 of streptavidin with its free carboxylate group. This finding explained why the use of theStrep-tag in fusions with recombinant proteins was restricted to their carboxyl end. Employing a synthetic peptide spot assay, the variantStrep-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 × 104M−1was determined for theStrep-tag peptide, and slightly tighter binding was seen when theStrep-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.
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An impedance aptasensing method based on the strep-tag system integrated apta-magnetic probe for acetamiprid determination
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Present addresses: T. G. M. Schmidt, Institut für Bioanalytik GmbH, Rudolf-Wissell-Str. 28, D-37079 Göttingen, Germany; A. Skerra, Institut für Biochemie, Technische Hochschule, Petersenstr. 22, D-64287 Darmstadt, Germany.
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