Abstract
Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin: streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.
Dokumententyp: | Zeitschriftenartikel |
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Fakultät: | Physik |
Themengebiete: | 500 Naturwissenschaften und Mathematik > 530 Physik |
ISSN: | 1748-3387 |
Sprache: | Englisch |
Dokumenten ID: | 47751 |
Datum der Veröffentlichung auf Open Access LMU: | 27. Apr. 2018, 08:13 |
Letzte Änderungen: | 04. Nov. 2020, 13:24 |