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A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking.

Protein Eng Des Sel.. 2017-12; 
Hinrichsen M, Lenz M, Edwards JM, Miller OK, Mochrie SGJ, Swain PS, Schwarz-Linek U, Regan L.
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… 4 M.Hinrichsen et al. Page 5. and probed using appropriate primary antibodies. Mouse anti-His6 (GenScript (Piscataway, NJ), Cat. # A00186-100), and mouse antiV5 (Invitrogen (Carlsbad, CA), Cat. # 46–0705) primary anti- bodies …

摘要

We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a cov... More

关键词

S. cerevisiae; SpyCatcher-SpyTag; membrane protein; protein engineering; single cell