Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells.
Site-specific protein labeling methods allow cell biologists to access the vast array of existing chemical probes for the study of specific proteins of interest in the live cell context. Here we describe the use of the transglutaminase enzyme from guinea pig liver (gpTGase), whose natural function is to cross-link glutamine and lysine side chains, to covalently conjugate various small-molecule probes to recombinant proteins fused to a 6- or 7-amino acid transglutaminase recognition sequence, called a Q-tag. We demonstrate labeling of Q-tag fusion proteins both in vitro and on the surface of living mammalian cells with biotin, fluorophores, and a benzophenone photoaffinity probe. To illustrate the utility of this labeling, we tagged the NF-kappaB p50 transcription factor with benzophenone, cross-linked with UV light, and observed increased levels of p50 homodimerization in the presence of DNA and the binding protein myotrophin.
Lin
Chi-Wang
CW
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Ting
Alice Y
AY
D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000737:chemistry; D002384:Catalysis; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006168:Guinea Pigs; D006367:HeLa Cells; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000032:analysis* / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism
C485182:Cyan Fluorescent Protein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D052002:NF-kappa B p50 Subunit; D049452:Green Fluorescent Proteins; D019809:Streptavidin; D011503:Transglutaminases; D002103:Cadaverine