Work

Transglutaminase-Catalyzed Site-Specific Conjugation of Small-Molecule Probes to Proteins in Vitro and on the Surface of Living Cells

Year: 2006

Type: article

Abstract: 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.

Source: Journal of the American Chemical Society

DOI: 10.1021/ja0604111

Authors: Chi-Wang Lin, Alice Y. Ting

Institution: Massachusetts Institute of Technology

Language: English

Topic: Click Chemistry and Applications

Subfield: Organic Chemistry

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: National Institutes of Health, Office of Naval Research, National Institute of General Medical Sciences

Award: Library-based development of new optical imaging probes