Site-specific biosynthetic incorporation of a fluorescent tag into proteins via cysteine-tRNA(Cys)

Site-specific incorporation of biophysical probes into proteins during translation can permit structure/function studies on selected proteins in heterogeneous environments. We report here a procedure for incorporating a fluorescent tag into proteins via Escherichia coli Cys-tRNA(Cys) during in vitro protein synthesis. Naturally occurring Cys-tRNA(Cys) is an attractive vehicle for fluorophore incorporation since it can be readily prepared in quantity and reacted with commercially available fluorophores. Moreover, proteins can often be constructed with a single Cys so that fluorophore incorporation results in a tag at a unique site.