Cell-free N-terminal protein labeling using initiator suppressor tRNA

A highly efficient method for the introduction of fluorophores and other markers at the N terminus of proteins produced in a cell-free extract has been developed. The method utilizes an amber (CUA) initiator suppressor tRNA chemically aminoacylated with a fluorophore-amino acid conjugate which is introduced into an Escherichia coli S30 cell-free translation system. The DNA template contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. Using this approach, the fluorophore BODIPY-F1 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionic acid) has been incorporated at the N terminus of several model proteins. The specific labeling achieved (27-67%) using this approach is much higher than that of wild-type tRNAs. Several potential biophysical and biotechnological applications of this new technology are described.