Abstract
The biotin-streptavidin association constant is considered to be the highest known in biochemistry. Correspondingly, in vivo biotinylation has been used for a large number of biological applications. Here we report an evolved pyrrolysyl-tRNA synthetase/tRNA pair, which allows the site-directed incorporation of various unnatural amino acids (UAAs) including three biotin derivatives. When incorporated in response to the amber stop codon, these derivatives open various applications in bacterial and mammalian cells based on their differences in binding behavior to streptavidin. Using the site-directed incorporation approach we were able to isolate the protein recA harboring a biotinylated UAA and identified 16 interaction partners through this pull-down assay. Further, the successful incorporation of biotin, iminobiotin and desthiobiotin derived UAAs in HEK-293 cells demonstrates that the in vivo biotinylation protocol can be transferred to mammalian cells. The small size of the UAA side chains, the different elution properties of the three analogues and the absence of a recognition sequence or peptide-tag make this approach an attractive tool. We demonstrated the applications of site-specific incorporation of biotin analogs for the purification, immobilization and the characterization of proteins.