Efficient introduction of aryl bromide functionality into proteins in vivo.

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.