Efficient introduction of aryl bromide functionality into proteins in vivo

Artificial proteins can be engineered to exhibit interesting solid state, l iquid crystal or interfacial properties and may ultimately serve as importa nt alternatives to conventional polymeric materials. The utility of protein -based materials is limited, however, by the availability of just the 20 am ino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into prote ins by biosynthetic means. In this study, we incorporate para-bromopbenylal hnine (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 DH FR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-B r-phe-supplemented minimal medium resulted in 88% replacement of phenylalan ine 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 anal og. Protein expression yields of 20-25 mg/l were obtained from cultures sup plemented with p-Br-phe; this corresponds to about two-thirds of the expres sion levels characteristic of cultures supplemented with phe. The aryl brom ide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated prot eins via metal-catalyzed coupling reactions. In addition, bromination may b e useful in X-ray studies of proteins via the multiwavelength anomalous dif fraction (MAD) technique. (C) 2000 Federation of European Biochemical Socie ties.