title: Efficient introduction of aryl bromide functionality into proteins in vivo.
abstract: 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.
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: N
authors.initials: N
authors.lastname: Sharma
authors.forename: R
authors.initials: R
authors.lastname: Furter
authors.forename: P
authors.initials: P
authors.lastname: Kast
authors.forename: D A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001966:Bromine / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C060302:4-bromophenylalanine; D010649:Phenylalanine; D013762:Tetrahydrofolate Dehydrogenase; D010652:Phenylalanine-tRNA Ligase; D001966:Bromine