Efficient introduction of aryl bromide functionality into proteins in vivo

FEBS Letters

Furter, Rolf³  Sharma, Nandita²  Tirrell, David A.¹  Kast, Peter⁴ 

[1] Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA;Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003, USA;Bank J. Vontobel and Co. Ltd, Bahnhofstr. 3, CH-8022 Zurich, Switzerland;Laboratorium für Organische Chemie, Swiss Federal Institute of Technology (ETH), CH-8092 Zurich, Switzerland

关键词: Amino acid analog;    Para-bromophenylalanine;    Multiwavelength anomalous diffraction;    Phenylalanyl-tRNA synthetase;    Phenylalanine replacement;    Protein engineering;   

DOI  :  10.1016/S0014-5793(00)01120-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown