Abstract

Until recently, all organisms from viruses to humans have used the same 20 amino acids to form proteins. In 2001, Peter Schultz's group reported the modification of the translation machinery of Escherichia coli to allow incorporation of additional novel amino acids into proteins [1xExpanding the genetic code of Escherichia coli. Wang, L. et al. Science. 2001; 292: 498–500Crossref | PubMedSee all References][1]. Now, this same laboratory has described the application of this technique to incorporate specifically a photocrosslinking amino acid, p-benzoyl-l-phenylalanine (pBpa), into proteins [2xAddition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Chin, J.W. et al. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11020–11024Crossref | PubMed | Scopus (278)See all References][2].First, the authors needed to select for an orthogonal aminoacyl-tRNA synthetase/tRNA pair that could incorporate pBpa specifically in response to the amber codon TAG. A strategy was devised to mutate the Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS). It was already known that this synthetase does not aminoacylate any endogenous E. coli tRNAs with tyrosine but can aminoacylate a mutant tyrosine amber suppressor. A library expressing mutated TyrRS was subjected to positive selection for chloramphenicol resistance based on the suppression of an amber stop codon in the gene encoding CAT. These cells were then challenged to grow in the presence of pBpa and chloramphenicol. At this point, these cells contained synthetases that could incorporate either a natural or unnatural (pBpa) amino acid. These synthetases were then transferred into cells containing the toxic barnase gene with amber mutations. Growth of these cells in the absence of pBpa provided selection against synthetases that could use natural amino acids. Five rounds of this positive and negative selection were performed. The sequences of six of these ‘selected’ synthetases were determined and used to model how pBpa complexes with the mutated TyrRS. Based on calculations related to the resistance to chloramphenicol in the presence or absence of pBpa, the authors conclude that they have achieved substantial in vivo specificity for the incorporation of pBpa in response to an amber codon using these mutated TyrRS.To prove the usefulness of this technology, the authors tested a modified Schistosoma japonicum glutathione S-transferase (SjGST). This protein is known to be a dimer of two identical subunits. By placing amber codons at specific sites in this gene, and using the modified TyrRS system, specific amino acids within SjGST were substituted with pBpa. One of these residues (Phe52) was known to interact between the SjGST subunits and the other (Tyr198) was known not to react. Upon irradiation, the purified SjGST (Phe-52-Bpa) was converted to a covalently linked homodimer as detected by SDS–PAGE, whereas the other purified SjGST (Tyr198-Bpa) remained as a monomer. This demonstrates the utility of this technique to incorporate this photocrosslinking agent specifically into proteins and crosslink them together. The goal of this technology is to increase the ability to identify specific amino acids involved in protein–protein interactions.

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