Prolegomena to Future Experimental Efforts on Genetic Code Engineering by Expanding Its Amino Acid Repertoire

Abstract

Protein synthesis and its relation to the genetic code was for a long time a central issue in biology. Rapid experimental progress throughout the past decade, crowned with the recently elucidated ribosomal structures, provided an almost complete description of this process. In addition important experiments provided solid evidence that the natural protein translation machinery can be reprogrammed to encode genetically a vast number of non-coded (i.e. noncanonical) amino acids. Indeed, in the set of 20 canonical amino acids as prescribed by the universal genetic code, many desirable functionalities, such as halogeno, keto, cyano, azido, nitroso, nitro, and silyl groups, as well as C=C or C[triple bond]C bonds, are absent. The ability to encode genetically such chemical diversity will enable us to reprogram living cells, such as bacteria, to express tailor-made proteins exhibiting functional diversity. Accordingly, genetic code engineering has developed into an exciting emerging research field at the interface of biology, chemistry, and physics.

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…24,65 However, recently Wilkins and coworkers could show that these amino acids can also be incorporated by in-frame stop codon suppression. The only prerequisite is a masking before incorporation.…”

“…28,29 Due to the advantages of the method, most experiments related to the incorporation of fluorinated amino acids were performed using this approach. 24,25,[30][31][32][33] The second approach, genetic code expansion, has the major advantage of being site-specific. This is achieved by manipulation of the genetic template in terms of inserting an in-frame stop codon into the coding sequence.…”

Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

“…An almost quantitative incorporation of both Met analogs, Nle and Mox, into rhPrP C was achieved via routine bio-expression protocols, based on the supplementation incorporation (SPI) method (37). The purified proteins were analyzed by SDS/PAGE gel electrophoresis and Coomassie staining.…”

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

“…Despite the specificity of these enzymes for their natural substrates, protein engineers have exploited the promiscuity of aaRS to incorporate a wide array of noncanonical amino acids into recombinant proteins (1,2). Elevation of the aaRS activity of the expression host can be critical for efficient in vivo production of proteins in which one of the natural amino acids is globally replaced by a noncanonical amino acid (3)(4)(5)(6).…”