Expanding the Genetic Code for Biological Studies

The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Chemistry & biology (Impact Factor: 6.65). 04/2009; 16(3):323-36. DOI: 10.1016/j.chembiol.2009.03.001
Source: PubMed


Using an orthogonal tRNA-synthetase pair, unnatural amino acids can be genetically encoded with high efficiency and fidelity, and over 40 unnatural amino acids have been site-specifically incorporated into proteins in Escherichia coli, yeast, or mammalian cells. Novel chemical or physical properties embodied in these amino acids enable new means for tailored manipulation of proteins. This review summarizes the methodology and recent progress in expanding this technology to eukaryotic cells. Applications of genetically encoded unnatural amino acids are highlighted with reports on labeling and modifying proteins, probing protein structure and function, identifying and regulating protein activity, and generating proteins with new properties. Genetic incorporation of unnatural amino acids provides a powerful method for investigating a wide variety of biological processes both in vitro and in vivo.

28 Reads
  • Source
    • "The methodology based on unnatural amino acids (UAAs) incorporation into desired loci of the protein of interest is widely used for understanding protein structure-function relationships, investigating protein-based biological processes, and generating proteins and organisms with new properties [1]. Over the past two decades, the most established methods to site-specifically incorporate UAA in vivo were based on genetic code expansion. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.
    PLoS ONE 07/2013; 8(7):e68363. DOI:10.1371/journal.pone.0068363 · 3.23 Impact Factor
  • Source
    • "Further progress came with the development of orthogonal tRNA(CUA) and aminoacyl-tRNA synthetase (aaRS) pairs, which produce an orthogonal tRNA(CUA) carrying a nonproteinogenic amino acid in vivo under multiple turnover conditions. This technology allows us to not only synthesize a protein carrying a nonproteinogenic amino acid at a specific position in various types of cells, but also obtain a protein containing a nonproteinogenic amino acid in a larger quantity than by in vitro genetic code expansion [31] [32] [33] [34]. The use of various orthogonal tRNA/aaRS pairs has allowed for the synthesis of proteins carrying various artificial functional groups, such as a biochemical group (e.g., sulfate, acetate, or methylate) [35] [36] [37] [38], fluorescent probe [39] [40] [41] [42] [43], photo-cross-linker [44– 49], photo-caged group [50] [51] [52] [53] [54] [55] [56], and bioorthogonal reactive group [57] [58] [59] [60] [61] [62] [63], for the study of protein structure and function. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.
    Journal of nucleic acids 10/2012; 2012:713510. DOI:10.1155/2012/713510
  • Source
    • "PGK1). Interestingly, correct processing of the tRNA, and not absolute expression levels, is essential to generate functional tRNA and increase NNAA incorporation (reviewed by Wang et al., 2009). "

    Protein Engineering, 02/2012; , ISBN: 978-953-51-0037-9
Show more

Similar Publications


28 Reads
Available from