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: 6.59). 04/2009; 16(3):323-36. DOI: 10.1016/j.chembiol.2009.03.001
Source: PubMed

ABSTRACT 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.

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    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
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    Protein Engineering, 02/2012; , ISBN: 978-953-51-0037-9
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    ABSTRACT: Posttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.
    FEBS letters 01/2012; 586(6):729-33. DOI:10.1016/j.febslet.2012.01.029 · 3.34 Impact Factor


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