Author : Jianming Xie
Publisher :
ISBN 13 :
Total Pages : 278 pages
Book Rating : 4.:/5 (763 download)
Book Synopsis Adding Unnatural Amino Acids to the Genetic Repertoire by : Jianming Xie
Download or read book Adding Unnatural Amino Acids to the Genetic Repertoire written by Jianming Xie and published by . This book was released on 2006 with total page 278 pages. Available in PDF, EPUB and Kindle. Book excerpt: This thesis focuses on expanding the genetic code of E. coli to include unnatural amino acids beyond the common 20. By means of an orthogonal pair of amber suppressor tRNA[Special characters omitted.] /tyrosyl-tRNA synthetase (TyrRS) imported from an archaea M. jannaschii, several novel amino acids were site-specifically incorporated into proteins in response to an amber codon in E. coli, with high fidelity and efficiency. Typically, large libraries of M. jannaschii TyrRS (Mj TyrRS) mutants were first generated by randomizing 6 residues in the amino acid binding pocket. The libraries were then subjected to alternating positive and negative selections, which may afford synthetase variants that could charge the cognate tRNA[Special characters omitted.] with the unnatural amino acid but not any of the 20 natural amino acids. In one project, a heavy-atom-containing amino acid, p -iodo-L-phenylalanine, was site-specifically incorporated into proteins. The anomalously scattering iodine atom could be used to facilitate X-ray structure determination by SAD phasing. In the second project, a phosphotyrosine analogue, p -carboxymethyl-L-phenylalanine, was cotranslationally incorporated into proteins. When this unnatural amino acid was used to replace Tyr701 in human STAT1, the resulting protein dimerized and bound the same DNA sequence as Tyr701-phosphorylated STAT1. Because this amino acid is resistant to protein tyrosine phosphatase, it should be useful in the generation of stable phosphoprotein analogues, or peptide-based inhibitors to SH2 domains. In another project, a metal-chelating amino acid, bipyridylalanine, was selectively incorporated into proteins. Genetically encoding bipyridylalanine and other metal-chelating amino acids should be useful for the generation of proteins with novel or enhanced properties, such as artificial enzymes, proteins that undergo metal-ion-assisted dimerization/trimerization or folding, proteins that contain redox-active metal-ligand complexes on their surface for use in the artificial electron transfer system, and proteins that bind fluorescent or luminescent metal ions as imaging agents, or bind radionuclides for therapeutic use. A final series of experiments involved attempts to incorporate N-methyl amino acids into proteins, which could be useful in generating protease-resistant proteins. While the N-methyl amino acid was N-demethylated by metabolic enzymes in E. coli, recent experiments suggest that such amino acids are stable in yeast, presenting a possible solution to this problem.
