Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis.
Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility...
| Publicado en: | Journal of the American Chemical Society Vol. 137; no. 13; pp. 4404 - 4414 |
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| Autores principales: | , , , , |
| Formato: | Artículo |
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American Chemical Society
4/8/2015
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| Materias: | |
| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=102711941&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 102711941 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 4/8/2015 vid: 137 iid: 13 pid: 997 pub: American Chemical Society artinfo: ui: 102711941 10.1021/ja5131963 ppf: 4404 ppct: 10 formats: tig: atl: Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis. aug: au: Zhenling Cui Stein, Viktor Tnimov, Zakir Mureev, Sergey Alexandrov, Kirill affil: Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia Australian Institute for Bioengeneering and Nanotechnology, University of Queensland, St. Lucia, Queensland 4072, Australia su: Genetic code Synthetic biology Protein engineering Transfer RNA Polypeptides sug: subj: Genetic code Synthetic biology Protein engineering Transfer RNA Polypeptides ab: Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and lie, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and lie tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly. pubtype: Academic Journal doctype: Article src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2015 holdings: @attributes: islocal: N |
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