TAILIEUCHUNG - Báo cáo khoa học: Cell-free protein synthesis

Cell-free protein synthesis using cell extracts from Escherichia coli, wheat germ and rabbit reticulocytes has been used for over 40 years to produce small amounts of radiolabeled proteins for identification of gene products and other applications. In the E. coli system programmed with plasmid DNA, the cell extract contains or is supplemented with an RNA po-lymerase to transcribe the gene, and the mRNAs are translated by a complex mixture that contains ribo-somes and a full complement of initiation,. | IFEBS Journal MINIREVIEW SERIES Cell-free protein synthesis Nicholas E. Dixon Research Schoolof Chemistry Australian National university Canberra ACT Australia Cell-free protein synthesis using cell extracts from Escherichia coli wheat germ and rabbit reticulocytes has been used for over 40 years to produce small amounts of radiolabeled proteins for identification of gene products and other applications. In the E. coli system programmed with plasmid DNA the cell extract contains or is supplemented with an RNA polymerase to transcribe the gene and the mRNAs are translated by a complex mixture that contains ribosomes and a full complement of initiation elongation and termination factors as well as a full set of amino-acyl-tRNA synthetases and other required enzymes. The presence of molecular chaperones protein disulfide and peptidyl-prolyl cis-trans isomerases generally ensures that proteins are correctly folded into soluble active forms. With the eukaryotic extracts it has additionally been possible to program protein synthesis directly with in vitro transcribed mRNA that can be produced directly from PCR products and the presence of factors responsible for post-translational modification of proteins ensures the production of fully functional gene products. Given the simplicity of these systems it is not surprising that considerable effort has been invested over the past two decades to improve their productivity and efficiency. The key breakthroughs were the development of methods for continuous feeding of amino acids and nucleoside triphosphates into the reaction mixtures and the use of efficient energy-regeneration systems. As a result cell-free synthesis can now be used routinely to rapidly make milligram quantities of proteins for a range of applications especially in structural and functional proteomics. Coupled with improvements in high-throughput protein isolation by use of purification tags and the efficiency and sensitivity of downstream applications like .

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• Report medicine
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• cell proliferation
• oxidation
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• analysis of cytoplasmic
• Crystal structure
• bacteria
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• chemical reaction
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