Báo cáo khoa học: Cell-free protein synthesis ppt

MINIREVIEW SERIES Cell-free protein synthesis Nicholas E. Dixon Research School of 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 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, 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 disul- fide and peptidyl-prolyl cis-trans isomerases generally ensures that proteins are correctly folded into soluble, active forms. With the eukaryotic extracts, it has addi- tionally been possible to program protein synthesis directly with in vitro transcribed mRNA that can be produced directly from PCR products, and the pres- ence of factors responsible for post-translational modi- fication of proteins ensures the production of fully functional gene products. Given the simplicity of these systems, it is not sur- prising that considerable effort has been invested over the past two decades to improve their productivity and efficiency. The key breakthroughs were the develop- ment 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 struc- tural and functional proteomics. Coupled with improve- ments in high-throughput protein isolation by use of purification tags, and the efficiency and sensitivity of downstream applications like protein crystallization and NMR spectroscopy, such quantities of proteins are now adequate for complete structure determin- ation. Especially exciting new developments are in the use of selected lipids and detergents in cell-free reactions to solubilize membrane proteins in fully functional membrane-inserted forms. That preparative cell-free protein production systems can now be programmed not only by plasmid DNAs, but also by PCR products and in vitro-transcribed mRNAs, has enabled rapid isolation of useful quanti- ties of proteins in high-throughput applications with- out ever having to resort to use of living cells. It is possible, starting with PCR products, to simulta- neously generate large numbers of genes or mutant versions of a single gene, express them in parallel, and analyse their functions within periods of days rather than months. While maximization of protein production is clearly a very useful outcome, it is not the only application of these systems. As an example, direct screening for genes that encode proteins with selected functions from large gene libraries (e.g., in directed molecular evolu- tion experiments or in functional genomics applica- tions) might require production of assayable quantities of an enzyme from a single copy of a gene in an artificial compartment (e.g., a droplet). For such appli- cations, attention has turned to the use of fully func- tional transcription ⁄ translation systems reconstituted with highly purified ribosomes, enzymes, and trans- lation factors. The expectation is that these ‘pure’ systems will enable new technologies, and should result in better understanding of the processes of translation that can be exploited to further manipulate reactions for better productivity in particular circumstances. This series of minireviews examines four different aspects of the use of cell-free translation systems. Each article describes the current state of the art and high- lights future prospects and challenges. In the first mini- review, Shimizu et al. give an overview of the various cell-free translation systems, and then highlight the roles of the chaperones and folding isomerases in pro- tein folding, and use of cell-free methods for capturing membrane proteins in liposomes, for insertion of un- natural amino acids into proteins, and for directed molecular evolution experiments using their recently developed fully reconstituted system. The second minireview by Klammt et al. gives a thorough overview of the use of cell-free systems for doi: 10.1111/j.1742-4658.2006.05445.x FEBS Journal 273 (2006) 4131–4132 ª 2006 The Author Journal compilation ª 2006 FEBS 4131 production of integral membrane proteins, both in insoluble forms that are often easily resolubilized in detergents and, in the presence of a range of deter- gents, in soluble form. This article gives an excellent guide to strategies for optimization of reactions with such challenging proteins. The next minireview by Ozawa et al. focuses on the use of cell-free protein synthesis for direct isotopic labeling of proteins for NMR applications. Because only the newly synthesized protein is labeled, NMR spectra can be run directly using the reaction mixture, and combinatorial [ 15 N]-labeling can be used to rapidly assign peaks in HSQC spectra to a particular amino acid type. This not only increases efficiency in NMR structure determination, but simplifies study of pro- tein–ligand interactions. Finally, Vinarov et al. describe how an optimized wheat germ system is being used in high-throughput formats to produce proteins for NMR structure determination as part of their eukaryotic structural genomics program. Their successes show that cell-free protein synthesis has come of age as an alternative to cell-based methods for high-throughput applica- tions. Nick Dixon received his PhD training in Biochemistry at the University of Queensland with Burt Zerner, and followed this with postdoctoral appointments in Chemistry with Alan Sargeson at the Australian National University (ANU) and in Biochemistry with Arthur Kornberg at Stanford. He established his research group at the ANU in 1986, where he is currently Professor of Biological Chemistry. His research focuses on the bac- terial DNA replication machinery as a model system to study the chemistry of protein–protein interactions in dynamic macromolecular machines. Cell-free protein synthesis N. E. Dixon 4132 FEBS Journal 273 (2006) 4131–4132 ª 2006 The Author Journal compilation ª 2006 FEBS . SERIES Cell-free protein synthesis Nicholas E. Dixon Research School of Chemistry, Australian National University, Canberra, ACT, Australia Cell-free protein. challenging proteins. The next minireview by Ozawa et al. focuses on the use of cell-free protein synthesis for direct isotopic labeling of proteins for