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Cell-free protein synthesis systems 总被引:1,自引:0,他引:1
Cell-free protein synthesis systems enable the direct in vitro expression of proteins from template DNA or RNA. Use of biochemical and bioengineering techniques has greatly improved the yields and productivities of cell-free systems. In some cases, the yields approach the in vivo levels. Moreover, in vitro systems are capable of rapidly providing artificial polypeptides that greatly facilitate protein engineering. Post-translational modification steps in cell-free systems also offer exciting possibilities as reviewed here. 相似文献
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Alison M Jackson Joe Boutell Neil Cooley Mingyue He 《Briefings in Functional Genomics and Prot》2004,2(4):308-319
The use of cell-free expression systems as an alternative to cell-based methods for protein production is greatly facilitating studies of protein functions. Recent improvements to cell-free systems, and the development of cell-free protein display and microarray technologies, have led to cell-free protein synthesis becoming a powerful tool for large-scale analysis of proteins. This paper reviews the most commonly used cell-free systems and their applications in proteomics. 相似文献
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Cell-free protein synthesis by rumen protozoa 总被引:1,自引:0,他引:1
J K Chesters 《The Journal of protozoology》1968,15(3):509-512
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Cell-free protein synthesis programmed with R17 RNA: identification of two phage proteins 总被引:26,自引:0,他引:26
M R Capecchi 《Journal of molecular biology》1966,21(1):173-193
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Glial fibrillary acidic (GFA) protein has been synthesized in an RNA-dependent cell-free system derived from rabbit reticulocytes. The cell-free synthesized product appears to have the same size as GFA protein isolated from bovine spinal cord, thus showing that GFA protein does not undergo detectable proteolytic processing. 相似文献
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We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. 相似文献
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T. Nojima T. Fujii K. Hosokawa A. Yotsumoto S. Shoji I. Endo 《Bioprocess and biosystems engineering》2000,22(1):13-17
Microbiochemical reactors having two inlet ports and one outlet port were fabricated on a silicon wafer by means of anisotropic etching in order to develop a parallel and automatic experimental system for cell-free translation. Using cell-free extract prepared from Escherichia coli, we tested the reactor for the translation of polyuridylic acid and MS2 phage RNA, and found that polypeptide and protein syntheses could be proceeded according to the genetic codes on the mRNAs. It indicates that the microfabricated reactor is useful for enzymatic reactions including complicated ones like cell-free translation. We also discuss the possibility of microsystems as advanced experimental tools for not only cell-free translation but also other various biochemical and biological research fields. 相似文献
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Background
Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS. 相似文献13.
Cell-free synthesis of bacteriophage f2 maturation protein 总被引:5,自引:0,他引:5
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Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels.
This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen
atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling
of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in
drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular
structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free
system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form. 相似文献
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James W. Whittaker 《Biotechnology letters》2013,35(2):143-152
Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids. 相似文献
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