|M.Sc Student||Ivanir Eran|
|Subject||Development of an Efficient Cell-Free Translation System|
|Department||Department of Biotechnology||Supervisor||Professor Yuval Shoham|
|Full Thesis text - in Hebrew|
The purpose of this work was to develop an efficient cell-free protein synthesis system. This type of system allows the production of proteins which cannot be overexpressed by in-vivo systems, such as membrane and regulatory proteins. Furthermore, it can be used for in-vitro gene regulation studies in systems where standard genetic tools are not applicable; high throughput screening techniques; and specific isotope labeling of proteins for structure-function studies. To date, in-vitro protein synthesis systems have not been widely accepted as a practical alternative for protein production, in part due to the poor yields. Currently, commercial kits of cell free protein synthesis produce relatively low amounts of proteins at high cost (e.g., 600$/mg protein); therefore there is a great interest in improving these systems and attaining high yields at low cost.
In this study we have optimized a cell free protein synthesis system based on E. coli MRE600 lysate. The model protein was the extracellular xylanase from G. stearothermophilus T-6, since its enzymatic activity can be easily followed using chromogenic substrates. The components that have been tested were magnesium acetate, potassium acetate, ammonium acetate, rifampicin, polyethylene glycol 6000, 3',5' cyclic AMP ,mixture of 20 tRNAs, folinic acid, DTT, 3-PGA, mixture of 20 amino acids, ATP, GTP, UTP, CTP, S-30 extract and the template DNA. The optimized system provided 180 mg protein per ml compare to 60 mg protein per ml in the initial system. Further improvement was obtained by prolonging the reaction time by increasing the concentration of the amino acid glutamine resulting in a final yield of 230 mg protein per ml.
The optimized system was tested for the production of GanR from the thermophilic bacterium Geobacillus Stearothermophilus T-6. GanR is a regulatory protein that cannot be over-expressed in E. coli due to its high toxicity. The protein was successfully synthesized in the optimized cell-free system, resulting in 210 mg of pure protein in a six ml reaction volume. When comparing our final results to commercial kits available, the improved cell free protein synthesis system appear to be 15 fold more economical.