Studies on Increasing Efficiency of Cell-free Protein Synthesis

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Cell-free protein synthesis from a cloned DNA fragment presents an alternative way to obtain the translated product without using living cells. It has a great advantage of making the system free from the constraint of maintaining machinary of life so that it offers a number of useful applications. However, relatively small amount of the product due to early stopping of the translational reaction has been the primary bottleneck for its application. To increase both the final concentration of the product and the efficiency of the cell-free protein synthesis, three approaches have been successfully taken.1. Development and optimization of novel minibioreactor systemsWe found that the major cause of the early stopping of the translation was depletion of biochemical energy supply, i.e.decreases in the concentrations of ATP and GTP.To supply enough ATP and GTP throughout the reation we developped two types of novel minibioreactor systems ; hollow fiber type and flat membrane type. In the for … More mer one, 1.2mg/ml chloramphenicolacetyltranslase (CAT) was produced in 2.5h using coupled transcription/translation of E.coli S30 fraction. In the latter one, 0.14mg/ml dihychofolate reductase was obtained using condensed wheat-germ extract as the translational machinary.2. Molecular engineering of 5'-UTR of cap-independent mRNA translation in wheat-germ extract systemWe demonstrated that 5'-UTR (144nt) of tobacco etch virus (TEV) worked effectively for cap-independent translation of dihydrofolate redactase (dhfr) -coding mRNA.Moreover, by truncating the full length 5'-UTR,We found that a fragment consising of only of 35nt had more effective than the full-length UTR for not only dhfr but also cat nand other gens.3. Separation and purification of the synthesized protein after ranslation reaction The translation reaction system contains a number of different protein amounting totally 1-10mg/ml, from which the synthesized protein (usually 0.1-1.0mg/ml) must be separated and purified if one wants to characterize it in terms of its various properties. We have tried it by tagging 5 histizine residues at N- or C- terminus of CAT,and applying nickel resin. Separation of CAT having His-tag at N-terminus was unsuccessful. However, the one having His-Tag at C-terminus was able to be effectively separated if all dithiothreitol was removed from the reaction mixture and the concentration of imidazole was gradually increased. Less