Studies on Increasing Efficiency of Cell-free Protein Synthesis

从克隆DNA片段合成无细胞蛋白提供了一种无需使用活细胞即可获得翻译产物的替代方法。它的一大优点是使系统摆脱了维护生命机器的束缚，从而提供了许多有用的应用。然而，由于平移反应过早停止而导致的产物相对较少一直是其应用的主要瓶颈。为了提高产物的最终浓度和无细胞蛋白合成的效率，已经成功地采用了三种方法。新型微型反应器系统的开发与优化我们发现，翻译早期停止的主要原因是生化能量供应的枯竭，即ATP和GTP浓度的降低。为了在整个过程中提供足够的ATP和GTP，我们开发了两种新型微型生物反应器系统；中空纤维型和平膜型。前者利用大肠杆菌S30片段的偶联转录/翻译，在2.5h内产生了1.2mg/ml的氯霉素乙酰基翻译酶（CAT）。以浓缩小麦胚芽提取物为转译载体，二氢叶酸还原酶的转化率为0.14mg/ml。烟草侵染病毒（TEV）的5′-UTR （144nt）可有效翻译二氢叶酸重羧酶（dhfr）编码的mRNA。此外，通过截断全长5'-UTR，我们发现仅包含35nt的片段不仅对dhfr，而且对cat和其他基因都比全长UTR更有效。翻译反应体系中含有许多不同的蛋白质，总量为1-10mg/ml，如果要对合成蛋白的各种性质进行表征，必须对合成蛋白（通常为0.1-1.0mg/ml）进行分离纯化。我们尝试在CAT的N或C端标记5个组氨酸残基，并应用镍树脂。在n端有his标记的CAT分离不成功。但是，如果将反应混合物中所有的二硫苏糖醇全部去除，并逐渐增加咪唑的浓度，则c端有His-Tag的一种可以有效分离。少

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