Studies on Increasing Efficiency of Cell-free Protein Synthesis

提高无细胞蛋白质合成效率的研究

• 批准号: 07455328
• 负责人: YAMANE Tsuneo
• 金额: $ 4.8万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07455328/
• 关键词: Cell-free protein synthesis; Wheat-germ extract; E.coli S30 fraction; Minibioreactor; cap-independent translation; Histidine tag; 遺伝子; 翻訳; 分子進化工学; バイオリアクター; mRNAの安定性; ATP; 無細胞蛋白合成

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

从克隆的DNA片段的无细胞蛋白质合成提供了一种不使用活细胞获得翻译产物的替代方法。它的一个很大的优点是使系统摆脱了维护生命机械的约束，从而提供了许多有用的应用。然而，由于翻译反应的早期停止，产物的相对少量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质合成的效率，已经成功地采取了三种方法。新型微型生物反应器系统的开发与优化我们发现，生物化学能量供应的耗尽，即ATP和GTP浓度的降低是导致翻译早期停止的主要原因，为了在整个反应过程中提供足够的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。在成形 ...更多信息 用大肠杆菌S30组分的转录/翻译偶联法，在2.5小时内产生1.2mg/ml氯霉素乙酰转移酶（CAT）。在后者中，以小麦胚浓缩液为翻译机器，获得了0.14mg/ml的二羟基叶酸还原酶.小麦胚芽提取物体系中帽非依赖性mRNA翻译的5 ′-UTR的分子工程我们证明烟草蚀纹病毒（tobacco etch virus，TEV）的5 ′-UTR（144 nt）能够有效地进行编码二氢叶酸还原酶（dihydrofolate redactase，dhfr）的mRNA的帽非依赖性翻译。我们发现一个仅由35 nt组成的片段比全长UTR对dhfr和cat基因都有更好的作用.翻译反应后合成蛋白质的分离和纯化翻译反应体系中含有大量不同的蛋白质，总量为1- 10 mg/ml，如果要对其各种性质进行表征，必须从中分离和纯化合成蛋白质（通常为0. 1 - 1. 0 mg/ml）。我们在CAT的N-或C-末端标记了5个组氨酸残基，并应用镍树脂进行了尝试。在N-末端具有His-标签的CAT的分离不成功。然而，如果从反应混合物中除去所有的二硫苏糖醇并且逐渐增加咪唑的浓度，则在C-末端具有His-Tag的一种能够被有效地分离。少

项目成果

Hideo Nakano: "Accumulation of translational inhibitors during multi-hour cell-free protein synthesis reaction using rabbit reticulocyte lysate" Journal of Fementation and Bioengineering. 83. 470-473 (1997)

Hideo Nakano：“使用兔网织红细胞裂解物进行多小时无细胞蛋白质合成反应期间翻译抑制剂的积累”发酵与生物工程杂志。

Yasuaki Kawarasaki: "Purification and some properties of wheat germ acid phosphatases" Plant Science. 119. 67-77 (1996)

Yasuaki Kawarasaki：“小麦胚芽酸性磷酸酶的纯化和一些特性”植物科学。

Yasuaki Kawarasaki: "A long-lived batch reaction system of cell-free protein synthesis" Analytical Biochemistry. 226・2. 320-324 (1995)

Yasuaki Kawarasaki：“无细胞蛋白质合成的长寿命批量反应系统”226・2（1995）。

Yasuaki Kawarasaki: "Prolonged cell-free protein synthesis in a batch system using wheat germ extract" Bioscince,Biotechnology and Biochemistry. 58・10. 1911-1913 (1994)

Yasuaki Kawarasaki：“使用小麦胚芽提取物在批量系统中延长无细胞蛋白质合成”生物科学、生物技术和生物化学 58・10 1911-1913（1994）。

Yasuaki Kawarasaki: "Prolonged cell-free protein synthesis in a batch system using wheat germ extract" Bioscience, Biotechnology and Biochemistry. 58. 1911-1913 (1994)

Yasuaki Kawarasaki：“使用小麦胚芽提取物在批量系统中延长无细胞蛋白质合成”生物科学、生物技术和生物化学。