A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based.
A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based.
复制标题
对体外系统中蛋白质合成的比较研究：从原核生物重构为基于真核生物提取物。
DOI：
10.1186/1472-6750-8-58
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发表时间：
2008-07-29
影响因子：
3.5
通讯作者：
Chong, Shaorong
中科院分区：
文献类型：
--
作者：
Hillebrecht, Jason R.;Chong, Shaorong
无细胞蛋白质合成技术不仅是一种快速、高通量的蛋白质合成技术，而且为研究蛋白质的翻译和折叠提供了一个体外平台。详细比较不同无细胞系统中的体外蛋白质合成，可能会为它们的应用提供生物学差异和指导。在体外重组原核系统，S30提取物为基础的系统和真核系统中的蛋白质合成进行了研究。与S30系统相比，重组系统中蛋白质合成的产量降低，并且对冷更敏感。用来自S30提取物的尺寸排阻分离的级分补充重构系统显著增加了产率和活性，达到接近S30系统的水平。虽然在原核和真核系统中的蛋白质合成没有表现出显着差异的真核报告蛋白，观察到巨大的差异时，人工融合蛋白在体外合成。原核系统不能合成和正确折叠大量的全长融合蛋白，即使补充真核细胞裂解物。只有在真核系统中才能合成有活性的全长融合蛋白。重组的细菌系统是足够的，但在蛋白质合成中效率不高。相比之下，S30系统含有能够增强蛋白质翻译和折叠的额外细胞因子。真核生物的翻译机制可能是从原核生物进化而来的，目的是将更复杂（难以翻译）的模板翻译成活性蛋白。
Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications. Protein synthesis was investigated in vitro in a reconstituted prokaryotic system, a S30 extract-based system and a eukaryotic system. Compared to the S30 system, protein synthesis in the reconstituted system resulted in a reduced yield, and was more cold-sensitive. Supplementing the reconstituted system with fractions from a size-exclusion separation of the S30 extract significantly increased the yield and activity, to a level close to that of the S30 system. Though protein synthesis in both prokaryotic and eukaryotic systems showed no significant differences for eukaryotic reporter proteins, drastic differences were observed when an artificial fusion protein was synthesized in vitro. The prokaryotic systems failed to synthesize and correctly fold a significant amount of the full-length fusion protein, even when supplemented with the eukaryotic lysate. The active full-length fusion protein was synthesized only in the eukaryotic system. The reconstituted bacterial system is sufficient but not efficient in protein synthesis. The S30 system by comparison contains additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate) templates into active proteins.
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影响因子：
56.9
作者：
Frydman, J;Hartl, FU
通讯作者：
Hartl, FU
影响因子：
4.1
作者：
Hino, Mami;Kataoka, Masatoshi;Baba, Yoshinobu
通讯作者：
Baba, Yoshinobu
DOI：
10.1016/j.bbrc.2004.06.095
发表时间：
2004-08-06
影响因子：
3.1
作者：
Ying, BW;Taguchi, H;Ueda, T
通讯作者：
Ueda, T
影响因子：
3.8
作者：
Underwood, KA;Swartz, JR;Puglisi, JD
通讯作者：
Puglisi, JD
DOI：
10.1016/j.bbrc.2006.11.017
发表时间：
2007-01-05
影响因子：
3.1
作者：
Ohashi, Hiroyuki;Shimizu, Yoshihiro;Ueda, Takuya
通讯作者：
Ueda, Takuya