Cell-free protein expression under macromolecular crowding conditions.
Cell-free protein expression under macromolecular crowding conditions.
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DOI：
10.1371/journal.pone.0028707
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Xu J
中科院分区：
文献类型：
--
作者：
Ge X;Luo D;Xu J
目前，由体外转录和翻译组成的无细胞蛋白表达（CFPE）是在相对稀的溶液中进行操作，其中活细胞中存在的大分子拥挤效应在很大程度上被忽略。这可能不仅会影响体外蛋白质合成的效率，还会限制我们对这一基本生物过程中生物分子的功能和相互作用的理解。以小麦胚芽提取物中海肾荧光素酶的无细胞合成为模型系统，我们研究了大分子拥挤环境下的 CFPE，模拟了三种不同的拥挤剂：PEG-8000、Ficoll-70 和 Ficoll-400，这三种拥挤剂的化学性质和分子大小各不相同。我们发现在大分子拥挤溶液中转录显着增强；在 20% (w/v) 的 Ficoll-70 存在下，检测到 mRNA 产量增加了 4 倍。相反，三种拥挤剂的添加通常会抑制翻译。这可能是由于 PEG 诱导的蛋白质沉淀以及翻译因子与 Ficoll 分子的非特异性结合。我们进一步探索了两阶段 CFPE，其中转录和翻译分别在高大分子拥挤条件和低大分子拥挤条件下进行。其产生的蛋白质产量比偶联的 CFPE 对照高 2.2 倍。随后，通过在大分子拥挤环境下无细胞合成约两倍大的蛋白质萤火虫荧光素酶，证实了 CFPE 的大分子拥挤效应。本研究中使用的三种大分子拥挤剂对转录和翻译具有相反的影响。这项研究的结果应该有助于研究人员选择大分子拥挤剂，并表明两级 CFPE 比耦合 CFPE 更有效。
Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process. Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments. Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.
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影响因子：
2.9
作者：
Kern, JA;Davis, RH
通讯作者：
Davis, RH
影响因子：
3.8
作者：
Jewett, MC;Swartz, JR
通讯作者：
Swartz, JR
影响因子：
2.9
作者：
Ballantyne, Kaye N.;van Oorschot, Roland A. H.;Koukoulas, Irene
通讯作者：
Koukoulas, Irene
影响因子：
14.8
作者：
Chattopadhaya, Souvik;Tan, Lay Pheng;Yao, Shao Q.
通讯作者：
Yao, Shao Q.
DOI：
10.1111/j.1432-1033.1996.0881u.x
发表时间：
1996-08-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Kim, DM;Kigawa, T;Yokoyama, S
通讯作者：
Yokoyama, S