Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins.,Biotechnology and Bioengineering

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Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins.
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-03-26 , DOI: 10.1002/bit.27326
Kae Higuchi ₁ , Takashi Yabuki _{1,

2} , Masahiro Ito ₁ , Takanori Kigawa ₁

Affiliation

Protein folding is usually slowed-down at low temperatures, and thus low-temperature expression is an effective strategy to improve the soluble yield of aggregation-prone proteins. In this study, we investigated the effects of a variety of cold shock proteins and domains (Csps) on an Escherichia coli cell extract-based cell-free protein synthesis system (CF). Most of the 12 Csps that were successfully prepared dramatically improved the protein yields, by factors of more than 5 at 16°C and 2 at 23°C, to levels comparable to those obtained at 30°C. Their stimulatory effects were complementary to each other, while CspD and CspH were inhibitory. The Csps' effects correlated well with their Pfam CSD family scores (PF00313.22). All of the investigated Csps, except CspH, similarly possessed RNA binding and chaperon activities and increased the messenger RNA amount irrespective of their effect, suggesting that the proper balance between these activities was required for the enhancement. Unexpectedly, the 5'-untranslated region of cspA was less effective as the leader sequence. Our results demonstrated that the use of the Csps presented in this study will provide a simple and highly effective strategy for the CF, to improve the soluble yields of aggregation-prone proteins.

中文翻译：

冷休克蛋白在易聚集蛋白的可溶性产率方面改善了大肠杆菌的无细胞合成。

蛋白质折叠通常在低温下减慢，因此低温表达是提高易聚集蛋白质的可溶性产率的有效策略。在这项研究中，我们研究了各种冷休克蛋白和结构域 (Csps) 对基于大肠杆菌细胞提取物的无细胞蛋白质合成系统 (CF) 的影响。成功制备的 12 个 Csps 中的大多数都显着提高了蛋白质产量，在 16°C 下提高了 5 倍以上，在 23°C 下提高了 2 倍，达到与 30°C 下获得的水平相当的水平。它们的刺激作用是互补的，而CspD和CspH是抑制作用。Csps 的效果与其 Pfam CSD 家族分数 (PF00313.22) 密切相关。所有研究的 Csps，除了 CspH，同样具有 RNA 结合和伴侣活性，并增加了信使 RNA 的数量，而不管它们的效果如何，这表明这些活性之间的适当平衡是增强所必需的。出乎意料的是，cspA 的 5'-非翻译区作为前导序列的效果较差。我们的结果表明，本研究中介绍的 Csps 的使用将为 CF 提供一种简单且高效的策略，以提高易聚集蛋白质的可溶性产量。

更新日期：2020-03-26

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