Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.
Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.
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DOI：
10.1021/bi300653m
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发表时间：
2012-09-18
期刊：
影响因子：
2.9
通讯作者：
Dunker AK
中科院分区：
文献类型：
--
作者：
Santner AA;Croy CH;Vasanwala FH;Uversky VN;Van YY;Dunker AK
本质上无序、高度带电的蛋白质序列充当熵刚毛（EB），当其翻译地与伙伴蛋白融合时，通过为水相互作用创造大的有利表面积和伙伴周围的大量排除体积，充当有效的增溶剂。通过远离伴侣并清除大分子，EB 可以使目标蛋白不受干扰地折叠。使用天然存在的和人工的多肽，我们证明了本质无序融合作为蛋白质增溶剂的成功实施。本文讨论的人工融合体具有低序列复杂性和高净电荷，但通过独特的氨基酸组成和长度而多样化。使用 6xHis 融合体作为对照，观察到 20 种蛋白质组合的可溶性蛋白质表达从 65% (EB60A) 增强到 100% (EB250)。此外，与常用的融合物（例如麦芽糖结合蛋白、谷胱甘肽 S-转移酶、硫氧还蛋白和氮利用物质 A）相比，这些 EB 能够更有效地溶解靶标。最后，尽管这些 EB 具有非常独特的理化特性，但它们不会干扰绿色荧光蛋白或谷胱甘肽 S-转移酶蛋白的结构、构象稳定性或功能。因此，这项工作说明了本质无序融合的成功从头设计，并为试图溶解顽固蛋白的研究人员提供了一种有前途的技术和补充资源。
Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can enable the target protein to fold free from interference. Using both naturally-occurring and artificial polypeptides we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have low sequence complexity and high net charge, but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently-used fusions such as maltose-binding-protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physio-chemical properties they did not perturb the structure, conformational stability nor function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically-disordered fusions, and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.
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影响因子：
4.8
作者：
Alsheikh, MK;Heyen, BJ;Randall, SK
通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
ZIPSER, D
DOI：
10.1016/j.bbrc.2005.05.164
发表时间：
2005-08-05
影响因子：
3.1
作者：
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通讯作者：
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