Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths.
Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths.
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DOI：
10.1002/biot.201600689
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发表时间：
2017-05
影响因子：
4.7
通讯作者：
Etsushi Yamamoto;S. Yamaguchi;Teruyuki Nagamune
中科院分区：
文献类型：
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作者：
Etsushi Yamamoto;S. Yamaguchi;Teruyuki Nagamune
从细菌包涵体中复性蛋白是生产重组蛋白的关键步骤，但由于聚集，复性步骤通常导致显著较低的产率。为了防止聚集，经常使用化学添加剂。然而，添加剂有效增加重折叠产率的能力是蛋白质依赖性的，因此，重要的是要了解添加剂的亚结构赋予蛋白质重折叠合适性质的方式。以聚乙二醇单油醚（PGME）系列非离子洗涤剂为研究对象，系统研究了2 ~ 90个聚乙二醇（PEG）长度的PGME对猪肌肉乳酸脱氢酶（LDH）、鸡蛋白色溶菌酶和酵母α-葡萄糖苷酶的复性影响。具有较长PEG长度的PGME如PGME 20、50和90抑制聚集，并增加重折叠产率。值得注意的是，PGME 20将LDH产率从没有添加剂的2.5%增加到56.7%。根据LDH的复性动力学分析，与PGME 50和90相比，在PGME 20溶液中的复性速率常数在较宽的浓度范围内保持相对较高，这是因为其参与折叠的分子内相互作用的空间位阻较弱，导致复性优于聚集。这些研究结果应提供基本的指导方针，以确定适当的PEG为基础的非离子洗涤剂的蛋白质复性。
Protein refolding from bacterial inclusion bodies is a crucial step for the production of recombinant proteins, but the refolding step often results in significantly lower yields due to aggregation. To prevent aggregation, chemical additives are often used. However, the ability of additives to effectively increase refolding yields are protein dependent, and therefore, it is important to understand the manner in which the substructures of additives confer suitable properties on protein refolding. We focused attention on nonionic detergents, the polyethylene glycol monooleyl ether (PGME) series, and systematically studied the influence of two to 90 polyethylene glycol (PEG) lengths of PGMEs on the refolding of pig muscle lactate dehydrogenase (LDH), hen egg white lysozyme, and yeast α-glucosidase. PGMEs with longer PEG lengths such as PGME20, 50, and 90 suppressed aggregation, and increased refolding yields. Notably, PGME20 increased the LDH yield to 56.7% from 2.5% without additives. According to the refolding kinetic analysis of LDH, compared with PGME50 and 90, the refolding rate constant in PGME20 solutions remained relatively high at a broad range of concentrations because of its weaker steric hindrance of intramolecular interactions involved in folding, leading to a preference for refolding over aggregation. These findings should provide basic guidelines to identify appropriate PEG-based nonionic detergents for protein refolding.