Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.
Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.
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DOI：
10.1371/journal.pone.0022981
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
Yang Z;Zhang L;Zhang Y;Zhang T;Feng Y;Lu X;Lan W;Wang J;Wu H;Cao C;Wang X
重组蛋白的大规模生产对蛋白质功能和结构的研究具有重要意义，尤其是利用大肠杆菌高效表达系统。然而，大约70%的重组蛋白以不溶性包涵体的形式过度表达。在此，我们提出了一种从包涵体中提取可溶性蛋白质的有效方法，即通过两步变性和一步复性。我们首先用三种不同折叠性质的蛋白质证明了这种方法比传统的一步变性和一步复性的方法更具优势。体外试验和生物测定表明，复性后的蛋白质具有活性。然后，我们通过分析来自人类和其他生物的88种蛋白质来测试这种方法的普遍适用性，所有这些蛋白质都以包涵体的形式表达。我们发现，这些蛋白质中约有76%被复性，平均得率为75%的可溶性蛋白质。这种“两步变性复性”(2DR)方法简单、高效、通用性强，可用于获得具有活性的重组蛋白，用于基础研究和工业应用。
The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.
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