State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai, China.
PLoS One. 2011;6(7):e22981. doi: 10.1371/journal.pone.0022981. Epub 2011 Jul 29.
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.
大规模生产重组蛋白对于蛋白质功能和结构研究非常重要，特别是使用大肠杆菌过表达系统；然而，约 70%的重组蛋白以不溶性包涵体的形式过量表达。在这里，我们提出了一种通过两步变性和一步复性从包涵体中生成可溶性蛋白的有效方法。我们首先通过使用具有不同折叠特性的三种蛋白质，证明了该方法相对于具有一步变性和一步复性的传统方法的优势。通过体外试验和生物测定发现，复性后的蛋白质具有活性。然后，我们通过分析来自人和其他生物体的 88 种蛋白质，这些蛋白质均以包涵体形式表达，测试了该方法的普遍适用性。我们发现，约 76%的这些蛋白质通过复性得到了折叠，可溶性蛋白的平均产率超过 75%。这种“两步变性和复性”（2DR）方法简单、高效且具有普遍适用性；它可以用于获得用于基础研究和工业目的的活性重组蛋白。
