Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

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.大规模制备重组蛋白是蛋白质功能与结构研究的重要基础，其中借助大肠杆菌（Escherichia coli）外源过表达系统开展相关研究尤为普遍；然而约70%的重组蛋白会以不溶性包涵体的形式被过量表达。本研究提出了一种可从包涵体中获取可溶性蛋白的高效方法，该方法采用两步变性与一步复性的工艺。我们首先选用三种具有不同折叠特性的蛋白质，对比验证了本方法相较于传统“一步变性-一步复性”流程的优势。经体外实验与生物活性测定证实，复性所得蛋白仍保留生物活性。随后，我们选取了来自人类及其他物种的88种均以包涵体形式表达的蛋白质，以此验证本方法的普适性。实验结果显示，其中约76%的蛋白质可成功复性，可溶性蛋白的平均回收率高于75%。这种“两步变性复性法（two-step-denaturing and refolding，2DR）”操作简便、效率极高且普适性强，可用于为基础研究与工业应用获取具有活性的重组蛋白。