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.

大规模生产重组蛋白在蛋白质功能与结构研究中具有重要意义，尤以大肠杆菌（Escherichia coli）过表达系统的应用最为广泛；然而，约70%的重组蛋白会以不溶性包涵体的形式过量表达。本研究提出了一种高效的包涵体可溶性蛋白制备方法，该方法采用两步变性、一步复性的实验流程。我们首先选取三种具有不同折叠特性的蛋白质作为模型，验证了该方法相较于传统一步变性-一步复性流程的优势。体外活性检测与生物测定结果表明，复性后的蛋白具备生物学活性。随后，我们通过分析来自人类及其他物种的88种以包涵体形式表达的蛋白质，验证了该方法的普适性：其中约76%的蛋白质成功实现复性，可溶性蛋白的平均得率超过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，可用于获取基础研究与工业应用所需的活性重组蛋白。