文献类型：期刊文献

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

作者：Yang, Zhong[1,3];Zhang, Linlin[2,3];Zhang, Yan[2];Zhang, Ting[3];Feng, Yanye[1,3];Lu, Xiuxiu[2];Lan, Wenxian[2];Wang, Jufang[4];Wu, Houming[2];Cao, Chunyang[2];Wang, Xiaoning[1,4]

机构：[1]Fudan Univ, State Key Lab Genet Engn, Dept Microbiol, Sch Life Sci, Shanghai 200433, Peoples R China;[2]Chinese Acad Sci, State Key Lab Bioorgan & Nat Prod Chem, Shanghai Inst Organ Chem, Shanghai, Peoples R China;[3]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Sch Biotechnol, Shanghai 200237, Peoples R China;[4]S China Univ Sci & Technol, Sch Biosci & Bioengn, Guangzhou, Guangdong, Peoples R China

年份：2011

卷号：6

期号：7

外文期刊名：PLOS ONE

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000293286500062)】；

基金：This work was supported by grants from the Ministry of Science and Technology of the People's Republic of China (No. 2009CB918600), the National Scientific Fundation Committee (No. 20872169 and 20921091), and the Pujiang Talents Awards from Science and Technology Commission of Shanghai Municipality (No. 08PJ1411700). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

语种：英文

摘要：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.