文献类型：期刊文献

英文题名：An Automatic Refolding Apparatus for Preparative-Scale Protein Production

作者：Feng, Yanye[1,2];Zhang, Ming[1];Zhang, Linlin[2];Zhang, Ting[1];Ding, Jianfeng[3];Zhuang, Yingping[1];Wang, Xiaoning[1];Yang, Zhong[2]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Sch Biotechnol, Shanghai 200237, Peoples R China;[2]Fudan Univ, State Key Lab Genet Engn, Dept Microbiol, Sch Life Sci, Shanghai 200433, Peoples R China;[3]Novoprotein Sci Inc, Dept R&D, Shanghai, Peoples R China

年份：2012

卷号：7

期号：9

外文期刊名：PLOS ONE

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000309517500044)】；

基金：This work was supported by National Basic Research Program of China (No. 2009CB918600). 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：Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.