Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro
- 作者：Man Luo ; Yi-Xin Guan ; guanyx@zju.edu.cn ; Shan-Jing Yao
- 关键词：DsbA ; Lysozyme ; Refolding ; Plackett&ndash ; Burman design ; Small central composite design ; Multiple objective programming
- 刊名：Process Biochemistry
- 出版年：2012
- 期刊代码：29_13595113
- 类别：bio
- 出版时间：August, 2012
- 卷：47
- 期：8
- 页码：1268-1276
- 文件大小：1387 K
摘要
DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett-Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7%protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 渭g/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.