Optimization of DsbA Purification from Recombinant Escherichia coli Broth Using Box-Behnken Design Methodolog

Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. Th...

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Published in中国化学工程学报：英文版 no. 2; pp. 185 - 191
Main Author LUO Man GUAN Yixin YAO Shanjing
Format Journal Article
LanguageEnglish
Published 2013
Subjects
DsbA蛋白
优化
发酵液
离子交换色谱法
纯化
肽质量指纹图谱
设计方法论
重组大肠杆菌
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Summary:Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was （377.9±1.7） mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.
Bibliography:disulfide bond formation protein A, protein purification, Box-Behnken experiment design, response surface methodology, multi-object programming
Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was （377.9±1.7） mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.
11-3270/TQ
ISSN:1004-9541
2210-321X