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Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions
Chen, Ying1,2; Wang, Qi1,2; Zhang, Chun1; Li, Xiunan1; Gao, Qiang3; Dong, Changqing1; Liu, Yongdong1; Su, Zhiguo1
2016-06-01
发表期刊PROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
卷号122期号:JUNE页码:1-7
摘要Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency. (C) 2016 Elsevier Inc. All rights reserved.
关键词Proinsulin Inclusion Bodies Additive Oxidative Refolding Diselenide Aggregate
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.pep.2016.01.015
收录类别SCI
语种英语
关键词[WOS]HUMAN INSULIN ; PROTEIN ; EXPRESSION ; DISELENIDES ; CATALYSTS ; CLEAVAGE ; ARGININE ; TRYPSIN ; PATHWAY
WOS研究方向Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS类目Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
项目资助者National Natural Science Foundation of China(21576267) ; Novo Nordisk - Chinese Academy of Sciences Research Fund(NNCAS-2014-02) ; Open Funding Project of the National Key Laboratory of Biochemical Engineering(2014KF-05)
WOS记录号WOS:000375172200001
引用统计
文献类型期刊论文
条目标识符http://ir.ipe.ac.cn/handle/122111/21029
专题生化工程国家重点实验室
作者单位1.Chinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing 100190, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Novo Nordisk Res Ctr China, Beijing 102206, Peoples R China
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GB/T 7714
Chen, Ying,Wang, Qi,Zhang, Chun,et al. Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions[J]. PROTEIN EXPRESSION AND PURIFICATION,2016,122(JUNE):1-7.
APA Chen, Ying.,Wang, Qi.,Zhang, Chun.,Li, Xiunan.,Gao, Qiang.,...&Su, Zhiguo.(2016).Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.PROTEIN EXPRESSION AND PURIFICATION,122(JUNE),1-7.
MLA Chen, Ying,et al."Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions".PROTEIN EXPRESSION AND PURIFICATION 122.JUNE(2016):1-7.
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