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	Different Effects of L-Arginine on Protein Refolding: Suppressing Aggregates of Hydrophobic Interaction, Not Covalent Binding
	Chen, Jing; Liu, Yongdong; Wang, Yinjue; Ding, Hong; Su, Zhiguo
刊名	BIOTECHNOLOGY PROGRESS
	2008-11-01
卷号	24期号:6页码:1365-1372
关键词	refolding arginine GFP CAB rhG-CSF aggregation
ISSN号	8756-7938
其他题名	Biotechnol. Prog.
中文摘要	Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins., which is not yet well explained. In this work, refolding of three proteins in the presence of 0-2 M arginine was investigated and compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was successfully refolded with the help of arginine. The refolding yield could reach almost 100% in the presence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG-CSF), containing five cysteines, could only achieve 65% refolding yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacetamide and subsequently refolding of the protein could reduce the aggregate formation substantially. Further investigation on recombinant green fluorescence protein (GFP), containing two cysteines, also revealed the accumulation of oligomers. The content of oligomers increased with the concentration of arginine, reaching about 30% at 2 M arginine. Comparison of reduced and nonreduced SDS-PAGE revealed that the oligomers were formed through intermolecular disulfide binding. Analysis of the refolding kinetics indicated that intermolecular disulfide bonds were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation.
英文摘要	Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins., which is not yet well explained. In this work, refolding of three proteins in the presence of 0-2 M arginine was investigated and compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was successfully refolded with the help of arginine. The refolding yield could reach almost 100% in the presence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG-CSF), containing five cysteines, could only achieve 65% refolding yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacetamide and subsequently refolding of the protein could reduce the aggregate formation substantially. Further investigation on recombinant green fluorescence protein (GFP), containing two cysteines, also revealed the accumulation of oligomers. The content of oligomers increased with the concentration of arginine, reaching about 30% at 2 M arginine. Comparison of reduced and nonreduced SDS-PAGE revealed that the oligomers were formed through intermolecular disulfide binding. Analysis of the refolding kinetics indicated that intermolecular disulfide bonds were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation.
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Biotechnology & Applied Microbiology ; Food Science & Technology
研究领域[WOS]	Biotechnology & Applied Microbiology ; Food Science & Technology
关键词[WOS]	GREEN FLUORESCENT PROTEIN ; CARBONIC ANHYDRASE-B ; ESCHERICHIA-COLI ; INCLUSION-BODIES ; PROPOSED MECHANISM ; RENATURATION ; LYSOZYME ; CHROMATOGRAPHY ; STABILIZATION ; PURIFICATION
收录类别	SCI
原文出处	://WOS:000262069100020
语种	英语
WOS记录号	WOS:000262069100020
公开日期	2013-10-08
内容类型	期刊论文
版本	出版稿
源URL	[http://ir.ipe.ac.cn/handle/122111/2724]
专题	过程工程研究所_研究所（批量导入）
作者单位	Chinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing 100190, Peoples R China
推荐引用方式 GB/T 7714	Chen, Jing,Liu, Yongdong,Wang, Yinjue,et al. Different Effects of L-Arginine on Protein Refolding: Suppressing Aggregates of Hydrophobic Interaction, Not Covalent Binding[J]. BIOTECHNOLOGY PROGRESS,2008,24(6):1365-1372.
APA	Chen, Jing,Liu, Yongdong,Wang, Yinjue,Ding, Hong,&Su, Zhiguo.(2008).Different Effects of L-Arginine on Protein Refolding: Suppressing Aggregates of Hydrophobic Interaction, Not Covalent Binding.BIOTECHNOLOGY PROGRESS,24(6),1365-1372.
MLA	Chen, Jing,et al."Different Effects of L-Arginine on Protein Refolding: Suppressing Aggregates of Hydrophobic Interaction, Not Covalent Binding".BIOTECHNOLOGY PROGRESS 24.6(2008):1365-1372.

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