Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis

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	Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis
	Yang, Q ; Hu, XJ ; Xu, XM ; Gao, XR ; An, LJ ; Su, ZG ; Janson, JC
刊名	PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS
	2002-06-01
卷号	29 期号:3 页码:390-393
关键词	thrombin-like enzyme cloning expression purification characterization
ISSN号	1000-3282
其他题名	Prog. Biochem. Biophys.
中文摘要	The cDNA of gloshedobin was synthesized and amplified by RT-PCR from the total RNA of snake (Gloydius shedaoensis) venom gland. The 711 bp nucleotide sequence, which encodes the mature gloshedobin, was, cloned into expression vector pPIC 9K and transferred into yeast Pichia Pastoris, strain GS115. Transfermants with phenotype His(+) Mut(+) were selected to study their expression. Recombinant protein was conveniently separated and purified from the supernatant by two chromatographic steps: ion exchange chromatography on Q-Sepharose FF and affinity chromatography on Benzamidine-Sepharose 4BCL. Like intact gloshedobin, the recombinant enzyme exhibited strong esterase activity using tripeptide p-nitroanilide derivatives as substrate, but hydrolyzed N-p-tosyl-L-arginine methyl ester (TAME) very weakly. The recombinant protein displayed extreme instability at 37degreesC in neutral buffer but higher stability at 0degreesC, and also, pH is not a key factor to affect its stability while the optimal pH for its enzymatic activity is pH 8.0.
英文摘要	The cDNA of gloshedobin was synthesized and amplified by RT-PCR from the total RNA of snake (Gloydius shedaoensis) venom gland. The 711 bp nucleotide sequence, which encodes the mature gloshedobin, was, cloned into expression vector pPIC 9K and transferred into yeast Pichia Pastoris, strain GS115. Transfermants with phenotype His(+) Mut(+) were selected to study their expression. Recombinant protein was conveniently separated and purified from the supernatant by two chromatographic steps: ion exchange chromatography on Q-Sepharose FF and affinity chromatography on Benzamidine-Sepharose 4BCL. Like intact gloshedobin, the recombinant enzyme exhibited strong esterase activity using tripeptide p-nitroanilide derivatives as substrate, but hydrolyzed N-p-tosyl-L-arginine methyl ester (TAME) very weakly. The recombinant protein displayed extreme instability at 37degreesC in neutral buffer but higher stability at 0degreesC, and also, pH is not a key factor to affect its stability while the optimal pH for its enzymatic activity is pH 8.0.
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Biochemistry & Molecular Biology ; Biophysics
研究领域[WOS]	Biochemistry & Molecular Biology ; Biophysics
关键词[WOS]	SNAKE-VENOM ; MOLECULAR-CLONING ; SEQUENCE-ANALYSIS ; CDNA ; PROTEASES ; BATROXOBIN ; RHODOSTOMA
收录类别	SCI
原文出处	://WOS:000176431800012
语种	英语
WOS记录号	WOS:000176431800012
公开日期	2013-11-12
内容类型	期刊论文
版本	出版稿
源URL	[http://ir.ipe.ac.cn/handle/122111/5665]
专题	过程工程研究所_研究所（批量导入）
作者单位	1.Dalian Univ Technol, Bioengn Inst, Dalian 116012, Peoples R China 2.Chinese Acad Sci, Inst Chem Met, State Key Lab Biochem Engn, Beijing 100080, Peoples R China 3.Biosurface Sci Ctr, S-75123 Uppsala, Sweden
推荐引用方式 GB/T 7714	Yang, Q,Hu, XJ,Xu, XM,et al. Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis[J]. PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS,2002,29(3):390-393.
APA	Yang, Q.,Hu, XJ.,Xu, XM.,Gao, XR.,An, LJ.,...&Janson, JC.(2002).Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis.PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS,29(3),390-393.
MLA	Yang, Q,et al."Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis".PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS 29.3(2002):390-393.