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Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis
Alternative TitleProg. Biochem. Biophys.
Yang, Q; Hu, XJ; Xu, XM; Gao, XR; An, LJ; Su, ZG; Janson, JC
2002-06-01
Source PublicationPROGRESS IN BIOCHEMISTRY AND BIOPHYSICS
ISSN1000-3282
Volume29Issue:3Pages:390-393
AbstractThe 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.; 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.
KeywordThrombin-like Enzyme Cloning Expression Purification Characterization
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
URL查看原文
Indexed BySCI
Language英语
WOS KeywordSNAKE-VENOM ; MOLECULAR-CLONING ; SEQUENCE-ANALYSIS ; CDNA ; PROTEASES ; BATROXOBIN ; RHODOSTOMA
WOS Research AreaBiochemistry & Molecular Biology ; Biophysics
WOS SubjectBiochemistry & Molecular Biology ; Biophysics
WOS IDWOS:000176431800012
Citation statistics
Cited Times:3[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/5665
Collection研究所(批量导入)
Affiliation1.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
Recommended Citation
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.
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