大连蛇岛蝮蛇类凝血酶基因在毕赤酵母中的克隆表达及分离纯化(英文)  

Expression, Purification and Partial Characterization of Recombinant Gloshedobin, a Thrombin-like Enzyme from The Venom of Gloydius shedaoensis

作　　者：杨青[1] 胡学军[1] 许小明[1] 高晓蓉[1] 安利佳[1] 苏志国[2] J.C.JANSON 

机构地区：[1]大连理工大学化工学院,大连116012 [2]中国科学院化工冶金研究所生物化学工程国家重点实验室,北京100080 [3]Biosurface Science Center

出　　处：《生物化学与生物物理进展》2002年第3期390-393,共4页Progress In Biochemistry and Biophysics

基　　金：辽宁省科学技术基金项目 (993 0 5 0 0 1);瑞典政府NUTEK基金资助 .~~

摘　　要：根据同源性分析设计引物 ,通过RT PCR方法从大连蛇岛蝮蛇毒腺总RNA中合成扩增出类凝血酶基因 ,之后将该基因克隆到表达载体 pPIC9K中 ,经电激转化后整合至毕赤酵母细胞基因组中 .经筛选得到甲醇快速生长型转化子His+ Mut+ 在 5 0 0ml摇瓶中培养 ,甲醇诱导分泌表达 .上清液中重组类凝血酶是通过两步柱层析得到 :QSepharoseFF和Benzamidine Sepharose 4BCL .与天然蛇毒类凝血酶一致 ,分泌表达的重组类凝血酶具有较强的酯酶活性 ,但精氨酸甲酯如TAME的水解活性较弱 .此重组类凝血酶在 37℃中性溶液中保存过夜将分解成小肽 ,但在 0℃下很稳定 .该酶的最适pH为 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.

关 键 词：大连蛇岛 蝮蛇 类凝血酶基因 毕赤酵母 克隆 表达 分离纯化 

分 类 号：Q786[生物学—分子生物学] TQ460.38[化学工程—制药化工]