嗜热菌Pyrococcus horikoshiiOT3蛋白酶基因的原核表达、 纯化和性质表征

J4 ›› 2010, Vol. 48 ›› Issue (06): 1065-1069.

嗜热菌Pyrococcus horikoshiiOT3蛋白酶基因的原核表达、纯化和性质表征

詹冬玲^1,2, 白挨玺¹, 白鹤¹, 韩葳葳¹, 冯雁¹

1. 吉林大学分子酶学工程教育部重点实验室, 长春 130012;2. 吉林农业大学食品科学与工程学院, 长春 130118

收稿日期:2010-03-18 出版日期:2010-11-26 发布日期:2010-11-26
通讯作者: 韩葳葳 E-mail:weiweihan1997@yahoo.com.cn

Prokaryotic Expression, Purication and Properties of a Proteasefrom Thermophilic Archaeon Pyrococcus horikoshii OT3

ZHAN Dongling^1,2, BAI Aixi¹, BAI He¹, HAN Weiwei¹, FENG Yan¹

1. Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun 130012, China|2. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Chin

Received:2010-03-18 Online:2010-11-26 Published:2010-11-26
Contact: HAN Weiwei E-mail:weiweihan1997@yahoo.com.cn

摘要/Abstract

摘要：

将来源于嗜热菌Pyrococcus horikoshii OT3的蛋白酶（PH1704）基因在大肠杆菌BLP-CodonPlus中高效表达. 采用超声、热失活和HiTrap
Q Sepharose柱层析等方法对产物进行分离纯化. 通过SDSPAGE, NativePAGE和Western-blot对表达产物进行鉴定, 发现该重组酶以十二聚体为主的寡聚体形式存在, 并具有SDS抗性. 蛋白酶活力染色表明, 其六聚体以上有活力. 以azo-gelation为底物, 对其酶学性质进行初步研究表明, 其最适温度为85 ℃, 最适pH=8.0, 并且具有良好的热稳定性.

关键词: 嗜热蛋白酶；表达；纯化；性质

Abstract:

Gene PH1704 from an archeaon Pyrococcus horikoshii OT3 was cloned and expressed at high levels in E.coli BLPCodonPlus. The recomb
inant enzyme was purified to homogeneity via ultransonic, heating and HiTrap Q Sepharose chromatography. Through SDSPAGE, NativePAGE, activitystaining and westernblotting, the protease was identified as homooligomers with dodecamer being the major polymer, and showed resistance to SDS. The enzyme was characterized for azogelation, its optimum temperature and pH were 85 ℃ and 8.0, respectively. The enzyme had a favourable heat resistance.

Key words: thermophilic protease, expression, purification, character

中图分类号:

Q786

詹冬玲, 白挨玺, 白鹤, 韩葳葳, 冯雁. 嗜热菌Pyrococcus horikoshiiOT3蛋白酶基因的原核表达、纯化和性质表征[J]. J4, 2010, 48(06): 1065-1069.

DAN Dong-Ling, BAI Ai-Xi, BAI He, HAN Wei-Wei, FENG Yan. Prokaryotic Expression, Purication and Properties of a Proteasefrom Thermophilic Archaeon Pyrococcus horikoshii OT3[J]. J4, 2010, 48(06): 1065-1069.

[1]	陈宏宇, 冯树丹, 刘相国, 尹悦佳, 于莹, 杨丽颖, 郝东云. NaCl和Na₂CO₃胁迫下的高粱MAPKs基因家族表达模式[J]. J4, 2012, 50(01): 139-146.
[2]	蒋世翠, 张明哲, 王义, 孙春玉, 李校堃, 张美萍. 人参SQS基因的干扰载体构建及转化人参愈伤组织[J]. J4, 2011, 49(06): 1136-1140.
[3]	王佳思, 金英花. 人突变型CDK2（F80A）稳定细胞系的建立[J]. J4, 2010, 48(05): 871-875.