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

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

作　　者：詹冬玲[1,2] 白挨玺[1] 白鹤[1] 韩葳葳[1] 冯雁[1] 

机构地区：[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

出　　处：《吉林大学学报（理学版）》2010年第6期1065-1069,共5页Journal of Jilin University:Science Edition

基　　金：国家自然科学基金(批准号:31070638);吉林省自然科学基金(批准号:20105109);吉林大学基本科研业务费专项基金(批准号:200810019)

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.Gene PH1704 from an archeaon Pyrococcus horikoshii OT3 was cloned and expressed at high levels in E.coli BLP-CodonPlus. The recombinant 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.

关 键 词：嗜热蛋白酶 表达 纯化 性质 

分 类 号：Q786[生物学—分子生物学]