hPin1原核表达、纯化及热稳定性研究  

Prokaryotic expression,purification and thermal stability of His-hPin1

作　　者：王敬章[1] 刘鑫[1] 惠心娣[1] 黄瑜[1] 黄新河[1] 杜林方[1] 

机构地区：[1]四川大学生命科学学院生物资源与生物环境教育部重点实验室,成都610065

出　　处：《四川大学学报（自然科学版）》2007年第6期1339-1343,共5页Journal of Sichuan University(Natural Science Edition)

基　　金：教育部新世纪人才计划(NCET-04-0861);科技部"十一五"支撑计划(2006BAF07B01)

摘　　要：利用E．coli BL21原核表达，纯化得到了His-hPin1融合蛋白，并研究了它的热稳定性．在中性条件下进行不同温度处理，SDS-PAGE分析表明，低于40℃处理不会影响His-hPin1的溶解性，50～100℃处理会使其部分变性而沉淀．荧光发射光谱研究表明，未处理的His-hPin1具有339nm的荧光发射峰；热处理使荧光发射峰的位置变化，低于40℃处理对His—hPin1的荧光强度影响较小，50～100℃热处理使HiS-hPin1的荧光强度明显降低．圆二色谱研究表明，热处理对His-hPin1的二级结构有影响，引起α-螺旋含量的明显减少，而无规卷曲增加．以上数据表明hPin1是一个相对热稳定的蛋白．The His-tagged hPin1 was expressed by the pET-19b-hPin1 plasmid in E. coli BL21 and was purified using Ni^2＋ -NTA-Sepharose. The thermal stability of the His-hPin1 was investigated. Heat treatment induced some of His-hPin1 dissolvability at 50-100℃. The maximum fluorescence emission of the His-hPin1 was at 339 nm, suggesting that tryptophan residues were located in a hydrophobic environment of the hPin1 molecule. Heat treatment resulted in shift of the maximum emission wavelength and decrease of the fluores- cence emission intensity, suggesting some conformation changes of the hPin1. Further Far-UV circular dichroism result showed that secondary structure of the His-hPin1 was altered after heat treatment, the content of a-helix remarkably decreased with increase of random coil. All the results suggest that hPin1 is a relative thermal stability protein.

关 键 词：hPin1 原核表达 热稳定性 荧光光谱 圆二色谱 

分 类 号：Q78[生物学—分子生物学]