丝氨酸蛋白酶催化结构域无活性突变体的表达、纯化与结晶  

Expression,Purification and Crystallization of Inactivated Serine Protease Domain of Matriptase

作　　者：郭团玉[1,2] 赵宝玉[3] 袁彩[3] 黄明东[3] 

机构地区：[1]宁德师范学院生物系,福建宁德352100 [2]特色生物化工材料福建省重点实验室,福建宁德352100 [3]中国科学院福建物质结构研究所,福建福州350007

出　　处：《过程工程学报》2017年第4期834-838,共5页The Chinese Journal of Process Engineering

基　　金：国家自然科学基金资助项目(编号:31170707;21473096)

摘　　要：构建了丝氨酸蛋白酶Matriptase的催化结构域无活性突变体质粒S195A,并在酵母中表达,通过阴离子柱粗捕获及分子筛和离子交换柱纯化得到高纯度重组蛋白,通过气相扩散法得到无活性突变体的晶体.结果表明,与野生型相比,单点突变极大程度提高了Matriptase的表达,Matriptase无活性突变体不具有水解底物活性,但仍可与抑制蛋白HAI-1结合.晶体分辨率为1.48?,195A突变没有影响Matriptase的构象.An inactivated mutant of matriptase serine protease domain （S 195A） was constructed, and the recombinant protein in Pichia pastoris was expressed. After captured by anion exchange chromatography, the recombinant protein was further purified by gel-filtration chromatogram column and resource Q anion exchange column with high purity. High quality crystals of this inactivated protein were obtained by sitting-drop vapor diffusion. The results showed that the single point mutant increased the expression level of matriptase compared with the wild type. This inactivated mutant forms stable complex with its inhibitor HAI-1, although it does not possess catalytic activity. Crystals of this inactivated protein were diffracted to 1.48 A. The mutant has the same conformation as the wild type.

关 键 词：丝氨酸蛋白酶 表达 晶体生长 

分 类 号：Q556.9[生物学—生物化学]