摘要
采用定位突变和重叠延伸PCR方法扩增尿激酶催化结构域基因片段,将其克隆至表达载体pPICZαA上,转化毕氏酵母X-33。重组蛋白通过阳离子琼脂糖柱纯化、凝胶层析柱检测,纯度达到99%以上,该重组的尿激酶催化结构域(C279A/N302Q/S356A)不具有丝氨酸蛋白酶活性。用气相扩散法获得蛋白晶体,其衍射分辨率达1.77(?),结构解析发现在其复合物结构中有三个抑制剂分子苯甲醚。
The gene fragment of urokinase catalytic domain mutant(C279A/N302Q/S356A) was amplified by the site-mutated PCR method and was cloned into pPICZαA secretory expression plasmid.The recombinant plasmid was transformed into yeast X-33. The recombinant protein was captured by the cation exchange chromatography and purified to 99%purity as analyzed by gel-filtration chromatogram column Superdex75.The recombinant mutant protein was inactive,as expected,and crystallized by the method of sitting-drop vapor diffusion.These crystals diffracted to 1.77A in our house X-ray.The structure analysis of the uPA-benzamidine reveals that the presence of three benzamidine molecules and three SO4 ions right next to uPA.This three-dimensional structure provides the basis for the design of small molecules.
出处
《生物技术通报》
CAS
CSCD
北大核心
2009年第S1期336-339,共4页
Biotechnology Bulletin
基金
基金委(30625011
30811130467)
中科院创新基地(KSCX2-YW-R-082)
科技部863项目(2006AA02A313)
福建省青年人才项目(2007F3119)
关键词
尿激酶
定位突变PCR
表达
纯化
结晶
Urokinase
Site-mutated PCR
Expression
Purification
Crystallization
