摘要
利用重叠延伸PCR方法扩增尿激酶催化结构域的突变体基因片段,将其克隆至表达载体pPICZαA上,转化酵母X-33,用Zeocin筛选高拷贝数的酵母菌落.重组蛋白通过阳离子琼脂糖柱纯化,纯度达到99%,该仅含尿激酶催化结构域的突变体(C279A/N302Q),无需激活即具有尿激酶活性.用气相扩散法获得蛋白质晶体,其衍射分辨率达1.45!.
The gene fragment of urokinase catalytic domain mutant （C279A/N302Q） was amplified by the si te-mutated PCR method and was cloned into pPICZαA secretory expression plasmid. The recombinant plasmid was transformed into yeast X-33 and selected with Zeocin. The recombinant protein was captured by the cationexchange chromatography SPFF and was purified to 99% of purity. The recombinant mutant protein was crystallized by the method of sittifig-drop vapor diffusion. These crystals diffracted to 1.45A° with synchrotron X-ray.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2006年第4期377-381,共5页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金重点资助项目(30430190)
结构化学国家重点实验室项目(021061)
中国科学院百人计划资助项目.
