【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!.更多还原

【Abstract】 The gene fragment of urokinase catalytic domain mutant (C279A/N302Q) 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 and selected with Zeocin. The recombinant protein was captured by the cation exchange chromatography SPFF and was purified to 99% of purity. The recombinant mutant protein was crystallized by the method of sitting-drop vapor diffusion. These crystals diffracted to 1.45?魡 with synchrotron X-ray.更多还原