钙离子通道膜蛋白DdMCU的酵母表达

Yeast Expression of Calcium Channel Membrane Protein DdMCU

Mitochondrial Ca^(2+)Uniporter(MCU)作为细胞钙离子通道uniporter复合物的重要组分发挥了关键作用.近期研究发现将盘基网柄菌(Dictyostelium discoideum)DdMCU重构于酵母系统进一步证明了MCU是线粒体正常发挥uniporter性的最基本原件.为了深入研究DdMCU结构与功能,本研究通过合成盘基网柄菌的MCU基因,利用PCR得到目的DNA片段,克隆到表达载体pPICZX,构建DdMCU-GFP融合蛋白表达质粒pPICZX-DdMCU.重组质粒转化酵母X33,经博莱霉素梯度筛选,得到8个酵母重组子,且PCR检测目的基因已与酵母基因组整合成功.酵母重组子进行甲醇诱导表达,经激光共聚焦显微镜与western检测,均表明蛋白已成功表达.

Ca（2＋） Uniporter Mitochondrial（MCU） plays a key role as a component of the uniporter complex.In recent studies,the DdMCU（Dictyostelium discoideum） reconstruction in the yeast system further shows that MCU is indeed channel forming subunit and the basic component of the activity of uniporter.In the present study,the target DNA fragment was amplified by PCR when using the synthetic DdMCU gene as the template and then was subcloned into pPICZX.As a result,the expression plasmid pPICZXDdMCU of the fusion protein DdMCU- GFP was constructed.This plasmid transformed into X33.After zeocin gradient screening,eight yeast recombinants were obtained and PCR further prove that target gene has been successfully integrated with the yeast genome.Afterward recombinant expressed by methanol inducement and proteins have been successfully expressed,which was testified by laser scanning confocal microscopy and western assay.