人源FCHo1蛋白μHD结构域的表达、纯化及晶体生长  

Expression,purification and crystallization of Human FCHo1 μHD domain

作　　者：李夫[1] 夏鹏飞[1] 戴雪辰 石康[1] 熊鹰[1] 刘映乐[1] 

机构地区：[1]武汉大学生命科学学院病毒学国家重点实验室,武汉430072

出　　处：《生物学杂志》2015年第5期7-10,共4页Journal of Biology

基　　金：国家"973计划"项目(No.2011CB504900)

摘　　要：人FCHo1蛋白(Fer/Cip4 homology domain only proteins 1)是网格蛋白介导的内吞途径中的成核剂,其碳端μHD结构域(AP2-μhomology domain)对于FCHo1在披网格蛋白小泡(clathrin-coated vesicle)形成过程中的正确定位非常重要。为揭示人FCHo1碳端μHD结构域的作用机制,制备可衍射的μHD结构域蛋白质晶体并解析其结构至关重要。通过构建p ET15b原核表达载体,并转化大肠杆菌BL21(DE3),诱导表达。利用亲和层析、离子交换层析和分子筛层析技术,制备得到纯度95%以上的蛋白。通过多次优化结晶条件,获得衍射率约2.1的μHD结构域蛋白晶体,为解析其晶体结构奠定了良好的基础。The human FCHo1 protein （Fer/Cip4 homology domain only proteins 1） is nucleator of clathrin-mediated endocytosis and its C terminalμHD domain （ AP2-μhomology domain） is critical to the correct positioning of FCHo1 in the formation of clathrin-coated vesicles of clathrin-mediated endocytosis.To determine the crystal structure and understand the structural mechanism of human FCHo1μHD domain,it is essential to obtain high resolution protein crystal which can be analyzed by X-Ray.The human FCHo1 μHD was cloned into the expression vector pET15b and verified by bacteria liquid PCR and gene sequencing.Proteins were overexpressed in E. coli BL21 （DE3） which induced by 0.2 mmol/L IPTG at 16℃overnight.The recombined proteins were purified by Ni-affinity chro-matography （ Ni-NTA） , ion exchange chromatography （ IEC） and size exclusion chromatography （ SEC） , respectively.Proteins were detected by SDS-PAGE and the purity was over 95%.Crystal screen kits from Hampton Research were applied to the preliminary screening ofμHD protein by setting-drop method.The human FCHo1μHD protein crystal at about 2.1 ？resolution was acquired after optimizing the condition of crystallization by hanging-drop vapor-diffusion method and data were collected in Shanghai synchrotron radia-tion facility （ SSRF） .This study has built a good foundation for determining the crystal structure of human FCHo1 μHD domain.

关 键 词：FCHo1 μHD结构域 表达 纯化 结晶 衍射 

分 类 号：Q786[生物学—分子生物学]