牙龈卟啉单胞菌磷酸酶SerB的表达、纯化和晶体衍射  

Expression,purification and crystal diffraction of phosphatase SerB from Porphyromonas gingivalis

作　　者：高洁 杨金敏 杨孟钰 王艳秋 张修月[2] 葛燕 刘彬 吴桃清 GAO Jie;YANG Jinmin;YANG Mengyu;WANG Yanqiu;ZHANG Xiuyue;GE Yan;LIU Bin;WU Taoqing(Sichuan Key Laboratory of Medicinal Periplaneta americana,Chengdu 610031;Key Laboratory of Bioresources and Ecoenvironment(Ministry of Education),College of Life Sciences,Sichuan University,Chengdu 610065;Sichuan Good Doctor Zhongzang Pharmaceutical Co.Ltd.,Chengdu 610404,China)

机构地区：[1]药用美洲大蠊四川省重点实验室,四川成都610031 [2]四川大学生命科学学院,生物资源与生态环境教育部重点实验室,四川成都610065 [3]四川好医生中藏药业有限公司,四川成都610404

出　　处：《生物技术》2025年第1期14-19,共6页Biotechnology

基　　金：国家自然科学基金区域创新发展联合基金项目(U21A20409)。

摘　　要：[目的]原核表达和纯化牙龈卟啉单胞菌SerB蛋白,并进行晶体筛选和X射线衍射。[方法]以生物信息学手段预测SerB蛋白基本特点,借助无缝克隆技术构建pET22b-serB重组质粒,并在大肠杆菌BL21(DE3)中表达,采用镍柱亲和层析和分子排阻层析纯化重组SerB蛋白,孔雀绿显色法检测磷酸酶活性,以及筛选蛋白结晶条件并收集X射线衍射数据。[结果]SerB蛋白由N端的ACT结构域和C端的丝氨酸磷酸酶结构域组成,分子量为45.93 kDa。成功构建pET22b-serB重组质粒及获得分子量正确、浓度为6 mg/mL的目的蛋白。孔雀绿显色法显示SerB具有丝氨酸磷酸酶活性。在0.1 mol/L C_(3)H_(2)Na_(2)O_(4)、pH 8.0,0.1 mol/L Tris-HCl pH 8.0,30%(W/V)PEG1500条件下获得了较好的SerB晶体,分辨率为3.84A。[结论]成功表达具有丝氨酸磷酸酶活性的牙龈卟啉单胞菌SerB蛋白并结晶,收集到分辨率为3.84?的衍射数据。[Objective]To purify the recombinant SerB of Porphyromonas gingivalis via prokaryotic expression,and perform crystal screening and X-ray diffraction.[Method]Bioinformatics was employed to predict the basic characteristics of SerB protein.The recombinant plasmid pET22b-serB was constructed using seamless cloning method,and expressed in Escherichia coli BL21(DE3).The recombinant SerB protein was purified with Ni-NTA and size exclusion chromatography.Phosphatase activity was assessed via a malachite green procedure.Conditions for SerB crystallization were screened,and X-ray diffraction data were collected.[Result]Overall structure of SerB consists of an N-terminal ACT domain and a C-terminal serine phosphatase domain,with a molecular weight of 45.93 kDa.The pET22b-serB plasmid was successfully constructed,and the SerB protein was obtained at a concentration of 6 mg/mL with proper molecular weight.Considerable serine phosphatase activities of purified SerB were observed via malachite green assay.The crystals of SerB were obtained under conditions of 0.1 mol/L C_(3)H_(2)Na_(2)O_(4) pH 8.0,0.1 mol/L Tris-HCl pH 8.0,and 30%(W/V)PEG1500,further used for X-ray diffraction with a resolution of 3.84A.[Conclusion]The P.gingivalis SerB,exhibiting serine phosphatase activity,was successfully expressed and crystallized,with initial X-ray diffraction data collected to 3.84A.

关 键 词：牙龈卟啉单胞菌 SerB 磷酸酶 诱导表达 纯化 酶活检测 晶体 X射线衍射 

分 类 号：Q71[生物学—分子生物学]