摘要
目的构建人CD147胞内结构域(CD147 intracellular domain,CD147ICD)融合蛋白质粒,表达、提取、纯化CD147ICD蛋白并进行蛋白质互作分析。方法将CD147ICD的cDNA片段插入pET-32a(+)质粒载体后,转化大肠杆菌Origami B(DE3)感受态细胞,大量培养后用异丙基硫代-β-d-半乳糖苷诱导蛋白表达,超声破碎法裂解细胞,采用Ni-Sepharose FF预装柱纯化TrxA-His6-CD147ICD融合蛋白,经凝血酶酶切、超滤后获得CD147ICD蛋白。结果CD147ICD融合蛋白质粒经测序比对后表明构建成功;采用Ni-Sepharose FF预装柱纯化的TrxA-His6-CD147ICD融合蛋白纯度和浓度均较高,经凝血酶酶切、超滤后获得的CD147ICD蛋白纯度在95%以上,蛋白浓度为0.74 mg/mL。结论纯化出了高纯度的、具有生物学活性的CD147ICD蛋白,为CD147ICD互作分子的鉴定及结构解析奠定了基础。
Objective To express and purify the recombinant intracellular domain of human CD147(CD147ICD)and to study the interacting proteins of CD147ICD.Methods cDNA of CD147ICD was ligated into pET-32a(+)plasmid vector,then transformed into E.coli Origami B(DE3)competent cells and the transformants were induced with isopropyl-β-d-thiogalactoside.The fusion proteins were purified with Ni-Sepharose FF column in combination with thrombin digestion and ultrafiltration.Results The recombinant plasmid was confirmed with sequencing.Purified TrxA-His6-CD147ICD protein was obtained with Ni-Sepharose FF column.After thrombin digestion and ultrafiltration,the purity of CD147ICD protein was above 95%,and the protein concentration was 0.74 mg/mL.Conclusion CD147ICD protein was purified with high purity and activity,which would provide a basis for the future studies on characterization of CD147ICD interacting proteins and structure determination.
作者
吴东
南刚
李佳悦
刘芬玲
崔洪勇
WU Dong;NAN Gang;LI Jiayue;LIU Fenling;CUI Hongyong(The First Student Battalion,School of Basic Medical Sciences,Air Force Medical University,Xi’an Shaanxi 710032,China;Department of Cell Biology,School of Basic Medical Sciences,Air Force Medical University,Xi’an Shaanxi 710032,China)
出处
《转化医学杂志》
2019年第6期326-328,338,共4页
Translational Medicine Journal
基金
国家自然科学基金(31601127)
关键词
CD147
胞内结构域
表达与纯化
CD147
Intracellular domain
Expression and purification
