大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定  

Eukaryotic Expression,Purification and Activity Identification of Rat His-Akt1 Recombinant Protein

作　　者：孟利 杜彩萍 MENG Li;DU Cai-ping(Research Center for Biochemistry and Molecular Biology,Jiangsu Key Laboratory of Brain Disease Bioinformation,Xuzhou Medical University,Xuzhou 221004)

机构地区：[1]徐州医科大学,江苏省脑病生物信息重点实验室生物化学与分子生物学研究中心,徐州221004

出　　处：《生物技术通报》2020年第12期98-103,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(81100852);江苏省徐州市科技计划项目(KC18205)。

摘　　要：构建His-Akt1-pcDNA3.1真核表达载体,于HEK293细胞进行表达,Ni-NTA亲和纯化His-Akt1,并对Akt1进行活性鉴定。以Akt1(全长)-pcDNA3.1重组体为模板,采用PCR扩增目的基因,然后克隆入真核表达载体pcDNA3.1;将His-Akt1-pcDNA3.1转染HEK293细胞表达,Ni-NTA纯化His-Akt1,用SDS-PAGE、考马斯亮蓝染色及免疫印迹鉴定蛋白纯度;蛋白经透析后,免疫印迹检测Akt1 Ser473和Thr308磷酸化水平。His-Akt1-pcDNA3.1真核表达载体构建成功,在HEK293细胞中高效表达;考马斯亮蓝染色和免疫印迹结果显示,取2 mg过表达His-Akt1的蛋白混合液经Ni-NTA纯化后,加入含100 mmol/L咪唑的洗脱液洗脱,可获得高纯度His-Akt1重组蛋白。上述His-Akt1重组蛋白经透析后,免疫印迹结果显示His-Akt1 Ser473和Thr308磷酸化水平(活化水平)均呈高水平。重组His-Akt1-pcDNA3.1真核表达载体成功构建,且蛋白高表达,His-Akt1经提取纯化后具有较高的酶活性。To obtain rat His-Akt1 recombinant protein with high activity,His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed firstly,and then transfected into HEK293 cells.Next,the His-Akt1 was purified by Ni column affinity chromatography(Ni-NTA),and its activity was then identified.His-Akt1 cDNAs were amplified from the full-length recombinant Akt1-pcDNA3.1 by PCR and cloned into eukaryotic expression vector pcDNA3.1.The HEK293 cells transfected with His-Akt1-pcDNA3.1 plasmid were collected and sent to Ni-NTA.His-Akt1 purity was identified by SDS-PAGE,Coomassie blue staining and Western blot.Dialytically purified His-Akt1 finally was subjected to Western blot analysis to detect Ser473 and Thr308 phosphorylation levels.His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed successfully and efficiently expressed in HEK293 cells.The Coomassie blue staining and Western blot results showed that high-purity His-Akt1 recombinant protein was obtained by purifying 2 mg overexpressing His-Akt1 protein samples with Ni-NTA and eluting with 100 mmol/L imidazole.In addition,the Ser473 and Thr308 phosphorylation(i.e.activation)of the His-Akt1 after dialysis presented both high levels.The recombinant His-Akt1-pcDNA3.1 eukaryotic expression vector is constructed successfully,and the protein highly expressed,and the purified His-Akt1 recombinant protein demonstrates high enzymatic activity.

关 键 词：AKT1 真核表达载体 蛋白纯化 活性鉴定 

分 类 号：Q786[生物学—分子生物学]