可溶性TCR蛋白复合物原核表达与纯化的研究  

Expression and Purification of Soluble TCR Protein Complex

作　　者：汤磊 李梦媛 蒋小猛 郭薇[1] TANG Lei;LI Meng-yuan;JIANG Xiao-meng;GUO Wei(School of Life Science&Technology,China Pharmaceutical University,Nanjing 210009,China;Department of Gastroenterology,Sir Run Run Hospital,Nanjing Medical University,Nanjing 211666,China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]南京医科大学附属逸夫医院,江苏南京211166

出　　处：《药物生物技术》2021年第5期441-445,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.81973221);中国肝炎防治基金会天晴肝病研究基金(TQGB20210089);2020年中国药科大学创新创业菁英培养计划。

摘　　要：T细胞受体(T cell receptor,TCR)是通过二硫键连接α链和β链或者γ链和δ链的异二聚体蛋白复合物。本研究使用原核系统体外分别表达两条单链,摸索并确定快速获得一定量的TCR蛋白的复性条件,实验发现TRA、TRB蛋白变性液按照2:1(mg/mg)的比例混合均匀后缓慢滴加到4℃搅拌的复性液(50 mmol/L Tris、0.4 mol/L L-精氨酸、5 mol/L脲、5 mmol/L EDTA、10 mmol/L还原型谷胱甘肽、2 mmol/L氧化型谷胱甘肽,pH=7.6)中复性24 h,使用Hitrap^(TM)Capto^(TM)Q初步分离纯化,去除部分不溶性及等电点相差较大的杂质蛋白,Superdex^(TM)75Increase 10/300 GL进一步精细分离获得纯度在90%以上的TCR复合物蛋白。实验通过还原型和非还原型电泳及Western blot对纯化后的TCR蛋白进行分子量及空间结构鉴定,证明了TCR二聚体的原核表达与纯化的技术可行性。该研究为后续快速获得高纯度且正确折叠的TCR蛋白、进一步研究其功能和结构打下了夯实的基础。T cell receptor(TCR)is a heterodimeric protein complex that links alpha chain and beta chain or gamma chain and delta chain by disulfide bonds.At present,there are two main ways to study the structure of TCR:the first method is to express TCR alpha chain and beta chain respectively,then refold to form a TCR heterodimer complex,while the second method is to express TCR alpha chain and beta chain linked by a short peptide,then restore the natural conformation of TCR by renaturation.The authors utilized the former way to respectively express the chains in vitro and renaturate TCR’s conformation.The basic experimental conditions are as follows:Mix TRA protein:TRB protein=2:1(mg/mg)in 30 mL 8 mol/L urea,stay at 37℃at least for 30 min,and drip the mixture into the refolding solution(50 mmol/L Tris,0.4 mol/L L-arginine,5 mol/L urea,5 mmol/L EDTA,10 mmol/L reduced glutathione,2 mmol/L oxidized glutathione,pH=7.6)at 0.05 mL/min flow rate to refold for 24 h.The final concentration of refolding protein is not higher than 0.1 mg/mL.After that,20 mmol/L Tris(pH=7.6)was used for continuous dialysis for 72 h at 4℃,and the dialysate was replaced every 24 h.The authors also determined the methods of preliminary purification and fine purification utilizing Hitrap^(TM)Capto^(TM)Q and Superdex^(TM)75 Increase 10/300 GL,and the conditions for eluting the target protein and eliminating the impurity protein.Finally,a TCR heterodimer with a purity greater than 90%was obtained.The structure of the TCR was identified by reduced and non-reduced SDS-PAGE and Western blot,which proves the feasibility of expression of protein TRA and TRB by Escherichia coli,as well as the renaturation and purification of TCR dimer complex.Articles based on previous work established a laboratory-grade purification method of obtaining soluble TCR heterodimer protein complex,providing a feasible technical route for quick access to other TCR heterodimer with specific sequence.It lays the foundation of subsequent research on TCR protein structure and protein

关 键 词：T细胞受体 复性 表达纯化 包涵体 异二聚体 还原、非还原型SDS-PAGE电泳 

分 类 号：Q51[生物学—生物化学]