ProNGF在大肠杆菌中的表达、纯化和复性  

Expression,Purification and Renaturation of ProNGF in Escherichia coli

作　　者：江汉民[1] 柴新君[1] 何冰[1] 赵娟[1] 俞新大[1] 

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《生物工程学报》2008年第3期509-514,共6页Chinese Journal of Biotechnology

基　　金：天津市自然科学基金资助(No.033605211)~~

摘　　要：将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中,转化大肠杆菌BL21(DE3)pLysS,经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右,表达蛋白主要以包涵体的形式存在。用6mol/L的盐酸胍溶解包涵体后,通过Ni2+-NTA柱纯化,获得纯化的目标融合蛋白,电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示,表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白,分子量为27kD,100mL表达菌液可获得13.1mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠,复性率为18%,在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定,结果显示复性后的proNGF蛋白具有良好的生物活性。Nerve growth factor （NGF） promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor-proNGF in vivo. In this paper, a pET-proNGF prokaryocyte expression vector was constructed and transformed into E. coli BL21（DE3）pLysS. The proNGF was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was successfully purified by Ni^2＋-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment. The protein was dialyzed in a redox system containing reduced and oxidized glutathione. RP-HPLC was used to analysis the result of the refolding. The refolded proNGF protein can induce neurite outgrowth of PC12 cells, which indicated that pro-form of NGF we obtained had biological activity.

关 键 词：神经生长因子 表达 前导肽 蛋白复性 生物活性 

分 类 号：Q78[生物学—分子生物学] S884.51[农业科学—特种经济动物饲养]