ProNGF在大肠杆菌中表达、纯化和复性.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, March 25; 24(3): 509-514 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 技术与方法 ProNGF 江汉民, 柴新君, 何冰, 赵娟, 俞新大 , 300071 : 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 6 mol/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 27 kD, 100 mL表达菌液可获得 13.1 mg 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。 : 神经生长因子, 表达, 前导肽, 蛋白复性, 生物活性 Expression, Purification and Renaturation of ProNGF in Escherichia coli Hanmin Jiang, Xinjun Chai, Bing He, Juan Zhao, and Xinda Yu College of Life Sciences, Nankai University, Tianjin 300071, China Abstract: 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. SDSrevealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was success