人α防御素5在大肠杆菌中的可溶性表达及纯化研究.PDF

生 物 工 程 学 报 Chin J Biotech 2008, February 25; 24(2): 291-296 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 α5 王艾平, 粟永萍, 程天民, 邹仲敏, 王军平 , , 400038 : 采用PCR 扩增大肠杆菌偏好的人α防御素5 成熟肽(mHD-5)密码子序列, 并将其克隆至pMAL-p2x 质粒, 构建 pMAL-p2x-mHD-5 表达载体, 转化大肠杆菌BL (DE ), 诱导表达, SDS分析目的蛋白表达量并优化表达条件。经 21 3 亲和层析、酶切和离子交换层析等方法分离、纯化重组mHD-5(rmHD-5) 多肽。采用浊度法测定rmHD-5 对细菌的抑制 活性。通过优化表达条件, 获得约 30%的可溶性目的蛋白表达量, 并成功纯化 rmHD-5 。rmHD-5 对大肠杆菌标准菌株 (ATCC25922)具有较强的抑制活性, 在终浓度为 62.5μg/mL 时, 90%以上的细胞被抑制。结果表明采用可溶性融合表达 策略, 在原核表达系统中诱导表达并纯化具有生物活性的防御素是可行的途径之一。 : 防御素, 原核表达, 纯化, 生物活性 Soluble Expression and Purification of Human Alpha- Defensin-5 in Escherichia coli Aiping Wang, Yongping Su, Tianmin Cheng, Zhongmin Zou, and Junping Wang Institute of Combined Injury of the People ’s Liberation of Army; National Key Laboratory of Trauma, Burn and Combined Injury, Third Military Medical University , Chonqqing 400038, China Abstract: DNA fragment containing human alpha-defensin 5 mature peptide (mHD-5) coding sequence with biased codons of E. coli was amplified by PCR, which was subsequently cloned into the plasmid pMAL-p2x in order to create pMAL-p2x-mHD-5 ex- pression vector. The plasmid pMAL-p2x-mHD-5 was transferred into engineered strain BL (DE ) to express heterogeneous fusion 21 3 protein (MBP-mHD-5). The soluble MBP-mHD-5 targeted