人α 防御素5 在大肠杆菌中的可溶性表达与纯化研究

作者：王韵壹、李英、熊慧卿

生物工程学报 Chin J Biotech 2008, February 25; 24(2): 291-296

journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061

Received: April 26, 2007; Accepted: July 3, 2007

Supported by: Sci & Tech Research Foundation of PLA “Eleventh Five-Year Plan” (No. 06G076), the National Natural Science Foundation of China

(No. 30771892), and Academician Fund of Chongqing City (No. CSTC, 2007AB5022).

Corresponding author: Junping Wang. Tel: +86-23- 68752883; E-mail: wangjunp@yahoo.com

军队“十一五”科技功关项目(No. 06G076)、国家自然科学基金 (No. 30771892)和重庆市院士基金(No. CSTC, 2007AB5022)资助。

研究报告

人α 防御素5 在大肠杆菌中的可溶性表达与纯化研究

王艾平, 粟永萍, 程天民, 邹仲敏, 王军平

第三军医大学军事预防医学院全军复合伤研究所, 创伤、烧伤与复合伤国家重点实验室, 重庆 400038

摘要: 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BL21(DE3), 诱导表达, SDS-PAGE 分析目的蛋白表达量并优化表达条件。经亲和层析、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。

关键词: 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 expression vector. The plasmid pMAL-p2x-mHD-5 was transferred into engineered strain BL21(DE3) to express heterogeneous fusion protein (MBP-mHD-5). The soluble MBP-mHD-5 targeted protein inducible expressed by IPTG was accounted for about 30% under optimized conditions. The recombinant mHD-5 (rmHD-5) peptide was successfully purified through a separation process including affinity chromatography, Factor Xa digestion and ion exchange chromatography. The bioactivity of rmHD-5 was examined by bacteria-inhibition tests in liquid culture. The growth of E. coli ATCC25922 was dramatically suppressed with an inhibition rate of 90%, with the presence of 62.5μg/mL rmHD-5 in the media. These results indicate that the strategy of soluble expression of fusion protein in E. coli can be a useful and practical way to produce bioactive defensins.

Keywords: defensin, prokaryotic expression, purify, bioactivity

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