β-琼脂糖酶Ⅰ DagA的原核表达和活性鉴定  

Expression of β-agarase Ⅰ DagA in Prokaryotic Cell and Its Activity Identification

作　　者：周雁胜[1] 王保莉[1] 曲东[2] 

机构地区：[1]西北农林科技大学生命科学学院 [2]西北农林科技大学资源环境学院,杨凌712100

出　　处：《农业生物技术学报》2009年第1期132-137,共6页Journal of Agricultural Biotechnology

基　　金：西北农林科技大学创新团队项目资助

摘　　要：采用PCR技术从假别单胞菌（Pseudoalteromonas atlantica）19262基因组DNA中获得β-琼脂糖酶I基因（dagA）及去除信号肽的编码序列dagA（↓△），分别与载体pET21a连接后转入大肠杆菌（Escherichia coli）ER2566中，共表达分子伴侣Ds-bc及FkpA，筛选出以包涵体为主要表达形式的高效表达体系：ER2566-pET21a-dagA（↓△）-DsbC菌株。包涵体蛋白达到菌体总蛋白的60％左右。包涵体用8mol／L尿素溶解、镍离子亲和层析纯化和梯度稀释复性。SDS-PAGE检测表明，复性后的DagA蛋白相对分子质量约为30.8kD，且具有水解琼脂糖的生物活性。酶学特性分析表明，在pH4.8-6.8范围内，DagA蛋白活性保持60％以上，最适pH5.8；在温度37-60℃均有活性，最适温度为55℃。dagA gene and dagA （↓△） which is a dagA gene encoding sequence without signal peptide were cloned from genome DNA ofPseudoalteromonas atlantica 19262 by PCR. After ligation with pET21 vactor, dagA and dagA （↓△） were expressed in E. coil ER2566, respectively, using molecular chaperone DsbC and FkpA. Strain ofER2566- pET21a-dagA （↓△）-DsbC was screened as high effective expressing system, in form of the inclusion body in which had the target protein up to 60 % of total bacterial protein. DagA protein was renaturated and purified by dissolving in 8 mol/L of urea, Ni-NTA resin affinity chromatography and refolding by urea gradient method. DagA was about 30.8 kD identified by SDS-PAGE and had the ability to digest agarose. At the range of pH 4.8 to 6.8, DagA could hold a bio-activity of beyond 60 %, with 5.8 as optimum pH value and its activity was available under 37 to 60℃, with 55℃ as optimum temperature.

关 键 词：β-琼脂糖酶ⅠdagA 原核表达 包涵体复性 生物活性 

分 类 号：S188[农业科学—农业基础科学]