在大肠杆菌中的表达
王朝霞1,2，江昌俊3*，余有本4，李 娟3
（1. 安徽教育学院生物系，安徽 合肥 230061；2. 安徽农业大学农业部茶叶生物化学与生物技术重点开放实验，安徽 合肥 230062；3. 安徽农业大学生物技术中心，安徽 合肥 230062；4. 西北农林科技大学茶叶研究所，陕西 杨陵 712100）
摘 要：通过RT-PCR法扩增出茶树中巯基蛋白酶抑制剂基因（Tea cystatin,TC）cDNA编码区序列,定向克隆到表达载体pET 32a(+)上, 得到重组质粒pET-TC，在表达宿主菌BL21trxB(DE3)中高效表达，产生分子量约为31.8 kDa的特异融合蛋白。实验表明：随着诱导时间的延长，表达的融合蛋白Trx-TC的产量逐渐增加，特异蛋白的表达量最高可占菌体总蛋白的35. 4 ％；在15 ℃、25 ℃、30 ℃和37 ℃四个不同诱导温度下，均能诱导产生融合蛋白；IPTG终浓度在0.5 mmol/L -5 mmol/L范围内均能诱导产生融合蛋白，表达的产物主要以可溶性蛋白形式存在。另外，体外酶促反应表明表达的融合蛋白Trx-TC对木瓜蛋白酶有明显的抑制活性。
|
|
Expression of Tea Cystatin in E.coli
WANG Zhao-xia1,2 , JIANG Chang-jun3* , YU You-ben4 , LI Juan3
(1. Department of Biology, Anhui Institute of Education, Hefei 230061, Anhui, China; 2.Key Laboratory of Tea Biochemistry & Biotechnology, Ministry of Agriculture, Anhui Agriculture University, Hefei 230036, Anhui, China; 3. Biotechnology Center, Anhui Agricultural University, Hefei 230036, Anhui, China; 4. Tea Research Insititute , Northweast A&F University, Yanglin 712100, Shannxi, China.)
Abstract: The coding region of the tea cystatin was amplified by RT-PCR and cloned into expression vector pET-32a(+). The tea cystatin expression plasmid pET-TC was thus obtained. Expression of the plasmid in Escherichia coli BL21trxB(DE3) resulted in the production of a special fusion protein(ca.31.8kDa)． With the inducing time increasing, the amount of fusion protein also increased, the highest content amounted to 35.4% of gross proteins. Different temperature( 15 ℃, 25 ℃, 30 ℃and 37 ℃) and different density of IPTG from 0.5 mmol/L to 5 mmol/L all could induce expression of fusion protein, and most of them was soluble. Furthermore, the results of in vitro enzymatic reaction indicated that the special fusion protein had biological-activity and exhibited inhibitory activity toward papain.
|
