人组织激肽释放酶成熟蛋白在大肠杆菌中的高效表达  

Overexpression of Mature Human Tissue Kallikrein Fusion Protein in E. coli

作　　者：李体远[1] 杜珙[1] 蔡筱彦[1] 石之磷[1] 黄瑞芳[1] 陈德珩[1] 戴勇[1] 肖德明[1] 

机构地区：[1]暨南大学医学院第二附属医院

出　　处：《中国生物化学与分子生物学报》2003年第3期312-316,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：深圳市科技局资助课题 (No .19990 60 14);广东省十五攻关课题 (No .C1180 1)~~

摘　　要：将编码人组织激肽释放酶成熟蛋白的基因片段扩增并分别克隆到原核表达载体pET2 8(b)及分泌型表达载体pET2 0 (b)中 ,使其C端融合 6×HisTag序列 .转化不同受体菌 ,IPTG诱导表达后利用SDS PAGE、免疫印记等方法对重组蛋白进行分析 .在 6株基因工程菌株中 ,均表达出分子量约30kD的激肽释放酶融合蛋白 ,其中激肽释放酶在pET2 8载体中的表达水平高于pET2 0载体 .pET2 8和pET2 0载体表达的重组激肽释放酶蛋白分别占菌体总蛋白约 2 6 %和 10 % .Western印迹分析表明 ,目的蛋白可与抗人血清KK单克隆抗体发生特异性反应 .未经纯化的激肽释放酶融合蛋白具有一定的水解苯甲酰精胺酸乙酯 (BAEE)的能力 .在大肠杆菌中获得了人组织激肽释放酶的高效表达 ,表达产物具有免疫原性和生物活力 。Human tissue kallikrein gene fragment encoding mature kallikrein was amplified and cloned into pET 28(b) and pET 20(b) plasmid with C terminal 6×His Tag. The recombinant fusion protein was overexpressed in BL21(DE3), BL21(DE3)plysS and HMS174(DE3) under the induction of IPTG. SDS PAGE showed that the kallikrein fusion protein were produced at a level of approximately 26% and 10% of the total cellular protein in E. coli BL21(DE3) plysS by pET 28(b) and pET 20(b), respectively. The relative molecular weight of the expressed protein was 30 kD. Western blot analysis indicated that the expressed kallikrein had the antigenicity to human serum kallikrein. The kallikrein fusion protein in the un purified solution showed low activity to hydrolysis BAEE by using potentiometric method. The overexpressed human tissue mature kallikrein could be used for the development of genetic engineering products and further study of its biological property.

关 键 词：人组织激肽释放酶 大肠杆菌 表达 基因 克隆 

分 类 号：Q786[生物学—分子生物学]