人组织激肽释放酶成熟蛋白在大肠杆菌中的高效表达

ISSN 1007 7626CN 11 3870 Q

中国生物化学与分子生物学报

Chinese Journal of Biochemistry and Molecular Biology

2003年6月19(3):312~316

人组织激肽释放酶成熟蛋白在大肠杆菌中的高效表达

李体远, 杜 珙*

, 蔡筱彦, 石之磷, 黄瑞芳, 陈德珩, 戴 勇, 肖德明

(暨南大学医学院第二附属医院,深圳市人民医院临床医学研究中心,深圳 518020)

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pE T28(b)及分泌型表达载体pE T20(b)中,使其C 端融合6 His Tag 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pET28载体中的表达水平高于pET20载体.pE T28和pE T20载体表达的重组激肽释放酶蛋白分别占菌体总蛋白约26%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(B AEE)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,这为研究其生物功能和开发基因工程药物奠定基础.关键词 人组织激肽释放酶,大肠杆菌,高效表达中图分类号 Q78

Overexpression of Mature Human Tissue Kallikrein

Fusion Protein in E .coli

LI Ti yuan,DU Gong *

,C AI Xiao yan,SHI Zhi lin,HUANG Rui fang,

C HE N De heng,DAI Yong,XI AO De ming

(Me dical Researc h Cente r ,Shenzhe n People s Hospital ,Medic al Colle ge ,Jinan U nive rsity ,Shenzhe n 518020,China )

Abstract Human tissue kallikrein gene fragment encoding mature kallikrein was amplified and cloned into pE T 28(b)and pET 20(b)plasmid with C terminal 6 His Tag.The recombinant fusion protein was overe xpressed in B L21(DE3),B L21(DE3)plysS and HMS174(DE3)under the induction of IPTG.SDS PAGE showed tha t 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 30kD.Western blot analysis indicated that the expressed kallikrein had the antigenicity to human serum kallikrein.The kallikrein fusion protein in the un purified solution sho wed low activity to hydrolysis B AEE by using potentiometric method.The overe xpressed human tissue mature kallikrein could be used for the development of gene tic engineering products and further study of its biological property.

Key words kallikrein,overexpression,E .coli 收稿日期:2002 05 28,接受日期:2002 10 28

基金项目:深圳市科技局资助课题(No.199906014),广东省十五攻关课题(No.C11801)

李体远,男,1964年1月生,博士,副研究员,现在美国Ohi o 州立大学做博士后研究工作*

联系人:Tel:0755 ******** 2599,Fa x:0755 ********E mail:tiyuanl@

Supported by Shenzhen Bureau of Science and Technology (No.199906014)and the 10th Fi ve Year Plan Special Research Programs of Guangdong Province (No.C11801)

Recei ved:May 28,2002;Accepted:October 28,2002*Corres pondi ng author Tel:0755 ******** 2599,Fax:0755 ********E mail:tiyuanl@

高血压为最常见的心血管疾病,同发达国家一样,我国的发病率呈逐年升高趋势.据全国统计资料显示,我国现有高血压患者已达一亿,每年新增300万以上.肾脏等疾患同样威胁着广大人民的健康.研制开发新的抗高血压、改善肾脏功能等药物具有广阔的经济价值及深远的社会效益.

激肽释放酶(kallikrein,KK)具有广泛的药用价值,除降血压之外,还可以用于治疗糖尿病肾病、改善肾脏功能,治疗心、脑血管疾病等

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.猪源激肽