一种新型改良硫氧还蛋白融合表达体系的建立及cathelicidin家族Lf-CATH2的表达  

Construction of novel thioredoxin fusion protein expression system and the production of recombinant Lf-CATH2

作　　者：鲁一灵 高久香 乔雪[1] 王义鹏[2] 于海宁[1] 

机构地区：[1]大连理工大学生命科学与技术学院,辽宁大连116024 [2]苏州大学药学院,江苏苏州215123

出　　处：《生物工程学报》2015年第3期403-410,共8页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.41206153);辽宁省高等学校杰出青年学者成长计划(No.LJQ2013010);大连市优秀青年科技人才基金(No.2013J21DW013)资助~~

摘　　要：通过改良硫氧还蛋白融合表达体系,原核表达cathelicidin家族抗菌肽Lf-CATH2。首先在Lf-CATH2基因上游加入凝血酶位点,并去除p ET32α载体的凝血酶序列和S标签序列,构建优化的Lf-CATH2-p ET32α-TS载体,于大肠杆菌中表达。产物融合蛋白经凝血酶切割释放Lf-CATH2,纯化后进行抗菌活性检测。结果表明改良的硫氧还蛋白融合表达体系显著提高酶切效率达37%,Lf-CATH2在新体系中获得了可溶性高表达,且保留了抗菌活性。因此该新型硫氧还蛋白融合表达体系,有望为cathelicidin家族及其他阳离子活性肽提供更好的原核表达载体工具。The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-p ET32α-TS was successfully constructed by firstly deleting the thrombin site and S tag from the p ET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.

关 键 词：CATHELICIDIN Lf-CATH2 硫氧还蛋白融合表达 凝血酶切割 

分 类 号：Q78[生物学—分子生物学]