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

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pET32α载体的凝血酶序列和S标签序列，构建优化的Lf-CATH2-pET32α-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。

Abstract:

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-pET32α-TS was successfully constructed by firstly deleting the thrombin site and S tag from the pET-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.