颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达

Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli

为高效表达颗粒裂解肽G13结构域并避免G13对宿主菌的毒性,将人工合成的编码G13的基因片段,PCR扩增后克隆于原核表达载体pThioHisA中,构建了重组表达载体pThioHisA-G13,将其转化于大肠杆菌BL21(DE3)中,经IPTG诱导表达融合蛋白Trx-G13,表达产物以包涵体的形式存在,其表达量约占细菌总蛋白的58%。包涵体蛋白经8 mol/L尿素溶解后,再经CNBr切割,阳离子交换层析,得到纯化的重组G13结构域。琼脂糖扩散法检测表明重组G13结构域多肽具有抗菌活性。

The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin （Trx） gene to construct the recombinant expression vector （pThioHisA-G13）. A cyanogen bromide （CNBr） cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 （DE3）. Upon induction by IPTG, Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% （W/W） of total cellular proteins. The inclusion body was solved by urea （8 mol/L） and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.