颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达-生物谷.doc

颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达 刘小强, 查向东, 肖亚中, 杨金环, 李能树 安徽大学生命科学学院合肥 230039: 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。 关键词: 颗粒裂解肽, G13结构域, 融合表达, 阳离子抗菌肽 Efficient fusion expression of G13 domain derived fromgranulysin in Escherichia coli Xiaoqiang Liu, Xiangdong Zha, Yazhong Xiao, Jinhuan Yang, and Nengshu Li Key Laboratory of Ecological Engineering and Biotechnology of Anhui Province, School of Life Science, Anhui University, Hefei 230039, China Abstract: 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. Keywords: Granulysin, G13 domain, fusion expression, cationic antimicrobial peptide 抗菌肽(Antimicrobial peptide)是生物体内产生