Wang Xiuqing, Zhu Mingxing, Yang Guimao, Su Chunxia, Zhang Aijun, Cao Ruibing, Chen Puyan
School of Clinical Laboratory of Medicine;
Exp Ther Med. 2011 Jul;2(4):655-660. doi: 10.3892/etm.2011.262. Epub 2011 May 9.
Natural cecropin B (CB), consisting of 35 amino acids, is a member of the cecropin family with the highest antibacterial activity. Here, a novel approach for the generation of recombinant CB in the methylotrophic yeast Pichia pastoris was explored. For this purpose, the CB gene was amplified by recursive PCR (rPCR) and cloned into the pPICZαA vector. The SacI-linearized plasmid pPICZαA-CB was transformed into P. pastoris SMD1168 by electroporation. The expression of recombinant CB was induced with 1.0% methanol at pH 5.0 for 60 h at 28°C. Recombinant CB was purified using cationic exchange chromatography; 5.0 mg of pure active CB was obtained from 100 ml of culture broth supernatant. Antimicrobial assays demonstrated that CB has a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional CB for both research and industrial purposes.
天然天蚕素B（CB）由35个氨基酸组成，是天蚕素家族中抗菌活性最高的成员。在此，探索了一种在甲基营养型酵母毕赤酵母中产生重组CB的新方法。为此，通过递归PCR（rPCR）扩增CB基因并将其克隆到pPICZαA载体中。通过电穿孔将经SacI线性化的质粒pPICZαA-CB转化到毕赤酵母SMD1168中。在28°C下，用1.0%甲醇在pH 5.0条件下诱导重组CB表达60小时。使用阳离子交换色谱法纯化重组CB；从100 ml培养液上清液中获得了5.0 mg纯活性CB。抗菌试验表明，CB对革兰氏阳性菌和革兰氏阴性菌均具有广谱抗菌活性。我们的结果表明，毕赤酵母表达系统可用于大量生产具有完全功能的CB，用于研究和工业目的。
