Li Gui-ping, Chen Yi-ben
South China Sea Institute of Oceanology, Guangzhou 510301, China.
Wei Sheng Wu Xue Bao. 2007 Feb;47(1):115-20.
To enhance the antibacterial ability of Magaininl-12, its N side was joined with an alkaline peptide named Hexapeptide( RRWQWR), which would make Magaininl-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris, a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin( 1-12). Synthesized through gene splicing by overlap extension, the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation, the hybrid antibacterial peptide was expressed on high level. The new peptide, which has a weight of 2.3kDa, could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay, Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.
为增强Magaininl - 12的抗菌能力，将其N端与一种名为六肽（RRWQWR）的碱性肽相连，这会使Magaininl - 12更紧密地附着在细菌细胞膜上。根据巴斯德毕赤酵母的偏爱密码子，基于六肽和Magainin（1 - 12）的序列设计了一种新的杂合抗菌肽Hex - Mag。通过重叠延伸基因拼接合成后，将杂合基因克隆到pPIC9中构建表达载体pPIC9 - HM。经限制性内切酶分析和纯化后，将pPIC9 - HM转化到巴斯德毕赤酵母GS115中。通过表型筛选出的阳性克隆用甲醇诱导。优化摇瓶培养发酵条件后，杂合抗菌肽得以高水平表达。这种新肽分子量为2.3kDa，在沸水中处理3小时以上仍能保持其抑制活性。通过琼脂扩散试验检测，Hex - Mag不仅对革兰氏阴性菌而且对革兰氏阳性菌都显示出广谱抗菌能力。抗菌实验证实了增加正电荷的作用，结果表明Hex - Mag的活性明显强于Magainin1 - 12。
