Meng De-Mei, Li Wen-Juan, Shi Lin-Yue, Lv Yu-Jie, Sun Xue-Qing, Hu Jin-Cheng, Fan Zhen-Chuan
State Key Laboratory of Food Nutrition and Safety, China International Scientific & Technological Cooperation Base for Health Biotechnology, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
Tianjin Haifa Sea Food Industrial Development Co., LTD, People's Republic of China.
Protein Expr Purif. 2019 Aug;160:19-27. doi: 10.1016/j.pep.2019.03.007. Epub 2019 Mar 21.
Hispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6 × His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell factory. Results showed that the E. coli-derived recombinant Hispidalin did not show any antimicrobial activity against all the tested strains, whereas the P. pastoris-derived recombinant Hispidalin (rHispidalin) showed a broad antibacterial spectrum against five pathogenic bacteria of both Gram-negative and Gram-positive. rHispidalin also has bactericidal activity and completely killed all of the Staphylococcus aureus within 40 min. Additionally, rHispidalin showed a broad range of thermostability and pH stability, and a hemolytic activity of less than 2% even at a concentration of 300 μg/ml; it was resistant to trypsin and proteinase K, but was moderately sensitive to pepsin and papain. Moreover, rHispidalin effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. After an initial optimization was performed, the amount of rHispidalin accumulation could reach as high as 98.6 μg/ml. These results indicate that Hispidalin could be produced on a large scale by P. pastoris and has a great potential to be utilized as a new antibacterial agent for further development.
丝瓜抗菌肽是从冬瓜种子中分离出的一种新型抗菌肽，据报道它对多种细菌和真菌病原体具有广泛的抗菌活性。为了大量生产丝瓜抗菌肽，首次成功地在大肠杆菌或毕赤酵母细胞工厂中表达了一种带有N端6×组氨酸标签和肠激酶序列的重组丝瓜抗菌肽。结果表明，大肠杆菌来源的重组丝瓜抗菌肽对所有测试菌株均未显示出任何抗菌活性，而毕赤酵母来源的重组丝瓜抗菌肽（rHispidalin）对革兰氏阴性和革兰氏阳性的五种病原菌均表现出广泛的抗菌谱。rHispidalin还具有杀菌活性，能在40分钟内完全杀死所有金黄色葡萄球菌。此外，rHispidalin表现出广泛的热稳定性和pH稳定性，即使在浓度为300μg/ml时溶血活性也小于2%；它对胰蛋白酶和蛋白酶K具有抗性，但对胃蛋白酶和木瓜蛋白酶中度敏感。此外，rHispidalin能有效通透细胞质膜并破坏靶向细菌细胞的形态。经过初步优化后，rHispidalin的积累量可高达98.6μg/ml。这些结果表明，毕赤酵母可大规模生产丝瓜抗菌肽，并且它作为一种新型抗菌剂具有巨大的进一步开发潜力。
