The high-level expression and production of antimicrobial peptides Epinecidin-1 and Lactoferricin B in E. coli and the assessment of their efficacy and safety

• Main Authors: Chung-Yiu Hsieh, 謝宗佑
• Other Authors: Hung-Jen Liu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/3m4ttq

碩士 === 國立中興大學 === 分子生物學研究所 === 100 === Adding a variety of antibiotics in animal feed is a common use in animal husbandry to prevent the infectious diseases of animals. It might be the reasons of leading to the increases of drug-resistance bacteria. To overcome this circumstance, the cationic antimicrobial peptides (AMPs) show the potential to become novel antibiotics with new modes of actions. The AMPs show broad spectra of antimicrobial activities against gram-negative bacteria, gram-positive bacteria, fungi, and viruses. The purpose of this study is to produce high-level expression of antimicrobial peptide in E. coli, in a low-cost and effective method. In this study, two candidates of AMPs, epinecidin-1 (Epi-1) and bovine lactoferricin (LfcB) were produced. Besides, two different protein expression systems were developed and employed for expression of these two AMPs. In the first protein expression system, the AMPs to be expressed were fused with the sortase A (SrtA) of Staphylococcus aureus at the N terminus. The AMPs can be released from expressed SrtA-AMPs by SrtA self-cleavage reaction which is induced in the presence of Ca2+. Second, the mIFc2 (7 kDa) with negative charge was coexpressed with the AMPs, making AMPs expressed in insoluble form and thus reducing their toxicity to E. coli host. In the SrtA-fusion strategy, both SrtA-Epi-1 and SrtA-LfcB recombinant genes were amplified by over-lap PCR and cloned into the expression vectors of pET32a and pET21d. Furthermore, the S-Epi-1 and S-Lfc fusion proteins were expressed in E. coli and then purified. The yields of S-Epi-1 and S-Lfc are 0.8 mg/100 mL and 3.6 mg/100 mL, respectively. The antibacterial activity assay showed that after SrtA-mediated cleavage, the Epi-1 inhibited the growth of E. coli DH5a and S. typhimurium, indicating Epi-1 posseses the antibacterial activity; in the mIFc2 co-expression strategy, both mIFc2 and the recombinant AMP gene were amplified by the over-lap PCR and then cloned into the expression vector pET21b. The expressed LfcB successfully formed inclusion body with mIFc2, and the yield of LfcB is 1.61 mg/500 mL. After changing the gene order of mIFc2 and LfcB, the yield of LfcB was elevated up to 3.6 mg/500 mL. However, the expression of Epi-1 caused the lysis of the E. coli host when induction with IPTG. The LfcB expressed via the mIFc2 co-expression system exhibited antibacterial activity to the gram-negative and gram-positive bacterium, the range of the MIC was between 37-75 ug/mL. A part of the inclusion body becomes soluable form after the heat treatment, and exhibited antibacterial activity to Salmonella choleraesuis. In comparison with these two expression systems, the mIFc2 co-expression system shows higher efficiency for AMP production than SrtA-fusion system.