Expression of recombinant magainin-2 in eukaryotic systems and evaluation of its biological activity

• Main Author: 張銘倫
• Other Authors: 邱繡河
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/76061628218879978640

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 93 === The purposes of this study are to express magainin-2 in eukaryotic expression systems and to evaluate its biological activities. Magainins are antimicrobial peptides isolated from skin of African frog (Xenopus laevis). Based on the amino acid sequence, magainins can be divided into magainin-1 and magainin-2. Both consist of 23 amino acid residues, forming an amphipathic α-helix structure. Of these two, magainin-2 exhibits broader spectrum of antimicrobial activity against many microorganisms, including bacteria, parasites, fungi, and viruses. It possesses low toxicity for normal eukaryotic cells. Therefore, magainin-2 may have potential to be exploited as anti-microbial reagent for solving the present problem of antibiotics-resistant bacteria. In this study, the in vitro antimicrobial activity of synthetic magainin-2, either used alone or in combination with amoxicillin, was investigated against the clinical isolated antibiotic resistant pathogen Salmonella Choleraesuis. This bacteria isolate was susceptible to magainin-2 at a minimal inhibitory concentration of 128 µg/ml. Synergy occurred when magainin-2 was combined with amoxicillin. In order to produce magainin-2 in large quantity, the recombinant plasmid pYES3/M23 carrying magainin-2 gene was transformed into Saccharomyces cerevisae cells. After galactose induction, we observed an expressed peptide of 2.45 kDa by SDS-PAGE, which was the expected molecular weight of magainin-2. We further employed high-cell-density cultivation technique to improve the expression of recombinant magainin-2. Under 30oC growth temperature, the yield of the 2.45 kDa peptide reached 2g/L when growth conditions were set at an air flow rate of 3 vvm, 60% dissolved oxygen, and without controlling pH. The yield reached 8g/L when air flow rate was set as 3vvm, agitation speed was 300 rpm, and pH was not controlled. Therefore, in this study, yeast expression system under high-cell-density cultivation allowed us to express recombinant magainin-2 in large quantity. In addition, we also constructed the recombinant plasmid pVaxIsMagHis and it will be coordinated with mammal cell expression system to expect the expressing product to be similar natural magainin-2.