| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 95 === AMPs (antimicrobial peptides) are an important part of innate immunity in all living things. In recent years, there was increase drug resistance of pathogenic microbes to many antibiotics due to antibiotics abuse. Researchers then change their research targets to develop AMPs which have unique bioactivity and different function mechanism compared to antibiotics. Extraction of antimicrobial peptide from organisms is complicated and little amount was obtained. The cost of chemical synthesis of peptides is high. To obtain large amount of AMPs, recombinant expression of AMPs would be the best choice. In this research, we have developed an E. coli fusion expression system to produce AMPs. NNVB2 was used as the fusion partner. The coding sequence of AMPs was cloned into pET29b(+) vector and expression as a B2-C-AMP (C presents a chemical cleavage site) fusion protein in E coli. Three different chemical cleavage reagents, cyanogen bromide, formic acid and hydroxylamine were used instead of protease to cleave off AMPs from fusion proteins, and the use of chemical cleavage reagents was cost less. Three AMPs, apidaecin Ia, calcitermin and CAP 7 (expression as fusion protein B2-M-Cal, B2-D-Cal, B2-NG-Cal, B2-M-Api, B2-D-Api and B2-M-CAP) have been successfully expressed using this expression system and the resulting expression level of fusion proteins reached up to 73.2~ 247.1 mg per liter cell culture. All fusion proteins were expressed as inclusion bodies in E. coli. 90% AMPs can be cleaved off from fusion protein by cyanogen bromide and 46% by formic acid. No cleavage product was found treated with hydroxylamine. The antimicrobial activities of the AMPs, purified by C18 reverae-phase column FPLC, were measured and are identical to that of the native extracted peptides. Recombinant apidaecin Ia and CAP 7 both inhibited the growth of E. coli and clear inhibition zones can be seen in inhibition zone assay. Recombinant calcitermin inhibited the growth of E. coli temporarily similar to native extracted calcitermin. In this research, we have developed successfully a high production system of antimicrobial peptides using NNVB2 as fusion partner. E. coli expression system that expression of antimicrobial peptides by a fusion partner reinforcing formation of inclusion bodies can increase the level of productions.
|
|---|
