| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 104 === Bacteriocins are proteins or peptides produced by bacteria that have bactericidal activities and often against the closely related bacteria. In our previous studies, the genes of GlyA and GlyB from plant pathogen X. axonopodis pv. glycines YR32 (Xag YR32), were linked together and is capable of forming an insoluable fusion protein ALB after expression in E. coli. In addition, during renaturation of the the ALB fusion protein, a processed mature mALB protein with bactericidal activity was observed. In this study, expression and purification of ALB, mALB, and antimicrobial peptide cecropin A in Escherichia coli were carried out. To enhance the soluability of the ALB and mALB proteins, the thioredoxin-, CBD-intein-, and MalE- fused proteins were constructed. Soluable form fusion proteins were obtained for MalE-fused ALB and mALB; however, these fusion proteins convert to inclusion bodies after removing of the MalE fusion tag by TVMV protease digestion. Expression of the mALB in X. axonopodis pv. glycines 12609 (Xag 12609) showed no difference in intercellular and extracellular bactericidal activities against Xv66. Enhancing the intercellular but not extracellular bactericidal activity was observed for ALB expressed in Xag 12609. In the second part of this study, four plasmids containing the antimicrobial peptide cecropin A and a self-cleavageable CBD-intein as fusion tag were constructed. Results showed that the CBD-intein fusion proteins expressed from pTWIN-cecA-SN and pTWIN-cecA-SC plasmids have a higher self-cleavage activity. After Ni2+-NTA column purification, cecropin A with detectable bactericidal activity against DH5αwas obtained. Moreover, in order to improve the production of active cecropin peptide, expression plasmids containing one to seven copies of tandem cecropin A genes and individually fused with thioredoxin, SUMO, and CBD-intein were constructed. Results showed that the majority of the fusion proteins expressed from tandem multimers of cecropin A with SUMO and thioredoxin fusion tags were soluble and the fusion monomer and dimer had the highest expression level at about 13.2 % of the total protein.
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