Browsing by Subject "expression plasmid"
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(2019)Finding and developing new antimicrobial compounds against clinically important antimicrobial drug resistant bacterial pathogens is necessary to counter the threats to global health, food security and development caused by these organisms. One potential source for leads for novel antimicrobial agents are bacteriophages, whose genomes hold vast numbers of genes encoding for proteins that are able to inhibit bacteria in yet uncharacterized ways. Characterization of these proteins and their functions is likely to aid the discovery of new antimicrobial drugs. This study aimed to optimize the heterologous production of three bacteria-inhibiting proteins from bacteriophage φR1-RT for the characterization of the proteins and their interactions with the bacterial host cell. Expression plasmids were successfully constructed for the heterologous production of the proteins in both Lactococcus lactis and Escherichia coli -based expression systems. The L. lactis expression system utilized a tightly regulated nisin-controlled promoter and featured a lactococcal SSusp45 secretion leader to target the produced protein to extracellular secretion. The E. coli expression system used a tightly regulated arabinose-inducible promoter to control the expression of the bacteriotoxic proteins. Despite the successful construction of the expression plasmids, the bacteriophage φR1-RT proteins were not able to be produced in quantities suitable for protein purification in either of the expression systems used in this study. The lack of protein expression is likely due to either codon bias or the harmful effects of the bacteriotoxic proteins that build up inside the bacterial cells. Codon optimized genes or a eukaryotic expression system could be tried to produce enough protein for purification and further characterization.
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