Optimization of recombinant expression of the Mycobacteria tuberculosis rhomboid protease 1 using Escherichia coli overexpression plasmid

Abstract

The mycobacteria Rhomboid proteases (1 & 2) have been identified as novel targets for drug targeting in mycobacteria giving a ray of hope for the realisation of new drug candidates for the management not only multidrug-resistant TB but also Buruli ulcers, leprosy, to mention but a few. In a bid to find out the native peptide substrates for these rhomboid proteases, biochemically significant amounts of the proteins have to be produced through recombinant technologies. The main objective of this study was to establish the optimum conditions for the recombinant expression of MTB Rho 1 using E. coli overexpression plasmid. In the study, E. coli BL21 (DE3) cells transformed with the MTB Rho 1 DNA having the pMAL c5X plasmid were expressed in three media LB, 2xYT and Terrific broth. The media that gave the best expression was tested at temperatures of 370C, 300C, and 260C. The best expression temperature using this media was then tested over times of 1 hour, 2 hours, and 4 hours. Induction of each expression was done with 0.5mM IPTG inducer concentration after which 1.5ml aliquots were removed at and analysed by SDS-PAGE for the presence of the target protein. We found out that terrific broth was the best medium for the expression of MTB Rho 1 and the optimum temperature was 370C and the best time after induction was 4 hours. In conclusion, biochemically significant amounts of MTB Rho 1 are obtained when expression is done in E. coli BL21 (DE3) with terrific broth at 37 0C for 4 hours.