Cloning, Expression And Characterization Of ClpX And ClpP2 Protease From Mycobacterium Tuberculosis

Tuberculosis remains one of the major threats to human health, Mycobacterium tuberculosis kills two or three million people per year. M. tuberculosis is a strict and successful intracellular pathogens, which survives and replicates in macrophages. Protease has been considered an important virulence factor for pathogens. Comparative genomics found there were 16 families of proteases in M. tuberculosis, Many members of these were involved in pathogenicity and persisitence of M. tuberculosis. The degradation of protein was essential for all physiological regulation and protein quality control. Investigating the intracellular protein quality control mechanism will give clues to understand the pathogenesis of M. tuberculosis and find new pharmaceutical targets.The main stresses which M. tuberculosis have to face in the macrophages are nutrient deficiency, oxidizing stress, low pH and high temperature. These will make protein unfolding and aggregation. The degradation of these misfolded proteins is very important for M. tuberculosis under the stress environment. In bacteria, there are four other ATP-dependent proteases which carry out intracellular proteolysis, compartmentalized proteases of the ClpAP/XP, HslUV, FtsH and Lon families. Mtb is unique for lacking HslUV and Lon. Therefore, proteasome must play more important functions in Mtb.The genes encoding ClpX and ClpP2 were amplified from M. tuberculosis H37Rv genomic DNA, the PCR products were respectively cloned into pET28a(+) and pET32a(+) vector, PCR and sequencing identified pET28-clpX and pET32-clpP2. Recombinant plasmids were constructed successfully. The recombinant plasmids were transformed into competent E. coli BL21 (DE3). We studied the effect of overexpression ClpX and ClpP2 on host strains. Researches showed that Overexpression ClpX made bacterial grew fast and overexpression ClpP2 cells are resistant to hydrogen peroxide. Overexpression ClpP2 at 30℃over night, there were two different bands of the purified ClpP2 protein. The two bands was analysed with MALDI-TOF-TOF. Bioinformatics analysis suggested that ClpP2 protease had Amidation sites, CAMP phosphorylation site and so on which can make a different of protein molecular weight. Online database was employed to predicted ClpX or ClpP2 interaction network, which will help to understand the Physiological function ClpXP. All the results suggested the M. tuberculosis ClpX and ClpP2 involved in bacteria growth and oxidative stress tolerance.