AMPLIFICATION, CLONING AND EXPRESSION OF A PROBABLE GMP SYNTHASE ENZYME FROM MYCOBACTERIUM TUBERCULOSIS H37RV

Tathyana Mar Amorim Franco (INCT-TB PUCRS); Cristopher Zandoná Schneider (INCT-TB PUCRS); Luiz Augusto Basso (INCT-TB PUCRS); Diógenes Santiago Santos (INCT-TB PUCRS)

Tuberculosis (TB) is a serious infectious disease mainly caused by Mycobacterium tuberculosis. Epidemiologists estimate that around one-third of the world population is latently infected with M. tuberculosis. Since global incidence of TB has increased in recent years, there is an urgent need for developing new anti-TB drugs and vaccines. Guanosine monophosphate synthase (GMPS), is a key enzyme in the purine biosynthetic pathway, where it catalyzes the conversion of xanthosine monophosphate to guanosine monophosphate. The guaA (Rv3396c) gene encoding GMPS (EC 6.3.5.2) was identified by sequence homology in the M. tuberculosis H37Rv genome. The objectives of this study were the gene amplification, cloning, expression, purification and characterized of the probable GMPS from M. tuberculosis. Oligonucleotides complementary to the 5' and 3'ends of the Rv3396c coding sequence were designed and used to amplify the guaA gene from M. tuberculosis H37Rv genomic DNA. The PCR product (1578 bp) was gel-purified and cloned into the pCR-Blunt vector. Then, it was cleaved with NdeI and BamHI restriction enzymes and subcloned into the pET-23a(+) expression vector. Automatic DNA sequencing of the cloned fragment confirmed both identity and absence of gene mutations. Different protein expression conditions and Escherichia coli host strains were tested for production of M. tuberculosis GMPS. After introducing the recombinant expression plasmid into E. coli, 1.5 ml of culture were collected at specific time points (3, 6, 9, 24 and 48 hours of incubation), centrifuged and cells were disrupted by sonication. Soluble and insoluble fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. The M. tuberculosis enzyme was successfully expressed in the soluble fraction of E. coli C41(DE3) grown at 37ºC in LB medium containing 1 mM of isopropyl-b-D-thiogalactopiranoside. The next steps of this work will be the purification, biochemical and kinetic characterization of GMPS, aiming at developing new drugs to treat TB.