AMPLIFICATION, CLONING, EXPRESSION AND PURIFICATION OF HOMOSERINE DEHYDROGENASE FROM MYCOBACTERIUM TUBERCULOSIS

Gregório Henrique Salles Laseck (CPBMF/INCT-TB/PUCRS); Anne Drumond Villela (CPBMF/INCT-TB/PUCRS); Luiz Augusto Basso (CPBMF/INCT-TB/PUCRS); Diógenes Santiago Santos (CPBMF/INCT-TB/PUCRS)

Tuberculosis (TB) is a disease usually caused by the bacillus M. tuberculosis. The World Health Organization estimates that 9.27 million new cases of TB occurred in 2007, causing around of two million deaths annually. The TB treatment currently recommended is long and involves undesirable side effects to the administered drugs which result in low patient's adherence to treatment. Thus, it is essential the development of new drugs to improve the current treatment. The understanding of essential M. tuberculosis metabolic routes as the aspartate pathway and the characterization of the enzymes involved in this pathway are an important step to the development of anti-TB agents. Homoserine dehydrogenase (HSD) catalyzes the NADPH-dependent reduction of aspartate-4-semialdehyde into homoserine, which is a precursor of threonine, isoleucine and methionine. HSD is not found in humans, it is therefore an attractive target due to the possibility to find a selective inhibitor to be used as anti-TB agent. The thrA gene (Rv1294) encoding M. tuberculosis HSD (EC 1.1.1.3) was identified by homology in the genome of M. tuberculosis H37Rv. Two oligonucleotide primers complementary to regions 5' and 3' of thrA gene were design and thrA gene was amplified from the M. tuberculosis H37Rv genomic DNA by Polymerase Chain Reaction (PCR). The PCR product (1326 bp) was cloned into pCR-Blunt vector and subcloned into pET-23a(+) expression vector. Automatic DNA sequencing confirmed both identity and integrity of thrA gene of M. tuberculosis H37Rv. Electrocompetent E. coli strains were transformed with the recombinant plasmid by electroporation. Different expression conditions were employed in order to obtain HSD expression in the soluble fraction. The expression of HSD was observed in the insoluble fraction of Rosetta(DE3) and BL21-Lys(DE3) E. coli strains at 37°C. A small amount of protein could also be found in the soluble fraction of BL21-Lys(DE3) at 30°C, though the major amount of protein was yet expressed in the insoluble fraction. The next step of this work will be the optimization of the HSD expression in the soluble fraction and the protein purification. Availability of homogeneous M. tuberculosis HSD protein will allow the biochemical and kinetic characterization of the enzyme which is an important step toward the development of new drugs to treat TB.