25º Congresso Brasileiro de Microbiologia

25º Congresso Brasileiro de Microbiologia

ResumoID:2132-1

Área: Micobacteriologa ( Divisão C )

CYTIDINE 5'-TRIPHOSPHATE SYNTHETASE FROM MYCOBACTERIUM TUBERCULOSIS H37RV: CLONING, EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN

Jacqueline Gonçalves Rehm (INCTT); Cristopher Zandoná Schneider (INCTT); Luiz Augusto Basso (INCTT); Diógenes Santiago Santos (INCTT)

Resumo

Human tuberculosis (TB) is an infectious bacterial disease mainly caused by Mycobacterium tuberculosis. Approximately 9 million people develop active TB each year. High rates of mortality (2 million/year) and HIV-TB co-infection, as well as emergence of resistant strains of M. tuberculosis, have increased the need for developing new and less toxic anti-TB drugs that can shorten the duration of current therapy and be effective against latent bacilli. Thus, it is necessary to identify promising targets for the development of novel anti-TB agents. The cytidine 5'-triphosphate synthetase (CTPS) enzyme from M. tuberculosis, encoded by pyrG gene, is an attractive target because it catalyzes the last step in the de novo pyrimidine pathway, forming CTP from UTP in an ATP-dependent reaction and using either L-glutamine or ammonia as the nitrogen source. Thus, the main goal of this study is the kinetic and structural characterization of the CTPS enzyme from M. tuberculosis H37Rv. Synthetic oligonucleotide primers were designed according to genome sequencing data, and M. tuberculosis H37Rv genomic DNA was used as a template for pyrG amplification by the PCR technique. The PCR product was cloned into the pCR-Blunt vector and subcloned into the pET-23a(+) expression vector. Protein expression tests were performed in different conditions using Escherichia coli as a host. Expression of CTPS was observed in the soluble fraction of E. coli BL21(DE3) cells transformed with pET-23a(+)::pyrG recombinant plasmid and grown at 37°C in LB medium with or without 1 mM IPTG induction. Protein purification protocols using High Performance Liquid Chromatography are currently underway to obtain quantities of purified CTPS enzyme for future determination of kinetic parameters and structural studies. This work will provide data that may prove useful in the rational design of M. tuberculosis CTPS inhibitors.

Palavras-chave: cytidine 5'-triphosphate synthetase, Mycobacterium tuberculosis, pyrimidine metabolism

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