| Área: Genética e Biologia Molecular ( Divisão N ) BIOCHEMICAL AND KINETIC STUDIES OF POLYNUCLEOTIDE PHOSPHORYLASE (EC 2.7.7.8) FROM MYCOBACTERIUM TUBERCULOSIS Leonardo Martinelli (CPBMF INCT-TB PUCRS); Luiz Augusto Basso (CPBMF INCT-TB PUCRS); Diógenes Santos (CPBMF INCT-TB PUCRS)
ResumoTuberculosis (TB) is the leading cause of death in the world due to infection with a single microbial agent, being mainly caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO), one third of the world population is infected with the tubercle bacillus, nearly 2 billions worldwide, however only 5-10% of the infected people will developed the disease. The co-infection with the human immunodeficiency virus (HIV) increases greatly the chances of the TB reactivation. Regardless of the availability of effective short-course chemotherapy (DOTS), which is preconized by the WHO, and the Bacille Calmette-Guérin (BCG) vaccine, the bacillus continues to claim more lives than any other infectious agent. Due to the outburst of resistant MDR-TB and XDR-TB strains to the classics agents of treatment, a novel line of treatment should be developed with the conception of a new drug and/or vaccine. The enzyme polynucleotide phosphorylase, PNPase (EC 2.7.7.8), encoded by gpsI gene, catalyses the de novo synthesis of polyribonucleotide using nucleoside diphosphates as substrates. Once the role of the PNPase in M. tuberculosis has not been determined yet, it becomes necessary to study this enzyme and its possible role in the bacillus invasion and persistence. The objective of this work is the amplification and cloning of the gene gpsI, the overexpression, purification and characterization of recombinant enzyme to determine its role in M. tuberculosis.
The full-length gpsI coding region (2259bp) was PCR amplified from M. tuberculosis H37Rv genomic DNA. The PCR fragment was cloned into vector pCR-Blunt® (Invitrogen) and then subcloned into expression vector pET-23a(+) (Novagen). The resulting pET-23a(+)::gpsI plasmid was sequenced and have shown its integrity and the absence of mutations. The recombinant protein was obtained in a soluble form when plasmid was transformed into E. coli BL21(DE3) pLysS strain and the colonies were grown in TB medium at 37°C with induction of 1mM IPTG (isopropyl-β-D-thiogalactopyranoside) after 5 hours of growth.
After obtaining the recombinant protein in its soluble form, preliminary trials will be conducted to purify the PNPase enzyme using different chromatographic techniques, such as anion exchange, size exclusion and hydrophobic interaction. Next it is necessary the PNPase enzyme kinetic characterization, N-terminal sequencing and mass spectrometry to confirm its identity.
Palavras-chave: tuberculosis, polynucleotide phosphorylase, expression, purification, kinetic |