44
IV Mostra de Pesquisa
da Pós-Graduação
PUCRS
Cloning, expression, purification and kinetics studies of the enzime
polynucleotide phosphorylase (PNPase, EC 2.7.7.8) from
Mycobacterium tuberculosis
Leonardo Krás Borges Martinelli1,2, Luiz Augusto Basso1, Diógenes Santiago Santos1
1 -Centro de Pesquisa em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e
Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul, Av.
Ipiranga 6681, Porto Alegre, RS, Brasil
2-Programa de Pós-Graduação em Biologia Celular e Molecular, Faculdade de Biociências, PUCRS,
Introduction
Tuberculosis (TB) is the leading cause of death in the world due to infection with a
single microbial agent, being mainly caused by Mycobacterium tuberculosis (Zahrt, 2003).
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 (WHO,
2005). 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 (Cole et al, 1998). 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
(Rv2783c), catalyses the de novo synthesis of polyribonucleotide using nucleoside
diphosphates as substrates (Littauer, 2005). 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.
IV Mostra de Pesquisa da Pós-Graduação – PUCRS, 2009
45
Materials and methods
- Amplification of the gpsI gene with NdeI and HindIII restriction site by PCR method
from M. tuberculosis H37Rv genomic DNA;
- Cloning of the amplicons into cloning vector pCR-Blunt® (Invitrogen) and
expression vector pET-23a(+) (Novagen);
- Sequencing of the plasmid pET-23a(+) containing the gpsI gene;
- Overexpression of the recombinant protein in Escherichia coli system, using
different strains of E. coli, different medium and temperature, to obtain the enzyme PNPase in
its soluble form;
Results
The full-length gpsI coding region (2259bp) was PCR amplified from M. tuberculosis
H37Rv genomic DNA, with NdeI and HindIII restriction sites. The PCR fragment was cloned
into vector pCR-Blunt® (Invitrogen) and then subcloned at the respective restriction sites of
the 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.
Conclusion
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.
IV Mostra de Pesquisa da Pós-Graduação – PUCRS, 2009
46
References
1- Cole ST, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete
genome sequence. Nature, 1998; 393: 537-544.
2- Littauer UZ. From polynucleotide phosphrylase to neurobiology. J Biol Chem, 2005; 280
(47): 38889-38897.
3- WHO/Stop TB Partnership. The Global Stop TB 2006-2015;
4- Zahrt TC. Molecular mechanisms regulating persistent Mycobacterium tuberculosis
infection. Microbes Infect, 2003; 5: 159-167.
IV Mostra de Pesquisa da Pós-Graduação – PUCRS, 2009