Topic:

Tuberculosis and other mycobacterial infections

Abstract No.:

ISE.292

Title:

Purification and biochemical characterization of a putative monooxygenase (Rv3083 / mymA gene) of Mycobacterium tuberculosis

Author(s):

I. Saraav, P. Kanvatirth, S. Sharma, M. Sharma, S. Sharma; miranda house, zoology , Delhi, DELHI/IN

Abstract:

Background: Mycobacterium tuberculosis is among the microorganisms most successful at adapting to long term residence in macrophage phagosomes. The biosynthesis of fatty acids plays an essential role in the formation of cell wall components; in particular mycolic acids, which have been targeted by many of the drugs used to treat M. tuberculosis infection. It was observed that several of the early up regulated genes after infection of macrophages belong to the mymA operon (Rv3083 to Rv3089).Here we report mymA/Rv3083 gene, the bioinformatics study and biochemical assay revealed its function as a probable monooxygenase and homology modelling predicted its protein structure.Purification of Rv3083 gene expressed protein and designing target assay can be used to look for modulators of target function and immunological characterization for further studies. 
Methods: For protein expression, extracted DNA from M. tuberculosis H37Rv was used to clone mymA gene into E.coli DH5α using T/A cloning. After sequencing confirmation the desired product was ligated into pET vector, initially transformed into E.coli DH5α following BL-21(DE3) for recombinant protein expression. Recombinant protein was purified using Ni²⁺-NTA affinity chromatography, SDS-PAGE and immunoblotting was performed to confirm 55.5 kDa protein.Bionformatics study of the gene was done using multiple sequence alignment and homology modelling for protein structure prediction.Monooxygenase assay ELISA kit was used to confirm its given putative function.
Results: The bioinformatics study of mymA/Rv3083 revealed its function as a probable monooxygenase, highly similar to other putative monooxygenase flavin binding family as confirmed by multiple sequence alignment. Further, homology modelling predicted its protein structure having similarity to crystal structure of PQSL, a probable fad-dependent monooxygenase from Pseudomonas aeruginosa. 55.5kDa recombinant protein was expressed and purified.
Conclusion: The bioinformatics study of the gene and biochemical assay revealed its function as a probable monooxygenase. Rv3083 gene was successfully cloned and expressed 55.5kDa recombinant protein.