ADP-glucosa pirofosforilasa de Mycobacterium tuberculosis: Clonado molecular del gen y caracterización de la proteína recombinante.

A.M. DEMONTE; D. GONZÁLEZ; D.G. ARIAS; S.A. GUERRERO; A.A. IGLESIAS

Buenos Aires – Argentina

Congreso; XVII Congreso Latinoamericano de Microbiología – X Congreso Argentino de Microbiología.; 2004

ADP-glucose pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27) is the regulatory enzyme in the glycogen and starch synthesis pathways in bacteria and plants, respectively. Unlike to Gram negative bacteria, works on the characterization of enzymes involved in the polysaccharide metabolism in Gram positive bacteria are scarce. Glycogen accumulation in Mycobacterium tuberculosis would be important for the bacterium viability after hypobiosis periods. In this way, to understand how the glycogen is synthesized in this microorganism and its relationships with nutritional conditions, it is relevant to study the occurrence and regulation of glycogen metabolism. Here, we show the molecular cloning of the glgC gene coding for ADPGlcPPase by using genomic DNA and primers designed after the sequence available from the genome of M. tuberculosis H37Rv. The amplified gene identity was confirmed by sequencing. The glgC gene was subcloned in the pET19b vector, to express a recombinant protein mainly recovered in inclusion bodies. MALDI-TOF analysis revealed the correctness of the recombinant ADPGlcPPase. Optimization of the enzyme expression in heterologous cells is a key alternative tool to characterize ADPGlcPPase in Gram positive bacteria, as well as to determine the actual relevance of storage polysaccharyde metabolism in these microorganisms.