Overexpression and purification of Rhizobium etli glutaminase a by recombinant and conventional procedures - A comparative study of enzymatic properties

Rhizobium etli glutaminase A was purified to homogeneity by conventional pr ocedures that included ammonium sulfate differential precipitation, ion-exc hange chromatography, hydrophobic interaction chromatography, gel filtratio n, and dye-ligand chromatography. Alternatively, the structural glsA gene t hat codifies for glutaminase A was amplified by PCR and cloned in the expre ssion vector pTrcHis. The recombinant protein was purified to homogeneity b y affinity chromatography. This protein showed the same kinetic properties as native glutaminase A (K-m for glutamine of 1.5 mM and V-max of 80 mu mol ammonium min-l mg protein(-1)). Physicochemical and biochemical properties of native and recombinant glutaminase were identical. The molecular mass o f recombinant glutaminase A (M-r 106.8 kDa) and the molecular mass of the s ubunits (M-r 26.9 kDa) were estimated by mass spectrometry. These results s uggest that R. etli glutaminase A is composed of four identical subunits. T he high-level production of recombinant glutaminase A elevates the possibil ities for determination of its three-dimensional structure through X-ray cr ystallography. (C) 2001 Academic Press.