Overexpression and purification of Rhizobium etli glutaminase A by recombinant and conventional procedures.
Rhizobium etli glutaminase A was purified to homogeneity by conventional procedures that included ammonium sulfate differential precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, and dye-ligand chromatography. Alternatively, the structural glsA gene that codifies for glutaminase A was amplified by PCR and cloned in the expression vector pTrcHis. The recombinant protein was purified to homogeneity by 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 micromol ammonium min(-1) mg protein(-1)). Physicochemical and biochemical properties of native and recombinant glutaminase were identical. The molecular mass of recombinant glutaminase A (M(r) 106.8 kDa) and the molecular mass of the subunits (M(r) 26.9 kDa) were estimated by mass spectrometry. These results suggest that R. etli glutaminase A is composed of four identical subunits. The high-level production of recombinant glutaminase A elevates the possibilities for determination of its three-dimensional structure through X-ray crystallography.
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, C.P. 04510, México, D.F., México.
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Huerta-Saquero
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Calderón
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Arreguín
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Calderón-Flores
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Durán
D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005972:Glutaminase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D020539:Sequence Analysis, Protein; D013696:Temperature
D011993:Recombinant Fusion Proteins; D005972:Glutaminase