The goal of the present work was to produce glyceraldehyde-3-phospate dehydrogenase from M. tuberculosis in E. coli cells in soluble and catalytically active form and to elaborate a method for the purification of the recombinant enzyme. The His-tagged recombinant enzyme (Mtb-GAPDH_His) was shown to be inactive and insoluble. The untagged enzyme (Mtb-GAPDH) was catalytically active and exhibited higher solubility. Mtb-GAPDH was purified from the cell extract using ammonium sulfate fractionation and ion-exchange chromatography. The presence of glycerol was necessary for isolation of Mtb-GAPDH, presumably, to facilitate folding of the recombinant enzyme. The yield of Mtb-GAPDH constituted 1.3 mg per 10 g of the cell biomass. The specific activity of the purified Mtb-GAPDH was 55 ± 5 μmol NADH/min per mg protein (pH 9.0, 22 °C) that exceeded the activity of the previously described preparation of His-tagged recombinant GAPDH from M. tuberculosis that was co-expressed with GroEL/ES chaperone by approximately 5-fold. The results suggest that the folding of the recombinant GAPDH is hindered by the His-tag, which may result in the production of insoluble protein or in isolation of the preparation with decreased specific activity.
Keywords: Enzyme kinetics; Glyceraldehyde-3-phosphate dehydrogenase; M. tuberculosis; Protein folding; Recombinant proteins.
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