title: Host vector system for high-level expression and purification of recombinant, enzymatically active alanine dehydrogenase of Mycobacterium tuberculosis.
abstract: The 40-kDa antigen of M. tuberculosis, which is an alanine dehydrogenase, is a species-specific antigen that is potentially useful for strain identification. Large quantities of the purified protein are required for immunological, as well as for detailed biochemical and structural, characterization. The AlaDH gene was cloned by PCR from H37Rv (virulent) and H37Ra (partially attenuated) strains of M. tuberculosis, and their DNA sequence was determined. A host-vector system suitable for the production of sufficient quantities of the recombinant AlaDH antigen was developed. The AlaDH gene was expressed under the control of strong, transcriptional (bacteriophage pLpR) and translational (atpE) signals. High-level expression of soluble AlaDH was obtained using the recombinant E. coli K-12 strain CAG629 [pMSK12], which is deficient in Lon protease and the heat-shock response. A simple two-step procedure for the rapid purification of the recombinant protein was developed. The protein was purified to near homogeneity, and the purified AlaDH showed a specific enzyme activity comparable to the native protein isolated from M. tuberculosis. In addition, the product showed an expected amino acid sequence and reacted strongly to the 40-kDa (AlaDH)-specific mAb HBT-10. Furthermore, the epitope of the mAb HBT-10 was mapped to a 12-amino-acid region. Contrary to the published results, we show that the AlaDH and the PNT (pyridine nucleotide transhydrogenase) of M. tuberculosis do not share common epitopes reacting to the species-specific mAb HBT-10. The availability of highly purified AlaDH should now enable a detailed biochemical and structural characterization of this important enzyme of M. tuberculosis.
authors.affiliation: GBF-German National Research Center for Biotechnology, Braunschweig, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Hutter
authors.forename: M
authors.initials: M
authors.lastname: Singh
mesh_terms: D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D009250:NADP Transhydrogenases / Q000276:immunology; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D009250:NADP Transhydrogenases