title: The Mycobacterium tuberculosis 38-kDa antigen: overproduction in Escherichia coli, purification and characterization.
abstract: The 38-kDa protein (Ag38) of the Gram+ bacterium, Mycobacterium tuberculosis H37Rv, is an immunodominant antigen of potential utility for diagnosis and vaccine development. Assessment of this potential requires large amounts of the purified protein that would be difficult, if not impossible, to obtain from M. tuberculosis itself. The gene coding for Ag38 had been previously cloned and in the present study was expressed as an unfused protein in Escherichia coli under the control of strong transcriptional (bacteriophage lambda pLpR) and translational (atpE) signals. Fermentation of the recombinant E. coli K-12 strain CAG629[pMS9-2], which is deficient in Lon protease and the heat-shock response, produced recombinant Ag38 (reAg38) at high levels (about 10% of total cellular protein). The reAg38, which accumulated as inclusion bodies, was completely solubilized in 6 M guanidine.HCl, refolded and purified to apparent homogeneity. The product showed the expected amino acid composition and M(r), and had similar reactivities as the native protein with three different mAb. Polyclonal antibodies raised against reAg38 reacted strongly with the native antigen in enzyme-linked immunosorbent assay. These results demonstrate that reAg38, which cannot be distinguished antigenically from the native protein of M. tuberculosis, can be prepared in quantity from E. coli.
authors.affiliation: Department of Microbiology, GBF-National Research Center for Biotechnology, Braunschweig, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Singh
authors.forename: A B
authors.initials: AB
authors.lastname: Andersen
authors.forename: J E
authors.initials: JE
authors.lastname: McCarthy
authors.forename: M
authors.initials: M
authors.lastname: Rohde
authors.forename: H
authors.initials: H
authors.lastname: Schütte
authors.forename: E
authors.initials: E
authors.lastname: Sanders
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005285:Fermentation; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012329:RNA, Bacterial; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: C075966:Ag38 antigen, Mycobacterium tuberculosis; D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D008074:Lipoproteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins