title: Design and optimization of a recombinant system for large-scale production of the MPT64 antigen from Mycobacterium tuberculosis.
abstract: Early clinical trials of a potential new tuberculosis (TB) diagnostic, the Patch Test for Active TB (PTAT), used MPB64 protein that was purified from the spent medium of Bacillus Calmette-Guérin (BCG) Tokyo 172 vaccine production. The yield was poor, 0.05 mg/L, and the process for purification of the protein was complex, requiring four chromatographic steps. The combination of yield and purification complexity compromised the ability to produce the PTAT diagnostic in quantities sufficient for larger clinical trials and commercialization. We report here a highly efficient method for the overexpression and purification of recombinant MPT64 from Escherichia coli (rMPT64) based upon a mild insolubility of rMPT64 following induction, and scalable anion-exchange and gel filtration chromatographies. Yields of protein were improved substantially to approximately 250 mg/L, and resulted in a preparation greater than 98% pure. Quantitative release assays were developed and used with MALDI-TOF mass spectrometry to confirm the identity of rMPT64. Using a guinea pig model of active TB, we found that rMPT64 elicited a specific immune response indistinguishable from that of MPB64 purified from BCG Tokyo culture filtrates. These results describe the first efficient and scalable protocol for production of rMPT64, demonstrate its activity in an animal model of active TB, and lay the foundation of ongoing and future use of the PTAT in clinical settings.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, USA.
authors.forename: Brian V
authors.initials: BV
authors.lastname: Geisbrecht
authors.forename: Boris
authors.initials: B
authors.lastname: Nikonenko
authors.forename: Rowena
authors.initials: R
authors.lastname: Samala
authors.forename: Reiko
authors.initials: R
authors.lastname: Nakamura
authors.forename: Carol A
authors.initials: CA
authors.lastname: Nacy
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Sacksteder
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015183:Restriction Mapping; D012882:Skin Tests; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000942:Antigens, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; C510537:MPT64 protein, Mycobacterium tuberculosis; D010446:Peptide Fragments; D011994:Recombinant Proteins