title: High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris.
abstract: Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc- (His)6 tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein. We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested posttranslational modifications may contribute to the size difference. The NH2-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.
authors.affiliation: Molecular Biotechnology Group, Laboratory of Immunology, Institute Pasteur de Tunis, BP74, 1002, Tunis, Tunisia.
authors.forename: C
authors.initials: C
authors.lastname: Benabdesselem
authors.forename: M R
authors.initials: MR
authors.lastname: Barbouche
authors.forename: M A
authors.initials: MA
authors.lastname: Jarboui
authors.forename: K
authors.initials: K
authors.lastname: Dellagi
authors.forename: J L
authors.initials: JL
authors.lastname: Ho
authors.forename: D M
authors.initials: DM
authors.lastname: Fathallah
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012698:Serologic Tests; D014376:Tuberculosis / Q000175:diagnosis
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C480221:Cfp32 protein, Mycobacterium tuberculosis; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins