title: High-level production and purification of P30P2MSP1(19), an important vaccine antigen for malaria, expressed in the methylotropic yeast Pichia pastoris.
abstract: P30P2MSP1(19) is a recombinant subunit vaccine derived from merozoite surface protein 1 (MSP1) of Plasmodium falciparum, the causative agent of malaria. P30P2MSP1(19) consists of two universal T-cell epitopes fused to the most C-terminal 19-kDa portion of MSP1, and this protein has previously shown promising potential as a vaccine for malaria. However, previous attempts at producing this molecule in Saccharomyces cerevisiae resulted in the production of a truncated form of the molecule missing most of the universal T-cell epitopes. Here, we report the production of full-length P30P2MSP1(19) in Pichia pastoris. As salt precipitation is a common problem during P. pastoris high-density fermentation, we utilized an alternative low-salt, fully defined medium that did not reduce growth rates or biomass yields to avoid precipitation. A total of 500 mg/L of secreted purified protein was produced in high cell density fermentation and the protein was purified in one step utilizing nickel-chelate chromatography. P30P2MSP1(19) produced in Pichia was reactive with monoclonal antibodies that recognize only conformational epitopes on correctly folded MSP1. Rabbits immunized with this molecule generated higher and more uniform antibody titers than rabbits immunized with the protein produced in Saccharomyces. P30P2MSP1(19) produced in Pichia may prove to be a more efficacious vaccine than that produced in Saccharomyces and Pichia would provide a system for the cost-effective production of such a vaccine.
authors.affiliation: Malaria Vaccine Development Unit, Laboratory of Parasitic Diseases, Rockville, Maryland 20852, USA.
authors.forename: C P
authors.initials: CP
authors.lastname: Brady
authors.forename: R L
authors.initials: RL
authors.lastname: Shimp
authors.forename: A P
authors.initials: AP
authors.lastname: Miles
authors.forename: M
authors.initials: M
authors.lastname: Whitmore
authors.forename: A W
authors.initials: AW
authors.lastname: Stowers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors; D017780:Malaria Vaccines / Q000276:immunology*; D016778:Malaria, Falciparum / Q000517:prevention & control; D020066:Merozoite Surface Protein 1 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000276:immunology*; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014170:Transformation, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D018984:Epitopes, T-Lymphocyte; D017780:Malaria Vaccines; D020066:Merozoite Surface Protein 1; D011993:Recombinant Fusion Proteins