title: Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials.
abstract: In previously published studies, Saccharomyces cerevisiae recombinant protein expression systems have been employed to express the malaria parasite antigen Pfs25, a candidate transmission-blocking vaccine antigen against Plasmodium falciparum malaria. However, despite having been in two Phase 1 trials, the recombinant Pfs25 so produced (previously called TBV25H) exists as a mixture of two monomeric protein conformational forms, Pfs25H-A and Pfs25H-B. In this study, we optimized the expression and purification of the two Pfs25H conformers in S. cerevisiae, and characterized their biochemical and antigenic properties, immunogenicities, and transmission-blocking activities. Pfs25H-A is apparently homogeneous, and has the correct conformation as measured by monoclonal antibody recognition. It is, however, expressed at a low yield of only 0.19mg/l. By contrast, Pfs25H-B is produced as a heterogeneous population of molecules that do not seem to have the correct conformation. Nonetheless, both forms appear equally effective in their ability to produce transmission-blocking antibodies in mice. To address the low yield seen with S. cerevisiae, we also expressed Pfs25 in Pichia pastoris. P. pastoris is apparently superior to S. cerevisiae in producing higher yield, immunologically more potent, biologically more active Pfs25H-A.
authors.affiliation: Malaria Vaccine Development Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
authors.forename: Lanling
authors.initials: L
authors.lastname: Zou
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Miles
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Stowers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002986:Clinical Trials as Topic / Q000379:methods; D009033:Culicidae / Q000276:immunology / Q000469:parasitology; D005260:Female; D015966:Gene Expression Regulation, Fungal / Q000276:immunology; D006801:Humans; D017780:Malaria Vaccines / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use*; D016778:Malaria, Falciparum / Q000276:immunology* / Q000469:parasitology* / Q000635:transmission; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010843:Pichia / Q000276:immunology / Q000378:metabolism*; D010957:Plasmids / Q000276:immunology; D010963:Plasmodium falciparum / Q000276:immunology; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000276:immunology / Q000627:therapeutic use
chemical_list: D017780:Malaria Vaccines; C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic