Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media

Abstract

Plasmodium falciparum merozoite surface protein 3 (MSP3) is a leading blood-stage malaria vaccine candidate. Vaccination with Pichia pastoris derived recombinant MSP3 protected Aotus nacymai monkeys from the parasite's lethal challenge and the post-challenge antibody titer against MSP3 correlated with protection. In our preliminary attempts to produce this vaccine in fermentors, little or no expression of MSP3 was observed in chemically defined media, although the same P. pastoris strain produced MSP3 in complex media. Our goal is to develop a Phase I/II clinical manufacturing process in completely chemically defined media because of the concern of potential prion contamination in complex media containing animal derived products. Here, we report our investigations into various factors to improve the yield of MSP3 in defined media. We found that an induction pH (pH(i)) 6.8 yielded MSP3 at 434 mg/L whereas there was no product at pH(i)< or = 5, though cell growth was the same in all pH(i) levels examined. High levels of NH(4) (+) consumed at pH(i) 6.8 were directly correlated to the enhanced MSP3 production. Furthermore, an additional 3.5-fold increase in the yield of MSP3 was obtained by addition of casamino acids at pH(i) 6.8. No direct correlation was observed between protease activity in the culture supernatants and lack of MSP3 expression. Neither high P. pastoris biomass generated at a high specific growth rate (0.04/h) nor low induction temperatures during induction improved yield. Nitrogen source was the most important factor affecting expression of MSP3 in defined media.

Genetic Diversity of Polymorphic Vaccine Candidate Antigens (Apical Membrane Antigen-1, Merozoite Surface Protein-3, and Erythrocyte Binding Antigen-175) in Plasmodium falciparum Isolates from Western and Central Africa

“…The genetic diversity and relatively balanced distribution of allele classes provides evidence that an effective vaccine should necessarily account for different allelic classes in combination, although this could be challenging with regards to product development 44. If vaccines based on universally conserved subunit targets were to be used, field monitoring of known and novel allelic polymorphisms associated with the target before and after vaccination will provide important information on vaccine epidemiology and immune selection.…”

Plasmodium falciparumMerozoite Surface Protein 3 Is a Target of Allele‐Specific Immunity and Alleles Are Maintained by Natural Selection

“…If the full-length MSP3 molecule is developed as a vaccine, it would be preferable to include both major allelic forms (at least one representative allele of both K1-like and 3D7-like types), although this adds to the challenging process of product development [31]. MSP3 appears to be under natural immune selection, so such a vaccine might protect infants and younger children in areas of endemicity (a minority of those with P. falciparum infection although the majority of those with clinical cases) without greatly increasing the overall selection pressure on the locus.…”

Enrichment of fermentation media and optimization of expression conditions for the production of EAK16 peptide as fusions with SUMO

“…Amino acids and their concentration or other micronutrients in the supplied medium are usually very important on inducing recombinant protein expression. For instance, Wang et al (2005) reported that the addition of either a cocktail of five amino acids or casamino acids to a defined medium for the production of Plasmodium falciparum merozoite surface protein 3 (MSP3) by recombinant Pichia pastoris resulted in varying production of the protein. Future experiments should focus on the identification of nutrients essential for protein expression.…”