title: Overproduction of Pichia pastoris or Plasmodium falciparum protein disulfide isomerase affects expression, folding and O-linked glycosylation of a malaria vaccine candidate expressed in P. pastoris.
abstract: Production of recombinant malaria proteins in the methylotrophic yeast Pichia pastoris has been difficult due to constraints in transcription, translation and/or post-translation controls. Use of codon-optimized genes has resolved many of the transcriptional controls; however, efforts to overcome translational and post-translational modifications involving disulfide bond formation and glycosylation have been mostly restricted to knocking-out putative N-linked glycosylation sites. We report now on the effect of overproduction of P. pastoris protein disulfide isomerase (PpPDI) and Plasmodium falciparum (PfPDI) on production of a disulfide-rich P. falciparum transmission-blocking vaccine candidate, Pfs25. Pfs25 is expressed in P. pastoris as two isoforms (A and B); the A form has been selected for Phase I human studies. Overproduction of PpPDI in the P. pastoris Pfs25 production clone markedly enhanced the expression level of Pfs25(A) and (B) by 3-fold, while overproduction of PfPDI increased the proportion of Pfs25(A) compared to (B). The resultant Pfs25 products were purified and fully characterized biochemically. In addition to differences in production levels, the mass spectra of PpPDI-Pfs25(A) compared to Pfs25(A) and PfPDI-Pfs25(A) were different due to the pattern and level of O-linked glycosylation. The overproduction of PpPDI or PfPDI provides new platforms for expression of disulfide-rich malaria proteins.
authors.affiliation: Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Disease, National Institutes of Health, 5640 Fishers Lane, Rockville, MD 20852, USA.
authors.forename: Chiawei W
authors.initials: CW
authors.lastname: Tsai
authors.forename: Peter F
authors.initials: PF
authors.lastname: Duggan
authors.forename: Richard L
authors.initials: RL
authors.lastname: Shimp
authors.forename: Louis H
authors.initials: LH
authors.lastname: Miller
authors.forename: David L
authors.initials: DL
authors.lastname: Narum
mesh_terms: D000818:Animals; D015870:Gene Expression; D006031:Glycosylation; D017780:Malaria Vaccines / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D017510:Protein Folding; D046188:Protein Modification, Translational / Q000235:genetics; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017780:Malaria Vaccines; C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases