title: Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase.
abstract: A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.
authors.affiliation: Biological Process Development Facility, Department of Chemical Engineering, University of Nebraska, Lincoln, 207P Othmer Hall, Nebraska 68588-0643, USA. minan2@unl.edu
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
authors.forename: Dinesh
authors.initials: D
authors.lastname: Aryasomayajula
authors.forename: Jayanta
authors.initials: J
authors.lastname: Sinha
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics; D017221:Necator americanus / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding
chemical_list: C099548:ASP protein, Ancylostoma caninum; D015801:Helminth Proteins; D019704:Protein Disulfide-Isomerases