Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase

Inan M., ARYASOMAYAJULA D., Sinha J., Meagher M.

BIOTECHNOLOGY AND BIOENGINEERING, vol.93, no.4, pp.771-778, 2006 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 93 Issue: 4
  • Publication Date: 2006
  • Doi Number: 10.1002/bit.20762
  • Journal Name: BIOTECHNOLOGY AND BIOENGINEERING
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.771-778
  • Keywords: Pichia pastoris, protein secretion, protein disulficle isomerase, Necator americanus secretory protein, Na-ASP1, protein overexpression, secretion bottleneck, PANCREATIC TRYPSIN-INHIBITOR, SACCHAROMYCES-CEREVISIAE, ENDOPLASMIC-RETICULUM, YEAST, EXPRESSION, GENE, COEXPRESSION, DEGRADATION, PDI, ER
  • Akdeniz University Affiliated: No

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 PD1 proteins. (c) 2005 Wiley Periodicals, Inc.