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

• Published in: Biotechnology and bioengineering Vol. 93; no. 4; pp. 771 - 778
• Main Authors: Inan, Mehmet, Aryasomayajula, Dinesh, Sinha, Jayanta, Meagher, Michael M.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05-03-2006; Wiley; Wiley Subscription Services, Inc
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cloning, Molecular; Endoplasmic Reticulum - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Helminth Proteins - biosynthesis; Helminth Proteins - genetics; Infections; Na-ASP1; Necator americanus; Necator americanus - metabolism; Necator americanus secretory protein; Pichia - genetics; Pichia - metabolism; Pichia pastoris; protein disulfide isomerase; Protein Disulfide-Isomerases - biosynthesis; Protein Disulfide-Isomerases - chemistry; Protein Disulfide-Isomerases - genetics; Protein Folding; protein overexpression; protein secretion; Proteins; Saccharomyces cerevisiae; secretion bottleneck; Vaccines; Yeast; Intramolecular oxidoreductases; Necator americanus secretory protein; Enzyme; Secretion; protein overexpression; protein disulfide isomerase; Gene overexpression; Na-ASP1; Pichia pastoris; protein secretion; Fungi; Isomerases; secretion bottleneck; Secretory protein; Helmintha; Protein disulfide-isomerase; Ascomycetes; Nemathelminthia; Necator americanus; Recombinant protein; Invertebrata; Nematoda; Thallophyta
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20762

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 α‐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. © 2005 Wiley Periodicals, Inc.