Enhancement of Protein Secretion in Pichia pastoris by Overexpression of Protein Disulfide Isomerase

Authors

Mehmet İnan, University of Nebraska-Lincoln
Dinesh Aryasomayajula, University of Nebraska-Lincoln
Jayanta Sinha, University of Nebraska-Lincoln
Michael M. Meagher, University of Nebraska-Lincoln

Date of this Version

9-26-2005

Citation

Biotechnology and Bioengineering (2006) 93(4): 771-778

doi: 10.1002/bit.20762

Comments

Copyright 2005, Wiley. Used by permission

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 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.