Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

Abstract

Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 11-05-2018Abstract We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomyces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved -HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (P MOX ), on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low P MOX induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains.

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

“…Un-spliced HAC1 mRNA was detected under normal growth conditions and splicing of HAC1 mRNA was only detected when cells were grown in presence of dithiothreitol (DTT) to activate the UPR. It should be mentioned, though, that sometimes overexpression of folding helpers actually reduced protein secretion or did not have any effect (van der Heide et al 2002). …”

Engineering of chaperone systems and of the unfolded protein response

“…Elevated BiP levels may increase Cytotechnology (2008) (Morris et al 1997). These vesicles may be similar to 'BiP bodies' observed in yeast in which ER-to-Golgi transport is blocked (Nishikawa et al 1994) and function in normal cells as cargo selection sites for ER-to-Golgi traffic (Nishikawa et al 1994) or sites that target proteins for degradation (van der Heide et al 2002). Increasing expression levels of calnexin/calreticulin improved secretion of heterologous proteins in insect cells and Chinese hamster ovary (CHO) cells (Chung et al 2004;Kato et al 2005).…”

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

“…However, overexpression of Bip in the S. cerevisiae expression system is not always adaptable for the secretion of heterologous protein, even negatively impairing the r-protein production. For example, no or neglectable increase was observed in the secretion of several different r-proteins with overexpression of chaperone Bip in S. cerevisiae ( Robinson et al, 1996 ; Kauffman et al, 2002 ; van der Heide et al, 2002 ). A possible explanation is that Bip might more frequently bind to the unoptimized heterologous proteins, directing expressed proteins to degradation rather than to the secretory pathway ( Gasser et al, 2008 ).…”