Publication:


abstract

Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be Intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway.

Related Articles
Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure.
Microb Cell Fact. 2011
Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure.
Pfeffer M, Maurer M, Köllensperger G, Hann S, Graf AB, Mattanovich D. Microb Cell Fact. 2011 Jun 26; 10:47. Epub 2011 Jun 26.
Engineering of Pichia pastoris for improved production of antibody fragments.
Biotechnol Bioeng. 2006
Engineering of Pichia pastoris for improved production of antibody fragments.
Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D. Biotechnol Bioeng. 2006 Jun 5; 94(2):353-61.
Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris.
J Vis Exp. 2010
Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris.
Weidner M, Taupp M, Hallam SJ. J Vis Exp. 2010 Feb 25; (36). Epub 2010 Feb 25.
Review Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.
J Mol Recognit. 2005
Review Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.
Daly R, Hearn MT. J Mol Recognit. 2005 Mar-Apr; 18(2):119-38.
Review Secretory expression of human protein in the Yeast Pichia pastoris by controlled fermentor culture.
Recent Pat Biotechnol. 2010
Review Secretory expression of human protein in the Yeast Pichia pastoris by controlled fermentor culture.
Murasugi A. Recent Pat Biotechnol. 2010 Jun; 4(2):153-66.