title: Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation.
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.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pfeffer
authors.forename: Michael
authors.initials: M
authors.lastname: Maurer
authors.forename: Johannes
authors.initials: J
authors.lastname: Stadlmann
authors.forename: Josephine
authors.initials: J
authors.lastname: Grass
authors.forename: Marizela
authors.initials: M
authors.lastname: Delic
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
mesh_terms: D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055571:Secretory Pathway
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins