title: Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau.
abstract: It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.
authors.affiliation: Biological Process Development Facility, Department of Chemical Engineering, University of Nebraska-Lincoln, NE, USA. jsinha@unlnotes.unl.edu
authors.forename: Jayanta
authors.initials: J
authors.lastname: Sinha
authors.forename: Bradley A
authors.initials: BA
authors.lastname: Plantz
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D015153:Blotting, Western; D002458:Cell Fractionation; D003593:Cytoplasm / Q000201:enzymology; D005990:Glycerol / Q000378:metabolism; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D000432:Methanol / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010843:Pichia / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D011257:Pregnancy Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep; D013696:Temperature; D013997:Time Factors; D014617:Vacuoles / Q000201:enzymology
chemical_list: D007370:Interferon Type I; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau; D010447:Peptide Hydrolases; D005990:Glycerol; D000432:Methanol