title: Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica.
abstract: The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.
authors.affiliation: The Biotechnology Laboratory, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Brian E
authors.initials: BE
authors.lastname: Mayson
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Kilburn
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Zamost
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Raymond
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Lesnicki
mesh_terms: D019149:Bioreactors; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005246:Feedback; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D000432:Methanol / Q000378:metabolism* / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011786:Quality Control; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014168:Transferrin / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D014168:Transferrin; D000432:Methanol