title: High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system.
abstract: A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug. Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter. Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations. Continuous production of the enzyme by P. pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source. The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L. Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight [DCW]) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD). No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.
authors.affiliation: Genzyme Corp., One Kendall Square, Cambridge, Massachusetts 02139, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Goodrick
authors.forename: M
authors.initials: M
authors.lastname: Xu
authors.forename: R
authors.initials: R
authors.lastname: Finnegan
authors.forename: B M
authors.initials: BM
authors.lastname: Schilling
authors.forename: S
authors.initials: S
authors.lastname: Schiavi
authors.forename: H
authors.initials: H
authors.lastname: Hoppe
authors.forename: N C
authors.initials: NC
authors.lastname: Wan
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D010843:Pichia / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D013997:Time Factors
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; C017721:casamino acids; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002688:Chitinases; D000432:Methanol