High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system
✍ Scribed by J. C. Goodrick; M. Xu; R. Finnegan; B. M. Schilling; S. Schiavi; H. Hoppe; N. C. Wan
- Publisher
- John Wiley and Sons
- Year
- 2001
- Tongue
- English
- Weight
- 464 KB
- Volume
- 74
- Category
- Article
- ISSN
- 0006-3592
- DOI
- 10.1002/bit.1140
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✦ Synopsis
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. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 492–497, 2001.