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Current Topics in Biotechnology   Volumes    Volume 4 
Abstract
Optimized productions of recombinant human proteins in fermentor cultures of the yeast, Pichia pastoris
Akira Murasugi
Pages: 101 - 108
Number of pages: 8
Current Topics in Biotechnology
Volume 4 

Copyright © 2008 Research Trends. All rights reserved

ABSTRACT
 
Three human recombinant proteins, bile salt-stimulated lipase, midkine, and pleiotrophin, were successfully expressed under the control of the AOX1 promoter in Pichia pastoris. In the early stage experiments, human bile salt-stimulated lipase was induced by methanol at low cell density in the fermentor culture. The enzyme was scarcely accumulated in the medium. Therefore, the induction was started at the high cell density. Approximately 0.2 g/l of the enzyme was accumulated in the medium. Then, the cell growth condition was studied. In the final condition, the expression was continued for 15 days and 1 g/l recombinant enzyme was accumulated in the medium. In the expression of human midkine, secretion signal of itself was used. A half of the product was modified with yeast specific mannosylation. Then, the yeast α-mating factor secretion signal was used. Mannosylated product was not detected anymore. However, about 70% of the product was truncated. Therefore, pep4 mutant Pichia pastoris host strain was used, and the midkine was expressed at 20 °C and pH 3. In this condition, 360 mg/l of authentic recombinant human midkine was obtained at the end of one week induction. For the expression of recombinant human pleiotrophin, the yeast α-mating factor secretion signal was used, and the pleiotrophin was expressed in a fermentor at 20 °C and pH 5. Approximately 260 mg/l of non-mannosylated product was obtained at forth day of the induction. For the industrial productions of the recombinant proteins, optimizations of the fermentor production were necessary. However, the best condition of fermentor culture depends on the proteins to be expressed as described above. Optimization processes described here will be helpful for the fermentor expression of other proteins.
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