Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design
In: Journal of Biotechnology, Jg. 32 (1994-02-01), S. 249-259
Online
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Zugriff:
The conditions for the high cell density fed-batch culture of a Saccharomyces cerevisiae strain producing recombinant hirudin (rHV2-Lys47) have been established. A Leu+ derivative of S. cerevisiae cl3ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding rHV2-Lys47 and driven by the MFα1 promoter. In order to develop the fed-batch culture protocol, the recombinant strains' physiology was first of all investigated in chemostat culture. The maximum respiratory capacity of the recombinant strain was observed to be between dilution rates of 0.2 and 0.26 h−1, which is typical for laboratory strains as compared to values published for baker's yeasts. Furthermore, maximum biomass yield and product secretion were observed at a dilution rate of approx. 0.15 h −1. The plasmid segregational stability of the recombinant strain showed that the expression plasmid was stable, irrespective of the dilution rates used, for more than 80 generations of growth between dilution rates of 0.043 h−1 and 0.3 h−1. The chemostat data was used to define a fed-batch process. The fed-batch results demonstrated a biomass production of 60 g l−1 CDW and a high production level of recombinant hirudin of 500 mg l−1. Stability of the expression of the gene coding for rHV2-Lys47 was maintained during all the studied fed-batch conditions. The plasmid copy number in the fed-batch remained constant at approx. 43 at a specific growth rate of 0.12 h−1, whereas it increased by 60–95% at a lower dilution rate (μ = 0.06 h−1). Although a variation of the plasmid copy number could be expected, it was postulated from the experimental data that the observed amplification could have been influenced by an environmental effect due to an accumulation of medium components in the supernatant. The results presented here illustrate the importance of a well-balanced medium when considering the production of a recombinant protein in a high cell density cultivation process with high production levels.
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Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design
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Autor/in / Beteiligte Person: | Hebert, C. ; Mendoza-Vega, O. ; Brown, S.W. |
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Zeitschrift: | Journal of Biotechnology, Jg. 32 (1994-02-01), S. 249-259 |
Veröffentlichung: | Elsevier BV, 1994 |
Medientyp: | unknown |
ISSN: | 0168-1656 (print) |
DOI: | 10.1016/0168-1656(94)90211-9 |
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