| DOI | Resolve DOI: https://doi.org/10.1016/S0378-1097(03)00956-X |
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| Author | Search for: Bélanger, L.; Search for: Figueira, M. M.1; Search for: Bourque, D.1; Search for: Morel, L.1; Search for: Béland, M.1; Search for: Laramée, L.1; Search for: Groleau, D.1; Search for: Miguez, C. B.1 |
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| Affiliation | - National Research Council of Canada. NRC Biotechnology Research Institute
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| Format | Text, Article |
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| Subject | biotechnology |
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| Abstract | The green fluorescent protein (GFP) was used as a model protein to study the recombinant protein production by the strain Methylobacterium extorquens ATCC 55366. Scale-up from shake flasks to 20 l fed-batch fermentation was achieved using methanol as a sole carbon and energy source and a completely minimal culture medium. Two different expression vectors were used to express GFP. Clone PCM-GFP containing the vector pCM110 with native promoter of the methanol dehydrogenase PmxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter Plac. Several fed-batch fermentations with and without selective pressure (tetracycline) were run in a 20 l stirred tank fermenter using the two different clones of M. extorquens. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth. It was maintained at a non-toxic level of 1.4 g l(-1) with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (micro(max)) was 0.18 h(-1) with an overall yield (Y(X/S)) of 0.3 g g(-1) methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l(-1)) of GFP with the clone PCM-GFP. The maximum specific GFP production (Y(GFP/X)) with this clone was 80 mg g(-1) representing approximately 16% of the total cell protein. Additional feeding of pure oxygen to the fermenter permitted a longer phase of exponential growth but had no effect on the total yields of biomass and GFP. The specific GFP production of clone PCM-GFP remained unaffected in the presence or absence of selective pressure (tetracycline), within the initial 50 h of the fermentation culture. These results suggest that M. extorquens ATCC 55366 could be an interesting candidate for overexpression of recombinant proteins |
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| Publication date | 2004-02 |
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| In | |
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| Note | DA - 20040227IS - 0378-1097LA - engPT - Journal ArticleRN - 0 (Bacterial Proteins)RN - 0 (Luminescent Proteins)RN - 0 (Plasmids)RN - 147336-22-9 (green fluorescent protein)RN - 7782-44-7 (Oxygen)SB - IM |
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| Language | English |
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| NRC number | 37709 |
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| NPARC number | 3538605 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 20f54f65-5ddf-4ecc-a24e-421699641f14 |
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| Record created | 2009-03-01 |
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| Record modified | 2020-04-17 |
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