- Poster Presentation
- Open Access
Cost-effective production of labeled recombinant proteins in E. coli using minimal medium
© Rozkov et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Minimal Medium
- Shake Flask
- Rich Medium
Application of NMR spectroscopy in drug discovery often requires large quantities (hundreds mg) of recombinant proteins labeled with stable isotopes (nitrogen-15, deuterium and carbon-13 in various combinations). Expression of recombinant proteins in E. coli is traditionally carried out using rich media such as LB due to simplicity, low cost and familiarity. Use of commercial labeled rich medium with the same characteristics as a conventional LB is a very attractive option for small-scale production of labeled proteins. However, larger scale cultivation with such media could become cost-prohibitive. In this study we describe how minimal medium was used for large-scale cost-effective production of labeled recombinant proteins in E. coli.
Uniform high-level labeling was achieved by using a labeled substance as a sole source of the corresponding element, i.e. ammonium salts as a nitrogen-15 source and glucose as a carbon-13 source. In the experiments requiring deuteration, deuterium oxide (99.9%) and ortho-phosphoric acid were used as deuterium sources. The degree of deuterium incorporation in recombinant protein achieved by this method ranges from 84 to 89 per cent (remaining hydrogen atoms are derived from glucose). Use of these labeled substances allowed large savings of medium costs compared to commercially available rich medium. The medium cost savings were 6-fold for triple labeled (2H, 15N, 13C) and 20-fold for 15N-labeled medium. Achieved savings for production of 100 gram of triple-labeled biomass, for instance, can reach tens of thousands EUR.
Biomass yield coefficient (OD per g/L glucose) observed during production of 3 recombinant proteins in batch culture with minimal medium.
BL21(DE3) Protein A
BL21(DE3)* Protein B
BL21(DE3) Protein C
BL21(DE3) Protein C
1.5 (0.1mM IPTG)
1.3 (0.05mM IPTG)
0.9 (0.5mM IPTG)
The culture growing on minimal medium with glucose in shake flask is much less prone to oxygen limitation as opposed to rich medium due to a greatly reduced specific growth rate (1.5 vs. 0.5 1/h) . This makes a shake flask culture more suitable for small-scale experiments with a good potential for scale-up if needed.
Cells growing on minimal medium in batch culture metabolize glucose with a constant stoichiometry. Therefore, biomass growth is directly proportional to carbon dioxide evolution and oxygen and alkali consumption. These parameters can be used for indirect estimation of biomass in bioreactor and for triggering pre-programmed events such as induction and temperature changes. Unattended operation reduces a need for overtime and to increase reproducibility of the process.
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This article is published under license to BioMed Central Ltd.