Improved methods of cultivation and production of deuteriated proteins from E. coli strains grown on fully deuteriated minimal medium.
OBJECTIVE
The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance.


RESULTS
We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l-1). We also show that all E. coli cells are inherently capable of growth on deuteriated media.


CONCLUSIONS
We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains.


CONCLUSIONS
An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.
Paliy
O
O
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, UK.
Bloor
D
D
Brockwell
D
D
Gilbert
P
P
Barber
J
J
D000222:Adaptation, Physiological; D001431:Bacteriological Techniques; D003470:Culture Media; D003903:Deuterium / Q000378:metabolism* / Q000494:pharmacology; D004361:Drug Tolerance; D018274:Electroporation; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014170:Transformation, Genetic
D003470:Culture Media; D011994:Recombinant Proteins; D003903:Deuterium; D010444:Peptide Elongation Factor Tu