Robust high-yield methodologies for (2)H and (2)H/(15)N/(13)C labeling of proteins for structural investigations using neutron scattering and NMR.

We have developed a method that has proven highly reliable for the deuteration and triple labeling ((2)H/(15)N/(13)C) of a broad range of proteins by recombinant expression in Escherichia coli BL21. Typical biomass yields are 40-80g/L wet weight, yielding 50-500mg/L purified protein. This method uses a simple, relatively inexpensive defined medium, and routinely results in a high-yield expression without need for optimization. The key elements are very tight control of expression, careful starter culture adaptation steps, media composition, and strict maintenance of aerobic conditions ensuring exponential growth. Temperature is reduced as required to prevent biological oxygen demand exceeding maximum aeration capacity. Glycerol is the sole carbon source. We have not encountered an upper limit for the size of proteins that can be expressed, achieving excellent expression for proteins from 11 to 154kDa and the quantity produced at 1L scale ensures that no small-angle neutron scattering, nuclear magnetic resonance, or neutron crystallography experiment is limited by the amount of deuterated material. Where difficulties remain, these tend to be cases of altered protein solubility due to high protein concentration and a D2O-based environment.

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