Deuteration of human carbonic anhydrase for neutron crystallography: Cell culture media, protein thermostability, and crystallization behavior

Deuterated proteins and other bio-derived molecules are important for NMR spectroscopy, neutron reflectometry, small angle neutron scattering, and neutron protein crystallography. In the current study we optimized expression media and cell culture conditions to produce high levels of 3 different deuterated human carbonic anhydrases (hCAs). The labeled hCAs were then characterized and tested for deuterium incorporation by mass spectrometry, temperature stability, and propensity to crystallize. The results show that is possible to get very good yields (>10 mg of pure protein per liter of cell culture under deuterated conditions) and that protein solubility is unaffected at the crystallization concentrations tested. Using unlabeled carbon source and recycled heavy water, we were able to get 65-77% deuterium incorporation, sufficient for most neutron-based techniques, and in a very cost-effective way. For most deuterated proteins characterized in the literature, the solubility and thermal stability is reduced. The data reported here is consistent with these observations and it was clear that there are measurable differences between hydrogenous and deuterated versions of the same protein in Tm and how they crystallize.