High yield expression and efficient purification of deuterated human protein galectin-2

Structural studies of biological macromolecules often require deuterated proteins, necessitating an effective bioprocessing strategy for high yield deuteration and purification. The fermentation and bioseparation studies reported here concern deuterated human protein galectin-2 mutant C57M (hGal-2), a protein showing potential for therapeutic applications. Using the vector pET-28a and a defined D2O based minimal medium with glycerol as the sole carbon source and kanamycin for selection, we have demonstrated that a high density of Escherichia coli expressing deuterated protein at a bench bioreactor scale (7 L) can be achieved, with due attention to prevention of oxygen limitation. Yields achieved were 58 g/L biomass (wet weight) containing 0.7 g/L hGal-2. Affinity chromatography and ion-exchange chromatography were combined to achieve high purity as well as removal of hGal-2 aggregates, giving an overall yield of 1200 mg deuterated hGal-2. The deuterated hGal-2 was characterized and compared with the non-deuterated protein by size exclusion chromatography (SEC), HPLC, N-terminal sequencing, mass spectrometry (MS) and a dot blot immunoassay, showing that deuteration and subsequent purification did not impact the lactose binding and antibody recognition abilities of hGal-2. MS for both intact and trypsin-digested hGal-2 demonstrated that the extent of labeling of non-exchangeable hydrogen atoms by deuterium was (66 ± 1)%, which provides sufficient contrast variation for structural studies using small angle neutron scattering. The fermentation and bioseparation method established in this work can be applied to process other deuterated proteins with high yield and purity, opening the way to advanced structural studies.

► A defined D2O based minimal medium was used for fermentation of deuterated protein galectin-2.
► A high density of E. coli at a bench bioreactor scale was achieved.
► Affinity and ion-exchange chromatography were combined to achieve high purity.
► Overall yield is 1200 mg purified deuterated protein from 7 L bioreactor.
► Deuteration did not impact ligand binding and antibody recognition of the protein.