High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

Although affinity purification is often considered the best choice for the first purification step of a recombinant protein from a crude mixture, we selected an alternate approach to minimize sample handling that could lead to aggregation and precipitation, such as protein The relative signal of light scattering (red), refractive index (blue), and UV absorbance at 280 nm (black) are represented by solid lines. The molar mass distribution is shown with green dots. A small amount of aggregated protein can be seen mainly from the light scattering signal. Peak integration of the UV absorbance trace shows that the main peak contains ≥98% of the eluted protein. The molar mass calculated from MALS analysis was normalized against BSA to give an average molecular weight of 224 ± 2 kDa with a polydispersity index of 1.02 ± 0.01 over the entire eluted peak. (b) Negative stain TEM. Left panel Typical grid imaging of negatively-stained WT Dicer. Right panels Main 2D class averages of WT Dicer dialysis, desalting, and concentration. Following cell lysis in low-salt buffer, the cytoplasmic fraction was first loaded on an anion-exchange column. The large volume and high salt concentration of the elution fractions from this first purification step is compatible with direct loading onto the HisTrap affinity column. Subsequently, the small elution volume from the second purification step allowed for a single injection on the preparative size-exclusion column without any additional manipulations. Overall, this strategy allows for multi-step purification of milligram amounts of recombinant Dicer in a fast and efficient manner. In fact, the entire purification procedure can be conveniently completed in a single work day.