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

It is important to note that the last step of purification can be performed either in the presence or absence of reagents (sucrose and DDM) that were found to prevent aggregation and precipitation at high protein concentration. These reagents are not required if the concentration of the protein eluted from the size-exclusion column, generally between 0.5-1 μM, is sufficient for subsequent applications. However, if higher protein concentrations are required and/or long-term storage is desirable, these reagents should be included in the buffer used for size-exclusion chromatography and final protein concentration. The SEC-MALS results indicate that the purified Dicer remains monomeric over six months of storage in sucrose/DDM-supplemented buffer. In agreement with this observation, similar thermodynamic and kinetic parameters (within two-fold) were measured over a period of nine months.