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

We have achieved large-scale expression of recombinant human Dicer in a human cell line. The expression in 293-6E cells coupled with our streamlined purification protocol yields up to 9 mg of protein per L of cell culture, which is higher than with previously reported protocols. The purification scheme was carefully optimized to minimize aggregation and maximize monomeric purity and stability of the final sample. To achieve these objectives, we minimized sample manipulation which, in turn, reduced the duration of the purification to a single work day. The purified enzyme adopts the typical L-shape architecture and binds a pre-let-7a-1 substrate in the low nM range, as previously observed. Steady-state kinetic studies yielded higher k cat and K M values than previously reported due to the use of experimental conditions that respected the steady-state assumption. Together, these studies provide an efficient method for high-yield production of human Dicer as well as revised kinetic parameters for Dicer cleavage that should prove useful for future structural and mechanistic studies of Dicer aimed at better understanding how the catalytic production of miRNAs is achieved and regulated.