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

To characterize the cleavage activity of the purified Dicer enzyme, we determined the steady-state kinetic parameters k cat and K M under multiple turnover conditions. To ensure that the initial cleavage rates were measured under steady state conditions, the enzyme concentration ([E]; 0.35 nM to 3.75 nM) was varied along with the initial substrate concentration ([S]; 0.08 μM to 7.68 μM) to maintain [S]/[E] ≥ 200, and time points were collected after at least one turnover of the enzyme pool at up to 10% substrate cleavage, as previously reported for steadystate kinetic studies of E. coli RNase III [38] . For each substrate concentration, the cleavage reaction of 32 P-labeled pre-let-7a-1 was monitored by denaturing gel electrophoresis to quantify the percentage of cleavage (Fig. 5a) . From this data, the concentration of product normalized against enzyme concentration ([let-7a-1]/[Dicer]) was plotted as a function of time, and the slope of the resulting time course was fitted by linear regression to derive the turnover frequency, expressed as v o /[E] t (Fig. 5b) . By plotting