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

From kinetic studies of pre-let-7a-1 cleavage by Dicer, we obtained a k cat value of 7.2 ± 0.5 min − 1 and a K M value of 1.2 ± 0.3 μM (k cat /K M = 6 ± 1 min − 1 μM − 1 ). These steadystate kinetic parameters are much higher than those derived from previous cleavage studies of the same pre-miRNA substrate (25-375 nM) by Dicer (5 nM): k cat = 0.45 min − 1 and K M = 20-30 nM (k cat /K M~1 8 min − 1 μM − 1 ) [20] . Notably, we found similar values (k cat = 0.8 ± 0.1 min − 1 and K M = 66 nM ± 2 nM; k cat /K M = 12 ± 2 min − 1 μM − 1 ) when performing kinetic experiments under similar conditions (5 nM Dicer and 2.5-1000 nM substrate; Additional file 1: Figure S2 ). However, the resulting curve did not fit a hyperbolic model, which is understandable given that the following condition, [S] ≫ [E], was not respected for every substrate concentration and, thus, did not agree with the steady-state assumption [44] . By selecting experimental conditions that ensured the validity of the steady-state assumption ([S]/[E] ≥ 200), much higher k cat and K M values are obtained, even though the k cat /K M is not substantially affected (Additional file 1: Table S1 ).