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

Here, we describe an efficient and fast procedure to obtain milligram quantities of pure, monomeric and stable recombinant human Dicer. To overcome the challenge of Dicer over-expression, we optimized transfection and expression in an HEK293-EBNA1 cell line. This allowed for purification of up to 9 mg of Dicer per liter of culture. A purification protocol was designed to minimize sample handling as well as to provide maximal recovery, purity and enzymatic activity. Although we noticed that human Dicer forms multimers at high concentration, our final three-step purification yields highly-pure monomeric Dicer. The structure and activity of the purified Dicer were assessed by size-exclusion chromatography coupled to multi-angle light-scattering and refractive index (SEC-MALS/RI), negative stain transmission electron microscopy (TEM), binding assay and steady-state kinetics. Our kinetic analysis for Dicer cleavage of the pre-let-7a-1 substrate were performed under strict steady state conditions and reveals k cat and K M values that are much higher than previously reported. Overall, this new method will facilitate future biochemical, biophysical and structural characterization of Dicer, including its interaction with RNA substrates and protein regulators.