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

Human WT Dicer final purification product was analyzed by SEC-MALS/RI using an AKTA micro FPLC (GE Healthcare) coupled to a multiple-angle light scattering and refractive index system (MALS/RI; Wyatt Dawn HELEOS II and OptiLab T-rEX). A volume of 100 μL (75-80 μg) of purified Dicer was loaded on a Superdex 200 Increase 10/300 (GE Healthcare) column equilibrated in sucrose/DDM-supplemented storage buffer at a flowrate of 0.3-0.5 mL/min. The SEC-MALS/RI data was collected and analysed with ASTRA chromatography software package, version 6.1.6.5 (Wyatt Technology). The MALS detector was normalized using the monomeric and dimeric signals of bovine serum albumin (BSA; Fisher scientific BP1605). For the Dicer sample purified in DDM-free buffer, the molar mass was determined by the dual detection method (RI/LS signals) implemented in the conjugated analysis mode of the ASTRA software, using a refractive index increment (dn/dc) of 0.190 mL/g [53] . A similar method was used for the Dicer sample purified in DDM-containing buffer, however the dual detection method used the UV and LS signals, because the RI signal may be perturbed by DDM-Dicer interactions.