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

Size-exclusion chromatography coupled to multi-angle light-scattering and refractive index (SEC-MALS/RI) is a powerful tool to characterize purified biomolecules in solution; it allows accurate molecular weight determination as well as the determination of the oligomeric state in solution. In the SEC-MALS/RI experiments performed here, SEC served only as a separation step, whereas the LS and RI detectors were used to calculate the molar mass of Dicer after purification and concentration in a sucrose/ DDM-free buffer (Fig. 3a) . Although a small percentage of multimeric protein appears to be present in the void volume of the SEC (elution volume~8 mL), the purified Dicer elutes predominantly as a single peak that corresponds to ≥98% of the total protein in the injected sample. The derived molecular weight for this peak based on light-scattering data is 224 ± 2 kDa, which essentially matches the theoretical molecular weight of the Histagged monomeric protein (221 kDa). The polydispersity index of 1.02 ± 0.01 over the entire eluted peak indicates that the main form of purified Dicer is monodisperse, i.e. homogeneous with respect to molar mass. Moreover, SEC -MALS/RI analysis of a purified Dicer sample stored for 6 months at − 80°C in sucrose/DDM-containing buffer shows that the purified protein remains almost exclusively monomeric ( ≥ 94%), indicating that the protein is intact after long-term storage (Additional file 1: Figure S1 ).