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

is an efficient tool to evaluate the quality of a protein sample, providing information on the homogeneity and the three-dimensional shape of the biomolecule. TEM micrographs of negative stain preparation of the purified Dicer using uranyl formate illustrate particles of roughly equal size throughout the field of view, providing additional evidence of the homogeneity of the purified sample (Fig. 3b) . Model-free 2D class averages allowed us to observe the characteristic L-shape architecture of Dicer, with two main unique subpopulations representing different forms of the enzyme, in agreement with previous studies [27, 30, 37] . Taken together, the SEC-MALS/RI and negative stain TEM studies validate the monomeric L-shape architecture of the purified Dicer and indicate that the conformational properties of the enzyme are preserved after long-term storage.