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

By optimizing transfection conditions, we have achieved high-level expression of recombinant human Dicer using a 293-6E cell expression system. In addition, a fast and efficient purification procedure was developed that yields up to 9 mg of pure, monomeric and stable recombinant human Dicer per liter of culture. To our knowledge this yield is higher than previously reported for human Dicer, and it may be possible to obtain even higher yields by further optimizing transfection conditions, expressing Dicer as a secreted protein, and/or developing an efficient stable transfection clone [31, 35, 39, 40] . The reported purification protocol was designed to prevent protein aggregation and precipitation, since we observed that Dicer tends to form multimers and precipitate at high-concentration or when it contacts metallic surfaces. Nevertheless, the purified protein was shown to be monomerically pure and highly active.