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

Although bacterial overexpression systems generally provide a simple and fast method to obtain significant amount of recombinant proteins, it has not proven practical for human Dicer (219 kDa). Eukaryotic expression systems offer an important alternative for expression of such a large eukaryotic protein because they allow for proper folding and post-translational modifications (reviewed in [13] ). Over the past 15 years, purification of recombinant human Dicer following expression in insect cells (Sf9) infected by baculovirus has been achieved with yields up to 0.5-1 mg/L culture [14] [15] [16] . This has allowed for the in vitro characterization of human Dicer's enzymatic activity [14, 15, [17] [18] [19] [20] [21] [22] [23] [24] [25] and provided the first descriptions of its three-dimensional structure by cryo-electron microscopy (cryo-EM) at 20-30 Ã…-resolution [26] [27] [28] . Subsequently, the production method in insect cells was improved by systematic optimization of the overexpression and purification steps to yield milligram amounts (3-4 mg/L culture) of highly pure Drosophila melanogaster Dicer-2 (dmDicer-2) [29] , which was used for its structure determination by cryo-EM at 7-Ã… resolution. More recently, expression in HEK293 cells grown in suspension was reported as part of a cryo-EM study of human Dicer that allowed structural reconstruction at 4.4-Ã… resolution [30] . This study provided unprecedented details into Dicer's domain organization as well as its interaction with TRBP and a pre-miRNA substrate. However, the optimization of Dicer production and the yields obtained were not reported. Therefore, it is possible that large-scale expression from mammalian cells grown in suspension could be optimized to provide pure protein at higher yields than currently reported. Such a procedure could be useful for future biochemical and structural investigations as there is still no X-ray or cryo-EM structure of human Dicer at atomic resolution and there are many questions that remain about its mechanism and regulation by protein factors.