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

Large-scale expression of His-tagged Dicer (400 mL to 1 L) was typically achieved in a 2.8-L glass Fernbach flask and harvested 72 hpt. For cell lysis, the washed cell pellet was resuspended in a low salt buffer containing 0.1% NP-40, allowing for lysis of the plasma membrane while leaving the nuclei intact. Nuclei and cell debris were then removed by centrifugation and filtering of the resulting supernatant. The first step of purification consisted of an anion-exchange chromatography (Q Sepharose Fast Flow), which helped remove protein and nucleic acid contaminants (Fig. 2c ). The addition of 10% glycerol in the elution buffers was found critical to purify Dicer free of contamination with Hsp70 (~70-kDa intense band in lane 1 of Fig. 2f ), which is one of the most abundant protein in the HEK-293 cell line [36] . The Dicer-containing fractions were pooled and directly loaded on a HisTrap HP column for purification by immobilized metal affinity chromatography (IMAC; Fig. 2d ). This step, which was carried out in the presence of 0.5 M NaCl and in the absence of glycerol, allowed for (Fig. 2f ) . The Dicer fractions were then loaded on a size-exclusion column (SEC) to isolate the major monomeric population from multimeric contaminants. When this monomeric population of Dicer was concentrated by ultrafiltration under standard conditions (e.g. 50 mM Tris pH 8.2, 10 mM NaCl/KCl 24:1, 0.5 mM MgCl 2 and 0.5 mM TCEP), it resulted in a significant loss of protein via precipitation. In addition, when Dicer was concentrated prior to SEC purification, substantial amounts of Dicer multimers were detected in the SEC elution profile. This led us to identify conditions that would allow for maximum protein recovery following concentration by ultrafiltration. We found that addition of sucrose and the non-ionic detergent n-dodecyl-β-Dmaltoside (DDM) helped stabilize the monomeric form. Thus, these reagents were added to the mobile phase during SEC (Fig. 2e , f ), and fractions containing monomeric Dicer could be readily concentrated at up to 22.5 μM (5 mg/mL) without precipitation. Analysis of the purified protein by SDS-PAGE gels stained with Coomassie Blue shows greater than 90% purity (Fig. 2f) , whereas Western blot analysis indicate that~50% of the protein is recovered from the cytoplasmic fraction (Fig. 2g ). Yields of 4-9 mg of human Dicer per liter of culture were typically obtained from large-scale preparations.