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Fig. 2 | BMC Biotechnology

Fig. 2

From: High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Fig. 2

Dicer expression and purification. (a) Domain architecture of Dicer. Domains were positioned using InterPro [56] and refined using available structural data [30, 57,58,59]. (b) Flowchart of the Dicer expression and purification protocol. (Day 1) Cells are passaged at 0.8 × 106 cells/mL and incubated 24 h before transfection. (Day 2) The transfection mix is prepared and added to the cell culture, which is incubated for 72 h (37 °C, 5% CO2) with shaking (100 RPM). (Day 5) Cells are harvested by centrifugation at 200×g and rinsed 3 times in cold PBS. After lysis, the cytoplasmic fraction is clarified by centrifugation, filtered and loaded on a 60-mL Q Sepharose Fast Flow column for ion-exchange chromatography. Fractions containing Dicer are pooled and loaded directly on a 5-mL HisTrap HP column for purification by immobilized metal affinity chromatography (IMAC). The Dicer-containing fractions are loaded directly on a 120-mL Superdex 200 column for purification by size-exclusion, and the fractions containing homogeneous Dicer are concentrated and stored at − 80 °C. (c-e) Typical chromatograms from the (c) ion-exchange, (d) affinity and (e) size-exclusion purifications, showing the UV absorbance at 280 nM and 260 nM along with the gradient trace. The selected fractions are highlighted by the grey area. (f-g) SDS-PAGE summary of the purification viewed by (f) Coomassie stain and (g) Western blot. Each lane is loaded proportionally to reflect the yield at each step (Lane 1: clarified lysate; Lane 2: ion-exchange fraction pool; Lane 3: affinity fraction pool; Lane 4: size-exclusion fraction pool; and Lane 5: concentrated protein). Yields were quantified from Western blot analysis, with loaded quantities of Dicer being in the linear range of detection

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