Gstrap affinity column

The protein constructs of interest (with either His or GST tags) were overproduced in E. coli BL21 (DE3) competent cells. Cell cultures were grown in the LB medium at 30°C overnight, and shaken at 180 rpm. 1% (w/v) lactose monohydrate was used for induction. Cells were harvested, lysed by microfluidizer (M110-L, Microfluidics), and centrifuged to pellet cell debris. The supernatant was then loaded onto a GE Healthcare HisTrapFF affinity column (for His-tagged proteins) or a GSTrap affinity column (for GST-tagged proteins). For the His-tagged proteins, the lysis and wash buffer contained 20 mM HEPES pH 8.0, 250 mM NaCl, 20 mM KCl, 20 mM MgCl₂, and 40 mM imidazole, while the imidazole concentration in the elution buffer was increased to 500 mM. For the GST-tagged proteins, the lysis and wash buffer contained 20 mM HEPES pH 7.5, 200 mM NaCl, 20 mM KCl, 20 mM MgCl₂, and the elution buffer additionally contained 50 mM Tris-HCl pH 8.0 and 20 mM of L-glutathione. After elution, the protein was purified by size exclusion chromatography (SEC) using an S200 Sepharose column and GE Lifesciences ÄKTA Prime and Purifier systems. After purification, the proteins were concentrated using the Amicon Ultra-15 spin concentrators (10 kDa cutoff point) and flash-frozen to be used in in vitro GST pulldown assays.

GST proteins fused with or without 150–313 amino acid residues of Ouib (GST-Ouib-Zf) containing 5 zinc finger domains were expressed using pGEX-4T-3 vector system (GE Healthcare) in Escherichia coli BL-21 strain. E. coli cells were harvested and crashed with sonication. GST alone and GST-Ouib-Zf were purified from the supernatant with AKTA start equipped with GSTrap affinity column (GE Healthcare).