MBP-tagged CLAMP DBD was expressed and purified as described previously (Kaye et al., 2018 (link)). MBP-tagged (pTHMT, Peti and Page, 2007 (link)) FL CLAMP protein was expressed in Escherichia coli BL21 Star (DE3) cells (Life Technologies). Bacterial cultures were grown to an optical density of 0.7–0.9 before induction with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 4 hr at 37°C.
Cell pellets were harvested by centrifugation and stored at −80°C. Cell pellets were resuspended in 20 mM Tris, 1 M NaCl, 0.1 mM ZnCl2, and 10 mM imidazole pH 8.0 with one EDTA-free protease inhibitor tablet (Roche) and lysed using an Emulsiflex C3 (Avestin). The lysate was cleared by centrifugation at 20,000 rpm for 50 min at 4°C, filtered using a 0.2 μm syringe filter, and loaded onto a HisTrap HP 5 ml column. The protein was eluted with a gradient from 10 to 300 mM imidazole in 20 mM Tris, 1.0 M NaCl pH 8.0, and 0.1 mM ZnCl2. Fractions containing MBP-CLAMP FL were loaded onto a HiLoad 26/600 Superdex 200 pg column equilibrated in 20 mM Tris, 1.0 M NaCl, pH 8.0. Fractions containing FL CLAMP were identified by SDS-PAGE and concentrated using a centrifugation filter with a 10-kDa cutoff (Amicon, Millipore) and frozen as aliquots.
Cell pellets were harvested by centrifugation and stored at −80°C. Cell pellets were resuspended in 20 mM Tris, 1 M NaCl, 0.1 mM ZnCl2, and 10 mM imidazole pH 8.0 with one EDTA-free protease inhibitor tablet (Roche) and lysed using an Emulsiflex C3 (Avestin). The lysate was cleared by centrifugation at 20,000 rpm for 50 min at 4°C, filtered using a 0.2 μm syringe filter, and loaded onto a HisTrap HP 5 ml column. The protein was eluted with a gradient from 10 to 300 mM imidazole in 20 mM Tris, 1.0 M NaCl pH 8.0, and 0.1 mM ZnCl2. Fractions containing MBP-CLAMP FL were loaded onto a HiLoad 26/600 Superdex 200 pg column equilibrated in 20 mM Tris, 1.0 M NaCl, pH 8.0. Fractions containing FL CLAMP were identified by SDS-PAGE and concentrated using a centrifugation filter with a 10-kDa cutoff (Amicon, Millipore) and frozen as aliquots.
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