Superdex s75 10 60 column

Plasmids encoding N-terminally His₆-tagged kinase domains of individual NAKs or GST-tagged CLINT1(N), CLINT1(C) were electroporated into Rosetta strain BL21 E. coli cells using Gene Pulser Xcell Electroporation Systems (Bio-Rad) with 1800V. Protein expression was induced with 0.5 mM IPTG at 20 °C overnight. Cells were then harvested and resuspended in lysis buffer composed of 50 mM Hepes (pH 7.5), 500 mM NaCl, 5 mM imidazole, 5% glycerol, and 0.5 mM TCEP [tris-(2-carboxyethyl) phosphine]. Following sonication and spinning at 48,400g at 4 °C for 60 min, the supernatant was harvested. NAKs were purified via Ni-affinity followed by tobacco etch virus protease digestion to remove the HIS₆ tag. NAKs were further purified by size-exclusion chromatography via Superdex S75 10/60 column on AKTA pure (GE Lifesciences). Proteins were concentrated via 10-kDa Amicon centrifugal filters (Merck) and suspended in storage buffer composed of 10 mM Hepes (pH 7.5), 300 mM NaCl, 5% glycerol, and 0.5 mM TCEP at −80 °C. Clint1(N) and Clint1(C) were purified by glutathione sepharose beads and eluted with 10 mM glutathione. His₆-tagged recombinant RbC (aa 771-928) was purified by cobalt–sepharose affinity chromatography (GE Sepharose cat#17-0575-01) and eluted with 200 mM imidazole. Histone H1 protein was from EMD Millipore (cat#14-155).