Hitrap chelating hp 5 ml column

E. coli BL21(DE3) cells carrying the pET28a-MetaChi18A plasmid were
grown in 200 mL of LB medium at 37°C until an OD600 of 0.5, and induced by the
addition of isopropyl-β-D thiogalactopyranoside (IPTG) to a final concentration of
0.3 mM. The induced culture was incubated for a further 3 h at 30°C before the
harvesting of the cells by centrifugation (10,000 g for 5 min) at
4°C. The cell pellet was suspended in 20 mL of lysis buffer (20 mM Tris-HCl, pH 8.0,
150 mM NaCl) and disrupted by ultrasonication in an ice bath (10 cycles of 40 s
pulses, 90 W, with 20 s intervals), using a Sonicator® XL 2020 (Heat
Systems-Ultrasonics Inc., USA). The crude extract was then centrifuged at 20,000
g for 30 min at 4°C to pellet the cell debris. The supernatant
containing the His-tagged protein was loaded onto a HiTrap Chelating HP 5 mL column
(GE Healthcare, USA), previously loaded with NiCl₂ 100 mM and equilibrated
with lysis buffer, using an ÄKTA basic chromatography system (GE Healthcare). The
column was washed with 5 volumes of the lysis buffer. The His-tagged protein was
eluted with an increasing gradient of imidazole up to 500 mM in elution buffer. The
elution of protein was monitored at 280 nm and protein fractions were analyzed by
SDS-PAGE, pooled, dialyzed (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 50% (v/v) glycerol)
and stored at −24°C until use.

hMLC was cloned into the His-bsSumo (K151) expression vector using Transfer-PCR. An Ala residue was incorporated at the N-terminal part of hMLC in order to facilitate cleavage of hMLC from the His-bdSumo tag. Expression was performed in E coli Bl21(DE3) cells. Protein production was induced by addition of 0.2 mM IPTG at mid-log phase and the cells were grown overnight at 15 °C. Cells were lysed using a cell disrupter (Constant Systems) at 4 °C in buffer A (50 mM Tris pH 8, 0.5 M NaCl, 20 mM Imidazole with EDTA-free protease-inhibitor tablet, 0.5 mg/ml lysozyme, and DNaseI). After centrifugation, the clarified supernatant was passed over a HiTrap chelating HP 5 ml column (GE, Healthecare). 1 ml bdSumo protease, bdSENP1, without His tag was added to the column which was left at 4 °C overnight. The cleaved protein was collected by washing the column with 10 ml buffer A. hMLC was further purified by size-exclusion chromatography (HiLoad_16/60_superdex75 prepgrade, GE Healthcare) equilibrated with 50 mM Tris pH 8, 0.1 M NaCl.
DAPK2 K42A and K42A S289A were cloned into the pET28-TevH expression vector using Transfer-PCR. Plasmids were expressed in Escherichia coli BL21(DE3) cells. Proteins were purified using excess peptide.