Histrap 5 ml column

CENP-V was fused to an N-terminal His-Tag and/or monomeric GFP, expressed from a baculovirus vector in SF9 insect cells (Expression Systems, cat# 94-001 F, https://expressionsystems.com/product/insect-cells/). Three days after infection, cells were harvested and lysed in buffer A (20 mM Hepes, 1 M NaCl, 20 mM imidazole, 1 mM DTT, pH 7.5) supplemented with EDTA-free Protease Inhibitor Cocktail (Roche) using an Emulsiflex C5 (Avestin). The lysate was cleared by centrifugation (18.000 g, JA-25.50 rotor (Beckman Coulter), 60 min, 4 °C). Soluble material was applied to a HisTrap 5 ml column (GE Healthcare) and washed with 10 column volumes (CV) buffer A, 5 CV ATP-buffer (5 mM ATP in 2xPBS 10 mM MgCl₂) and with buffer A containing 40 mM imidazole. Finally, the protein was eluted by applying the elution buffer (20 mM Hepes, 1 M NaCl, 300 mM imidazole, 1 mM DTT, pH 7.5). The eluted fraction was concentrated by centrifugation using an Amicon concentrator (10 kDa cutoff), and subjected to size exclusion chromatography using a Superdex-200 16/60 column (GE Healthcare) equilibrated with 20 mM Hepes, 300 mM NaCl, pH 7.0 at room temperature, using an Akta Pure chromatography system (GE Healthcare). Pooled samples were concentrated by centrifugation in Amicon tubes and snap frozen in liquid nitrogen.

Recombinant SH2-CD c-Abl, T231R, and YopH were co-overexpressed and SH2-CD c-Abl was purified from E. coli as previously described (77 (link)). Briefly, cells were grown in 2 liters of TB media until the OD reached 1.0, after which the cultures were cooled down and induced overnight at 18 °C with 0.2 mm isopropyl 1-thio-β-d-galactopyranoside. The cells were then lysed by sonication in His-tag binding buffer HA (50 mm Tris, pH 8, 500 mm NaCl, 5% glycerol, 25 mm imidazole) and centrifuged twice at high speed (50,000 × g, 20 min, 4 °C), before being injected in 5 ml of His-tag columns (histrap 5-ml column, GE Healthcare; buffer HA, same as above; buffer HB, same as HA with 0.5 m imidazole). Selected fractions were desalted using two HiPrep 26/10 desalting columns (GE Healthcare) in series (desalting buffer: 20 mm Tris, pH 8, 50 mm NaCl, 5% glycerol, 1 mm DTT), combined and further purified by anion-exchange chromatography using a MonoQ 5/50 GL column (GE Healthcare; buffer A, 20 mm Tris, 5% glycerol, 1 mm DTT, pH 8.0; buffer B, same as buffer A with 1 m NaCl). The final concentration of the c-Abl kinase was determined using the UV absorbance at 280 nm (m = 46797 g mol⁻¹ and ϵ₂₈₀ = 80,010 m⁻¹ cm⁻¹).

The EgTrp recombinant proteins were produced and purified as previously described [20 (link)]. Briefly, pQIA-EgTrp was expressed in the M15 (pREP4) (Qiagen) Escherichia coli strain. Bacterial cells containing the relevant plasmid were cultured at 37°C in yeast extract and tryptone medium supplemented with ampicillin/kanamycin, up to an OD600 nm of 0.5 to 0.7. EgTrp expression was induced by the addition of 1.5 mM isopropyl β-D-1-thiogalactopyranoside, and the bacteria were incubated for an additional 3h. The bacteria were then lysed in cell lysis buffer (50 mM phosphate buffer pH 8, 300 mM NaCl, 20 mM imidazole) and ruptured by sonication. Cellular debris were removed by centrifugation at 15 000 x g for 30 minutes at 4°C, and the supernatant was loaded onto a His-Trap 5 mL column (GE Healthcare). Elution was performed with a buffer containing increasing concentrations of imidazol (100 mM, 200 mM, 300 mM, and 500 mM). The fractions were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry.

For the production of CjSL, strain E. coli M15 (pREP4) transformed with pQE::cjsl plasmid was grown at 37 °C with agitation at 250 rpm to a cell density of 0.2–0.25 (A₆₀₀). Then, 0.2 mM isopropyl-β-D-thiogalactopyranoside and 1 mM CuSO₄ were added to culture medium. After that, the cells were first incubated for 18 h at 20 °C with agitation at 100 rpm, then they were incubated for 24 h at 25 °C without shaking. Cells were collected by centrifugation at 4,000 g for 30 min, suspended in 20 ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.5 M NaCl and 1 mM imidazole (buffer A) and disrupted by sonication. Cell debris was removed by centrifugation (40 min at 8.000 g). The protein was purified by affinity chromatography on a HisTrap 5-ml column (GE Healthcare, Chicago, IL, USA). Cell extract was loaded onto a HisTrap column equilibrated with buffer A. After loading, the column was washed with four volumes of the buffer A and then washed with four volumes of the buffer B (20 mM Tris-HCl, 0.5 M NaCl, 50 mM imidazole, pH 8.0). Active fractions containing the enzyme were eluted with buffer C (20 mM Tris-HCl, 0.5 M NaCl, 300 mM imidazole, pH 8.0). After the chromatography stage, the protein was dialyzed against 20 mM Tris-HCl buffer (pH 7.5) with 0.1 M NaCl, and then against 20 mM Tris-HCl buffer pH 7.5 without NaCl.

His
₆-MBP-CRY1(PHR) and His
₆-MBP-CRY2(PHR) were expressed in Sf9 (
Spodoptera frugiperda) insect cells (Invitrogen) via baculovirus infection as previously described.
^{13 (link)}
Cell pellets were resuspended in lysis buffer (1x PBS, 50 mM NaNO
₃, 1% (v/v) glycerol, 0.1% Triton X-100, and Complete Protease Inhibitor Cocktail (Roche); pH 7.4) and purified according to our previously determined method.
^{14 (link)}
Briefly, cells were sonicated on ice, centrifuged at 19,000 × g for 90 min at 4°C, and the supernatant, containing target CRY proteins, was purified via a high-performance liquid chromatography (HPLC) system using a HisTrap 5 ml column (GE Healthcare). After tobacco etch virus (TEV) protease cleavage of the His
₆-MBP tag, further purification was performed via a HiTrap Heparin HP column (GE Healthcare), amylose resin (E8021, New England Biolabs), and a gel filtration chromatography Superdex 75 16/60 column (GE Healthcare). Purified proteins were buffer-exchanged (see Protein crystallization and structure determination section) and concentrated using an Amicon Ultra (Merck) concentrator.