Ni nta resin

The thawed cell pellet was resuspended in 120 mL of buffer A [50 mM sodium phosphate (NaPi) (pH 7.5), 5% (v/v) glycerol, 200 mM sodium chloride (NaCl)], with 2 mM phenymethylsulfonylfluoride, 2 mM magnesium chloride, lysozyme (30 mg) and DNAse (10 mg) at 4 °C, and disrupted by sonication (Branson Ultrasonic). The cell lysate was clarified by centrifugation at 16,000 × g and 4 °C for 1 hour, and the supernatant was loaded onto the Ni-NTA resin (EMD Millipore) and washed with buffer containing 50 mM NaPi (pH 7.5), 500 mM NaCl, 5–20 mM imidazole, 5% (v/v) glycerol. Protein was eluted with buffer A, containing 300 mM imidazole, then concentrated and digested overnight at 4 °C with thrombin (Biopharma, 10 unit/mg protein). His-tag free protein was loaded again on Ni-NTA resin and the pass-through was collected and concentrated to 10–20 mg/ml. For final purification, proteins were separated on a Superdex200 column (GE Healthscience) in crystallization buffer [20 mM TrisHCl (pH 8), 100 mM NaCl], yielding greater than 95% pure samples, as judged by SDS-PAGE. Protein was concentrated to 10–20 mg/ml, flash-frozen in liquid nitrogen, and frozen at −80 °C until use. Mutant proteins were expressed and purified in the same manner as wild-type proteins with no modifications.

The DNA sequence encoding the HAT domain of yGCN5 (99‐262) was amplified by PCR and was sub‐cloned into a pET‐28a vector for overexpression. The plasmid was transformed into the Escherichia coli strain BL21(DE3) and was overexpressed by induction with 0.5 mM isopropyl β‐d‐thiogalactoside and incubated at 15℃. The protein was purified via sequential use of Ni‐NTA resin (Merck Millipore) and Superdex 75 (GE) gel filtration chromatography. Purified protein was concentrated to 10 mg/ml by using a Centriprep 10 concentrator. 50 μM Gcn5 protein (residues 99‐262) in 20 mM sodium phosphate, 0.15M NaCl, and pH8.0 was incubated with EGCG at room temperature for 30 min at different concentrations, and then, the mixtures were analyzed by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE).

The BLUF–exonuclease construct was ordered as a synthetic gene (Eurofins Genomics, Germany) and cloned into an expression vector (pQE-30-UA-mCherry-GFP, in-house modified plasmid from Qiagen, United States). The protein was expressed as described above. Purification was performed using the Ni-NTA resin (Merck, Germany), following the manufacturer’s instructions. For the activity assay, 1 µM of Cy-5 hexamer (AAAAAA) was mixed with 10 U of exonuclease I (NEB, Germany) as the positive control, or 1 µl of BLUF–exonuclease I. The mixture was incubated for 30 min at 37°C and then heat-inactivated. Samples were mixed with TBE-Urea sample buffer (Thermo Fisher Scientific, Germany) and resolved on 10% TBE-Urea gel. The gel was scanned by Odyssey (LI-COR, Germany).

Equal amounts (2 μg) of GST-CUL4A and His-DDB1 proteins were mixed to form the CUL4A-DDB1 complex at 4°C for 30 min. The CUL4A-DDB1 complex was then treated with DMSO, 0.2 μM, 2.0 μM or 20 μM NSC1892 at 4°C for 6 hrs, followed by incubating with the Ni-NTA resin (Sigma, #70666-3) at 4°C for 4 hrs. The Ni-binding proteins were washed with a buffer containing 50 mM Tris (pH 8.0), 75 mM NaCl, 75 mM imidazole, and 1 mM DTT. The pulldown proteins were determined by loading into a 10% SDS-PAGE gel and then stained with Coomassie blue.

SILAC labelling of blood-stage parasites was performed as previously outlined [41 (link)]. Briefly, parasites were grown for three cycles in heavy or light isoleucine media, and synchronized with sorbitol. Age-matched 20–28 hpi trophozoites were magnet purified and mixed in equal ratios. Equinatoxin in PBS was used to lyse the RBC membrane [42 (link)] and supernatant was collected. Haemoglobin was removed using Ni+ NTA resin (Sigma) prior to mass spectrometry performed exactly as described previously [16 (link)].

100 µg of recombinant protein was allowed to bind with Ni-NTA resin (Sigma) followed by blocking with 5% BSA in TBS. After washing, increasing concentration of cholesterol ((Himedia; 10–400 µM in binding buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.004% NP-40)) was allowed to bind with protein immobilized resin for 4 hrs. Resin was washed with binding buffer, and cholesterol bound protein was eluted with elution buffer (binding buffer + 250 mM imidazole). Concentration of bound cholesterol was estimated by Zak’s method⁴⁸ using a standard curve for cholesterol. Briefly, protein present in the sample was precipitated with FeCl₃-CH₃COOH solution (0.05%) followed by incubation with concentrated H₂SO₄ for 20 minutes at RT. Red color developed and absorbance was measured at 560 nm. Concentration of free cholesterol was calculated by subtracting bound concentration from total cholesterol added in the assay. K_d was determined by fitting a hyperbola directly to the saturation isotherm using GraphPad Prism 7.0 software (GraphPad Software, CA, USA).