Hitrap heparin hp

The IMPACT protein expression system including pTYB1 vector and chitin beads was provided by NEB. Target protein was cleaved from the intein-CBD fusion by DTT cleavage overnight and eluted from a chitin column. Enzymes were further purified by chromatography through a heparin column (5 ml HiTrap Heparin HP, GE Healthcare). Proteins were concentrated by low-speed centrifugation in protein concentrators (10 kDa cut-off) and resuspended in enzyme storage buffer (200 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol) and kept in a −20°C freezer. Small-scale protein purification was carried out using chitin magnetic beads from 1 ml cell lysate prepared from 20 ml IPTG-induced cells. E. coli T7 express (B strain) was used for protein expression. T7 Express cells carrying a restriction gene in a plasmid was cultured at 37°C to late log phase and enzyme production was initiated by addition of 0.5 mM IPTG final concentration and cells were induced at 18 to 20°C overnight in a temperature-controlled shaker. Cells were lysed by sonication in a chitin column buffer (20 mM Tris-HCl, pH 8, 0.5 M NaCl) at 4°C.

DNA sequences encoding wild-type or mutant AsCas12a (Supplementary Table 11) were cloned into pET28a vector by Gibson assembly^{11 (link)}. For protein expression, a single transformed E. coli BL21(DE3) colony was inoculated into 20 mL LB medium supplemented with 50 μg/mL kanamycin, and grown overnight at 37 °C, 250 rpm. The overnight culture was transferred to 1 l TB medium with kanamycin, grown at 37 °C, 250 rpm for ~2–3 h until OD600 reached 0.6. The culture was chilled at 4 °C for 30 min prior to induction with 1 mM IPTG, and further incubated at 18 °C, 250 rpm for 12~18 h.
The recombinant AsCas12a protein was purified as previously described for SpCas9^{11 (link)}. Briefly, E. coli cells were harvested by centrifugation, and homogenized with Emulsiflex-C3 high-pressure homogenizer (Avestin, Ottawa ON, Canada). The AsCas12a protein in clarified lysate was sequentially purified using immobilized metal affinity chromatography (HisTrap HP, GE Healthcare) and heparin chromatography (HiTrap Heparin HP, GE Healthcare). Purified protein was concentrated and dialyzed against storage buffer (20 mM TrisHCl, 300 mM NaCl, 0.1 mM EDTA, 50% Glycerol, 1 mM DTT, pH 7.4) overnight. The protein concentration was measured by NanoDrop using extinction coefficient at 143,940 M⁻¹cm⁻¹, diluted to 60 μM, and stored at −20 °C.

TFAM was cloned into a modified pET22 vector using EcoRI and XhoI. The vector harbored an N-terminal histidine-tagged maltose binding protein (MBP) cleavable by TEV protease. The resulting plasmid was transformed into ArcticExpress (DE3), and cells were grown in YB medium at 37°C until OD₆₀₀ reached ∼0.8. The expression of TFAM (aa 43–246) was induced by the addition of 0.3 mM IPTG at 16°C for 15 h. The cells were pelleted by centrifugation at 6500 rpm for 15 min, and lysed in lysis buffer (20 mM HEPES pH 8.0, 1 M KCl, 20 mM Imidazole) by sonication. The lysate was cleared by centrifugation at 13 000 rpm for 1 h, and the supernatant was applied to a Ni-NTA column equilibrated in the lysis buffer. The sample was eluted using lysis buffer containing 500 mM Imidazole. The histidine-tagged MBP was cleaved by TEV at 4°C overnight. TFAM was further purified using HiTrap Heparin HP (GE healthcare) equilibrated in 20 mM HEPES pH 8.0 and 1 mM DTT, and eluted by a linear KCl gradient from 0 to 1000 mM. As a final step, TFAM was applied to a Superdex 200 16/600 GL column (GE healthcare) equilibrated in 20 mM HEPES pH 8.0, 150 mM KCl and 1 mM DTT. The TFAM peaks were pooled and concentrated using an Amicon Ultra-15 concentrator (Millipore, 10k cutoff) to ∼15 mg/ml and stored at −80°C. The TFAM mutant, S61A, was generated by site-directed mutagenesis, and purified using the same procedure.

We used a thermostable and cysteine-modified mutant of human p53 (C124A, C135V, C141V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, C275A, C277A, and K292C)^{20 (link)}. The expression and purification of p53 were conducted by following our reported method^{20 (link)}. Briefly, p53 with the GST tag was expressed in Escherichia coli BL21 (DE3) pLysS cells. The cells were lysed by sonication, and the supernatants were loaded onto a GST column (GSTrap FF; GE Healthcare, Tokyo, Japan). The GST tag was cleaved using PreScission Protease (GE Healthcare), and samples were collected. The samples were purified further using a heparin column (HiTrap Heparin HP; GE Healthcare). DNA-binding ability of the purified sample was confirmed using a titration measurement based on fluorescence anisotropy^{8 (link)}. The purified p53 was labeled with ATTO532 using maleimide chemistry, as reported in our previous study^{20 (link)}. The p53 sample labeled with ATTO532 was purified using a cation exchange column (HiTrap SP HP; GE Healthcare). The labeling ratio was determined to be 0.8 dyes/monomer.

The dialysed crude extract was loaded onto a 1 ml HiTrap™Capto™Q (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) column, and purified by using FPLC®System (Pharmacia Biotech AB, Uppsala, Sweden) at a flow rate of 1 ml/min at 4°C. The unbound fractions were collected, and the column was washed with 10 column volumes (CV) of buffer A. The bound proteins were then eluted with 15 CV of 0-100% KCl linear gradient in buffer A. Both unbound and bound fractions were assayed for UDG enzymatic activity. The fractions containing PfUDG were pooled and dialysed at 4°C overnight against buffer B containing 50 mM HEPES pH 8.5, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.01% NP-40, 5% sucrose, and 20% glycerol. The dialysed fraction was then loaded onto a 1 ml Hitrap™ Heparin HP (GE Healthcare Bio-Sciences AB) column at a flow rate of 0.5 ml/min at 4°C. The unbound proteins were collected and the column was washed with 10 CV of buffer B. The column was then eluted with 15 CV of 0-100% KCl linear gradient in buffer B. Fractions containing PfUDG were pooled and termed native PfUDG.