Recombinant N-terminally histidine-tagged Φ6 RdRp was expressed from plasmid pAA5 in Escherichia coli BL21 (DE3) (38 (link)) and purified using Ni-NTA affinity column (Qiagen), HiTrapTM Heparin HP column and HiTrapTM Q HP column (GE Healthcare) as previously described (39 (link)). The purified protein was stored in 50% glycerol, 140 mM NaCl, 50 mM Tris–HCl pH 8.0, 0.1 mM EDTA, 0.1% Triton-X 100 at −20°C.
Hitraptm heparin hp column
Manufactured by GE Healthcare
Sourced in United Kingdom
The HiTrapTM Heparin HP column is a chromatography column for the purification of heparin-binding proteins. It is designed to provide a simple, reproducible, and scalable method for the purification of a wide range of biomolecules, including enzymes, growth factors, and coagulation factors.
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4 protocols using hitraptm heparin hp column
1
Purification of Recombinant Φ6 RdRp
2
Recombinant Protein Expression and Purification
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The pET15bM plasmids carrying hjc or the mutant genes were transformed into E. coli BL21 (DE3)-CodonPlus-RIL for protein expression. The procedure for protein induction and purification in E. coli cells was the same as previously described (Huang et al., 2017 (link)). Briefly, after induction, the cells were harvested and resuspended in buffer A (50 mM Tris–HCl pH 8.0, 200 mM NaCl, and 5% glycerol) for lysis by sonication. The soluble fractions were heated at 70°C for 30 min and after centrifugation the supernatants were purified by HitrapTM Heparin HP column and SuperdexTM 200 10/300 column sequentially (GE Health, United Kingdom). The protein concentration was determined by the Bradford method with bovine serum albumin (BSA) as the standard. Protein kinases were purified as described previously (Huang et al., 2017 (link)).
3
Synthesis and Purification of K63-Linked Polyubiquitin
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Lys63-linked ubiquitin chains (K63-polyUbn) were generated from a reaction containing 1 mM ubiquitin, 1 μM mE1, 10 μM UbcH13 and 10 μM Uev1A in buffer (10 mM ATP, 20 mM PC, 1.2 U/ml IPP, 1.2 U/ml CPK, 50 mM Tris pH 7.5, 10 mM MgCl2, 2 mM DTT). The K63-polyUbn synthesis reaction was performed at 37°C for 3–6 h. Synthesized polyUbn chains were purified as described previously (21 (link)). Briefly, ubiquitin chains were diluted 5-fold into 50 mM ammonium acetate, pH 4.5, and 0.1 M NaCl and separated over a 0.1–1.0 M NaCl gradient in 50 mM ammonium acetate, pH 4.5, using a HiTrapTM Heparin HP column (GE Healthcare). Fractions were applied to a Superdex™ 200 Increase 10/300 GL column equilibrated with buffer (50 mM Tris, pH 7.5, 2 mM DTT) for final purification to homogeneity.
4
Fractionation and Purification of Nuclear Extracts
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Crude nuclear fractions were prepared from mouse VV3 ESCs by homogenization in SHE buffer (10 mM HEPES pH 7.4, 0.21 M mannitol, 0.07 M sucrose, 0.1 M EDTA, 0.1 M EGTA, 0.15 mM spermine, 0.75 mM spermidine). The supernatant obtained by centrifugation (900 g, 10 min) was re-centrifuged (2000 g, 10 min). The obtained pellets (crude nuclear) were suspended in nuclear extraction buffer (50 mM HEPES pH 7.4, 0.3 M NaCl, 0.2% NP40, 1× cOmplete protease inhibitor cocktail [Roche]) and sonicated for 15sec. The suspension was centrifuged again (12,000g, 10 min). The supernatant (nuclear extract) was dialyzed against buffer A (50 mM Tris-HCl pH7.5, 50 mM NaCl, 0.2% NP40). Crude nuclear extracts were separated into four fractions (A—D) by step-gradient (0.05, 0.3, 0.6, 1.0 M NaCl) on HiTrapTM Heparin HP column (GE healthcare, HPLC system: Bio-Rad Biologic HR workstation). Each fraction was concentrated and desalted by centrifugal a filter unit Amicon ultra-4-10k (Millipore) and further separated into four fractions (I—IV) by step-gradient (0.05, 0.3, 0.6, 1.0 M NaCl) on HiTrapTM Q HP column (GE healthcare). Each fraction was concentrated and desalted by a centrifugal filter unit Amicon ultra-4-10k (Millipore).
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