The E. coli C41(DE3) strain was used as the host for protein expression. The cells were grown in lysogeny broth (LB) medium supplemented with ampicillin (100 μg ml−1) to an absorbance of ∼0.8 and expression was induced overnight at 20 °C by addition of 0.1 mM isopropyl-β-D -thiogalactopyranoside. The cells were pelleted by centrifugation (5,000g, 20 min), resuspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM imidazole and Complete Protease Inhibitor (Roche Diagnostics)), then disrupted by sonication. After centrifugation (20,000 r.p.m., 30 min, 4 °C), the bacterial lysate was applied onto a Ni2+−NTA column (Qiagen) for affinity purification. After extensive washing (50 mM Tris pH 8.0, 150 mM NaCl and 20 mM imidazole), the protein was eluted in presence of 300 mM imidazole. The fractions containing MsVps4ΔMIT were pooled and incubated overnight at room temperature with TEV (0.2 mg per 10 mg of protein) and dialysed against 25 mM HEPES, 150 mM NaCl and 10 mM imidazole. The TEV protease and uncleaved protein were removed by passing the solution through Ni2+-NTA column (Qiagen) and collecting the unbound protein. Cleaved MsVps4ΔMIT was concentrated to 80–150 μM using an Amicon 400 concentrator with 10-kDa cutoff membrane and purified by Superose 6 SEC on an ÄKTA FPLC system (GE Healthcare) in a buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM dithiothreitol.
Ni2 nta column
Manufactured by Qiagen
Sourced in United States, Germany
The Ni2+-NTA column is a chromatography column used for the purification of recombinant proteins containing a histidine-tag. The column matrix is coated with nickel ions (Ni2+) which bind to the histidine-tag on the target protein, allowing it to be separated from other cellular components during the purification process.
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Lab products found in correlation
Superdex 75, by GE Healthcare (5 mentions)
Superdex 200 column, by GE Healthcare (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Bl21 de3 cells, by New England Biolabs (1 mentions)
Glutathione sepharose 4b column, by Cytiva (1 mentions)
Bca kit, by Sangon (1 mentions)
Kta fplc system, by GE Healthcare (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Glutathione sepharose chromatography, by Cytiva (1 mentions)
Ni nitrilotriacetic acid nta beads, by Qiagen (1 mentions)
Pet his6 proteing tev lic vector 2p t, by Addgene (1 mentions)
E coli bl21 de3 cells, by Agilent Technologies (1 mentions)
Superdex 200 26 60 size exclusion column, by GE Healthcare (1 mentions)
Glutathione sepharose 4b resin, by GE Healthcare (1 mentions)
E coli bl21 de3 strain, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Nickel nitrilotriacetic acid beads, by Qiagen (1 mentions)
P 81 phosphocellulose, by Cytiva (1 mentions)
Glutathione sepharose 4b beads, by Merck Group (1 mentions)
28 protocols using ni2 nta column
1
Purification of MsVps4ΔMIT Protein from E. coli
2
Production and Purification of 6xHis-CHLH Protein
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A 6xHis-full-length CHLH protein was produced exactly as previously described (Wu et al., 2009 (link)). Briefly, cDNA encoding full-length CHLH was amplified by PCR using primers CHLH-Z1-F and CHLH-Z1-R. The PCR product was digested with EcoRI and SalI and then cloned into pET48b(+) at the EcoRI and SalI sites. The recombinant CHLH protein was expressed in E. coli Rosetta gami2 (DE3) (Novagen) and purified on a Ni2+-NTA column, as described in the manual provided by the manufacturer (Qiagen). Antiserum against 6xHis-full-length CHLH was raised by immunizing rabbits according to a standard protocol (Kiwa Laboratory Animals Co., Ltd). The primer sequences are listed in the Supplementary Table 1 .
3
Recombinant CYP158A3 Protein Production
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The expression and purification of CYP158A3 were carried out as previously described, with some modifications (Lim et al., 2012 (link); Park et al., 2014 (link)). Briefly, Escherichia coli DH5α cells transformed with pCW (CYP158A3) and pGroEL/ES vectors were inoculated in Luria-Bertani (LB) broth containing 50 μg/mL ampicillin and 20 μg/mL kanamycin; cells were then pre-cultured overnight at 37°C. LB cultures were then seeded into 500 mL of Terrific broth (TB) expression medium containing 50 μg/mL ampicillin (1:100 dilution). The expression cultures were grown at 37°C, with shaking (220 rpm), until the optical density at 600 nm reached around 0.6 (Zhao et al., 2007 (link)). Following supplementation (0.5 mM 5-aminolevulinic acid, 1.0 mM IPTG, 1.0 mM thiamine, and trace elements), the cultures were incubated for a further 22–24 h at 28°C, with shaking (190 rpm). The bacterial soluble fractions containing CYP158A3 were isolated and prepared from the TB expression cultures. CYP158A3 was purified using a Ni2+-NTA column (Qiagen, Valencia, CA, USA), as described previously (Kim et al., 2005 (link); Park et al., 2011 (link)). The dialyzed fraction was further concentrated using an Amicon Ultra-15 centrifugal filter unit (Millipore, Billerica, MA, USA) and dialyzed against 10 mM potassium phosphate buffer containing 10% glycerol.
4
Heterologous Expression and Purification of CalR3
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A DNA fragment containing intact calR3 was amplified using KOD-plus DNA polymerase from S. chartreusis NRRL 3882 genomic DNA with primers 28aR3-F1 and 28aR3-F2. The obtained PCR fragment was double-digested using NdeI and EcoRI and then ligated with linearized vector pET28a(+) (Novagen) at the corresponding restriction sites to generate pET28a-calR3. After sequencing validation, pET28a-calR3 was introduced into E. coli BL21(DE3)/plysE (Stratagene) for protein over-expression. Cell growth, induction, and harvest conditions were performed as previously described (Wu et al., 2013 (link)). Collected cells were re-suspended in binding buffer (20 mM Tris-Cl, pH 8.0, 300 mM NaCl) and disrupted by ultrasonication. After centrifugation, the supernatant was loaded onto a Ni2+-NTA column (Qiagen, Germany) that had been pre-equilibrated with binding buffer. The elution condition was a linear-gradient of imidazole from 0 to 500 mM. The purified protein was desalted by a HisTrap desalination column with buffer C (100 mM Tris-HCl, pH 8.0) and measured using SDS-PAGE. Finally, the purified His6-tagged CalR3 protein was stored in stock buffer containing 10% glycerol at -80°C.
5
Purification of VopK Protein by Ni2+-NTA Chromatography
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VopK protein was purified by Ni2+-nitrilotriacetic acid (Ni2+-NTA) chromatography by following a published protocol [12 (link)]. The wild-type gene was cloned into the NdeI-BamHI site of the pET15b vector (Novagen) to generate an N-terminal His6-VopK fusion protein. The clone was confirmed by sequencing and transformed into E. coli BL21 (DE3). After induction with 0.4mM IPTG (isopropyl1-thio-β-D-galactopyranoside), VopK protein was purified through Qiagen Ni2+-NTA column. The purified protein was dialyzed overnight in a solution of buffer A containing 10mM Tris, pH 7.9, 100 mM KCl, 0.1mM EDTA, 0.1mM DTT, 5% glycerol. Protease assay was done essentially as described earlier [13 (link)].
6
cGAS Full-Length and Catalytic Domain Purification
7
Cloning and Purification of M. tuberculosis PanD
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The M. tuberculosis panD gene was PCR amplified from the genome of type strain H37Rv using the primers: panD F: 5′-TAT CCA TGG GCA TGT TAC GGA CGA TGC TGA AG-3′ and panD R: 5′-TAT CTC GAG TCC CAC ACC GAG CCG GGG GT-3′. The PCR product was digested with NcoI and XhoI and cloned into the pET28a plasmid vector digested with the same enzymes. The correct clones were confirmed by DNA sequencing. The PanD protein was purified as described.17 (link) Briefly, log phase E. coli strain BL21(DE3) containing pET28a-panD was induced with 0.5 mM IPTG at 37 °C for 4 h. The cells were harvested, resuspended in buffer A (10 mM Tris-HCl, pH 7.5, 150 mM NaCl), and lysed by sonication. The PanD protein was purified by using a Ni+2-NTA column (Qiagen, Valencia, CA, USA) and dialyzed against 10 mM Tris, pH 8.0. The protein fractions were eluted with imidazole and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
8
Purification of Ari-1 and KoiU2 Proteins
9
Purification and Interaction of HSP90 and USP19
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The His-tagged HSP90 (in pET-28a) and GST-fused CS1 (residues 75–209) or CS2 (273–393) domain of USP19_b (in pGEX-4T-3) were expressed in E. coli BL21 (DE3) strain. The His-tagged HSP90 was purified through a Ni2+-NTA column (Qiagen), while GST-fused proteins were purified using the glutathione Sepharose 4B column (Amersham Bioscience). GST or GST-fused proteins were incubated with the glutathione Sepharose 4B beads in a PBS buffer (10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4), and the suspension was agitated at 4°C for 30 min. The beads were washed three times in the same buffer to remove any unbound protein. An equal molar amount of HSP90 was added, and the suspension was agitated at 4°C for about 2 hrs. After excessive washing, the beads were re-suspended in the sample buffer and subjected to SDS-PAGE followed by Coomassie staining.
10
Heterologous Expression of Echinacea Enzymes
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Full-length CDS of EpHTT, EpHQT, and EpHCT were amplified from Echinacea purpurea hairy root cDNA using primers listed in Supplementary Table 4 and cloned into the pDEST17 plasmid with 6 x His at the N-terminus53 (link). The recombinant plasmids were transformed into E. coli BL21(DE3) which was grown on LB solid medium (50 μg/mL ampicillin). Overnight cultures were transferred to 200 mL LB liquid medium containing antibiotics until the OD600 reached 0.5–1.0. Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 0.4 mM, and the cultures were grown overnight at 16 °C with shaking at 200 rpm. Cells were harvested by centrifugation at 5,000 × g for 5 min.
Cells were resuspended in lysis buffer (25 mM Tris–HCl 8.0, 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine). ATP (1 mM) and PMSF (1 mM) were also added. The cells were broken using a disruptor with 600 bar. After centrifugation at 20,000 × g at 4 °C for 30 min, the supernatant was collected. Protein was purified using a Ni2+-NTA column (Qiagen) according to the manufacturer’s instructions. Imidazole was removed using FPLC coupled with HiTrap Q HP column for purification of enzymes from crude extracts. The protein concentration was determined using a BCA kit (Sangon Biotech, China). Recombinant proteins were stored in PBS (pH7.0 with 10% glycerol) at -80 °C before use.
Cells were resuspended in lysis buffer (25 mM Tris–HCl 8.0, 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine). ATP (1 mM) and PMSF (1 mM) were also added. The cells were broken using a disruptor with 600 bar. After centrifugation at 20,000 × g at 4 °C for 30 min, the supernatant was collected. Protein was purified using a Ni2+-NTA column (Qiagen) according to the manufacturer’s instructions. Imidazole was removed using FPLC coupled with HiTrap Q HP column for purification of enzymes from crude extracts. The protein concentration was determined using a BCA kit (Sangon Biotech, China). Recombinant proteins were stored in PBS (pH7.0 with 10% glycerol) at -80 °C before use.
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