The whole sequence of Rv1566c and the targeted sequence Rv1566c-444n were amplified by polymerase chain reaction (PCR) with H37Rv genome DNA as a template. The sequences were then respectively ligated into plasmid pET-32a after digestion with EcoRI and HindIII and then transformed into E. coli BL21 (DE3). One mM isopropyl β-D-thiogalactoside (IPTG) was used in the medium to induce the expression of the proteins. After incubating for 3.5 h at 37°C with shaking the cells were harvested by centrifugation (4,000 rpm, 10 min, 4°C). The expression level and form of Rv1566c and Rv1566c-444 in E. coli were evaluated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then the inclusion body proteins were denatured and used for protein purification by Ni-NTA chromatography (GE Healthcare, Pittsburgh, PA, USA), followed by renatured in 20mM Tris-HCL (pH 8.0). Endotoxin from the recombinant protein was removed using a ToxinEraser endotoxin removal kit (GenScript, Piscataway, NJ, USA) and then the solution was sterile filtered using a 0.22-mum filter followed by quantification with a bicinchoninic acid kit (Transgen Biotech, Beijing, China). The preparation of Ag85B was done as described previously by our laboratory colleagues (18 (link), 19 (link)).
Ni nta chromatography
Manufactured by GE Healthcare
Sourced in United States
Ni-NTA chromatography is a purification technique used to isolate and purify recombinant proteins that have been engineered to contain a histidine-tag. The nickel-nitrilotriacetic acid (Ni-NTA) resin specifically binds to the histidine-tag, allowing the target protein to be separated from other cellular components. This method provides a simple and efficient way to obtain highly pure protein samples for further analysis and applications.
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Lab products found in correlation
Superdex 200, by GE Healthcare (2 mentions)
Bicinchoninic acid kit, by Transgene (1 mentions)
Toxineraser endotoxin removal kit, by GenScript (1 mentions)
Gel filtration standard mixture, by Bio-Rad (1 mentions)
Mouse anti ha antibody, by Merck Group (1 mentions)
Hitrap heparin hp affinity column, by GE Healthcare (1 mentions)
Mono q anion exchange chromatography, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
6 protocols using ni nta chromatography
1
Recombinant Rv1566c and Rv1566c-444 Proteins Purification
2
Purification of Recombinant M. mazei NTR Protein
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The full-length NTR of M. mazei strain Gö1 (MM2353) was cloned into pET28a, resulting in a cleavable N-terminal 6xHis-tagged fusion protein. Protein was produced in BL21pRIL Escherichia coli cells upon induction with 0.2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) at 22 °C. Cells were disrupted by sonication in 20 mM Tris-HCl buffer, pH 7.6, containing 300 mM NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride. The crude extract was clarified by centrifugation at 40,000× g for 1 h. The enzyme was purified using nickel-nitriloacetic acid (Ni-NTA) chromatography (GE Healthcare) and digested with thrombin (EMD Chemicals, San Diego, CA, USA) in buffer, 20 mM Tris-HCl pH 8, 150 mM NaCl, and 2 mM CaCl2, at room temperature overnight. The last step of purification involved gel filtration chromatography (Sephacryl S-300) using 10 mM Tris-HCl pH 7.6, 100 mM NaCl, and 2 mM 2-mercaptoethanol buffer. For protein molecular weight estimation by size exclusion chromatography, the column was calibrated with the Gel Filtration Standard mixture from Bio-Rad.
3
Purification and Cross-linking of Mgm1
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C-terminal HA-tagged s-Mgm1 was PCR amplified and inserted into pET28a. To avoid nonspecific cross-linking, four exposed Cys (C341, C676, C709, C763) were mutated into Ser. All other point mutants, including E751C, S397C, R712C, were also generated by site-directed mutagenesis from s-Mgm1. The proteins were purified from E. coli BL21 (DE3) cells. The harvested cells were resuspended in lysis buffer (40 mM Hepes-KOH, pH 7.4, 250 mM NaCl, 250 mM KCl, 1 mM DTT) and lysed by sonication at 4 °C. Cell lysates were clarified by centrifugation at 9,000 × g for 10 min, and the supernatant was collected. The protein was isolated by Ni-NTA chromatography (GE Healthcare) and eluted with elution buffer (20 mM Tris⋅HCl, pH 8.0, 150 mM NaCl, 300 mM imidazole). The protein was then further purified by HiTrap Heparin HP affinity column (GE Healthcare) and gel filtration chromatography (Superdex-200; GE Healthcare). Purified protein (0.6 μM) was incubated with 5 μM BMH (Thermo Scientific) at room temperature for 20 min in 25 mM Hepes, pH 7.4, 250 mM KCl, 250 mM NaCl. The samples were analyzed by 6% Tris-Acetate SDS/PAGE and immunoblotted with mouse anti-HA antibody (Sigma-Aldrich). HiMark LC5699 (Invitrogen) and 26619 (Thermo Scientific) were used as PAGE Rulers.
4
Recombinant AlkB Protein Purification
5
Purification and Cloning of Fluorescent Biosensor
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The fluorescent biosensor pRSET-FLII12Pglu-700μδ6 [15 ] was acquired from Addgene (Addgene plasmid # 13568). For bacterial expression, chemically competent NEB T7 Express (BL21 strain) cells were transformed with pRSEt-FLII12Pglu-700μδ6. Protein was expressed using methods adapted from [18 (link)]; briefly, cells were grown in LB broth at 30°C to density of OD600 ~0.6 and then induced with 0.7mM IPTG and grown at 18°C for 24 hours. Cells were then harvested and lysed via French press and protein purified using Ni-NTA chromatography (GE; Pittsburg, PA). Purified protein was exchanged into PBS with 20%v/v glycerol using a PD-10 desalting column (GE; Pittsburg, PA) and then stored at -80°C until further use. FLII12Pglu-700μδ6 was cloned into the trypanosome expression vectors pXS2 and pXS6 using BamHI and HindIII restriction sites.
6
Overexpression and Purification of IniA Protein
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The IniA constructs were transformed into E. coli strain BL21 (DE3) for protein overexpression. The bacteria were cultured in Luria-Bertani media at 37 °C to an OD600 of 0.6. Protein expression was induced by the addition of 0.2 mM IPTG for 20 h at 16 °C. Cells were harvested after centrifugation at 4000 g for 30 min, resuspended in Buffer A (20 mM Tris, pH 8.0, 150 mM NaCl), lysed under ultra-high pressure, and centrifuged at 39,000 g for 30 min to remove cell debris. The supernatant was then collected. The soluble protein was isolated by Ni-NTA chromatography (GE Healthcare) and further purified by gel filtration chromatography (Superdex-200 10/300GL increase; GE Healthcare) in Buffer A. Fractions containing IniA were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C for later use.
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