The expression and purification of Renilla luciferase was similar to previous studies [20 (link),21 (link)]. Briefly, The Renilla luciferase coding sequence was inserted into pET-21a (+) plasmid and transformed into Escherichia coli BL21 (DE3) PLYS strain (Novagen, USA). The transformed cells were cultured in 10 mL Luria-Broth (LB) supplemented with ampicillin (100 μg mL−1), induced with 0.5 mM isopropyl-beta-d -thiogalactopyranoside (IPTG, Sigma-Aldrich), and incubated at 30 °C for 16 h to express Renilla luciferase. The cells were lysed in lysis buffer (50 mM Tris-HCl and Triton 0.01 %, pH 8.0) via sonication and the lysate was centrifuged at 8000 rpm at 4 °C for 8 min to obtain the supernatant. The protein was purified using HisPurTM Ni-NTA Resin (Thermos scientific, USA).
Hispurtm ni nta resin
Manufactured by Thermo Fisher Scientific
Sourced in United States
HisPur Ni-NTA Resin is a nickel-nitrilotriacetic acid (Ni-NTA) matrix used for the purification of histidine-tagged recombinant proteins. It is designed for efficient and gentle capture and purification of these proteins from a variety of sample sources.
Automatically generated - may contain errors
Lab products found in correlation
Ni nta resin, by Thermo Fisher Scientific (3 mentions)
Anti flag antibody, by Merck Group (2 mentions)
Endo h denaturing buffer, by New England Biolabs (2 mentions)
Endo h, by New England Biolabs (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)
Pregnenolone, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
17α oh progesterone, by Merck Group (1 mentions)
4 androstene 3 17 dione, by Merck Group (1 mentions)
3 3 cholamidopropyl dimethylammonio 1 propanesulfonate chaps, by GoldBio (1 mentions)
Dehydroepiandrosterone dhea, by Avanti Polar Lipids (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
E pulkp, by Megazyme (1 mentions)
E isamy, by Megazyme (1 mentions)
Magicmedia, by Thermo Fisher Scientific (1 mentions)
Microcon centrifugal filter device, by Merck Group (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
28 protocols using hispurtm ni nta resin
1
Renilla Luciferase Expression and Purification
2
Purification of Steroid Metabolizing Enzymes
3
Recombinant Protein Purification from Sf9 Cells
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To purify the rVP1 or rchIL-12, Sf9 cells at a density of 106/mL were suspension-cultured and infected with B-VP1/B-VP2 or B-chIL-12 at a MOI of 1. At 96 hpi, the cells were lysed using Insect PE LBTM lysis buffer (G-Bioscience, USA), and were sonicated. The sample was then mixed with HisPurTM Ni-NTA Resin (ThermoFisher, USA) and loaded into a column. The column was washed with a washing buffer (20 mM pH 7.9 Tris, 30 mM imidazole, 500 mM NaCl), and the bound protein was eluted with an elution buffer (25 mM Tris, 200 mM imidazole, 500 mM NaCl). The elute was collected and verified with Western blotting. The protein concentration was determined using a Protein Assay kit (Bio-Rad, USA).
4
Purification of His-Tagged Proteins
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From an overnight culture, 10 mL were inoculated into 1 L of LB medium supplemented with 50 μg mL–1 kanamycin and grown at 37°C, 250 rpm until reaching an OD600 of 0.6. Induction was performed with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 4 h. Bacteria harvesting was carried out by centrifugation at 10,000 rpm, 4°C, 15 min. Cell pellets were resuspended in 20 mL native binding buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 8). Pellets were kept on ice, treated with 1 mg mL–1 lysozyme and 1 mM PMSF for 30 min, and lysed by sonication. Lysates were clarified by centrifugation at 15,000 rpm, 4°C, 30 min, and purified using HisPurTM Ni-NTA Resin (Thermo Fisher Scientific). Lysates were loaded onto the purification column (previously equilibrated with native binding buffer), unspecific binding was removed by washing (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole), and proteins were eluted (50 mM NaH2PO4, 300 mM NaCl, and 150 mM imidazole). Protein identity was confirmed by western blotting using a monoclonal Anti-His IgG1 − from mouse (Sigma).
5
Recombinant PlOMT4 Protein Expression and Purification
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The coding region of PlOMT4 was cloned into the Escherichia coli expression vector pET28a (Novagen, Darmstadt, Germany) with its N-terminus fused with a 6 × HIS tag. The primers used for the cloning are listed in Supplementary Table S1 . The expression of the recombinant PlOMT4 was induced with 0.5 mM isopropyl-D -thiogalactopyranoside (IPTG) at 16°C for 14 h. The recombinant proteins were purified using HisPurTM Ni-NTA Resin (Thermo) following the provided protocol. The purified proteins were analyzed by SDS-PAGE and protein quantification was determined by Bradford assays.
To check the enzyme activity, enzyme assays were performed in a 200 μl of the reaction mixture consisting of 2 μg of the purified recombinant PlOMT4, 50 mM Tris–HCl (pH 8.0), 2 mM S-adenosyl methionine, 2 mM dithiothreitol (DTT) and 100 μM acceptor substrates. The reactions were carried out at 37°C for 20 min, stopped with the addition of 200 μl of methanol, and 15 μl of the reaction mixture was directly used for HPLC analysis.
To check the enzyme activity, enzyme assays were performed in a 200 μl of the reaction mixture consisting of 2 μg of the purified recombinant PlOMT4, 50 mM Tris–HCl (pH 8.0), 2 mM S-adenosyl methionine, 2 mM dithiothreitol (DTT) and 100 μM acceptor substrates. The reactions were carried out at 37°C for 20 min, stopped with the addition of 200 μl of methanol, and 15 μl of the reaction mixture was directly used for HPLC analysis.
6
Overexpression and Purification of Recombinant Proteins
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Proteins were overexpressed from Escherichia coli BL21 (DE3) (Novagen) cells transformed with the corresponding expression plasmids (Table S6 ). Cultures were grown at 37°C in LB medium supplemented with 100 μg ml-1 ampicillin to an OD600 of 0.5–0.6. Then, protein expression was induced with 1 mM IPTG at 16°C for 16 h. Cells were harvested by centrifugation at 4°C, 4000 rpm for 30 min, resuspended in lysis buffer (100 mM Tris pH = 8, 300 mM NaCl, 1 mM TCEP) and lysed by sonication. The soluble fractions were obtained by centrifugation at 4°C, 15000 rpm for 1h and loaded onto a pre-equilibrated Nickel affinity gravity column (HisPurTM Ni-NTA Resin; Thermo Fisher). After two washes with 20 mM (40x bed volume) and 50 mM imidazole (30x bed volume), the proteins were eluted with lysis buffer containing 250 mM imidazole. The fractions were analyzed by SDS-PAGE and those fractions showing purest protein were selected, concentrated, and stored at −80°C.
For anomalous X-ray diffraction and phasing, RppC was selenomethionine-labeled (SeMet) by expressing the protein in SelenoMethionine Medium Complete (Molecular Dimensions Ltd; MD 12-500), according to the manufacturer instructions, and purified as described previously.
For anomalous X-ray diffraction and phasing, RppC was selenomethionine-labeled (SeMet) by expressing the protein in SelenoMethionine Medium Complete (Molecular Dimensions Ltd; MD 12-500), according to the manufacturer instructions, and purified as described previously.
7
Enzymatic Synthesis of Maltodextrin
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Linear maltodextrin MD18 (Maltooctadecaose) was purchased from CarboExpert (Yuseong-gu, Republic of Korea). Isoamylase was obtained from Pseudomonas sp. (E-ISAMY, 200 U/mL) and pullulanase M1 from Klebsiella planticola (E-PULKP, 650 U/mL) were obtained from Megazyme (Bray, Ireland). Glycogen branching enzymes of Escherichia coli (EcGBE13) was purchased from Creative Enzymes (Shirley, NY, USA), and MagicMedia and HisPurTM Ni-NTA Resin were purchased from ThermoFischer Scientific (Waltham, MA, USA). All chemicals were of analytical grade or higher.
8
Endoglycosidase Assay for sHF-LIPA
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sHF-LIPA glycosylation assay was performed as previously described (Jin et al., 2018 (link)) with minor modifications. Briefly, WT and QKO CHO cells were co-transfected with sHF-LIPA and control pEGFP-N1 vector or pEGFP-N1-MAN1A1 constructs as indicated and cultured for 72 h. 10 mL of the conditioned medium was collected and spun at 1000 g for 3 min at 4°C to remove dead cells, followed by incubation with 100 μL of prewashed HisPurTM Ni-NTA Resin (Thermo, 88222) with gentle rotation at 4°C for 2 h. Ni-NTA Resin were washed with His-buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 2 mM DTT, 40 mM imidazole) and sHF-LIPA were eluted from the resin using 200 mM imidazole in pH 7.4 PBS. Eluted proteins were denatured by a 10× Endo H denaturing buffer (New England Biolabs) at 95°C for 5 min. Each denatured sample was divided equally to two parts, untreated or treated with 250 U Endo H or PNGase F (New England Biolabs) following the manufacturer’s instructions. Both parts were incubated on a 37°C shaker for 1 h. Reactions were terminated by adding a 6× SDS loading buffer and heating at 95°C for 3 min. Samples were then analyzed by SDS-PAGE and Western blot. sHF-LIPA was detected using a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, M1804, 1:2,000).
9
Affinity Purification of Fc, CH2-H, and CH2
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For Fc, CH2-H, and CH2, cell culture supernatant was decanted from culture flasks 4 days post-transfection and centrifuged at 2000 × g for 5 min to pellet any residual cells. The supernatant was then passed through a 0.22-μm filter and diluted 1:1 with binding buffer (PBS, 10 mm HEPES, pH 8.0).
The protein of interest was then purified by immobilized metal affinity chromatography using HisPurTM Ni-NTA resin (ThermoScientific). Eluted fractions containing the target protein were concentrated using Vivaspin20 concentrators (10-kDa molecular mass cutoff for Fc, 3-kDa molecular mass cutoff for CH2-H and CH2) for further purification by size exclusion chromatography. Samples were applied to a pre-equilibrated Superdex200 HiPrep 16/60 (Fc) or Superdex75 HiPrep 16/60 (CH2-H, CH2) column. Protein was eluted with 10 mm HEPES, pH 8.0, 150 mm NaCl. The absorbance at 280 nm (A280) of the eluate was monitored to determine the presence of protein. Protein purity was assessed by SDS-PAGE.
The protein of interest was then purified by immobilized metal affinity chromatography using HisPurTM Ni-NTA resin (ThermoScientific). Eluted fractions containing the target protein were concentrated using Vivaspin20 concentrators (10-kDa molecular mass cutoff for Fc, 3-kDa molecular mass cutoff for CH2-H and CH2) for further purification by size exclusion chromatography. Samples were applied to a pre-equilibrated Superdex200 HiPrep 16/60 (Fc) or Superdex75 HiPrep 16/60 (CH2-H, CH2) column. Protein was eluted with 10 m
10
Purification of His-BrdA Proteins
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His-BrdA proteins in the crude extract were purified using HisPurTM Ni-NTA Resin (Thermo Fisher Scientific, Yokohama, Japan), according to the manufacturer’s protocol, and the sample in the elution buffer was exchanged into 50 mM Tris-HCl buffer using a Microcon centrifugal filter device (Millipore, Bedford, MA, USA). The concentration of purified proteins was measured using a Qubit protein assay kit and Qubit 2.0 (Invitrogen, Carlsbad, CA, USA). The expression and purity of each His-BrdA protein were assessed by SDS-PAGE.
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