H5 cells at 80% confluence were infected (MOI = 1) with rBV-3a (constructed as specified in the “Generation of recombinant viruses” section above) and incubated at 22°C for 72 h. Cells were harvested and resuspended in lysis buffer 1 (50 mM Tris-HCl, 300 mM NaCl, 0.5% Triton X-100, pH 7.5) supplemented with 1% protease inhibitor cocktail (Sigma). Protein extracts were centrifuged at 12,000 × g for 10 min at 4°C, and the pellets were resuspended in lysis buffer 2 (8 M urea, 50 mM Tris-HCl, 300 mM NaCl, 1% IGEPAL, 1 mM β-mercaptoethanol, 10 mM imidazole, pH 7.5). Each sample was sonicated three times for 20 s each time and centrifuged at 12,000 × g for 10 min at 4°C. Finally, the protein present in the supernatant was purified through metal affinity chromatography (IMAC) using cobalt resin (Clontech) following the manufacturer’s instructions. Every fraction from the purification process was analyzed in 12% polyacrylamide gels using Coomassie Blue EZBlue gel staining reagent (Sigma). Then, the obtained protein was desalted using a PD-10 desalting column (GE Healthcare) and eluted in PBS.
Cobalt resin
Manufactured by Takara Bio
Sourced in United States
Cobalt resin is a purification media used for the affinity chromatography of histidine-tagged proteins. It utilizes the interaction between the histidine tag and immobilized cobalt ions to capture and purify the target protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Terrific broth media, by Thermo Fisher Scientific (2 mentions)
E coli bl21 de3, by New England Biolabs (2 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Casein powder, by Merck Group (1 mentions)
Glucose oxidase from aspergillus niger, by Merck Group (1 mentions)
Bovine liver catalase, by Merck Group (1 mentions)
Escherichia coli, by Takara Bio (1 mentions)
Bira biotin ligase, by Avidity (1 mentions)
Superose 6 column, by GE Healthcare (1 mentions)
Gelcode blue, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
7 protocols using cobalt resin
1
Purification of Recombinant Baculovirus Proteins
2
Fluorescent Kinesin-1 Construct Purification
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Unlabeled and labeled (biotin and tetramethylrhodamine (TRITC)) lyophilized tubulin from porcine brain were purchased from Cytoskeleton. Neutravidin was purchased from Life Sciences (St. Louis, MO). Casein powder, glucose oxidase from Aspergillus niger, and bovine liver catalase were purchased from Sigma (St. Louis, MO). Mouse monoclonal anti-6xHis antibody was purchased from Abcam (Cambridge, MA). Biotinylated mouse anti-5xHis antibody was purchase from QIAGEN (Hilden, Germany). Mouse anti-tubulin β3 antibody from Bio-Rad Laboratories (Hercules, CA). We used a 6xHis-GFP-labeled truncated human kinesin-1 heavy chain construct (aa 1–560 (15 (link))) with an AviTag sequence susceptible to biotinylation added at the C-terminus (16 (link)). The protein was expressed in Escherichia coli, purified using cobalt resin from Clontech Laboratories (now Takara Bio USA, Mountain View, CA) (15 (link)). Biotin ligase BirA (Avidity) was used to biotinylate AviTag-kinesin (17 (link)).
3
Purification of EpsE-Hcp1 Fusion Proteins
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Constructs for purification were introduced into pET21(d)epsE(100‐503)‐8aa‐hcp1‐his6 (ΔN1‐EpsE‐Hcp1) (Lu et al. 2013 ) or pET21(d)epsE(1‐503)‐8aa‐hcp1‐his6 (full‐EpsE‐Hcp1) and expressed in E. coli BL21(DE3) under IPTG‐inducing conditions. Proteins were purified using metal‐affinity chromatography on cobalt resin (Talon, Clontech, Mountain View, CA, USA). Subsequently, size‐exclusion chromatography was performed using a Superose 6 column (GE Healthcare) as described previously (Robien et al. 2003 ; Lu et al. 2013 ) and compared to known protein standards. Gel filtration fractions containing protein peaks were analyzed using SDS‐PAGE and visualized by staining the gel with Gel Code Blue (Thermo Scientific, Waltham, MA, USA).
4
Tandem Affinity Purification of TRPM7 Complexes
5
Purification of Mutant hNNMT Enzyme
6
Recombinant Staphopain A Production and Purification
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Recombinant Staphopain A was produced using the cloning method in pET26b vector and E. coli BL21 as an expression host. Primers for cloning were designed based on the sequence of the scpA gene in the Gene Bank. Two restriction enzymes, BamHI and XhoI (Thermofisher, USA), were used to provide sticky ends in vector and insert sequences. The double-digested sequences were ligated using T4 ligase (Thermofisher, USA). Then, the ligated vector was transformed to the competent E. coliTop10. The recombinant vector was proved using colony PCR and sequencing the extracted recombinant vector.
The isolated recombinant vector was transformed to the expression host (E. coli BL21), and the protein was expressed in exposure to Isopropyl ß-D-1-thiogalactopyranoside (IPTG). The recombinant protein was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Anti His-tagged western blotting. Finally, staphopain A was purified using immobilized metal affinity chromatography by cobalt resin (Talon, Takara). The protein purity was measured by High-Performance Liquid Chromatography (HPLC), and the functionality of the recombinant enzyme was determined using the casein digestion spectrophotometric method. The detailed description of cloning, expression, protein purification, and enzyme confirmation is present in supplementary file (Supplementary file1 ).
The isolated recombinant vector was transformed to the expression host (E. coli BL21), and the protein was expressed in exposure to Isopropyl ß-D-1-thiogalactopyranoside (IPTG). The recombinant protein was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Anti His-tagged western blotting. Finally, staphopain A was purified using immobilized metal affinity chromatography by cobalt resin (Talon, Takara). The protein purity was measured by High-Performance Liquid Chromatography (HPLC), and the functionality of the recombinant enzyme was determined using the casein digestion spectrophotometric method. The detailed description of cloning, expression, protein purification, and enzyme confirmation is present in supplementary file (Supplementary file
7
Purification of Mutant hNNMT Enzyme
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Human NNMT containing three alanine mutations (Addgene 40734) was cloned into the pET28-lic vector and used as a template for site-directed mutagenesis. A100K, A101E, and A103K present in the wild-type form of hNNMT as well as four additional His residues in N-terminus His-tag were introduced. The resulting vector was transformed into chemocompetent E. coli BL21 (DE3; NEB). 1L of Terrific Broth media (Invitrogen) was inoculated with overnight pre-cultures and grown to OD600 0.6 at 37 °C, 220 rpm; growth was continued at 17 °C overnight. Bacterial pellets were collected, resuspended in 50 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 0.5 mM PMSF and lysed by sonication. Protein from the supernatant of centrifuged sonicate was purified by immobilized metal affinity chromatography using cobalt resin (Takara). Pooled fractions were further purified by size exclusion chromatography (SEC) on HiLoad 16/600 Superdex 75 pg, equilibrated with 50 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP. Purity of protein fractions was assessed by SDS-PAGE. Selected, homogenous SEC fractions were pooled and protein concentrated to 10 mg/mL using 10 kDa MWCO centrifugal filter units (Amicon).
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