Recombinant GST-Tev-TNPO1 was purified as described previously (Chook and Blobel, 1999 (link)) with modifications. In brief, E. coli BL21-CodonPlus(DE3)-RIL cells (Agilent) were transformed with GST-Tev-TNPO1 plasmid and expression was induced overnight at 25°C with 1 mM IPTG. Cells were pelleted and resuspended in Tris buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% (v/v) glycerol, 2 mM DTT, supplemented with protease inhibitors), then lysed by sonication. Cell lysate was then loaded onto glutathione Sepharose™ 4 Fast Flow resin (GE Healthcare), and washed with Tris buffer, followed by ATP buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EGTA, 0.5 mM MgCl2, 5 mM ATP, 20% glycerol, 2 mM DTT, supplemented with protease inhibitors), then washed and eluted with Buffer A (20 mM imidazole, 75 mM NaCl, 1 mM EDTA, 20% (v/v) glycerol, 2 mM DTT). Finally, the protein was cleaved with Tev protease and purified on a HiTrap Q HP column (GE Healthcare) using a salt gradient. Purified protein was concentrated, flash frozen, and stored at −80°C.
Glutathione sepharose 4 fast flow resin
Manufactured by GE Healthcare
Sourced in United States, Sweden
Glutathione Sepharose 4 Fast Flow resin is a pre-packed chromatography medium designed for the affinity purification of glutathione S-transferase (GST) fusion proteins. The resin consists of cross-linked agarose beads to which the glutathione ligand is covalently coupled. This provides a high-capacity, fast-flow matrix for the efficient capture and purification of GST-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap q hp column, by GE Healthcare (2 mentions)
Bl21 codonplus de3 ril cells, by Agilent Technologies (2 mentions)
Pgex 4t 1, by Cytiva (2 mentions)
Thrombin, by GE Healthcare (2 mentions)
Sypro ruby protein gel stain, by Bio-Rad (2 mentions)
L glutathione, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Ja25.50 rotor, by Beckman Coulter (1 mentions)
Overnight express instant tb medium, by Merck Group (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Amylose agarose resin, by New England Biolabs (1 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
E coli bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 3, by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Glucagen, by Novo Nordisk (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
15 protocols using glutathione sepharose 4 fast flow resin
1
Recombinant GST-Tev-TNPO1 Protein Purification
2
Purification of GST-tagged bacterial pilins
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All E. coli strains and plasmids used in this experiment are listed in Table 1 Table 3
3
Yeast Nap1 Variant Binding Assay
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About 10 μM GST‐tagged yNap1 variants were incubated for 30 min at 4°C with 40 μl of 50% Glutathione Sepharose 4 Fast Flow resin (GE Healthcare), pre‐equilibrated in buffer 3 (20 mM Tris, pH 8.0, 500 mM NaCl, 1 mM DTT, 0.1% Tween‐20). The resin was then washed three times with buffer 3, and H2A–H2B dimers were added and incubated overnight at 4°C. The resin was then washed five times with buffer 3, and bound protein was detected by boiling the resin in SDS–PAGE loading buffer, followed by SDS–PAGE and staining with Coomassie blue.
4
Recombinant MOAG-2/LIR-3 Purification
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Recombinant moag-2/lir-3 was expressed and purified fused to the glutathione S-transferase (GST) from the pGEX-6-P1 vector in E. coli BL21 (DE) gold strain (Stratagene). Cells were grown in Overnight Express Instant TB Medium (Merck Millipore) supplemented with ampicillin (100 μg/ml) overnight at 30°C under constant shaking at 250 rpm. The cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA and EDTA-Free protease inhibitor cocktail (Complete, Roche) and lysed by sonication. The cell debris was removed by centrifugation at 18 000 rpm (JA-25.50 rotor, Beckman Coulter). The supernatant was loaded onto a column containing Glutathione Sepharose 4 Fast Flow resin (GE Healthcare LifeSciences) and equilibrated with lysis buffer. The column was washed with 40 CV of lysis buffer and the protein was eluted in 50 mM Tris pH 8, 10 mM reduced glutathione and dialyzed in 50 mM Tris pH 7.4, 150 mM NaCl for the aggregation experiments.
5
Purification of S. pombe E1, E2 Enzymes and Nse Proteins
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The S. pombe E1 protein Uba1 and E2 enzymes (Ubc1, Ubc2, Ubc4, Ubc7, Ubc8, and Ubc13) were expressed in E. coli strain BL21 (DE3) RIL and purified similarly as described [21 (link)]. Cultures containing the Mms2-pGEX-6P-1 plasmid were grown at 37 °C to OD600 ~ 1 and induced by 1 mM IPTG at 37 °C for 3 h. The cell pellet was resuspended in buffer A containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors. The cells were lysed by sonication, centrifuged, and supernatant mixed with Glutathione Sepharose 4 Fast Flow resin (GE Healthcare, Chicago, IL, USA) at 4 °C for 1 h. Proteins were eluted by gravity using buffer A containing 10 mM glutathione. The GST tag was cleaved off using PreScission protease overnight at 4 °C. The protein mixture was then subjected to gel filtration on Superdex 75 (GE Healthcare) in buffer A. Fractions containing Mms2 were concentrated and snap-frozen in liquid nitrogen. The Nse1/3/4 trimer and its vRING mutant were prepared as described [22 (link)]. The S. pombe Nse1 protein, its mutants, and Nse1/3 dimer were expressed and purified similarly as in the case of human Nse1/3 dimer [22 (link)].
6
Cloning and Purification of Mhp271 and GRP78 Proteins
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The Mhp271 gene was PCR amplified from M. hyopneumoniae, the Mhp271 gene lacking the R1‐2 region was generated by overlap PCR and the R1‐2 region of Mhp271 cDNA was prepared by Comate Biotech Company (Jilin, China). These cDNAs were cloned into the pMAL‐c5x vector with the restriction enzymes Sal I and Bam HI.
The GRP78 gene was PCR amplified from PTECs and cloned into the pGEX‐GST vector with the restriction enzymes Bam HI and Sal I.
These recombinant plasmids were transformed into E. coli BL21(DE3) cells. The level of recombinant protein expression was analyzed by SDS‐PAGE. Subsequently, the Mhp271‐MBP, Mhp271(ΔR1‐2)‐MBP, and Mhp271 R1‐2‐MBP fusion proteins were purified by using amylose agarose resin (New England Biolabs) according to the manufacturer's instructions. The prurition of GRP78‐GST was performed using Glutathione Sepharose 4 Fast Flow resin (GE Healthcare).
The endotoxin concentration of the fusion proteins (<0.04 endotoxin units/ml) was verified using the ToxinSensor chromogenic LAL endotoxin assay kit (GenScript, China).
The GRP78 gene was PCR amplified from PTECs and cloned into the pGEX‐GST vector with the restriction enzymes Bam HI and Sal I.
These recombinant plasmids were transformed into E. coli BL21(DE3) cells. The level of recombinant protein expression was analyzed by SDS‐PAGE. Subsequently, the Mhp271‐MBP, Mhp271(ΔR1‐2)‐MBP, and Mhp271 R1‐2‐MBP fusion proteins were purified by using amylose agarose resin (New England Biolabs) according to the manufacturer's instructions. The prurition of GRP78‐GST was performed using Glutathione Sepharose 4 Fast Flow resin (GE Healthcare).
The endotoxin concentration of the fusion proteins (<0.04 endotoxin units/ml) was verified using the ToxinSensor chromogenic LAL endotoxin assay kit (GenScript, China).
7
Recombinant Viral Capsid Protein Production
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The cDNAs encoding the capsid P domain were cloned into the expression vector pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ, United States) between BamHI and NotI sites. After sequence confirmation, P proteins were expressed in Escherichia coli following previously described procedures (Tan et al., 2008 (link)). Briefly, The BL21 cultures were initiated by 0.5 mM IPTG (isopropyl-β-D -thiogalactopyranoside) at a temperature of 22°C overnight. The recombinant P domain-GST fusion proteins were purified using Glutathione Sepharose 4 Fast Flow resin (GE Healthcare Life Sciences, NJ, United States). The GST was removed from the P proteins by thrombin (GE Healthcare Life Sciences, Princeton, NJ, United States) cleavage on beads at 22°C overnight.
8
Cloning and Purification of Mouse BMAL1 Fragments
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cDNAs encoding amino acid residues 1–134 for F1, 135–265 for F2, 266–399 for F3, 400–506 for F4, and 507–626 for F5 from mouse BMAL1 [National Center for Biotechnology Information (NCBI) reference sequence, NM_007489.4] were cloned into the vector pGEX4T1 (GE Healthcare) with a TEV-cleavable N-terminal glutathione S-transferase (GST) tag and a C-terminal HA tag using Eco RI/Not I restriction sites. All constructs were confirmed by DNA sequencing. Expression and purification of GST-tagged constructs were done as described previously (73 (link)). Briefly, fusion proteins were expressed in Escherichia coli BL21 (DE3) and purified using glutathione Sepharose 4 Fast Flow resin according to the manufacturer’s protocol (GE Healthcare).
9
Recombinant Glucagon Expression and Purification
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Glucagon PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, USA) and the FUSION Taq High-Fidelity enzyme was purchased from Finnzymes, Thermo Scientific (Vantaa, Finland). The glucagon expression vector pGEX4t-3 and the Glutathione Sepharose 4 Fast Flow resin were purchased from GE Healthcare (Inc, Sweden). E. coli BL21 (DE3) cells (Invitrogen) were kindly provided by Embrapa Cenargen, Brazil. The expression inductor Isopropyl β-d -1-thiogalactopyranoside (IPTG) was acquired from Invitrogen (Carlsbad, CA., USA). Glucagen®, used as positive control, was obtained from Novo Nordisk A/S (Denmark). Polyclonal anti-glucagon antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA., USA).
10
Production and Purification of Norovirus P Proteins
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The P proteins of different GII.4 strains were made as described previously [19 (link)]. A cysteine-containing peptide was linked to the N (CNGRC-P) or C (P-CDCRGDCFC) terminus of the P domains to enhance P-particle formation. The cDNAs encoding the capsid P domain without the hinge were cloned into the expression vector pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ) between Bam HI and Not I sites. After sequence confirmation, the P proteins were expressed in E. coli following previously described procedures [35 (link),39 (link),40 (link)]. Briefly, the BL21 cultures were induced by IPTG (isopropyl-β-D-thiogalactopyranoside) (0.4 mM) at room temperature (22°C) overnight. The recombinant P domain-GST fusion proteins were purified using Glutathione Sepharose 4 Fast Flow resin (GE Healthcare life Sciences, NJ, USA) according to the manufacturer’s instructions. GST was removed from the P proteins by thrombin (GE Healthcare life Sciences, NJ, USA) cleavage on beads at room temperature overnight. The P-particle formation was confirmed by gel filtration, using a Superdex 200 (GE Healthcare Life-Sciences, Piscataway, NJ) size exclusion column, during which the P particles formed a peak at ~830 kDa [18 (link)].
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