mSH2, cSH2 and tSH2-WT were cloned from the yeast display vector into the pET42 vector using the restriction sites SpeI and XhoI to generate GST-tagged fusions. Primers FP2 and RP2 were used to introduce the restriction sites by PCR to facilitate cloning of mSH2 and cSH2 whereas primers FP3 and RP2 were used for cloning of tSH2-WT. Sequences were verified by colony sequencing using a T7 reverse primer by Genewiz. Subsequently, plasmids were introduced into Rosetta Cells™ (EMD Biosciences). Protein expression was induced by addition of 1mM IPTG followed by incubation at 20 °C for 12 hours with shaking at 250 rpm. After induction, cells were harvested by centrifugation (4800g, 10min), resuspended in PBS with 0.2mM PMSF (Sigma Aldrich) and lysed by sonication (2 sec ON, 5 sec OFF) for 7 minutes. The lysate was cleared by centrifugation and filtration through a 0.22 μm filter and purified using a GSTrap FF column (GE Healthcare Bio-Sciences) on a Bio-Rad Biologic LP system using a linear glutathione gradient. The eluted fractions were analyzed by SDS PAGE and pure fractions were combined and dialyzed into 4L of PBS using a 3.5kDa MWCO filter (Thermo Fisher). Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo scientific).
Gstrap ff column
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Spain
The GSTrap FF column is a chromatography column designed for the purification of glutathione S-transferase (GST) fusion proteins. It is composed of an agarose-based resin that has been coupled with glutathione, allowing for the efficient capture and separation of GST-tagged proteins from complex mixtures.
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Lab products found in correlation
Histrap hp column, by GE Healthcare (2 mentions)
Resource q column, by GE Healthcare (2 mentions)
Histrap ff, by GE Healthcare (2 mentions)
Histrap ff column, by GE Healthcare (2 mentions)
Emulsiflex c3 homogenizer, by Avestin (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protev protease, by Promega (2 mentions)
E coli bl21 krx l3002, by Promega (2 mentions)
Enrich q, by Bio-Rad (2 mentions)
Ge 200, by Hoefer (2 mentions)
Mwco filter, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Gstrap ff, by GE Healthcare (1 mentions)
Codon plus, by Agilent Technologies (1 mentions)
Prescission protease, by Beyotime (1 mentions)
Mut express 2 fast mutagenesis kit v2, by Vazyme (1 mentions)
Protease inhibitor cocktail, by Sangon (1 mentions)
Ritonavir, by Selleck Chemicals (1 mentions)
35 protocols using gstrap ff column
1
Cloning and Purification of SH2 Domains
2
Purification of Recombinant Proteins from E. coli
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Escherichia coli strain BL21 (DE3) or Rosette2 (DE3) were transformed with plasmids and cultured at 37°C. When the absorbance at 600 nm of the cultures reached 0.4, IPTG and ethanol were added to a final concentration of 1 mM and 2%, respectively for induction, and cells were cultured at 15°C for 16–24 h. Cells were harvested and disrupted by sonication to obtain cell free extracts. Recombinant full-length FD protein was purified from the inclusion body under denaturation conditions using Ni-NTA agarose (QIAGEN) according to the manufacture's instructions. Recombinant GST-tagged and His-tagged proteins were purified from cell-free extracts with a GSTrap FF column (GE Healthcare) and HisTrap HP column (GE Healthcare), respectively. The purities of the recombinant proteins were verified by SDS-PAGE and staining with Rapid Stain CBB (Nacalai tesque).
3
Purification and Phosphorylation of PINK1 Substrates
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Pediculus humanus GST-PINK1 (128-C) was purified on a GSTrap FF column (GE Healthcare) using 50 mM Tris, 250 mM NaCl, 1 mM DTT at pH 7.5 and eluted with freshly prepared 50 mM Tris, 250 mM NaCl, 1 mM DTT and 10 mM glutathione at pH 7.5. GST-PINK1 (10 μM) was incubated with either Ub (500 μM) or UblD (500 μM) and dialysed against buffer containing 50 mM Tris, 0.5 mM DTT, 10 mM MgCl2 and 5 mM ATP (pH 7.5) for 2 h at 25°C. The reaction was monitored to completion using Phos-Tag™ SDS–PAGE. Following the reaction, GST-PINK1 was removed from the reaction using a GSTrap FF column, collecting the flow-through fractions containing pUb or the pUbl domain. The phosphorylated proteins were separated from unphosphorylated species on a HiTrap Q column using 20 mM Bis-Tris propane (pH 8.7) and a 2-h 0–100% 0.5 M NaCl elution gradient. Unphosphorylated species were found in the flow through, while pure pUb and pUblD eluted around 20–40% of the salt gradient.
4
Purification of GST-tagged MDM2 Proteins
5
Purification of SARS-CoV-2 Papain-Like Protease
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The pGEX6P-1 bacterial expression vector carrying PLpro was kindly provided by Prof. Lei Liu (Tsinghua University, Beijing, China). PLpro C191S, C194S and C191S/C194S mutants were generated from wild-type PLpro plasmid using Mut Express II Fast Mutagenesis Kit V2 (Vazyme Biotech, Nanjing, China) and verified by DNA sequencing. The BL21(DE3)
E. coli competent cells (Sangon Biotech, Shanghai, China) were transformed with the plasmid, cultured in 2×YT medium at 37°C until the OD
600 reached 0.6. Then, protein expression was induced by the addition of 0.5 mM IPTG and overnight incubation at 18°C. The cells were harvested by centrifugation, resuspended in the lysis buffer supplemented with protease inhibitor cocktail (Sangon Biotech) and lysed by sonication. After centrifugation at 20,000
g for 1 h at 4°C, the supernatant was applied to a GSTrap FF column (5 mL; GE Healthcare, Sunnyvale, USA) and washed with the washing buffer. GST-tagged PLpro proteins were eluted with the elution buffer. PreScission Protease (Beyotime, Shanghai, China) was added to the eluate to remove the GST-tag, followed by dialysis overnight at 4°C. PreScission Protease and GST tags were removed by GSTrap FF column, after which purified wild-type PLpro or its mutants were quantified by SDS-PAGE.
E. coli competent cells (Sangon Biotech, Shanghai, China) were transformed with the plasmid, cultured in 2×YT medium at 37°C until the OD
600 reached 0.6. Then, protein expression was induced by the addition of 0.5 mM IPTG and overnight incubation at 18°C. The cells were harvested by centrifugation, resuspended in the lysis buffer supplemented with protease inhibitor cocktail (Sangon Biotech) and lysed by sonication. After centrifugation at 20,000
g for 1 h at 4°C, the supernatant was applied to a GSTrap FF column (5 mL; GE Healthcare, Sunnyvale, USA) and washed with the washing buffer. GST-tagged PLpro proteins were eluted with the elution buffer. PreScission Protease (Beyotime, Shanghai, China) was added to the eluate to remove the GST-tag, followed by dialysis overnight at 4°C. PreScission Protease and GST tags were removed by GSTrap FF column, after which purified wild-type PLpro or its mutants were quantified by SDS-PAGE.
6
Purification and Inhibition of SARS-CoV-2 Mpro
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Mpro was expressed and purified as reported in [17 (link)]. The pGEX-6P-1 vector to encode the SARS-CoV-2 Mpro (NC_045512) was purchased from GenScript (clone ID_M16788F). For Mpro C-terminal His-tag removal, the Prescission (1 U for 100 µg of protein) cleavage reaction was performed at 4 °C for 4 h, and Prescission protease was then removed by a GSTrap FF column (GE-Healthcare, Chicago, IL, USA). The Mpro solution was further purified by FPLC size-exclusion chromatography on Superdex 75 10/300 GL column. Lopinavir, Ritonavir, Nelfinavir, Carmofur, GC376, and NMV inhibitors were bought from Selleck Chemicals GmbH, Planegg, Germany.
7
Purification of GST-Tagged TIS7 Protein
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GST-TIS7 was expressed in E. coli BL21 by addition of IPTG to 0.1 mM final concentration and incubation at 20 °C overnight. The recombinant protein was purified using a GSTrapFF column with glutathione Sepharose beads (GE Healthcare). Elution was initiated with lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 10 μg/mL aprotinin, 1 μg/mL pepstatin, 1 μg/mL leupeptin, 0.4 mM Pefablock SC containing 10 mM glutathione (Sigma)). Additional purity was achieved via gel filtration, using a Sepharyl S200 column (GE Healthcare). The GST-TIS7 sample was dialyzed against 50 mM Tris pH 7.4, 150 mM NaCl.
8
Purification of CDTa Constructs
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Cell pellets were re-suspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA; 2 ml/g of pellet). Cells were passed through a homogeniser twice at a pressure of 20kpsi at 4 °C. The cell lysate was centrifuged at 25,000 rpm for 35 min at 4 °C and the resultant supernatant was filtered through both a Minisart® GF-pre-filter (Sartorius) and 0.45 µm millex-HA filter (Millipore). The filtered supernatant was loaded onto a 5 ml GSTrap-FF column (GE Healthcare) at 0.5 ml/min in lysis buffer. GST-tagged CDTa constructs were eluted at 0.5 ml/min in 20 mM reduced glutathione in lysis buffer. The GST-tag was removed during overnight dialysis at 4 °C against 50 mM Tris-HCl pH 8.0 and 20 mM NaCl, by adding PreScission™ protease (1U per 100 μg of protein, GE Healthcare) to the purified protein. Dialyzed protein was centrifuged at 4000g to remove any precipitated protein and re-loaded onto the GSTrap-FF column as above. Purified CDTa constructs were collected in flow through, concentrated to 5.0 mg/ml (Amicon Ultra-15, Millipore), flash frozen in liquid nitrogen and stored at −80 °C until further use.
9
Recombinant Protein Expression and Purification
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The pET28-syn-AtHEN1, pET28a-syn-AtMTase, and pET28-MpHEN1 plasmids were respectively transferred into E. coli. BL21 strain for recombinant protein expression. The bacteria pellets were collected from 400 mL bacterial culture with 0.125 mM Isopropyl, and β-d -1-thiogalactopyranoside (IPTG) induction was lysed with 150 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM DTT, and 10 mM imidazole) with freshly prepared 1 mM phenylmethylsulfonyl fluoride (PMSF). The recombinant proteins were purified with 1 mL HisTrap column (GE Healthcare, Chicago, IL, USA) by FPLC (GE Healthcare, Chicago, IL, USA). The collected fractions were checked on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dialyzed twice with dialysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 2 mM MgCl2). For the purification of GST-HC-ProR and GST-HC-ProK, the pGEX-HC_proR and pGEX-HC_proK plasmids were transformed into E. coli. BL21 strain and purified using the GSTrap FF column (GE Healthcare, Chicago, IL, USA).
10
Purification and Characterization of GST-SlCaM6 Fusion Protein
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pDEST15 vector expressing GST‐SlCaM6 was transformed into Rosetta™(DE3) competent cells, and the fusion protein was induced and purified with GSTrap column (GSTrap FF column, GE Healthcare) following the user manual. Native polyacrylamide gel electrophoresis (Native‐PAGE) was carried out as described previously (Niepmann & Zheng, 2006 ; Arndt et al., 2012 ). Equal volumes of 133 μM synthetic peptide and 50 μM GST‐ or His‐ SlCaM6 were mixed and incubated for 1 h at 4°C. Peptide–protein interactions were analysed on 12% tris‐glycine native gels (30 mM Tris‐HCl pH 7.5, 190 mM glycine, 5 mM DTT). NativeMark Unstained Protein Standard (Life Technologies) was used as a protein marker for the analysis. The electrophoresis was performed with the Mini‐PROTEAN Tetra Cell apparatus (BioRad) in native running buffer (25 mM Tris base, 192 mM Glycine) at a low current (10 mA) at 4°C. The proteins on the gel were visualized by Coomassie blue staining (Blakesley & Boezi, 1977 ).
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