DPYS was cloned into the pET-28a (+) (Novagen) vector containing an N-terminal 6×His tag. The protein was overexpressed in Escherichia coli BL21(DE3) host cells and induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich) for 3 h in presence of 1 mM ZnCl2. Cells were lysed with Buffer A (50 mM Potassium Phosphate pH 7.5, 150 mM NaCl, 0.1% NP40, 15 mM Imidazol (Sigma-Aldrich)) and protease inhibitors (Roche). Extracts were incubated for 30 min at 4°C and harvested at 28 000 rpm for 1 h. The soluble supernatant fraction was purified on a 5 ml HisTrap HP column (GE Healthcare) using the AKTA protein purification system (GE Healthcare). The column was washed with 10 column volumes of Buffer A with 60 mM Imidazol. Bound protein was eluted from the column using Buffer A with 250 mM Imidazol. The fractions corresponding to each peak in the chromatogram were dialysed against buffer containing 50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM DTT and 10% glycerol.
Imidazol
Manufactured by Merck Group
Sourced in United States, Germany
Imidazol is a chemical compound used in various laboratory applications. It is a heterocyclic aromatic organic compound composed of a five-membered ring containing two nitrogen atoms. Imidazol is often used as a buffer in biochemical and analytical procedures due to its ability to maintain a stable pH range.
Automatically generated - may contain errors
Lab products found in correlation
Histrap hp column, by GE Healthcare (1 mentions)
Akta protein purification system, by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Mtase glo methyltransferase assay kit, by Promega (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Cm5 sensor chip, by GE Healthcare (1 mentions)
Histrap ff crude, by GE Healthcare (1 mentions)
Discovery studio 2018r2, by Dassault Systèmes (1 mentions)
White 384 well plate, by Corning (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Complete his tag purification column, by Roche (1 mentions)
Bradford total protein assay kit, by ZellBio (1 mentions)
Kanamycin sulphate, by Merck Group (1 mentions)
Arachidonic acid, by Cayman Chemical (1 mentions)
Dithiotreitol, by Merck Group (1 mentions)
Eicosapentaenoic acid, by Cayman Chemical (1 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (1 mentions)
4 protocols using imidazol
1
Overexpression and Purification of DPYS
2
ZIKV Methyltransferase Functional Assay
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Dithiothreitol, imidazol, Tris-HCl and other common assay reagents were purchased from Sigma (St. Louis, MO, USA). The ZIKV cDNA was synthesized by Invitrogen (Waltham, MA, USA). The ZIKV RNA sequence for methyltransferase reaction was synthesized de novo by Genscript (Nanjing, Jiangsu, CHN). The RNA capping kit was purchased from New England Biolabs (Ipswich, MA, USA). MTase-Glo Methyltransferase Assay kit was purchased from Promega (Madison, WI, USA). HisTrap FF crude and CM5 sensor chip were purchased from GE (Uppsala, Sweden). 384-well white plates were obtained from Corning (Corning, NY, USA). Discovery Studio 2018 R2 software was provided by Accelrys (San Diego, CA, USA). The screening library was a natural product library provided by MedChem Express (Monmouth Junction, NJ, USA).
3
Recombinant Protein Production and Purification
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For production of milligram quantities of the recombinant protein, optimized conditions were scaled up accordingly. The E. coli BL21 harboring pET23a/rv1733 construct was grown in 200 ml of Terrific-Broth in 1 L baffled flask at 37°C to an OD
600
of 0.6, followed by induction with 0.4 mM IPTG for 10 h. The cells were then pelleted by centrifugation and stored at −20°C before being lysed.
The cell pellets were resuspended in the selected lysis buffer (20 ml/g cell pellet, 20 mM NaH
2
PO
4
, 500 mM NaCl and 6 M urea, pH 8) (Merck, Darmstadt, Germany) and incubated for 15 min on ice. The suspension was sonicated six times for 20 s and cell debris was removed by centrifugation (13000 g, 30 min, 4°C). The supernatant was passed through 0.45 μm filter before loading on complete His-Tag Purification Column (Roche, Mannheim, Germany). The column was washed extensively with washing buffer (20 mM NaH
2
PO
4
, 500 mM NaCl, pH 8) until the OD
280
of the buffer reached below 0.01. The proteins were then eluted using a column volume of 500 mM imidazol (Sigma-Aldrich), pH 8 containing complete protease inhibitor (Sigma-Aldrich). The eluted fractions were combined and dialyzed against PBS (pH 7.4), and quantified by Bradford total protein assay kit (ZellBio GmbH, Ulm, Germany). LPS contamination was evaluated by the Limulus amoebocyte lysate (LAL) assay (Cambre, USA).
600
of 0.6, followed by induction with 0.4 mM IPTG for 10 h. The cells were then pelleted by centrifugation and stored at −20°C before being lysed.
The cell pellets were resuspended in the selected lysis buffer (20 ml/g cell pellet, 20 mM NaH
2
PO
4
, 500 mM NaCl and 6 M urea, pH 8) (Merck, Darmstadt, Germany) and incubated for 15 min on ice. The suspension was sonicated six times for 20 s and cell debris was removed by centrifugation (13000 g, 30 min, 4°C). The supernatant was passed through 0.45 μm filter before loading on complete His-Tag Purification Column (Roche, Mannheim, Germany). The column was washed extensively with washing buffer (20 mM NaH
2
PO
4
, 500 mM NaCl, pH 8) until the OD
280
of the buffer reached below 0.01. The proteins were then eluted using a column volume of 500 mM imidazol (Sigma-Aldrich), pH 8 containing complete protease inhibitor (Sigma-Aldrich). The eluted fractions were combined and dialyzed against PBS (pH 7.4), and quantified by Bradford total protein assay kit (ZellBio GmbH, Ulm, Germany). LPS contamination was evaluated by the Limulus amoebocyte lysate (LAL) assay (Cambre, USA).
4
Peroxidase Activity Assay Protocol
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Dithiotreitol (DTT), N-ethylmaleimide (NEM), beta-mercaptoethanol (β-ME), reduced nicotinamide adenine dinucleotide phosphate (NADPH), isopropyl-β-d -thiogalactopyranoside (IPTG), kanamycin sulphate, ampicillin, 2-iodoacetamide, diethylenetriaminepentaacetic acid (DTPA), 8-anilino-1-naphtalene sulfonic acid (ANS) and imidazol were from Sigma-Aldrich (Darmstadt, Germany). H2O2 was obtained from Mallinckrodt Chemicals (St. Louis, MO, USA). 15(S)-HpETE (≥98% pure), 15(S)-HpEPE (≥98% pure), 9α,11α-epidioxy-15S-hydroperoxy-prosta-5Z,13E-dien-1-oic acid (PGG2, ≥95% pure), arachidonic acid (AA, ≥98% pure) and eicosapentaenoic acid (EPA, ≥98% pure) (Supplementary Figure S1 ) were obtained from Cayman Chemical (Ann Arbor, MI, USA) as ethanolic solutions (or in acetone solution in the case of PGG2) and stored under argon at −80 °C. All other reagents were obtained from standard commercial sources and used as received. Experiments were performed in 50 mM phosphate buffer plus 0.1 mM DTPA, pH 7.8 and 25 °C, except otherwise indicated.
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