FLAG tagged Dachs and Dlish fragment proteins were generated using Non-Radioactive TNT T7 Quick Coupled Transcription/Translation systems (Promega L1170). 300 μl of binding buffer (1X PBS, 0.1% NP40, 10% Glycerol, 0.5 mM DTT, protease inhibitor) PI, DTT, and 10 μl of the FLAG-tagged proteins were added to cleaned FLAG M2 beads (Sigma A2220), incubated at 4°C for 4 hr, and washed. pGEX2T-Dlish or -Dachs-1110-1232 was expressed in BL21(DE3)pLysS E. coli competent cells (Promega L1195), purified using their GST tags, and 4 μg incubated with protein-bound FLAG beads in binding buffer at 4°C for 2 hr, then washed and boiled for 8 min with 25 μl 2 × SDS/protein sample buffer prior to loading for SDS-PAGE.
E coli bl21 de3 plyss competent cells
Manufactured by Promega
Sourced in Malaysia, United States
E. coli BL21 (DE3) pLysS competent cells are a strain of Escherichia coli bacteria that have been genetically modified to enable efficient protein expression. These cells are designed for the high-level expression of recombinant proteins under the control of the T7 promoter.
Automatically generated - may contain errors
Lab products found in correlation
Flag m2 bead, by Merck Group (1 mentions)
Pet28b, by Thermo Fisher Scientific (1 mentions)
Hispur ni nta superflow agarose, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b bead, by Cytiva (1 mentions)
Pet32a expression vector, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by AppliChem (1 mentions)
Pet30a, by Merck Group (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Econo column chromatography column, by Bio-Rad (1 mentions)
9 protocols using e coli bl21 de3 plyss competent cells
1
Protein Binding Assay for Dachs and Dlish
2
Purification and Antibody Generation of Dlish
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The full length dlish cDNA was amplified from pUAST-attB-Dlish-FLAG, in-fusion cloned into pET-28b(+) (Invitrogen), transformed into BL21(DE3)pLysS E. coli competent cells (Promega L1195), and protein expressed induced with 100 μM IPTG at 25°C for 13 hr. His-Dlish protein was purified with HisPur Ni-NTA Superflow Agarose (Thermo 25214), extracted, run on SDS-PAGE, and Coomassie stained. 4–5 mg of His-Dlish protein on the gel was used by Genemed Synthesis Inc. to immunize rabbits. Antiserum was affinity purified using GST-Dlish-coupled Sepharose.
3
Recombinant Sol g 2.1 Protein Production
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The Sol g 2.1 coding sequence (GenBank: UYX46120.1) in the pProEx-HTB expression vector, which is composed of His6-tagged protein at the C-terminus, was expressed in E. coli BL21 (DE3) pLysS competent cells (Promega, Selangor, Malaysia), as described previously [22 (link)]. A single colony was inoculated in Luria–Bertani (LB) medium containing 50 μg/mL of Ampicillin at 37 °C overnight. The cell culture was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). After harvesting the cells, the cell pellets were extracted in a lysis buffer (80 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, and 4% glycerol, pH 7.2). The protein accumulated largely in insoluble inclusion bodies; it was then refolded using 8 N guanidinium HCl [23 (link)]. After denaturation and renaturation, the soluble protein solution was purified using a nickel affinity column (His-Bind resin, Novagen, Madison, WI, USA) using 20 mM Tris-HCl pH 7.4, 500 mM NaCl, and 20 mM imidazole as a binding buffer. His6-tagged proteins were eluted by increased concentrations of imidazole (50–500 mM) following the desalting step. For delipidation, the purified protein was incubated with methyl-functionalized methacrylate HIC resin (hydrophobic interaction chromatography, Bio-Rad, Hercules, CA, USA) in 50 mM Tris-HCl pH 7.4 at 4 °C for three days on a rotary mixer [28 (link)].
4
Purification of Recombinant Calmodulins from E. coli
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The resulting plasmids were introduced into E. coli BL21(DE3) pLysS-competent cells (Promega, Madison, WI, USA) in order to produce the fusion proteins with a glutathione-S-transferase (GST) affinity tag. The transformants were grown in LB medium (500 mL) containing ampicillin (100 μg/mL) and 2% glucose at 37 °C. Fusion protein expression was induced by adding isopropyl-ß-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM at OD600 = 0.6 and incubating the culture at 20 °C for 3.5 h. The bacteria were harvested by centrifugation, and the pellet was suspended in lysis buffer (50-mM Tris-HCl, pH 8.0, 150-mM NaCl, 5-mM EDTA, 5-mM EGTA, 1 % (v/v) Triton X-100, 1-mM PMSF and 0.2-mg/mL lysozyme) and disrupted by sonication. The cell extract was centrifuged at 18,000× g for 35 min, and the supernatant was loaded onto glutathione-Sepharose 4B beads (Cytiva, Uppsala, Sweden). Afterward, the column was washed multiple times with a buffer containing 50-mM Tris-HCl (pH 8.0) and 150-mM NaCl, and the GST fusion protein was eluted with 10-mM glutathione in 50-mM Tris-HCl (pH 9.0). The recombinant calmodulins (CaM1, 3, 7 and CML9) were purified according to Reference [32 (link)]. The homogeneity and purity of the eluted protein fraction was analyzed by SDS–PAGE electrophoresis (10% gel) with the Coomassie Blue gel staining.
5
Cloning and Expression of Sol g 4.1 Protein
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The Sol g 4.1 gene was subcloned from the pGEM-T easy vector into the pET-32a expression vector (Invitrogen, UK). Briefly, the vectors were double digested with NcoI and XhoI restriction enzymes, and the Sol g 4.1 gene was ligated into the same restriction sites of the pET-32a expression vector. Recombinant plasmids were transformed into E. coli BL21 (DE3) pLysS competent cells (Promega, Malaysia). A single colony from a freshly streaked plate was picked, inoculated in LB (Sigma-Aldrich, USA) starter media containing 50 μg/mL ampicillin and incubated at 37 °C overnight with shaking until the culture was turbid but not saturated.
The 5-mL starter cultures were transferred into 500 mL of LB expression medium containing 100 μg/mL ampicillin and incubated at 37 °C until the cell density reached to OD600 of ~ 0.5. Afterwards, the temperature was reduced to 30 °C and the culture was induced with 0.4 mM IPTG. The induced cultures were grown for 8 h. Cell pellets were collected and washed with 10 mL of lysis buffer (20 mM sodium phosphate, pH 7.4, 100 mM NaCl, 1 mM DTT, and 0.1 mM PMSF) and disrupted by sonication on ice. After centrifugation at 15,000×g for 20 min at 4 °C, the supernatants were separated on 13% SDS-PAGE gels.
The 5-mL starter cultures were transferred into 500 mL of LB expression medium containing 100 μg/mL ampicillin and incubated at 37 °C until the cell density reached to OD600 of ~ 0.5. Afterwards, the temperature was reduced to 30 °C and the culture was induced with 0.4 mM IPTG. The induced cultures were grown for 8 h. Cell pellets were collected and washed with 10 mL of lysis buffer (20 mM sodium phosphate, pH 7.4, 100 mM NaCl, 1 mM DTT, and 0.1 mM PMSF) and disrupted by sonication on ice. After centrifugation at 15,000×g for 20 min at 4 °C, the supernatants were separated on 13% SDS-PAGE gels.
6
Heterologous Expression of Polyketide Synthase
7
Cloning and Expression of JEV NS3 Helicase
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The NS3 helicase gene (amino acid residues 180-619 of NS3) was cloned from the JEV P3 strain (GenBank accession number U47032.1) and then subcloned into prokaryotic expression vector pET-30a (Merck, Darmstadt, Germany). The expression plasmid was transformed into E.coli BL21 (DE3) pLysS competent cells (Promega, Madison, WI, USA) for protein expression. Baby hamster kidney 21 (BHK-21) cells were cultured in growth medium at 37 °C with 5% CO2 and 80–95% humidity. The growth medium was Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (Invitrogen, Grand Island, NY, USA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Sigma-Aldrich). The JEV P3 strain was propagated in BHK-21 cells with maintenance medium containing 1% FCS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The compounds were purchased from Specs (Delft, Netherlands) and dissolved in dimethyl sulphoxide (DMSO) (Applichem, Darmstadt, Germany) to 10 mM.
8
Matk Protein Expression and Purification in E. coli
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The final expression clone containing the matK reading frame was heat shock transformed into E. coli BL21 DE3 pLysS competent cells (Promega) and protein induced using IPTG following standard transformation and induction protocols using IPTG at a final concentration of 500 µM. Induced cultures were shaken at 210 rpm at 35°C for an additional three hours before pelleting cultures at 10,000 rpm for 10 min for 4°C. Cold temperature spins were used to prevent protein degradation. Induction at 37°C was found to induce proteolysis of MatK protein in E. coli (data not shown); therefore, a lower temperature of 35°C was used for protein induction. Pre‐ and post‐induction samples were assayed for MatK expression by immune detection using one of two different anti‐MatK antibodies, a commercial antibody from Agrisera (AS132720) or an epitope‐derived antibody specifically designed against the MatK protein sequence in rice (Barthet & Hilu, 2007 ).
9
Recombinant His-AlkB Protein Purification
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The expression and purification of recombinant His-AlkB were performed as previously described with modifications 57 (link) . Briefly, the His-AlkB-AVA421 plasmid was transformed into E. coli BL21(DE3)pLysS competent cells (Promega, Cat# L1191) and cultured in Overnight Express™ Instant TB Medium (Sigma, Cat# 71491) at 37 ℃ to OD 600 1.2.
The cell pellet was resuspended and lysed by sonication and the cell debris was removed by centrifugation. Then, the clarified supernatant from the crude extract was carefully collected and mixed with TALON Metal Affinity Resin (Takara Bio, Cat# 635502) for 1 hour to allow for the binding of His-tagged AlkB protein to resin. After several washing steps, the resin-bind proteins were eluted and separated by Econo-Column® Chromatography Columns (BioRad, Cat# 7371512). The protein concentration of eight collected fractions was measured and the three protein-enriched fractions were pooled and dialyzed successively in Dialysis Buffer and Storage Buffer.
The dialyzed protein supernatant containing recombinant His-AlkB protein was validated by Coomassie Bule staining and western blotting, and used for ARM-seq.
The cell pellet was resuspended and lysed by sonication and the cell debris was removed by centrifugation. Then, the clarified supernatant from the crude extract was carefully collected and mixed with TALON Metal Affinity Resin (Takara Bio, Cat# 635502) for 1 hour to allow for the binding of His-tagged AlkB protein to resin. After several washing steps, the resin-bind proteins were eluted and separated by Econo-Column® Chromatography Columns (BioRad, Cat# 7371512). The protein concentration of eight collected fractions was measured and the three protein-enriched fractions were pooled and dialyzed successively in Dialysis Buffer and Storage Buffer.
The dialyzed protein supernatant containing recombinant His-AlkB protein was validated by Coomassie Bule staining and western blotting, and used for ARM-seq.
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