The gene encoding the BTN3A1-B30.2 domain (R322-A513) was cloned into pET26 with a C-terminal His tag, and transformed into BL21 (DE3) competent E. coli cells (New England Biolabs). Transformed E. coli were cultured at 37°C until an OD600 of 0.4-0.6 was attained. Protein expression was induced with 0.5 mM Isopropyl-β-D-1-Thiogalactopyranoside and cells were cultured for a further 18-20 hours at 18°C. The pelleted bacterial biomass was resuspended into 50mM Sodium phosphate pH 7.4, 500mM Sodium chloride, 50mM Imidazole, 0.5mM Tris (2-carboxyethyl) phosphine (TCEP) and COmplete EDTA free protease inhibitors (Sigma-Aldrich) and homogenised using an AVESTIN EmulsiFlex C3 and centrifuged at 19000rpm/4°C for 30 minutes. The harvested supernatant was loaded onto a 5ml Hi-Trap column (GE) overnight. The column was washed with 5 column volumes of resuspension buffer to prevent non-specific binding. His-tagged protein was eluted with resuspension buffer containing a high concentration of imidazole (300mM) and further purified by size exclusion chromatography using a HiLoad 26/60 Superdex 200 column pre-equilibrated with 50mM Sodium Phosphate pH 7.4, 150mM Sodium Chloride, 0.5mM TCEP and COmplete EDTA free protease inhibitors.
E coli bl21 de3 competent cells
Manufactured by New England Biolabs
Sourced in United States
E. coli BL21(DE3) competent cells are a laboratory strain of Escherichia coli bacteria that are commonly used for the expression of recombinant proteins. These cells contain the DE3 lysogen, which allows for the induction of protein expression from the T7 promoter. The BL21 strain is known for its high transformation efficiency and lack of certain proteases, making it well-suited for the production of proteins.
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Lab products found in correlation
Emulsiflex c3, by Avestin (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Xhoi, by New England Biolabs (2 mentions)
E coli dh5α competent cells, by New England Biolabs (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Wizard plus sv miniprep and sv gel and pcr extraction kits, by Promega (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Ampicillin trisodium salt, by Merck Group (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (2 mentions)
Luria broth, by Merck Group (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Complete edta free protease inhibitor, by Merck Group (1 mentions)
Pet 15b vector, by GenScript (1 mentions)
Sephacryl s 200 hr column, by GE Healthcare (1 mentions)
Ni nta chromatography, by Qiagen (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Plasmid miniprep kit, by Promega (1 mentions)
Bamhi and xhoi restriction enzymes, by New England Biolabs (1 mentions)
26 protocols using e coli bl21 de3 competent cells
1
Purification of BTN3A1-B30.2 Domain
2
Recombinant Expression and Purification of bm AANAT3 Protein
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Full-length bmAANAT3 cloned into a pET28a vector (thrombin cleavage site and kanamycin resistance) was expressed in BL21(DE3) competent E. Coli cells (New England Biolabs) as a fusion protein with an N-terminal His-tag. Cell growth was performed at 37°C and protein expression was induced at an OD600 = 0.5 by the addition of 1 mM IPTG, for 5 hours. Cells were harvested by centrifugation at 4°C, resuspended in 50 mM Tris-HCl, pH = 8.0, 20 mM imidazole, 500 mM NaCl, 1 mM Phenylmethylsulfonyl fluoride and 5 mM β-mercaptoethanol and lysed by sonication. Cell debris were removed by centrifugation at 35,000 x g (4°C). The clarified lysate was loaded on a Ni2+ affinity column, washed with 10 column volumes of cell lysis buffer and eluted with 400 mM imidazole in the same buffer. Fusion bmAANAT3 was further purified through a Superdex-75 gel-filtration column using a 50 mM Tris-HCl pH = 8.0, 150 mM NaCl, 0.5 mM EDTA and 5 mM β-mercaptoethanol buffer. The purification polyhistidine tag was removed by incubating bmAANAT3 with biotinylated thrombin (2U/mg). 15N- or 13C/15N-labeled bmAANAT3 was prepared using the same expression and purification protocol, except that cells were grown in M9 media containing 15N-NH4Cl and 13C-glucose as the sole source of nitrogen and carbon, respectively.
3
Recombinant expression of EHEC proteins
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EHEC (EDL933) DNA was isolated with a DNeasy Blood and Tissue Kit (Qiagen), following the manufacturer’s directions. To improve recombinant protein solubility, the N-terminus signal sequences of EscC (GenBank protein accession no. 12518466) and LomW (GenBank protein accession no. 12514345) were predicted using the SignalP 6.0 server, and the DNA segments without the predicted signal sequences were cloned into a pET30a(+) expression vector using NdeI and XhoI (New England BioLabs) restriction sites. The open reading frame for each protein was inserted in frame with a 6⨉His-tag on the C-terminus. Following ligation, pET30a(+)-EscC and pET30a(+)-LomW were transformed into DH5α competent E. coli cells following the manufacturer’s protocol (New England Biosciences). Upon confirmation of successful gene insertion via gel electrophoresis and directional sequencing (Azenta Life Sciences), plasmids were transformed into BL21 (DE3) competent E. coli cells (New England BioLabs) according to the manufacturer’s protocol.
4
Recombinant Protein Expression
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Double-stranded DNA
gBlocks gene fragments encoding ABD, anionic block, cleavable linker,
and C-terminal cysteine with flanking NcoI and XhoI restriction sites
were ordered from Integrated DNA Technologies (IA, U.S.A.). The gene
fragments were cloned into Novogen pET28a(+) vector at the NcoI and
XhoI restriction sites and transformed into DH5α competent E. coli cells (New England Biolabs Inc., MA, U.S.A.). Selection
of the correctly cloned bacterial colonies was confirmed by Sanger
sequencing (Quintara Biosciences, CA, U.S.A.). The corresponding plasmids
were harvested and transformed into BL21(DE3) competent E.
coli cells (New England Biolabs Inc., MA, U.S.A.) for protein
expression.
gBlocks gene fragments encoding ABD, anionic block, cleavable linker,
and C-terminal cysteine with flanking NcoI and XhoI restriction sites
were ordered from Integrated DNA Technologies (IA, U.S.A.). The gene
fragments were cloned into Novogen pET28a(+) vector at the NcoI and
XhoI restriction sites and transformed into DH5α competent E. coli cells (New England Biolabs Inc., MA, U.S.A.). Selection
of the correctly cloned bacterial colonies was confirmed by Sanger
sequencing (Quintara Biosciences, CA, U.S.A.). The corresponding plasmids
were harvested and transformed into BL21(DE3) competent E.
coli cells (New England Biolabs Inc., MA, U.S.A.) for protein
expression.
5
Bacterial Expression of His-Tagged Protein
6
Recombinant GAPDH Protein Purification
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The cDNA sequence of human GAPDH (OriGene, UK) was inserted into the pET-28b(+) vector (Novagen). Six residues of histidine were inserted at the N-terminus for purification by Ni-NTA chromatography and a FLAG-tag inserted at the C-terminus. The vector was transformed into BL21(DE3) competent E. coli cells (New
England Biolabs, UK) for expression. Sequences of GAPDH gene and primers used for cloning are given in Supplementary Table6 . 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma) was used to induce expression of the gene in cells cultured at 37 °C. GAPDH protein was purified by Ni-NTA chromatography (Qiagen) under native conditions, using standard protocols recommended by the manufacturer. Ice-cold sodium-phosphate buffer, pH 8.0 (50 mM Na2HPO4/NaH2PO4, 10 mM Tris-Cl, 300 mM NaCl, 5 mM imidazole) was used during resuspension and lysis of the BL21(DE3) cells. Purified GAPDH protein was eluted with 250 mM imidazole in sodium-phosphate buffer, pH 6.0. Protein samples were dialyzed and passed through a Sephacryl s-200 HR column (GE Health Care) for further purification. The protein was analysed by SDS-PAGE, and the size of the protein was confirmed by mass spectrometry, using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).
England Biolabs, UK) for expression. Sequences of GAPDH gene and primers used for cloning are given in Supplementary Table
7
Recombinant Expression of Human DNPH1 Proteins
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The DNA-encoding HsDNPH1
(UniProtO43598 ) and a truncated HsDNPH1 (HsDNPH1Trunc), each containing a TEVP-cleavable N-terminal His-tag
with restriction sites for NdeI and HindIII at the 5′- and
3′-ends, respectively, were purchased as codon-optimized (for
expression in E. coli) gBlocks (IDT). HsDNPH1- and HsDNPH1Trunc-encoding
gBlocks were polymerase chain reaction-modified and inserted into
modified pJexpress414 plasmids using Gibson Assembly12 (link) according to the manufacturer’s instructions (New
England Biolabs). Constructs were used to transform E. coli DH5α-competent cells (New England Biolabs),
were subsequently sequenced (Eurofins) to confirm the insertion of
the gene and that no mutations had been introduced, and then used
to transform E. coli BL21(DE3)-competent
cells (New England Biolabs). Transformed cells were grown independently
in lysogeny broth containing 100 μg mL–1 ampicillin
at 37 °C until an optical density at 600 nm (OD600) of 0.6–0.8 before expression was induced with 0.5 mM IPTG.
Cells were grown for an additional 3 h, harvested by centrifugation
(6774g, 15 min, 4 °C), and stored at −20
°C.
(UniProt
with restriction sites for NdeI and HindIII at the 5′- and
3′-ends, respectively, were purchased as codon-optimized (for
expression in E. coli) gBlocks (IDT). HsDNPH1- and HsDNPH1Trunc-encoding
gBlocks were polymerase chain reaction-modified and inserted into
modified pJexpress414 plasmids using Gibson Assembly12 (link) according to the manufacturer’s instructions (New
England Biolabs). Constructs were used to transform E. coli DH5α-competent cells (New England Biolabs),
were subsequently sequenced (Eurofins) to confirm the insertion of
the gene and that no mutations had been introduced, and then used
to transform E. coli BL21(DE3)-competent
cells (New England Biolabs). Transformed cells were grown independently
in lysogeny broth containing 100 μg mL–1 ampicillin
at 37 °C until an optical density at 600 nm (OD600) of 0.6–0.8 before expression was induced with 0.5 mM IPTG.
Cells were grown for an additional 3 h, harvested by centrifugation
(6774g, 15 min, 4 °C), and stored at −20
°C.
8
Cloning and Expression of AiiA Quorum-Quenching Enzyme
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The gene coding for AiiA was cloned into pET-47b(+) vector (BamHI-HindIII sites). The expression vector was transformed into E. coli BL21(DE3) competent cells (New England Biolabs, USA). The cells were grown in 2 L of LB media supplied with 35 mg/L Kanamycin at 37 °C. The expression of the protein was induced with 0.5 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) when OD600 reached 0.6. The cells were grown overnight at 18 °C. The harvested cells were resuspended in 50 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% (v/v) CHAPS, 10% (v/v) glycerol) and lysed by passing the homogenized cells through an Emulsiflex-C3 (Avestin, USA) high-pressure apparatus at 15,000 psi three times. The cell lysate was centrifuged at 25,000 g for 25 min. The supernatant was then applied to Ni-NTA gravity column (Bio-rad) equilibrated with lysis buffer. After extensive washing with lysis buffer, the bound proteins were eluted with lysis buffer containing increasing concentration of imidazole (0–300 mM). The eluted fractions were analyzed with 15% of SDS-PAGE and fractions containing the desired protein were pooled and dialyzed against lysis buffer. The final concentration of the protein was measured using Bradford Assay.
9
Recombinant Metalloprotein Expression and Purification
10
Engineered Human cMyBP-C Protein Variants
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pET45b vectors encoding Escherichia coli optimized codons for the C0–C1 or C0–C2 portion of human cMyBP-C with N-terminal His6 tag and tobacco etch virus protease cleavage site were obtained from GenScript (Piscataway, NJ). In addition, C0–C1 and C0–C2 mutants were generated with S133D substitutions to test the mimicry of GSK3β-mediated phosphorylation of the P/A linker. For testing the mimicry of PKA-mediated phosphorylation of the M-domain, C0–C2 mutants were generated with Asp for Ser substitutions in three PKA sites (3SD: S275D, S284D, and S304D). Mutations were engineered in the human cMyBP-C fragments using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA). All sequences were confirmed by DNA sequencing (Eton Bioscience, San Diego, CA). Protein production in E. coli BL21DE3-competent cells (New England Biolabs, Ipswich, MA) and purification of C0–C1 and C0–C2 fusion proteins using His60 Ni Superflow Resin was done as described (21 (link)). cMyBP-C (cleaved of the His-tag) was then concentrated, dialyzed to 50/50 buffer (50 mm NaCl and 50 mm Tris, pH 7.5), and stored at 4 °C. Proteins were typically used for experiments within 2 weeks.
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