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Escherichia coli bl21 de3

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Escherichia coli BL21 (DE3) is a commonly used bacterial strain for protein expression. It is a derivative of the E. coli B strain and contains the DE3 lysogen, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter. This strain is designed for the high-level expression of recombinant proteins under the control of the T7 promoter.

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Lab products found in correlation

Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b beads, by GE Healthcare (2 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (2 mentions) Lactulose, by Merck Group (1 mentions) Epilactose, by Merck Group (1 mentions) Ex taq polymerase, by Takara Bio (1 mentions) T4 dna ligase, by Takara Bio (1 mentions) Lactose, by Merck Group (1 mentions) Akta express, by GE Healthcare (1 mentions) Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions) Biotin protein ligase bira kit, by Avidity (1 mentions) Lb broth, by A&A Biotechnology (1 mentions) His select nickel affinity gel, by Merck Group (1 mentions) Lysozyme, by Merck Group (1 mentions) Dnase 1, by Merck Group (1 mentions) Profinity imac ni charged resin, by Bio-Rad (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) E coli m15, by Qiagen (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions)

Spelling variants of this product

BL21(DE3) (90 citations) Escherichia coli BL21(DE3) cells (13 citations) Escherichia coli BL21 (7 citations) Escherichia coli strain Bl21 (DE3) (6 citations) Escherichia coli BL21 (DE3) competent cells (5 citations) Escherichia coli (4 citations) Escherichia coli BL21 Star (DE3) (3 citations) Escherichia coli DH5αand BL21 (DE3) cells (2 citations) Escherichia coli strain BL21-Gold (DE3) (2 citations)

28 protocols using escherichia coli bl21 de3

1

Cloning and Characterizing Carbohydrate-Active Enzymes

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The restriction endonucleases NheI and EcoRI, T4 DNA ligase, and EX taq polymerase were obtained from Takara (Kyoto, Japan). The expression vector pET28a (+) was obtained from Novagen (Darmstadt, Germany). Plasmid DNA was purified by using DNA mini-preparation kits (Qiagen, Hilden, Germany). Escherichia coli BL21 (DE3) (New England Biolabs, Hertfordshire, UK) was used for protein expression. C. saccharolyticus DSM 8903 cell was purchased from DSMZ (Braunschweig, Germany). Standard sugars (lactose, epilactose, and lactulose) for high-performance liquid chromatography (HPLC) were purchased from Sigma-Aldrich (MO, USA).
Park A.R., Kim J.S., Jang S.W., Park Y.G., Koo B.S, & Lee H.C. (2017). Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus. Microbial Cell Factories, 16, 224.
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2

Recombinant Etf-1 Protein Production

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Avi-tagged full-length recombinant Etf-1 protein (rEtf-1) and truncated rEtf-1 proteins (primers shown in SI Appendix, Table S2) were expressed in Escherichia coli BL21(DE3) (New England Biolabs) and purified by affinity chromatography using HisPur Cobalt resin (Thermo Scientific) as described previously (7 (link)). The proteins were further purified by size-exclusion chromatography on an AKTA express (GE Healthcare) with a Superdex 200 Increase 10/300 GL column (GE Healthcare). For biotinylation, rEtf-1 was separated in Tris buffer (10 mM Tris, pH 8.0; 10 mM NaCl; 0.05% CHAPS; 0.05% sodium deoxycholate [NaDoc]) and concentrated to 2.2 mg/mL and was then biotinylated at the C-terminal Avi-tag using BirA Biotin-Protein Ligase Kit (Avidity). The reaction mixture was subjected to size-exclusion chromatography with a Superdex 200 Increase 10/300 GL column to remove BirA and d-biotin and then underwent a buffer exchange to PBS (8 mM Na2HPO4, 1.47 mM KH2PO4, 2.67 mM KCl, 137.9 mM NaCl, pH 7.4) containing 0.05% CHAPS and 0.05% NaDoc. The biotinylation of rEtf-1 was verified by Western blotting, and the labeled protein was then used for ELISA, panning, and OpenSPR analyses.
Zhang W., Lin M., Yan Q., Budachetri K., Hou L., Sahni A., Liu H., Han N.C., Lakritz J., Pei D, & Rikihisa Y. (2021). An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection. Proceedings of the National Academy of Sciences of the United States of America, 118(18), e2024102118.
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3

Recombinant Protein Isolation and Purification

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Genes coding for recombinant proteins were chemically synthesized with codon optimization for Escherichia coli host expression (GenScript, Piscataway, NJ, USA). Synthetic genes were cloned into pET-51b(+) vector using SalI and HindIII restriction sites yielding N-terminal Strep-Tag II Fusion and C-terminal 10xHis-Tag fusion. Obtained plasmids were transformed into competent Escherichia coli BL21(DE3) (#C2527 New England Biolabs, Hitchin, UK). All recombinant proteins were induced in 1000 mL LB broth (#2020, A&A Biotechnology) using 0.1 mM Isopropyl β-D-thiogalactoside at OD600 = 0.5; 37 °C for 3 h with 180 rpm shaking. Cell cultures were centrifuged and pellets were lysed in 50 mL buffer containing: 50 mM NaH2PO4, 300 mM NaCl, 10% Triton X-100, 70,000 U/mL lysozyme (#62971 Merck, Darmstadt, Germany), Dnase I (#10104159001, Merck Germany) 15 µg/mL Ph = 8.0. Recombinant proteins were isolated from the lysate using IMAC chromatography with His-Select Nickel Affinity Gel (#P6611 Merck, Germany). Column gravity approach with 1 mL of resin has been performed (Table 2).
Bigus D., Lewandowska W., Bięga E., Grela A., Siedlar A., Sosnowska M., Fabisiak M., Łęga T., Dashkievich Y., Nowacka-Dośpiał J., Palka K., Żołędowska S, & Nidzworski D. (2023). Ultra-Fast Impedimetric Immunoassay for Detection of Streptococcus agalactiae Using Carbon Electrode with Nanodiamonds Film. Micromachines, 14(5), 1076.
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4

Recombinant EV-A71 Protein Expression

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Recombinant EV-A71 proteins were expressed in Escherichia coli BL21 (DE3) (New England Biolabs, USA) competent cells transformed with the pET-52b(+)-2A expression plasmid. Protein expression was induced with isopropyl-beta-D-thiogalactopyranoside (Vivantis Technologies, Malaysia) and harvested at 4 hours 30 minutes post-induction. Cells were lysed with denatured lysis buffer (8 M urea, 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), followed by sonication, and cell debris was removed by centrifugation. The protein lysates were incubated with Profinity IMAC Ni-charged resins (Bio-Rad, USA) at 4°C for 30 minutes, washed twice with denatured washing buffer (8 M urea, 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and the protein was eluted with 4 ml elution buffer (8 M urea, 50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 8.0). Purified protein was then concentrated using Amicon Ultra centrifugal filters (Merck Millipore, USA) and stored at -20°C for western blot analysis.
Aw-Yong K.L., Sam I.C., Koh M.T, & Chan Y.F. (2016). Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients. PLoS ONE, 11(11), e0165659.
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5

Culturing Borrelia recurrentis and Escherichia coli

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Borrelia recurrentis strain A17 (human blood isolate, Addis Abeba, Ethiopia) (Cutler et al., 1994 (link)) was cultured at 33 °C until the mid-exponential phase (5 x 107 spirochetes per ml) in a modified MKP medium (Margos et al., 2015 (link)) containing 50% human serum (TCS, Biosciences Ltd., Buckingham, UK) and 5.5% of BSA (Sigma-Aldrich, St. Louis, MO). Cultures of Escherichia coli BL21 (DE3) (New England Biolabs, Ipswich, MA) and E. coli M15 (Qiagen, Hilden, Germany) harboring expression vectors for the production of different His-tagged proteins were propagated routinely at 37°C in yeast tryptone broth supplemented with ampicillin (50 µg/ml).
Röttgerding F., Njeru J., Schlüfter E., Latz A., Mahdavi R., Steinhoff U., Cutler S.J., Besier S., Kempf V.A., Fingerle V, & Kraiczy P. (2022). Novel approaches for the serodiagnosis of louse-borne relapsing fever. Frontiers in Cellular and Infection Microbiology, 12, 983770.
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6

Recombinant Peroxiredoxin Protein Expression

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The TgPrx1 and TgPrx3 genes were amplified from cDNA of the T. gondii PLK strain, as described in our previous studies [9 (link),10 (link)]. In brief, PCR products digested with BamHI and XhoI were inserted into the pGEX-4T3 plasmid vector (Table S1). Recombinant TgPrx1 and TgPrx3 were expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli BL21(DE3) (New England Biolabs Inc., Ipswich, MA, USA).
Fereig R.M, & Nishikawa Y. (2022). Genetic Disruption of Toxoplasma gondii peroxiredoxin (TgPrx) 1 and 3 Reveals the Essential Role of TgPrx3 in Protecting Mice from Fatal Consequences of Toxoplasmosis. International Journal of Molecular Sciences, 23(6), 3076.
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7

Cloning and Expression of Calpain Catalytic Core

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The rat CAPN1 WT and p.V301W catalytic core sequences were cloned into a pUC57 vector with an XhoI restriction site, with an amino-terminal thrombin cleavage site and a carboxy-terminal TEV protease cleavage site followed by a 6xHis tag, as previously described (Moldoveanu et al. 2002 (link); Gakhar et al. 2016 (link)). Calpain constructs were transferred into the pMAL-C5X vector to obtain an amino-terminal maltose-binding protein (MBP) as a fusion partner. Sequence of the calpain-flanking regions was confirmed by sequencing of constructs. Plasmids were amplified and isolated from DH5α cells and then were transformed into Escherichia coli BL21 (DE3; New England Biolabs).
Velez G., Bassuk A.G., Schaefer K.A., Brooks B., Gakhar L., Mahajan M., Kahn P., Tsang S.H., Ferguson P.J, & Mahajan V.B. (2018). A novel de novo CAPN5 mutation in a patient with inflammatory vitreoretinopathy, hearing loss, and developmental delay. Cold Spring Harbor Molecular Case Studies, 4(3), a002519.
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8

Bacteroides ADPRT Cloning and Mutagenesis

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The ADPRT sequence from Bacteroides stercoris (Uniprot: B0NRS8_BACSTE) was synthesized (Genewiz) into a pET28b vector with an IPTG inducible promoter to drive expression. A cleavable 6HIS-SNAP tag for purification and fluorescence labelling was fused to the coding sequence through a GGSGGS ENLYFQ linker which includes the TEV protease cleavable sequence. Point mutations in ADPRT were performed using QuikChange XL (Agilent) and verified by DNA sequencing (Genewiz). All residue numbers described in the manuscript correspond to the ADPRT sequence only. Expression plasmids were introduced into chemically competent Escherichia coli BL21(DE3) (NEB) following standard protocols. Disruption of bxa and tdk in Bacteroides stercoris was accomplished leveraging allelic exchange of the ADPRT gene with the suicide vector pKNOCK-bla-erm carrying 750 base pairs of homology with the bxa gene and tdk gene (Alexeyev, 1999 (link)). For each genetic mutant, the integration of the vector was confirmed by selection on erythromycin and disruption of the gene was confirmed via DNA sequencing (Genewiz).
Brown E.M., Arellano-Santoyo H., Temple E.R., Costliow Z.A., Pichaud M., Hall A.B., Liu K., Durney M.A., Gu X., Plichta D.R., Clish C.A., Porter J.A., Vlamakis H, & Xavier R.J. (2021). Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors. Cell host & microbe, 29(9), 1351-1365.e11.
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9

Enzymatic Synthesis of Phosphorylated Compounds

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Escherichia coli BL21 (DE3) was obtained from New England Biolabs. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 400 MHz system equipped with a broadband probe and sample changer. Spectrophotometric data were collected on a SpectraMax340 UV-visible plate reader. Unless otherwise noted, all compounds were purchased from commercially available sources. The phosphate colorimetric detection kit (MAK 030–1KT) was obtained from Sigma-Aldrich and used according to the manufacturer’s instructions. The chemical structures of the compound used for this investigation are presented in Figure 2. The chemical syntheses of compounds 7, R/S-8, R/S-9, 10, and 13 are presented in the Supplementary Information.
Compounds S-11 and 12 were synthesized enzymatically. The reaction for the preparation of 12 contained 25 μM Cj1438, 10 mM d-glucuronate, 10 mM ATP, 20 mM MgCl2, 5.0 mM ethanolamine phosphate (3), and 50 mM HEPES/K+ pH 8.0. The reaction mixture was allowed to incubate at 25 °C for 1 h before the Cj1438 enzyme was removed using a 10 kDa MWCO filter (Cytivia). This procedure was repeated for the synthesis of compound S-11 using 5.0 mM S-serinol phosphate (S-4) as the initial substrate.
Riegert A.S., Narindoshvili T., Platzer N.E, & Raushel F.M. (2022). Functional Characterization of a HAD Phosphatase Involved in Capsular Polysaccharide Biosynthesis in Campylobacter jejuni. Biochemistry, 61(21), 2431-2440.
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10

GST-TBX3 Protein Expression and Purification

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GST-TBX3(1–342) in pGEX-2TK and its mutant derivates were expressed in Escherichia coli BL21DE3 (New England Biolabs) by isopropyl-β-d-thiogalactopyranosid (IPTG) induction. Cleared sonified cell lysate was chromatographed on glutathione agarose (Cube Biotech, Monheim, Germany) following the company’s instructions1. Protein concentration in eluate fractions were measured by Bradford assay (Roti-Quant, Carl Roth, Karlsruhe, Germany).
Fischer K, & Pflugfelder G.O. (2015). Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression. Frontiers in Oncology, 5, 244.
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