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Escherichia coli bl21 gold de3

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The Escherichia coli BL21-GOLD (DE3) is a strain of bacteria commonly used in molecular biology and protein expression applications. It is designed for high-level expression of recombinant proteins.

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Lab products found in correlation

Pet15b vector, by Merck Group (3 mentions) Amicon ultra device, by Merck Group (3 mentions) Superdex 75 column, by GE Healthcare (2 mentions) Ni nitrilotriacetic acid nta column, by Qiagen (1 mentions) His tag purification resin, by Roche (1 mentions) Superdex 75, by GE Healthcare (1 mentions) Q exactive focus mass spectrometer, by Thermo Fisher Scientific (1 mentions) Noc 5 3 aminopropyl 1 hydroxy 3 isopropyl 2 oxo 1 triazene, by Merck Group (1 mentions) 600 mhz spectrometer, by Bruker (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) Miniprep reagents, by Qiagen (1 mentions) T5 exonuclease, by Illumina (1 mentions) Ni nta resin, by Qiagen (1 mentions) Fei vitrobot mark 4, by Thermo Fisher Scientific (1 mentions) R1 2 1, by Quantifoil (1 mentions) Thrombin, by Cytiva (1 mentions)

Close spelling variants of this product

Escherichia coli BL21-Gold (DE3) cells Escherichia coli BL21(DE3) BL21-Gold(DE3) BL21(DE3) BL21-Gold

7 protocols using escherichia coli bl21 gold de3

1

Purification of Human TFIIH p62 and RPB6

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Unlabeled or 13C/15N-labeled human TFIIH p62 PH-D (residues 1–108) and unlabeled or 13C/15N-labeled human RPB6 (residues 1–127) were prepared as previously described (5 (link)). In brief, p62 or RPB6 was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21 (DE3) Gold (Agilent Technologies). The lysed supernatant was loaded onto a Ni-nitrilotriacetic acid (NTA) column (QIAGEN), and the eluate was digested with thrombin to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).
Okuda M., Suwa T., Suzuki H., Yamaguchi Y, & Nishimura Y. (2021). Three human RNA polymerases interact with TFIIH via a common RPB6 subunit. Nucleic Acids Research, 50(1), 1-16.
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2

Expression and Purification of 13C/15N-labeled p62 Fragment

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The 13C/15N-labeled fragment of human p621–158 (residues 1–158) was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21 (DE3) Gold (Agilent Technologies). The cells were grown at 37°C in M9 minimal medium containing [15N]ammonium chloride and [13C]glucose. The product was expressed by induction with 1 mM isopropyl-β-d-thiogalactopyranoside. After 21 h of growth at 20°C, the cells were collected, resuspended in buffer A [20 mM Tris–HCl (pH 8.0), 10% glycerol, 1 M NaCl], lysed by sonication and then centrifuged. The supernatant was loaded onto a His-Tag Purification Resin (Roche) column equilibrated with buffer A, and the column was washed with buffer A containing 10 mM imidazole–HCl. The sample was eluted by 500 mM imidazole–HCl, peak fractions were pooled and the buffer was changed to thrombin cleavage buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 500 mM NaCl, 2.7 mM KCl, pH 7.3) by using an Amicon Ultra device (Merck Millipore). To remove the histidine tag, the sample was digested with thrombin for 63 h at 20°C. After concentration via an Amicon Ultra, the sample was applied to a Superdex75 (GE Healthcare) column equilibrated with 20 mM potassium phosphate (pH 6.8) and 500 mM NaCl.
Okuda M., Ekimoto T., Kurita J.I., Ikeguchi M, & Nishimura Y. (2020). Structural and dynamical insights into the PH domain of p62 in human TFIIH. Nucleic Acids Research, 49(5), 2916-2930.
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3

Synthesis and Characterization of Cyclic Dipeptides

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Molecular biology reagents and chemicals were purchased from Fisher Scientific, Sigma-Aldrich, or New England Biolabs, Inc if not specifically indicated. Primers were ordered from Sigma-Aldrich. 4-Me-d,l-tryptophan was from MP Biomedical (Santa Ana, CA), while NOC-5 (3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene) was purchased from EMD Millipore. Nine cyclic dipeptides used in this study were purchased from Chem-Impex Int’l, Inc. Brevianamide F was purchased from Ark Pharm, Inc. Escherichia coli BL21-GOLD (DE3) (Agilent) and BAP1 were used for routine molecular biology studies (Table S4) and protein expression, respectively, and were grown in Luria-Bertani broth or Terrific broth. DNA sequencing was performed at Eurofins. Primers used in this study were listed in Table S5. TB14 used in the study was reported in our previous study.[32 (link)] 1D and 2D NMR spectra of compounds were recorded in CD3OD on a Bruker 600 MHz spectrometer using 1.5mm High Temperature Superconducting Cryogenic Probe at the University of Florida, Gainesville, FL, USA. Spectroscopy data were collected using Topspin 3.5 software. All 1H NMR were water suppressed to get rid of high background water peak. HRMS data were obtained using a Thermo Fisher Q Exactive Focus mass spectrometer equipped with an electrospray probe on Universal Ion Max API source.
Jiang G., Zhang Y., Powell M.M., Hylton S.M., Hiller N.W., Loria R, & Ding Y. (2019). A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C-H Bonds of Aromatic 2,5-Diketopiperazines. Chembiochem : a European journal of chemical biology, 20(8), 1068-1077.
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4

Cloning and Expression of Recombinant Proteins

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Miller LB medium (BD Difco) was used for
growth of bacterial strains. Escherichia coli BL21
Gold (DE3) (Agilent) was used as the host strain for both cloning
and expression of recombinant proteins. Plasmid pET28a(+) was purchased
from Novagen. HotStart Taq Mastermix (Denville) was used for gene
amplification. Phusion DNA polymerase (Finnzymes), Taq DNA ligase
(MCLab), and T5 exonuclease (Epicenter) were purchased separately
and used to make the assembly master mix (AMM) used for cloning. Ni-NTA
resin and miniprep reagents were purchased from Qiagen. Primers were
synthesized by ValueGene. All other chemicals were purchased from
Sigma-Aldrich unless otherwise noted.
Vinokur J.M., Korman T.P., Cao Z, & Bowie J.U. (2014). Evidence of a Novel Mevalonate Pathway in Archaea. Biochemistry, 53(25), 4161-4168.
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5

Recombinant Apoferritin Expression and Purification

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Apoferritin was expressed in Escherichia coli BL21-Gold(DE3) (Agilent) from a plasmid encoding mouse heavy-chain ferritin provided by Dr. Haruaki Yanagisawa, the University of Tokyo. The protein was purified by heat treatment at 70 °C for 10 min, precipitation with ammonium sulfate, followed by gel filtration as described in the attached document. The apoferritin complex of ~500 kDa was eluted in 20 mM HEPES pH 7.5 and 300 mM NaCl, and the protein concentration was measured with the BCA assay (Pierce). Three μl of a sample solution containing apoferritin at a concentration of 3 or 4 mg ml−1 was applied onto a holey carbon-coated grid (Quantifoil R1.2/1.3, Quantifoil Micro Tools GmbH) with 200 mesh, and the grid was blotted off with filter paper for 4 s and immediately plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV (ThermoFisher Scientific) under 100% humidity at 4 °C.
Maki-Yonekura S., Kawakami K., Takaba K., Hamaguchi T, & Yonekura K. (2023). Measurement of charges and chemical bonding in a cryo-EM structure. Communications Chemistry, 6, 98.
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6

Preparation of Isotope-Labeled spPH Domain

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The 15N- or 13C/15N-labeled spPH domain (residues 1-108) was prepared by a previously described method [28 (link)]. In brief, the spPH domain was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21-Gold (DE3) (Agilent Technologies). The lysed supernatant was loaded on to a cOmplete His-Tag purification resin column (Roche), and the eluate was digested with thrombin (Cytiva) to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).
Okuda M, & Nishimura Y. (2023). Structural polymorphism of the PH domain in TFIIH. Bioscience Reports, 43(7), BSR20230846.
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7

Optimization of BM3 Variants for Steroid Hydroxylation

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Escherichia coli BL21‐Gold(DE3) (F−ompT hsdS(rB−mB−) dcm+TetRgalλ(DE3) endA Hte) was obtained from Agilent Technologies and used as recombinant host strain. As expression vector, we used the pMB1‐derived plasmid pETM11 carrying genes encoding the hydrophobic outer membrane pore AlkL and a BM3 variant (either KSA1, KSA2, KSA3, KSA14 or KSA14m; respective amino acid substitutions provided in Table S1) under control of the T7‐expression system (Bertelmann et al., 2022 ). 2β‐ and 15β‐hydroxytestosterone were acquired from Steraloids Inc. and Cfm Oskar Tropitzsch GmbH. All other chemicals were purchased from AppliChem, Carl Roth, Chemsolute or Sigma‐Aldrich in the highest purity available.
Bertelmann C., Mock M., Schmid A, & Bühler B. (2023). Efficiency aspects of regioselective testosterone hydroxylation with highly active CYP450‐based whole‐cell biocatalysts. Microbial Biotechnology, 17(1), e14378.
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