Human tau isoforms were cloned into the pRK172 bacterial expression vector using NdeI and EcoRI restriction sites as previously described [27 (link), 31 (link), 75 (link)]. Recombinant tau isoforms were expressed in Escherichia coli BL21 (New England Biolabs, Ipswich, MA) and purified. In brief, expression was induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) and high-salt 100 °C heat stable bacterial protein lysates were purified by MonoS cation exchange chromatography.
Escherichia coli bl21
Manufactured by New England Biolabs
Sourced in United States
Escherichia coli BL21 is a bacterial strain commonly used in molecular biology laboratories for the expression of recombinant proteins. It is a genetically modified variant of the E. coli bacteria, designed to enhance protein production and facilitate the purification process.
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Lab products found in correlation
Escherichia coli hb101, by Promega (2 mentions)
Escherichia coli dh10b, by New England Biolabs (2 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Gsh beads, by GE Healthcare (1 mentions)
Imagescanner, by GE Healthcare (1 mentions)
7 protocols using escherichia coli bl21
1
Recombinant Tau Isoform Expression
2
Escherichia coli Strains for Plasmid Preparation
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Escherichia coli HB101 (Promega) was used as the recipient strain for preparation of HIV-1 proviral vectors, whereas all of the other plasmid vectors were prepared from Escherichia coli DH10B (NEB). GST-fusion proteins were prepared from Escherichia coli BL21 (NEB). All these bacteria were cultured in LB broth in a shaking incubator at 37°C.
3
Recombinant Expression of p53 and Hsp40
4
Escherichia coli Strains for Plasmid Preparation
5
Purification and Characterization of BES1 and HSFA1a
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Recombinant proteins of BES1‐GST, BES1‐MBP, and HSFA1a‐MBP were expressed in Escherichia coli BL21 (New England Biolabs) using the expression vectors pGEX‐BES1 and pMAL‐BES1, pMAL‐HSFA1a, and subsequently purified using GSH beads (GE Healthcare) or Amylose resin (New England Biolabs). Protein elution from GSH beads was performed with elution buffer (150 mM NaCl, 5 mM DTT, 20 mM GSH reduced form, and 50 mM Tris–HCl, pH 8.0) and from Amylose resin (150 mM NaCl, 5 mM DTT, 10 mM Maltose, and 50 mM Tris–HCl, pH 8.0) concentrated using Roti‐spin MINI‐3 columns (Roth). HEX‐labeled probes for EMSAs were prepared by PCR and purified using a Phenol:Chloroform protocol (Unterholzner et al, 2017 (link)). The probes (0.5 pmol per reaction) were incubated with purified BES1‐MBP, HSFA1A‐MBP, or combinations of both proteins and separated on 3–6% PAGE gels. The bands were detected using a Molecular Imager FX Pro (Hercules, Bio‐Rad) equipped with a 532 nm laser for excitation and a 555 nm long pass emission filter as described previously (Unterholzner et al, 2017 (link)).
6
Recombinant Spirometra GST expression
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The first Spirometra GST gene deposited in GeneBank (AEI16476.1) was amplified by PCR with specific primers carrying BamHI and PstI restriction enzyme sites (underlined) (forward, 5′-ATGGATCC ATGGGTTC GCTCCCGGTTC-3′, and reverse, 5′-ATCTGCAG CTAAGCATCACCACGCCAG-3′), and the cycling protocol was as follows: 30 cycles of 95°C for 50 s, 60°C for 50 s and 72°C for 50 s. The final PCR products were purified, digested, and cloned into the pQE-80L vector (Ipswich, USA). The recombinant plasmid was then transformed into Escherichia coli BL21 (New England Biolabs, USA). Expression of rSmGST was induced by adding 0.5 mM IPTG at 37°C for 4 h. The rSmGST was purified by Ni2+ affinity chromatography (Shenggong Biotech, Shanghai, China) and identified by SDS-PAGE. Images of gels were recorded using ImageScanner (GE Healthcare, Fairfield, CT). Another gel was prepared by the same method and used for the western blotting analysis.
7
Bacterial Cloning and Protein Expression
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The bacterial strain XL1-Blue (DE3) (Stratagene, La Jolla, California, U.S.A.) was used during plasmid and PCR product cloning. Escherichia coli BL21 (New England Bio Labs, Ipswich, Massachusetts, U.S.A.) was used for the expression of GST fusion proteins. HeLa (CCL-2, LGC Promochem, Middlesex, United Kingdom). HEK-273-R1 (AMGEN, Thousand Oaks, California, U.S.A.) cells were maintained in DMEM with 5% fetal calf serum (FCS), transfected by calcium phosphate co-precipitation and stimulated with IL-1 or NDV as previously described [5] .
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