The CrvA gene without the signal sequence (encoding CrvAΔ(2-35)) was cloned into the pMALC-2 vector between the BamHI and SalI sites. This vector contains an N-terminal maltose-binding protein (MBP) tag and factor Xa cleavage site. The plasmid was transformed into BL21*(DE3) pLysS cells (Promega) and an overnight culture was diluted and grown to OD600 0.5. IPTG was added to a final concentration of 0.2 mM, at which time cells were grown for 20 hours with the temperature lowered to 18 °C. CrvAΔ(2-35) pellets were resuspended in low ionic strength “buffer A” (50 mM NaCl, 25 mM Tris, pH 7.2, 0.5 mM PMSF) and sonicated. The lysate was then centrifuged at 75,000 × g for 45 minutes at 4 °C in a Beckman Optima XE ultracentrifuge. The supernatant was loaded on pre-calibrated amylose resin high flow columns (NEB). Columns were washed with buffer A and eluted with buffer A with 10 mM maltose. Fractions were analyzed by SDS-PAGE and peak elution fractions were cleaved with 10 μg of Factor Xa (Novagen) overnight at 4 °C. Cleaved protein was assayed again by SDS-PAGE, dialyzed against buffer A overnight, and loaded again on an amylose column to remove the free MBP. The same purification scheme was also used to purify CrvAΔcc2 (CrvAΔ(2-35,143-182)). Purified protein was allowed to polymerize at 37 °C for one hour, and was stored at 20 °C prior to analysis (roughly ten minutes).
Bl21 de3 plyss cells
Manufactured by Promega
Sourced in Switzerland, United States, United Kingdom
BL21(DE3)pLysS cells are a laboratory strain of Escherichia coli bacteria commonly used for the expression of recombinant proteins. They are designed to facilitate the high-level expression of target proteins under the control of the T7 promoter system.
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Lab products found in correlation
Optima xe, by Beckman Coulter (1 mentions)
Hela cells atcc ccl 2, by American Type Culture Collection (1 mentions)
Pgex 6p 3 plasmid, by GE Healthcare (1 mentions)
Gibson assembly technique, by New England Biolabs (1 mentions)
Doxorubicin dox, by Merck Group (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Pgex 2t, by GE Healthcare (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Sephacryl s 300 resin, by GE Healthcare (1 mentions)
Oregon green 488 maleimide, by Thermo Fisher Scientific (1 mentions)
9 protocols using bl21 de3 plyss cells
1
Purification and Polymerization of CrvA Protein
2
Preparation of Competent Bacterial Cells
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Chemically competent E. coli DH5alpha and BL21(DE3)pLysS cells were purchased from Promega AG (Dübendorf, Switzerland). HeLa (ATCC CCL‐2) cells were obtained from ATCC (Manassas, VA).
3
Preparation of Scaffolded Fusion Proteins
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The cDNA encoding fusion proteins composed of a His-tag, RLuc, GGGS linker and scaffolds (in this order) used for BLI-ELISAs or the cDNA encoding His-tagged scaffolds used for ELISA were inserted into the multi-cloning site of the pGEX-6P-3 plasmid (GE Healthcare, Little Chalfont, UK) by the Gibson assembly technique (New England Biolabs, Ipswich, MA, USA). The cDNA encoding FLAP candidates were constructed by site-directed mutation of scaffold sequences. These plasmids were introduced into BL21 (DE3) pLys S cells (Promega, Fitchburg, WI, USA), after which the GST-tagged fusion proteins were expressed as described previously15 (link).
4
Purification and Expression of Cytotoxic Proteins
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cSBL was isolated from acetone-dried powder of unfertilized bullfrog body-cavity eggs using sequential chromatography with Sephadex G75, DEAE-cellulose, hydroxyapatite, and SP-Sepharose (Cytiva), as previously described (17 (link)). For the preparation of ONC, ONC cDNA was cloned into the pET-11d plasmid (Merck KGaA) in conjunction with the pelB sequence. BL21 (DE3) pLysS cells (Promega) were transformed with the plasmid, and its expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (0.2 mM) at 34°C for 72 h. ONC recombinant protein was purified from the culture liquid by sequential chromatography with Sephadex G75, DEAE-cellulose, hydroxyapatite, and SP-Sepharose. Doxorubicin (DOX) was purchased from Sigma-Aldrich. The anti-caspase-3 antibody (cat. no. #9662), peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies (cat. no. #7074 and #7076, respectively) were purchased from Cell Signaling Technology. The anti-aldo-keto reductase (AKR) 1B10 antibody (cat. no. ab96417) was purchased from Abcam (Cambridge, UK) The anti-β-actin antibody (clone AC-74, cat. no. A2228) was purchased from Sigma-Aldrich.
5
Recombinant FAP133 Protein Production
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Two constructs encoding amino acids 1–140 of FAP133 as fusion proteins were generated by PCR using the primers 5′-GGATCCATGCAAGAGGTCCCGCC-3′ and 5′-GAATTCGGTGGCGTTGAGGTCCTGG-3′, ligation into pGEM, digestion with BamHI and EcoRI, and religation into pET30A (Novagen, EMD Millipore, Billerica, MA) and pGEX-2T (GE Healthcare Life Sciences, Piscataway, NJ). Expression was induced in BL21(DE3) pLysS cells (Promega, Madison, WI), and soluble FAP133-hexahistidine fusion protein was purified with a nickel column (Novagen) and used as an antigen for polyclonal antibody production in rabbits (Spring Valley Laboratories, Sykesville, MD). Soluble FAP133-GST fusion protein was covalently cross-linked to glutathione–Sepharose 4B beads (GE Healthcare Life Sciences) according to manufacturer’s instructions. The FAP133 antibody was affinity purified on the FAP133-GST column as described in Perrone et al. (2003 (link)).
6
Viroporin-like protein activity in E. coli
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For the assessment of protein viroporin-like activity in E. coli, BL21(DE3)pLysS cells (Promega) were transformed with pOPT constructs, grown in the presence of ampicillin (100 µg mL−1) at 37 °C to an optical density at 600 nm (OD600) of 0.4–0.6, and then 1 mM isopropyl-β-D thiogalactopyranoside (IPTG) was added to induce protein expression. Subsequently, optical densities were measured for induced and non-induced samples in triplicate over a time course of 180 min post induction using a Spectra Max i3x (Molecular Devices) microplate reader. Non-induced, and 60 and 120 min post induction samples were also collected for protein detection by western blot.
7
Actin Purification and Labeling for Dynamic Polymerization
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Actin was extracted from chicken muscle acetone powder and purified by one cycle of polymerization and depolymerization (Spudich and Watt, 1971 (link)). Monomers were separated from oligomers and filaments via gel filtration using Sephacryl S-300 resin (GE Healthcare) in G-buffer (2 mM Tris, pH 8.0, 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2). For dynamic polymerization experiments, actin monomers were labeled with Oregon Green 488 maleimide (Thermo Fisher Scientific) prior to gel filtration. Human fascin-1 was expressed in BL21 (DE3) pLysS cells (Promega Corporation) from a pET21a plasmid that was modified to encode an N-terminal GST tag and a Tobacco Etch Virus (TEV) protease cleavage recognition sequence. Transformants were grown in 1 L of LB broth, induced at OD600 ∼0.6 with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and shaken at 16°C overnight. Following cell harvest, the protein was purified as previously described (Sherer and Courtemanche, 2022 (link)).
8
Expression and Purification of Human CDK2
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The gene encoding full‐length human CDK2 was cloned into the pGEX‐6P‐1 and transformed into BL21(DE3)pLysS cells (Promega, Southampton, UK). Cultures were grown for 2–3 h at 37 °C, and then, the temperature was decreased to 18 °C prior to induction with 0.25 mmol·L−1 IPTG at OD600 = 0.6. The cultures were allowed to grow for an additional 20–24 h at 18 °C and were harvested by centrifugation. All purification steps were performed by FPLC at 4 °C. Harvested cells were resuspended in 50 mmol·L−1 HEPES buffer (pH 7.5) containing 150 mmol·L−1 NaCl, 2 mmol·L−1 DTT, and 0.5 mg·mL−1 lysozyme at 4 °C for 1 h. After sonication and centrifugation (1 h at 29 000 g ), the supernatant was purified by immobilized glutathione Sepharose chromatography (GE LifeSciences, Little Chalfont, UK). Following incubation of peak fractions with 3C protease (20 : 1) at 4 °C, the cleaved GST‐tag was separated by size exclusion chromatography using a Superdex 75 (26/60) column and eluted with 50 mmol·L−1 HEPES buffer (pH 7.4) containing 150 mmol·L−1 NaCl and 1 mmol·L−1 DTT. Purified CDK2 was buffer‐exchanged into 100 mmol·L−1 Na/K phosphate buffer (pH 7.4) containing 1 mmol·L−1 DTT and concentrated to 10 mg·mL−1 for crystallization.
9
Purification and Binding Assay of Nck1
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E.coli BL21(DE3)pLysS cells (Promega) were transformed with plasmid pGEX-2TK-Nck1or pGEX-2TK. These were grown in Luria Bertani broth containing 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. Cells were induced at an OD600 of 0.5 with 0.5 mM IPTG at 18°C and cultured overnight. Cells were lysed in GST-lysis buffer (20 mM Tris-HCl [pH 7.5], 20 mM NaCl, 1 mM DTT, 1 mM PMSF, 1% Triton-X). Gst or Gst-Nck1 expressed in E. coli were bound with gentle rocking to glutathione Sepharose for 2 h at 4°C. The beads were washed 5× with Gst wash buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP40) to remove unbound proteins and stored at 4°C in the same buffer containing 20% glycerol and 1 mM phenylmethylsulfonyl fluoride. Cytoplasmic extracts were prepared 36 h after transfection of HEK293 with plasmids expressing bio-cCE or MS2-bio. Nonspecific proteins were removed by incubating each extract with glutathione-Sepharose, and 5 mg of pre-cleared extract was incubated with rocking for 2 h at 4°C with Gst- or Gst-Nck1-bound beads. These were recovered by centrifugation and unbound proteins were removed by five washes with Gst wash buffer. The bead-bound proteins were analyzed by Western blotting, and for guanylylation and capping activity [11] (link).
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E.coli BL21(DE3)pLysS cells (Promega) were transformed with plasmid pGEX-2TK-Nck1or pGEX-2TK. These were grown in Luria Bertani broth containing 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. Cells were induced at an OD600 of 0.5 with 0.5 mM IPTG at 18°C and cultured overnight. Cells were lysed in GST-lysis buffer (20 mM Tris-HCl [pH 7.5], 20 mM NaCl, 1 mM DTT, 1 mM PMSF, 1% Triton-X). Gst or Gst-Nck1 expressed in E. coli were bound with gentle rocking to glutathione Sepharose for 2 h at 4°C. The beads were washed 5× with Gst wash buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP40) to remove unbound proteins and stored at 4°C in the same buffer containing 20% glycerol and 1 mM phenylmethylsulfonyl fluoride. Cytoplasmic extracts were prepared 36 h after transfection of HEK293 with plasmids expressing bio-cCE or MS2-bio. Nonspecific proteins were removed by incubating each extract with glutathione-Sepharose, and 5 mg of pre-cleared extract was incubated with rocking for 2 h at 4°C with Gst- or Gst-Nck1-bound beads. These were recovered by centrifugation and unbound proteins were removed by five washes with Gst wash buffer. The bead-bound proteins were analyzed by Western blotting, and for guanylylation and capping activity [11] (link).
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