Recombinant sfMyt1 (N229A for the kinase-dead mutant or S75A mutant) cloned into the pGEX-4T-1 vector was expressed in Escherichia coli strain BL21 (DE3; Invitrogen) as an N-terminal GST-tagged protein, purified using glutathione-Sepharose 4B beads (GE Healthcare), and dialyzed against storage buffer (20 mM Pipes, pH 6.8, 200 mM sucrose, and 1 mM DTT). For His6-Cdc25 (WT or S188A mutant), E. coli strain BL21 (DE3) was transformed with the pET21a construct. Expressed His6-Cdc25 were purified from inclusion bodies under denaturing conditions (6 M urea) using Probond-Resin (Invitrogen) and then refolded via stepwise reduction of the urea concentration by dialysis using EasySep (TOMY) for 2 h against TBS (50 mM Tris and 150 mM NaCl, pH 7.5) containing 4 M urea, for 2 h against TBS containing 2 M urea, and for 2 h against TBS lacking urea. To prepare the recombinant GST-fused peptide substrates (AS peptide, AGRPRAATFIESG; sfMyt1-S75 peptide, ESRPRAVSFRQSE; sfCdc25-S188 peptide, AGRPRQISFIESG), E. coli strain BL21-CodonPlus-RIL (Agilent Technologies) was transformed with the pGEX-4T-1 constructs. The expressed GST-peptides were purified using glutathione-Sepharose 4B and dialyzed against PBS (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4, pH 7.4). A recombinant C-terminal 88-aa fragment of sfAkt was prepared as previously described (Okumura et al., 2002 (link)).
Bl21 codonplus ril
Manufactured by Agilent Technologies
Sourced in Germany, United States
The BL21-CodonPlus-RIL is a competent cell line designed for enhanced expression of recombinant proteins in Escherichia coli. It contains additional copies of the argU, ileY, and leuW tRNA genes, which help to improve the translation of proteins that require rare codons.
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Lab products found in correlation
Terrific broth, by Thermo Fisher Scientific (2 mentions)
Avanti j 26 xpi, by Beckman Coulter (2 mentions)
Probond resin, by Thermo Fisher Scientific (1 mentions)
Easysep, by TOMY (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Rosetta 2, by Merck Group (1 mentions)
Quikchange protocol, by Agilent Technologies (1 mentions)
Ni2 affinity chromatography, by GE Healthcare (1 mentions)
E coli top10, by Thermo Fisher Scientific (1 mentions)
Heparin sepharose, by GE Healthcare (1 mentions)
Thrombin protease, by GE Healthcare (1 mentions)
Glutathione affinity chromatography, by GE Healthcare (1 mentions)
9 protocols using bl21 codonplus ril
1
Recombinant Protein Expression and Purification
2
Recombinant Protein Expression in E. coli
3
Purification of recombinant transcription enzymes
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Recombinant thioredoxin-hexahistidine–tagged RPOTm, RPOTmp, and RPOTp investigated in in vitro transcription assays were expressed and purified under native conditions as described previously by Kühn et al. (2007 (link)). For mutagenesis experiments the wild-type sequence of RPOTmp encoding amino acids 107–1011 (locus tag At5g15700), was PCR-amplified with primers listed in Supplementary Table S3 and cloned via EcoRI/PstI- or SacI/XbaI restriction sites into the pCOLD DNAI vector (TaKaRa Bio Europe S.A.S., Saint-Germain-en-Laye, France) to result in construct pCold-His-RPOTmp. For mutagenesis, RPOTmp point mutations were introduced by site-directed mutagenesis using the PhusionTM Site-Directed Mutagenesis Kit (FINNZYMES, Thermo Fisher Scientific Biosciences GmbH, St. Leon-Rot, Germany) according to the manufacturer’s instructions. Used primers are listed in Supplementary Table S3. Recombinant hexahistidine-tagged enzymes were overexpressed in E. coli BL21 Codon Plus RIL (Agilent Technologies, Waldbronn, Germany) from resulting construct pCold-His-RPOTmp (RHR) in parallel with the wild-type enzymes. Protein expression was induced with a final concentration of 1 mM IPTG for 20–24 h at 15 °C. The purification of histidine-tagged proteins over Ni2+-NTA agarose was performed as described previously (Kühn et al. 2007 (link)).
4
AurA Kinase Domain Mutagenesis Protocol
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Site-directed mutagenesis of AurA kinase domain (122–403) in a pET30-based vector was carried out using a QuikChange protocol (Agilent). The mutations were C290A, C393A, and either F275C for the 275 construct or T288C and T287A for the 288 construct. Proteins were expressed in E. coli BL21-CodonPlus -RIL (Agilent) or Rosetta 2 (Merck) DE3 cells in LB medium, with initial growth at 37°C followed by overnight incubation at 21°C after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside. Bacterial pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2, 10% glycerol, 40 mM imidazole), supplemented with protease inhibitors and DNase (Roche), lysed by sonication and clarified by centrifugation. Lysates were filtered then subjected to Ni2+ affinity chromatography (GE Healthcare). Proteins were eluted in lysis buffer containing 250 mM imidazole, purified to homogeneity by S75 size exclusion chromatography (GE Healthcare) (gel filtration buffer: 50 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl2, 10% glycerol, 10 mM β-mercaptoethanol (BME)), then concentrated to ∼10 mg/mL and flash frozen for future use. Protein concentrations were measured in triplicate with a ND-1000 Spectrophotometer (NanoDrop) using molecular weights and extinction coefficients calculated by ProtParam (ExPASy).
5
Expression and Purification of Plant Proteins
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The RMS3 (residues 1–267), coding sequence from Pisum sativum, the AtD14 (At3g03990) (residues 1–267), the AtHTL (At4g37470) (residues 1–269) and the AtMES9 (At4g37150) (residues 1–256) coding sequences from Arabidopsis thaliana were amplified by PCR using adult leaves derived cDNA template and specific primers (see Supplementary Table 5
6
Expression and Purification of Vibrio parahaemolyticus Toxins
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Escherichia coli BL21 CodonPlus-RIL (Agilent Technologies, Inc., Santa Clara, CA, USA) and E. coli Top10 (Invitrogen, Carlsbad, CA, USA) were used for the transformation and replication, respectively, of the genes coding the toxins PirAvp and PirBvp and cloned into the pET system plasmid. The E. coli culture was maintained on Luria–Bertani (LB) agar plates supplemented with the appropriate antibiotics. For rPirAvp, kanamycin (50 μg/mL) and chloramphenicol (34 μg/mL) were used; for rPirBvp, ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) were used. For protein expression, cultures were grown in LB at 37 °C and induced at 30 °C with shaking (200 rpm).
The highly virulent M0904 strain of Vibrio parahaemolyticus (Vp), which causes AHPND in shrimp, was used. It was isolated from shrimp hepatopancreas [16 (link)]. A pure culture of VpM0904 was obtained by inoculation in tryptic soy broth (TSB, Bioxon, Mexico), according to Soto-Rodriguez et al. [16 (link)].
The highly virulent M0904 strain of Vibrio parahaemolyticus (Vp), which causes AHPND in shrimp, was used. It was isolated from shrimp hepatopancreas [16 (link)]. A pure culture of VpM0904 was obtained by inoculation in tryptic soy broth (TSB, Bioxon, Mexico), according to Soto-Rodriguez et al. [16 (link)].
7
Collagen-binding bFGF Fusion Proteins
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Four collagen-binding bFGF fusion proteins (CB-bFGFs) were used in this study (Figures 1 and 2 ). Two fusion proteins, bFGF-s3 and bFGF-s2b-s3, consisting of human bFGF and CBD or PKD-CBD derived from ColH, were constructed as previously described [10 (link), 12 (link)]. To construct bFGF-s3b, an expression plasmid, pCHG115, encoding a fusion protein between GST and a C-terminal CBD (s3b, ColG) [21 (link)] was digested with BamHI and EcoRI at the linker region and ligated with a hbFGF-encoding DNA fragment using BglII and EcoRI linkers. The ligation mixture was transformed into E. coli DH5α cells and the nucleotide sequence of the resulting plasmid (pCHG115-hbFGF) was confirmed by Sanger sequencing. The plasmid was transformed into E. coli BL21 CodonPlus RIL (Agilent Technologies, Santa Clara, CA) to express the GST-bFGF-s3b fusion protein. The protein was purified by glutathione affinity chromatography (GE Healthcare), the GST moiety was cleaved off using thrombin protease (GE Healthcare), and bFGF-s3b was further purified using an Heparin-Sepharose (GE Healthcare) affinity column as described previously [12 (link)]. Another fusion protein comprising bFGF and tandem CBDs derived from ColG (bFGF-s3a-s3b) was produced in the same manner using the pCHG112 expression plasmid [21 (link)].
8
Recombinant Protein Expression in E. coli
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The expression clone was introduced into E. coli strain BL21-CodonPlus-RIL (Agilent). Cells were grown at 25 °C in Terrific broth (Invitrogen) containing 100 μg/L ampicillin. When the OD660 of the cell culture reached ≈ 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a concentration of 100 μM to induce expression of the cDNA. Cells were grown at 18°C for an additional 18 h following IPTG induction and then collected by centrifugation at 4000 g for 15 min at 4 °C (Beckman Avanti J-26 XPI) and resuspended in extraction buffer (50 mM Tris–HCl (pH 7.7), 150 mM NaCl and 5 μM β- mercaptoethanol (β-Me)), which included 5% (v/v) glycerol.
9
Expression and Purification of Plant Proteins
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