Recombinant mouse OTX2 was produced and purified as described (Torero Ibad et al., 2011). Briefly, myc-tagged OTX2, with a 6 histidine tag for purification, at the c-terminal was cloned into a pTrac plasmid (Thermo Fischer, France). Transformed BL21Codon Plus RP (Stratagene/Agilent, Les Ulis, France) bacteria were grown overnight at 37°C in MagicMedium (Thermo Fischer, France) and the bacterial mass was resuspended with 10 mM Tris, 100 mM Na2HPO4, 20 mM imidazole, 6 M guanidine HCl (pH 8). Disrupted bacteria were centrifuged at 40,000 × g for 30 minutes and the supernatant recovered and loaded on a HisTrap HP affinity column (GE Healthcare, France). Protein elution was carried out with 6 M guanidine HCl, 0.2 M acetic acid and dialyzed against 20 mM phosphate, 0.5 M NaCl, and 25 μM ascorbic acid. A bacterial extract from an empty vector was obtained with the same procedure.
Bl21codon plus rp
Manufactured by Agilent Technologies
Sourced in France
The BL21-CodonPlus(DE3)-RP E. coli strain is a competent bacterial host designed for high-level recombinant protein expression. It is engineered to enhance the expression of proteins that require rare tRNAs found in E. coli.
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3 protocols using bl21codon plus rp
1
Recombinant Mouse OTX2 Protein Production
2
Production and Purification of Collagen NC1
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His-tagged (for production of antibody) and MBP-tagged (for immunoblotting) NC1 domains of type IV collagen α1-6 (V) were expressed in BL21-CodonPlus-RP (Agilent Technologies, Santa Clara, CA) transformed with pET-28a (Invitrogen) and pMAL (New England Biolabs, Beverly, MA), respectively. Each His or MBP fusion protein was purified through affinity chromatography with TALON metal affinity resin (Clonetech, Palo Alto, CA) or with amylose resin (New England Biolabs), respectively.
3
Production and Detection of AhGLK1 Protein
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This experiment was conducted using previously described methods56 (link). The AhGLK1 coding sequence were cloned in the pGEX4T-1 vector to allow production of GST-AhGLK1 fusion proteins after induction by isopropyl β-D-1-thiogalactopyranoside (IPTG). Expression of GST-AhGLK1 was induced in Escherichia coli BL21-Codon Plus-RP (Agilent Technologies) by adding IPTG to a final concentration of 0.5 mM at 37 °C for 9 h, after which the bacteria were transferred to 16 °C overnight. GST-AhGLK1 protein was purified and used for antibody production (polyclonal, rabbit). The antibody of PORA was obtained from Agrisera (http://www.agrisera.com ). Detection of AhGLK1 and AhPORA proteins by immunoblot analysis was carried out as previously described57 (link). Leaves (100 mg) of peanut or Arabidopsis were ground in liquid nitrogen and homogenized with 1 mL of sample buffer (50 mM Tris, pH 6.8, 2 mM EDTA, 10% w/v glycerol, 2% SDS and 6% 2-mercaptoethanol), then denatured at 100 °C for 5 min, and finally subjected to SDS-PAGE.
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