E. coli BL21 (DE3) cells for recombinant protein expression (Novagen, cat. no. 69450)
Erwinia carotovora carotovora 15 (obtained from Bruno Lemaitre Laboratory, EPFL, Switzerland)
Plasmid containing the full-length PGRP-LB gene
LB (Luria-Bertani Broth) MILLER (Novagen, cat. no. 71753)
Sodium phosphate monobasic (SIGMA Aldrich, cat. no. S0751)
Sodium phosphate dibasic (SIGMA Aldrich, cat. no. S0876)
NaCl (Fluka Analytical, cat. no. 71379)
Imidazole (ACROS Organics, cat. no. 122020020)
Dithiothreitol (DTT) (Fisher Scientific, cat. no. BP172-25)
β-Mercaptoethanol (β-ME) (Fluka Biochemika, cat. no. 63690)
Ampicillin sodium salt (SIGMA, cat. no. A9518)
Isopropyl-D-thiogalactopyranoside (IPTG) (Gold BioTechnology Inc, I2481C100)
Ni-NTA Agarose resin (Quiagen, cat. no. 30250)
TEV-protease (Invitrogen)
PBS
Methanol
Reagent setup
Ampicillin-LB broth: 2.5 % LB Broth MILLER. Autoclave for 30 min. Store at room temperature. Add ampicillin to a final concentration of 100 μg/ml before use.
Ampicillin-LB agar plates: 2.5 % LB Broth MILLER 1.5 % Bactor-Agar. Autoclave for 30 min. Allow LB agar to cool to 50 °C and then add ampicillin to a final concentration of 100 μg/ml. Dispense into sterile Petri dishes and allow to set at room temperature. Store the plates at 4 °C.
Cell lysis buffer: 20 mM sodium phosphate pH 7.6, 0.1 M NaCl and 2 mM β-ME.
40 mM imidazole wash buffer: Add 3 M imidazole pH 8.0 into the cell lysis buffer to a final concentration of 40 mM.
200 mM imidazole wash buffer: Add 3 M imidazole pH 8.0 into the cell lysis buffer to a final concentration of 200 mM.
Dialysis buffer: 20 mM sodium phosphate, pH 7.6, and 1 mM DTT.
1M NaCl wash buffer: Add 5 M NaCl to a final concentration of 1M into the dialysis buffer.