Escherichia coli rosetta 2

Polyhistidine-tagged B. burgdorferi SpoVG was purified essentially as described previously [1 (link),4 (link)]. Briefly, Escherichia coli Rosetta II (Invitrogen, MA) was transformed with pBLJ132, which consists of spoVG cloned into pET101 [1 (link)]. E. coli were then grown to an OD₆₀₀ of at least 1.0 in Super Broth (32 g Tryptone, 20 g Yeast Extract, and 5 g NaCl per liter), and recombinant SpoVG expression was induced for 1h by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM. Bacteria were harvested by centrifugation at 5400 × g for 30 minutes and frozen at −80°C until needed. Resuspended cells were lysed by sonication with the addition of B-PER bacterial protein extraction reagent to 2% v/v (Thermo-Fisher, MA). Recombinant proteins were purified using MagneHis nickel particles (Promega, WI), then dialyzed against EMSA buffer (50mM Tris-HCl, 25mMKCl, 10% glycerol (v/v), 0.01% Tween 20, 100nM dithiothreitol (DTT), and 1mM phenylmethanesulfonyl fluoride (PMSF)). Proteins were concentrated using 10kDa Amicon centrifugal units (MilliporeSigma, MA) and aliquots were stored at −80°C until needed. Protein purity and concentration were assessed by SDS-PAGE, Quick Start Bradford protein assay (Bio-Rad), and bicinchoninic acid assay (BCA) (Thermo-Fisher, MA).

Polyhistidine-tagged B. burgdorferi SpoVG was purified essentially as described previously [1 (link),4 (link)]. Briefly, Escherichia coli Rosetta II (Invitrogen, MA) was transformed with pBLJ132, which consists of spoVG cloned into pET101 [1 (link)]. E. coli were then grown to an OD₆₀₀ of at least 1.0 in Super Broth (32 g Tryptone, 20 g Yeast Extract, and 5 g NaCl per liter), and recombinant SpoVG expression was induced for 1h by adding isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Bacteria were harvested by centrifugation at 5400×g for 30 min and frozen at −80 °C until needed. Resuspended cells were lysed by sonication with the addition of B-PER bacterial protein extraction reagent to 2% v/v (Thermo-Fisher, MA). Recombinant proteins were purified using MagneHis nickel particles (Promega, WI), then dialyzed against EMSA buffer (50 mM Tris-HCl, 25mMKCl, 10% glycerol (v/v), 0.01% Tween 20, 100 nM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride (PMSF)). Proteins were concentrated using 10 kDa Amicon centrifugal units (MilliporeSigma, MA) and aliquots were stored at −80 °C until needed. Protein purity and concentration were assessed by SDS-PAGE, Quick Start Bradford protein assay (Bio-Rad), and bicinchoninic acid assay (BCA) (Thermo-Fisher, MA).