Bl21 star de3

AtHB13-GST fusion protein was purified from Escherichia coli expression strain BL21 Star (DE3) pRARE, which was generated by transforming the pRARE plasmid isolated from Rosetta (DE3) pRARE cells (Merck) into E. coli BL21 Star (DE3) (Invitrogen). Recombinant GST-fusion protein was purified using GST-agarose beads following the manufacturer’s instructions (Sigma–Aldrich, Taufkirchen, Germany). Electrophoretic mobility shift assays (EMSA) was performed as described (Wu et al., 2012 (link)) using the Odyssey Infrared EMSA kit (LI-COR). Sequences of 5′-DY682-labeled fragments are given in Supplementary Table S1.

Escherichia coli (BL21 Star (DE3)) (Merck Millipore, Billerica, Massachusetts, USA) was used as a host for protein expression. Overnight cultures of cells harboring the expression plasmids were prepared and diluted 1:100 in tryptic soy Broth (TSB) medium with yeast extract [31 (link)] and grown at 37 °C. When OD₆₀₀ reached approximately 1.5, protein expression was induced by addition of Isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. The cells were grown a further 2.5 h, after which they were harvested by centrifugation. The cell pellets were subsequently resuspended in TST-buffer (25 mM Tris(hydroxymethyl) aminomethane, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20 pH 8.0) supplemented with Complete EDTA-free protease inhibitor cocktail (Roche Diagnostic, Basel, Switzerland) and then broken by sonication. The fusion toxins were purified on an HiTrap NHS sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden) with immobilized HSA, as previously described [31 (link)]. The proteins were eluted with 0.5 M acetic acid (pH 2.6) followed by buffer exchange to phosphate buffered saline (PBS). The molecular masses of the eluted proteins were determined by liquid chromatography electrospray ionization mass spectrometry on a Bruker impact II instrument (Agilent Technologies, Santa Clara, CA, USA), as previously described [31 (link)].