Isopropyl b d 1 thiogalactopyranoside

The membrane topology and the boundaries the periplasmic sensing domain of CtaB (CtaB PTPSD, residues 32-272) from P. fluorescens Pf0-1 (UniProt ID Q3KK38) were predicted by TOPCONS server (http:// topcons.net/) (19) (Figure 1). The sequence encoding CtaB PTPSD was codon optimized for expression in Escherichia coli, synthesized and ligated into the pET151/D-TOPO vector (Invitrogen) by Genscript to generate an expression vector that harbors an N-terminal His6 tag followed by a TEV protease cleavage site. The expression vector was introduced into E. coli BL21 (DE3) (Novagen) and cells were grown in Luria-Bertani medium supplemented with 50 µg/mL ampicillin to an OD 600 of 0.6 at 310 K. Overexpression of CtaB PTPSD was induced with 0.5 mM isopropylb-D-1-thiogalactopyranoside (Thermo Scientific) and growth was continued for 3.5 h at 210 K. The cells were harvested by centrifugation at 6,000 g for 15 min at 277 K. The cells were resuspended in buffer A (10 mM Tris-HCl buffer pH 8.0 and 200 mM NaCl), lyzed by sonication and centrifuged at 10,000 g for 30 min at 277 K. SDS-PAGE gel electrophoresis of clarified supernatant and pellet confirmed that CtaB PTPSD expressed in inclusion bodies (IBs).

ELP expression vectors were transformed into OneTouch BL21 (DE3) E. coli (Invitrogen, Carlsbad, CA) cells, and 1 L cultures of these cells were grown in Terrific Broth (TB) (Thermo Fisher Scientific, Waltham, MA) supplemented with 100 µg/mL ampicillin (Thermo Fisher Scientific) and 10 mM L-proline (Sigma-Aldrich, St. Louis, MO). Expression was induced for 24 h using 2 mM isopropyl b-D-1-thiogalactopyranoside (Thermo Fisher Scientific).
Purification was achieved by first performing denaturing metal-affinity chromatography and eluting the ELPs using an imidazole step gradient. Buffered 8 M urea (Thermo Fisher Scientific) was used to lyse the cells and ensure the ELPs were fully soluble and free from any inclusion bodies. The urea was removed during the extensive column washing. Eluents were screened for the presence of ELPs by gel electrophoresis or by heating them to room temperature or 37°C, depending on the construct, and observing which samples turned reversibly cloudy. Samples confirmed to contain ELPs were combined and then subjected to one round of ITC for final purification. The temperature used to cause ELP aggregation varied depending on the construct. The purification was confirmed with denaturing PAGE (SDS-PAGE) and the ELP concentrations were measured by sample absorbance at 280 nm.