Complete edta free inhibitor cocktail

The full length BEND3 was expressed in both HEK293 cells and Escherichia coli. For HEK cells, the BEND3 cDNA was cloned into a pCPR0053 (in-house) LIC vector containing 6XHis and strep-I tags. For E. coli, the cDNA was cloned into a pNIC28-Bsa4 LIC vector containing sequences encoding 6xHis and Strep-II tags. Following recombinant protein expression, the cells were resuspended in lysis buffer containing 100 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 tablet/50 ml Complete Inhibitor cocktail EDTA Free (Roche), 1 mM PMSF, 50 U/ml Benzonase, 10% glycerol and 0.5 mM TCEP. The cleared cell lysate was passed through a 5ml Strep-Tactin^® column and the bound protein eluted with buffer containing 2.5 mM desthiobiotin (according to the IBA technologies manual). The eluted fractions were further purified using Heparin sepharose (GE Healthcare) and Superose 6 size exclusion columns (GE Healthcare) with the final buffer containing 25 mM Hepes–KOH, pH 7.5, 150 mM NaCl, 0.5 mM TCEP and 10% glycerol.

Fragments of the PICH and BEND3 cDNAs corresponding to different domains were cloned into pNIC28-Bsa4 LIC vector containing sequences encoding 6xHis and Strep-II tags at the N-terminus. The encoded domains were then expressed in E. coli Rosetta cells. The cells were grown to an OD 0.8 of 37°C, and expression was induced by 0.5 mM IPTG at 18°C for 26 h. Cells were then harvested and resuspended in lysis buffer (50 mM Na-phosphate, pH 7.5, 300 mM NaCl, 1 tablet/50 ml Complete Inhibitor cocktail EDTA-Free (Roche), 1× BugBuster (Novagen), 50 U/ml Benzonase, 10 mM Imidazole, 10% glycerol, 0.5 mM TCEP). The cell suspension was lysed using a French press, and the lysate was clarified by centrifugation. The cleared lysate was then purified using the combination of Ni-column chromatography, Strep tag and IMAC and further purified using heparin, anion and cation exchange chromatography. In each case, the final purification step was via size exclusion chromatography (Superdex 75 or Superdex 200; GE Healthcare). In each case, the column was equilibrated in 25 mM Hepes–KOH, pH 7.5, 150 mM NaCl, 0.5 mM TCEP and 10% glycerol. All purified proteins were stored in aliquots at –80°C, and were analyzed and validated using both SDS-PAGE and mass spectrometry. The correct folding of each of the isolated domains was also verified using circular dichroism.