Expression and Purification of GST-fusion Proteins

Expression and purification were performed as above, using the strain E. coli BL21 carrying either carrying pGEX4T3_stop or pGEX4T3_YgfB. Differing from above, the expression was carried out at 25 °C. For resuspension and lysis of the bacterial pellet, GST-A buffer (50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5) supplemented with lysozyme, Triton X-100, DNase and protease inhibitor was used. For purification, a GSTrap™ HP 1 ml column (Cytiva) connected to a peristaltic pump was used. After loading the column and collecting the flow through the column was washed using GST-A buffer and the protein eluted using GST-B-buffer (50 mM Tris, 150 mM NaCl, 10 mM reduced glutathione, pH 8). After column regeneration, the flow through was loaded on the column once again and also washed and eluted. The obtained eluate fractions were pooled and dialysed against 10 liter of PBS pH 7.4 and 0.5 mM DTT using a ZelluTrans (Roth) dialysis tube with a 3.4 kDa cutoff and frozen in dialysis buffer. Analysis by SDS-PAGE and protein storage was done as described above.