Figure 1.
On-column refolding is faster than solution based dialysis refolding. Refolding can be monitored using RP HPLC. As the disulfide bonds form in SDF-1α the retention time shift. A) RP HPLC traces of solution refolding SDF-1α taken from dialysis at the indicated time points. Solution refolding of SDF-1α after proteolytic removal of the hexahistadine tag is accomplished by diluting the TEV digestion reaction to 150 mL with 20 mM Tris pH 8.0 and dialysis against 4 L of the same buffer. B) RP HPLC traces of on-column refolding SDF-1α with time points as indicated. On-column refolding time points correspond to removal of metal affinity resin after binding solubilized SDF-1α inclusion bodies to the metal affinity resin (0 hours) and flowing a detergent buffer (0.25 hours), a oxidation buffer (0.5 hours), a wash buffer (0.75 hours), and an elution buffer (1.0 hours) over the column. The removed resin was eluted and the elution acidified to prevent further oxidation. Then refolding was monitored using RP HPLC.