Figure 1.

Schematic of the ITC protein purification method. First, cells are lysed and the cell debris is removed by centrifugation. NaCl is added to isothermally trigger the phase transition and the fusion protein is collected by centrifugation (hot spin). The supernatant is discarded and the pellet containing the fusion protein is resuspended in cold, low-ionic strength buffer. Some denatured contaminants may be trapped in the pellet, and therefore, a cold centrifugation step (cold spin) is carried out to remove the insoluble denatured contaminants; the fusion protein stays in solution during this step because the solution temperature is lower than the T_t of the fusion protein. The ITC process is repeated 3–5 times to obtain pure fusion protein.