The eluted recombinants from both methods were dialyzed against PBS (desalting step) using Amicon ultra 0.5 ml 3 kDa cutoff centrifugal filters (Millipore, Ireland). For that, the centrifugal filters were wet with 300 ml of PBS pH 7.0 before adding 200 ml of the eluted protein. After 30 min of centrifugation at 14,000 rpm at 4uC, the volume of the protein was reduced to approximately 100 ml and 400 ml of PBS pH 7.0 were added. This process was repeated four times and the resultant sample collected and stored at 220uC.