Artificial chaperone-assisted refolding of GuHCl-denatured alpha-amylase at low temperature: refolding versus aggregation.

Refolding of GuHCl-denatured alpha-amylase was investigated using the artificial chaperone-assisted method. Three different cationic detergents (CTAB, TTAB and DTAB) and two nonionic detergents (Tween 80 and Triton X-100) were evaluated as the capturing reagents along with alpha- and beta-CD as the stripping agents. The refolding yields, at a final protein concentration of 0.15 mg/ml, were 82, 71 and 66% in the presence of beta-CD and CTAB, TTAB or DTAB, respectively. To improve the refolding yield and to suppress the extent of aggregation, the initial rate of the stripping step was slowed down by maintaining the refolding environment at 4 degrees C for about 3 min followed by raising the temperature to 25 degrees C. Under this thermal procedure, the refolding yield and the extent of aggregation were changed from 82 and 25% at 25 degrees C to 94 and 7% at 4 degrees C, respectively. These findings may assist the activity recovery of recombinant proteins at relatively high concentrations.