Control of aggregate formation in refolding of carbonic anhydrase at high urea concentrations and effects of urea removal

Refolding of CAB was studied at high concentrations of protein and urea. Aggregate formation was decreased by using higher concentrations of urea in refolding buffer. Refolding yields of 60% were obtained at high concentrations of CAB by using urea concentrations of 3.0-4.0 M in renaturation mixture, Comparison of the refolding yield and the amount of soluble CAB in renaturation mixture indicated that the apparent specific activity of refolded CAB was lower than that of native one at higher concentrations of urea. Difference in the steric structures of native and refolded CAB was observed in CD spectra of these samples. However, removal of urea from loosely folded CAB solution by dialysis recovered the native structure of CAB, as well as its native specific activity. Hence the refolding efficiency after dialysis was improved to about 70-80% at CAB concentrations of 3-5 kg/m(3). Effect of incubation time of renaturation mixture before dialysis was studied, and it was shown that incubation time less than 3 hours increased aggregate formation and decreased the refolding yield. Therefore, formation of more stable intermediates in the refolding pathway decreases aggregate formation during urea removal. These results lead to a consecutive dilution-dialysis method for refolding at high concentrations of proteins.