Refolding of denatured/reduced lysozyme at high concentration with diafiltration

Refolding of reduced and denatured protein in vitro has been an important i ssue for both basic research and applied biotechnology. Refolding at low pr otein concentration requires large volumes of refolding buffer. Among vario us refolding methods, diafiltration is very useful to control the denaturan t and red/ox reagents in a refolding solution. We constructed a refolding p rocedure of high lysozyme concentration (0.5-10 mg/ml) based on the linear reduction of the urea concentration during diafiltration under oxygen press ure. When the urea concentration in the refolding vessel was decreased from 4 M with a rate of 0.167 M/h, the refolding yields were 85% and 63% at pro tein concentrations, 5 mg/ml and 10 mg/ml, respectively, after 11 h. This m ethod gave a high productivity of 40.1 mu M/h of the refolding lysozyme. Th e change in refolding yields during the diafiltration could be simulated us ing the model of Hevehan and Clark.