Refolding of denatured and reduced lysozyme with cysteine/cystine red/ox solution in 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. Diafiltrati on method is useful to control the denaturant and red/ox reagents in the re folding solution, We constructed a refolding procedure for high concentrati ons of reduced and denatured lysozyme of about 10 mg/ml (700 muM) on linear reduction of urea concentration in diafiltration, 0.8 mM cystine and 8 mM cysteine under nitrogen at 2 atm, This method can obtain about 90% refoldin g yield at 700 muM and almost 100% in 350 muM lysozyme, The refolding yield s during the diafiltration can be simulated using the competitive reaction between the refolding and aggregation. In the red/ox control with cysteine and cystine, a rate order of aggregation reaction of near 2 is obtained.