Refolding of denatured/reduced lysozyme at high concentration with diafiltration.
Refolding of reduced and denatured protein in vitro has been an important issue for both basic research and applied biotechnology. Refolding at low protein concentration requires large volumes of refolding buffer. Among various refolding methods, diafiltration is very useful to control the denaturant and red/ox reagents in a refolding solution. We constructed a refolding procedure of high lysozyme concentration (0.5-10 mg/ml) based on the linear reduction of the urea concentration during diafiltration under oxygen pressure. 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 protein concentrations, 5 mg/ml and 10 mg/ml, respectively, after 11 h. This method gave a high productivity of 40.1,microM/h of the refolding lysozyme. The change in refolding yields during the diafiltration could be simulated using the model of Hevehan and Clark.
Department of Biotechnology, Tottori University, Japan. yoshii@bio.tottori-u.ac.jp
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D000818:Animals; D002645:Chickens; D005374:Filtration / Q000295:instrumentation; D066298:In Vitro Techniques; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D014508:Urea
D014508:Urea; D009113:Muramidase