title: Effective renaturation of reduced lysozyme by gentle removal of urea.
abstract: To increase the folding yield of concentrated reduced lysozyme, we developed a renaturation method by means of dialysis from concentrated urea with redox agents. After lysozyme was incubated in the reducing buffer (8 M urea solution) with oxidized glutathione, renaturation of reduced lysozyme was started by dialysis against the dialyzing buffer containing 8 M urea with redox agents. The urea concentration of the dialyzing bottle was gradually diluted with dialyzing buffer without urea at a flow rate of 0.1 ml/min by high pressure pump. Using this systematic dialysis, a concentration as high as 5 mg/ml of reduced lysozyme could be renaturated in 80% yield, while the folding yield was < 5% even at a concentration of 1 mg/ml using a conventional rapid dilution method [Goldberg et al. (1991) Biochemistry, 30, 2790-2797]. Therefore, it was concluded that gentle removal of urea from denatured proteins, dissolved in concentrated urea solution, by means of dialysis should be useful to renature denatured proteins effectively.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Maeda
authors.forename: H
authors.initials: H
authors.lastname: Koga
authors.forename: H
authors.initials: H
authors.lastname: Yamada
authors.forename: T
authors.initials: T
authors.lastname: Ueda
authors.forename: T
authors.initials: T
authors.lastname: Imoto
mesh_terms: D002021:Buffers; D003956:Dialysis; D005978:Glutathione / Q000737:chemistry; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D012996:Solutions; D013696:Temperature; D014508:Urea / Q000032:analysis / Q000494:pharmacology
chemical_list: D002021:Buffers; D012996:Solutions; D008623:Mercaptoethanol; D014508:Urea; D009113:Muramidase; D005978:Glutathione