Effective renaturation of reduced lysozyme by gentle removal of urea.
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.
Maeda
Y
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Koga
H
H
Yamada
H
H
Ueda
T
T
Imoto
T
T
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
D002021:Buffers; D012996:Solutions; D008623:Mercaptoethanol; D014508:Urea; D009113:Muramidase; D005978:Glutathione