JOURNAL OF CHEMICAL ENGINEERING OF JAPAN
Print ISSN : 0021-9592
Biochemical, Food and Medical
Refolding of Denatured and Reduced Lysozyme with Cysteine/Cystine Red/Ox Solution in Diafiltration
HIDEFUMI YOSHIITAKESHI FURUTATAKAHIRO YONEHARADAISUKE ITOPEKKA LINKO
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2001 Volume 34 Issue 2 Pages 211-215

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Abstract

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. Diafiltration method is useful to control the denaturant and red/ox reagents in the refolding solution. We constructed a refolding procedure for high concentrations of reduced and denatured lysozyme of about 10 mg/ml (700 μM) 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% refolding yield at 700 μM and almost 100% in 350 μM lysozyme. The refolding yields 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.

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© 2001 The Society of Chemical Engineers, Japan
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