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  • Title: "Loose folding" and "delayed oxidation" procedures successfully applied for refolding of fully reduced hen egg white lysozyme.
    Author: Matsubara M, Nohara D, Kurimoto E, Kuroda Y, Sakai T.
    Journal: Chem Pharm Bull (Tokyo); 1993 Jul; 41(7):1207-10. PubMed ID: 8374991.
    Abstract:
    Several factors and/or procedures were examined quantitatively to improve the refolding yields of hen egg white lysozyme from its fully denatured and reduced state. Firstly, we found that refolding treatments were better conducted at lower lysozyme concentrations. The refolding yield decreased from 70% to less than 5% by increasing the lysozyme concentration from 1 to 36 microM in the refolding solution, probably due to aggregation. Secondly, in order to reduce the aggregation and improve the efficiency of refolding, we applied the "loose folding" procedure which required the incubation in the presence of about 2 M urea. The refolding of the lysozyme, studied at 17.4 microM, increased the yield to 80% yield in the presence of 2 M urea compared with a 30% yield in the absence of urea. Furthermore, we obtained a dramatic refolding yield of more than 95% in an experiment conducted at a concentration of 1.1 microM lysozyme, in the presence of 2 M urea. Finally, we examined the "delayed oxidation" procedure which meant that conformational folding preceded formation of disulfide bonds. The application of this procedure resulted in increases of 5-10% in the refolding yield. These procedures are expected to be useful in improving the refolding yield of precipitated proteins, for example, formed during recombinant DNA protein syntheses.
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