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The use of low concentrations of urea, guanidinium chloride or arginine has been reported in the literature to increase protein refolding and yield of active proteins by suppressing aggregate formation. However, no studies have yet examined whether these substances can exert synergistic or cooperative effects when used in combination. In this work, a comparative study was carried out on refolding of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the presence of different concentrations of urea, guanidinium chloride or arginine. All three folding aids could inhibit the formation of insoluble aggregates of rhG-CSF but with different efficacies. A low concentration of guanidinium chloride was found to denature protein, so that rhG-CSF was not fully or correctly folded even if concentration was reduced to 1M. Low concentration of urea (2M) or arginine (0.5M) did not cause rhG-CSF denaturation, but urea was unable to suppress the formation of soluble oligomers, which persisted at a level of about 30% in refolded soluble rhG-CSF. Arginine, in contrast, could inhibit formation of all soluble oligomers. Based on these phenomena, we tested rhG-CSF folding in a mixture of 2M urea and 0.5M arginine. Kinetic analysis indicated that urea aided in suppressing insoluble precipitates, while arginine prevented formation of soluble oligomers produced by hydrophobic interaction. With this combination system, the refolding yield of rhG-CSF could be increased 2-fold.
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| http://dx.doi.org/10.1016/j.pep.2009.02.004 | DOI Listing |
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