Refolding of denatured and denatured/reduced lysozyme at high concentrations.
Refolding of proteins at high concentrations often results in aggregation. To gain insight into the molecular aspects of refolding and to improve the yield of active protein, we have studied the refolding of lysozyme either from its denatured state or from its denatured/reduced state. Refolding of denatured lysozyme, even at 1 mg/ml, yields fully active enzyme without aggregation. However, refolding of denatured/reduced lysozyme into buffer that lacks thiol/disulfide reagents leads to aggregation. Thiol/disulfide redox reagents such as cysteine/cystine and reduced/oxidized glutathione facilitate the renaturation, with the yield depending on their absolute concentrations. We have obtained an approximately 70% renaturation yield upon refolding of lysozyme at 150 microgram/ml. The cysteine/cystine redox system is more efficient compared with the glutathione redox system. When lysozyme is refolded in the absence of redox reagents, a transient intermediate that has regained a significant amount of secondary structure is formed. The tryptophans in this intermediate are as exposed to water as in the fully unfolded protein. It shows increased exposure of hydrophobic surfaces compared with the native or completely unfolded enzyme. This aggregation-prone intermediate folds to active enzyme upon addition of oxidized glutathione before the aggregation process starts. These properties of the intermediate in the refolding pathway of lysozyme are similar to those proposed for the molten globule.
Raman
B
B
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Ramakrishna
T
T
Rao
C M
CM
D000818:Animals; D002021:Buffers; D002645:Chickens; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000187:drug effects; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics; D014364:Tryptophan; D014867:Water
D002021:Buffers; D014867:Water; D014364:Tryptophan; D009113:Muramidase; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide