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Heterologous expression of gamma E-crystallin produces protein with an aberrant tertiary structure
1Wolfson Laboratory, School of Animal and Microbial Sciences, University of Reading, United Kingdom.
Abstract:
Expression of complete rat gamma E-crystallin cDNA in Saccheromyces cerevisiae and in Escherichia coli at 25 degrees C produced soluble proteins similar to rat gamma E-crystallin but with an altered tertiary structure as judged by tryptophan fluorescence. Expression of rat gamma E-crystallin cDNA in E. coli at 37 degrees C produced insoluble inclusion bodies. Refolding of denatured rat gamma E-crystallin from these inclusion bodies produced a protein similar to rat gamma E-crystallin but with altered secondary and tertiary structure, judged by tryptophan fluorescence and circular dichroism. The secondary structure of the refolded gamma-crystallin was similar in beta-sheet content to the native gamma-crystallin structure but was somewhat shifted from the native spectrum, suggesting some alteration in the relative position of the beta-sheets in the refolded structure. These results have implications for crystallin folding, in particular the importance of lens-specific factors (alpha-crystallin, low water content, redox potential) and crystallin hydration.
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