Heterologous Expression of γE-Crystallin Produces Protein with an Aberrant Tertiary Structure

Expression of complete rat γE-crystallin cDNA in Saccheromyces cerevisiae and in Escherichia coli at 25°C produced soluble proteins similar to rat γE-crystallin but with an altered tertiary structure as judged by tryptophan fluorescence. Expression of rat γE-crystallin cDNA in E. coli at 37°C produced insoluble inclusion bodies. Refolding of denatured rat γE-crystallin from these inclusion bodies produced a protein similar to rat γE-crystallin but with altered secondary and tertiary structure, judged by tryptophan fluorescence and circular dichroism. The secondary structure of the refolded γ-crystallin was similar in β-sheet content to the native γ-crystallin structure but was somewhat shifted from the native spectrum, suggesting some alteration in the relative position of the β-sheets in the refolded structure. These results have implications for crystallin folding, in particular the importance of lens-specific factors (α-crystallin, low water content, redox potential) and crystallin hydration.