Heterologous expression of gamma E-crystallin produces protein with an aberrant tertiary structure.
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.
Wolfson Laboratory, School of Animal and Microbial Sciences, University of Reading, United Kingdom.
D
D
Goode
M J
MJ
Crabbe
D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003459:Crystallins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D010503:Periodic Acid-Schiff Reaction; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic; D014364:Tryptophan / Q000737:chemistry
D003459:Crystallins; D011994:Recombinant Proteins; D014364:Tryptophan