Attempts to construct an Amyloid fiber-plasmid model to study the effect of Thioredoxin and Flavin reductase on overexpression of the Curli Protein in Escherichia coli

Abstract

Alzheimer’s disease is a debilitating neurodegenerative disease characterized by the formation of insoluble amyloid plaques in the brain. Escherichia coli curli, encoded by the csg gene, is the prokaryotic equivalent of amyloid protein. To study the effect of a reducing protein on the solubility of curli, attempts were made to fuse curli to thioredoxin and flavin reductase. Both proteins are believed to enhance protein solubility by breaking disulfide bridges among thiol groups. Thioredoxin is featured in the Novagen pET32a(+) plasmid as a fusion protein that enhances solubility upon overexpression with a foreign protein. Three constructs were designed to study the curli-amyloid model: curli/thioredoxin, curli/flavin reductase, and curli alone. Plasmids from five potential pET32a(+) (cid:85) trxA/fre constructs, SW052 to SW056, were isolated by alkaline lysis method and analyzed by restriction digest with HincII, pFlMI, and BglI. No appropriate restriction endonuclease fragments were generated from the tested constructs, but a comparison of the linearized plasmids revealed the absence of an appropriate 790bp fre fragment. Another 67 clones, including SA067 to SA069 were generated for curli/thioredoxin, and 6 clones, SA061 to SA066 were generated for curli alone. Neither of the two clones gave appropriate bands in a simple restriction digest and gel electrophoresis experiment. More sequencing experiments and restriction digests must be done to assess the potential of the SA061 to SA069 constructs.