Attempted Cloning of Escherichia coli csg Into Flavin Reductase or Thioredoxin In The pET32a (+) Vector To Assess Disulfide Bond Effects on Amyloid Deposits Formation

Abstract

The decreased levels of thioredoxin activity within amyloid plaques in Alzheimer’s patients support the idea that the disease is initiated by reactive oxygen species. One suggestion for therapy is to create a reducing environment near the amyloid plaques, such as in the temporal or frontal lobe of the brain. The goal of this experiment was to determine if amyloid-like proteins called curli in Escherichia coli will express soluble subunits rather than amyloid-like fibrous tangles when fused with thioredoxin, a ubiquitous reducing enzyme. Fusing thioredoxin to a gene that tends to form inclusion bodies when overexpressed in Escherichia coli expression vectors produces viable, soluble protein with native conformation. An attempt was made to clone the curli gene, csg, into pET32a(+), a commercially available vector containing thioredoxin to determine if amyloid-like plaque formation is affected by disulfide linkages. A second objective was to determine if the NADPH:flavin oxidoreducatse can similarly produce soluble proteins. The project failed to yield any successful ligation of csg to either thioredoxin or NADPH:flavin oxidoreducatse.