Designing a De Novo Protein to Inhibit Shiga Toxin Expression in Pathogenic Escherichia coli
E. coli food poisoning is an issue well-known to the general population. Shiga toxin producing E. coli (STEC) are pathogenic strains of the bacteria often found at the root of outbreaks of foodborne illness. The Shiga toxin protein, encoded by the Stx gene in the bacterial chromosome, is responsible for the virulence of STEC food contamination. A natural repressor protein exists for the Stx gene, the lambda repressor protein (CI), but is cleaved by a variety of natural signals, notably by DNA damage which triggers the SOS response. This investigation aims to design and test several de novo proteins engineered to bind the Stx gene in E. coli, inhibiting the production of the Shiga toxin and reducing foodborne illness. A stable de novo four helix bundle protein from the Protein Data Bank serves as a base from which mutations are made. Mutations of our new inhibitor protein are made by applying knowledge of chemical principles in the program UCSF Chimera, and data evaluating protein binding is collected through Molecular Dynamics Simulations. The best protein designs will be tested in vitro to determine their binding constant relative to the target Stx gene. Due to the ongoing pandemic, only in silica design of the four helix bundle motif has been carried out. Thus far, 31 different mutated alpha helices have been created and are to be tested for their binding affinity using Chimera Molecular Dynamics Simulation. Our goal is to develop a protein that can bind to DNA tighter than the natural CI, minimizing food contamination.
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