Utilization of Polymer Based Protein Engineering and ATRP to Modulate Substrate Size Specificity of Avidin

Abstract

Avidin and biotin interact very strongly and are commonly used to tag and purify proteins or peptides for proteomic analysis. Using a pH dependent reversible biotin tag that targets primary amines, all the proteins can be tagged at a slightly basic pH. After labeling proteins with the biotin tag, any excess unreacted biotin left over in solution must be dialyzed away to prevent competition for binding spots on the Avidin beads. Tagged proteins can then be bound to a solid surface Avidin bead and contaminants washed away. Proteins can then be released from the beads in their original unmodified state by lowering the pH. While this workflow has been shown to be very useful for purifying proteomes, the requirement for dialysis after the initial tagging step makes this workflow laborious and difficult to automate. This step is time consuming, can result in protein loss or precipitation, and makes automation impossible. In this study we seek to utilize polymer based protein engineering to circumvent this issue.Atom transfer radical polymerization (ATRP) has been utilized in recent years to specifically modify and fine tune the behavior of proteins. By adding polymers of a specific composition and length in targeted areas of a protein, the behavior of a protein can be modified. In this study, we utilized ATRP to grow a dense polymer network off the surface of Avidin to act as a molecular sieve; The large polymers allow small molecules of biotin to bind to the binding site, while larger biotinylated proteins are excluded. The resulting “Caged Avidin” can then be used in the proteome purification workflow in the place of dialysis by binding and sequestering any unreacted free biotin, allowing the entire labeling, binding, washing, and eluting workflow to take place in a single tube with no bias and no sample loss. This can revolutionize sample preparation in biomarker and drug discovery by allowing for automation, and thus improved yield and reliability, of sample preparation.Support or Funding InformationThis work was supported in part by the Center for Polymer Based Protein Engineering at Carnegie Mellon UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.