About This Project

We are designing antibody domains stabilized by an internal isopeptide bond, an amide group between side chains. This structure is unusual for mammalian proteins, as the inside of proteins is hydrophobic. The motif is, however, common among certain prokaryotes. We will use a neural net to find residues to mutate to form isopeptide bonds. We hope if an internal isopeptide bond were inserted in a mammalian protein the half-life would be significantly greater, showing stabilizing.

Ask the Scientists

What is the context of this research?

Antibodies are frequently used in therapeutic contexts, as well as in assays during biochemical research. While they are relatively stable proteins, their half-life in the body (in the context of a biologic drug) could be extended if they weren't vulnerable to natural degradation mechanisms. We intend to insert an artificial bond into a single fragment of an antibody, as proof of concept that these proteins can be stabilized. This artificial 'isopeptide bond' is common to proteins extruded to the outside of some bacteria, but doesn't appear in mammalian proteins. This bond make the antibody less vulnerable to destruction in the body, which could create a longer lasting drug.

What is the significance of this project?

Antibody engineering has the potential to be an incredible tool both for pharmaceutical development and for biochemical tools such as 'enzyme linked immunosorbent assays.' The introduction of an isopeptide bond into a single domain of a mammalian antibody would be proof of concept for stabilization that would allow biologic drugs such as those used to treat autoimmune disease to last dramatically longer before being metabolized to inactive constituent parts. More stable antibodies also have the potential to create more robust assays for biochemical bench work, which would ultimately lower costs as the tests become less fragile.

What are the goals of the project?

This project will utilize a machine learning interface to identify the closest structural analogue to the antibody in question which already contains an isopeptide bond. Then we plan to use bioinformatic techniques to identify which residues must be altered to create a mutant antibody domain. Once these residues have been identified, we will express the mutated antibody fragments in E. coli, where they ought to remain soluble if they fold correctly. We will tag our antibodies with a fluorophore to see and quantify the soluble protein, which ought to contain the desired bond. We will test for the presence of this isopeptide bond within the folded antibodies using mass spectrometry.