Utilization of Synthetically Evolved Nanobodies to Target Small Molecules

Synthetically Evolved Nanobodies (SENs) are a novel molecular biology platform inwhich one can find binders to various small molecules at much faster rate than previous techniques. These nanobodies are produced through the use of directed evolution and flow cytometry. SENs are about 15kD in size and consist of antigen-binding heavy chain domains, originally found in Camelidae, that are structurally similar to human/mouse single chain variable fragments [1-3]. Additionally, SENs have quite robust biophysical properties, including thermodynamic stability, genetic encodability, and better protein folding reliability compared to ScFv’s. To produce these nanobodies, E. coli is engineered to display libraries of these nanobodies on the surface of the bacterial cell. From there, the library undergoes rapid evolution with the help of FACS to “evolve” the antibody-like scaffolds to recognize and bind specific antigens. Here, we skip the use of animals for antibody production and shift to a more automatable practice which takes a few days to weeks. For the purposes of this thesis, we target amyloid-beta protein, a major factor in the development of Alzheimer’s symptoms when misfolded in the human brain