Binding Affinities of Notch Delta Like Ligand 1 and Jagged 1 Variants Through Yeast Display
“Notch receptors are present in all multicellular organisms, but the binding affinities of DLL1 and Jagged1 ligands, or their subdomains, are not known. These ligands can bind to Notch receptors in a monomer form and act as inhibitors, but when multimerized the binding elicits an immune response that enhances T-cell proliferation and reduces size of tumors. The purpose for measuring the binding affinity is that DLL1 and Jagged1 consist of DSL and EGF repeats, the roles of which are not well know, and it is unclear if the ligands bind equally in mouse receptors and human receptors. We are examining several variants but the main focus is currently on a variant with DSL-EGF-EGF from mouse DLL1, one with MNNL-DSL-EGF-EGF-EGF (i.e., it contains two additional domains), and one with MNNL-DSL-EGF-EGF-EGF-EGF (i.e., it contains three additional domains). In addition, error-prone PCR libraries of these variants will be examined.
The presentation of these ligands will occur through yeast display. Through transformation of a shuttle plasmid pCTcon2, which contains a DNA insert that encodes the ligand, into EBY100 yeast cells, the ligand can be displayed on the surface of the cell. The transformed cells are then sorted with magnetic beads that allow for isolation of strongest binders to Notch receptors. These variants are then enriched using a fluorescent binding assay (FACS) that further isolates strong binding variants. Once the library has been sorted and enriched, a Surface Plasmon Resonance (SPR) assay will be used to determine the binding affinity of the variants.
The SPR assay demonstrated that the shortest construct binds to mouse receptors with a KD of 8.3 µM, which is consistent with previous literature, confirming our methodology for isolating the variants. The binding for construct with the additional MNNL and EGF domains had a KD of 67 nM indicating that the presence of the MNNL domain has a positive effect on binding affinity of mouse ligands to mouse receptors.”