Protein Binder Design Using RFDiffuse and AlphaFold
Presenter(s)
Alex Dahle and Henry Gramann
Abstract
Designing proteins with customized binding domains and structural geometries is a new capability in biochemistry. This study uses methods of protein design with molecular biology protocols to create and characterize novel protein structures. We used RFDiffusion to predict a high affinity protein binder for kinase Aurora-A (AurA), alongside a manual design of a custom protein scaffold of TPX2 (AurA's native binder). The resulting protein sequences were used to design codon optimized plasmids for expression in Escherichia coli. Gene blocks with flanking NheI and XhoI restriction sites, as well as a Tobacco Etch Virus (TEV) protease cleavage site, were ordered and purchased. We plan to insert these into a bacterial expression plasmid, adding an N-terminal 6xHis-tag for Ni-NTA protein purification. Protein purity and concentration will be assessed using SDS PAGE and Bradford assays, and if purification is successful, we plan to assess binding to AurA as well.
College
College of Science & Engineering
Department
Chemistry
Campus
Winona
First Advisor/Mentor
Emily Ruff
Location
Kryzsko Great River Ballroom, Winona, Minnesota; United States
Start Date
4-23-2026 2:00 PM
End Date
4-23-2026 3:00 PM
Presentation Type
Poster Session
Format of Presentation or Performance
In-Person
Session
2b=2pm-3pm
Poster Number
80
Protein Binder Design Using RFDiffuse and AlphaFold
Kryzsko Great River Ballroom, Winona, Minnesota; United States
Designing proteins with customized binding domains and structural geometries is a new capability in biochemistry. This study uses methods of protein design with molecular biology protocols to create and characterize novel protein structures. We used RFDiffusion to predict a high affinity protein binder for kinase Aurora-A (AurA), alongside a manual design of a custom protein scaffold of TPX2 (AurA's native binder). The resulting protein sequences were used to design codon optimized plasmids for expression in Escherichia coli. Gene blocks with flanking NheI and XhoI restriction sites, as well as a Tobacco Etch Virus (TEV) protease cleavage site, were ordered and purchased. We plan to insert these into a bacterial expression plasmid, adding an N-terminal 6xHis-tag for Ni-NTA protein purification. Protein purity and concentration will be assessed using SDS PAGE and Bradford assays, and if purification is successful, we plan to assess binding to AurA as well.
Comments
Dahle, Alex