Researcher(s)
- Lekha Antala, Biomedical Engineering, University of Delaware
Faculty Mentor(s)
- Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware
Abstract
A key regulator of the immune system is the interaction between MHC Class II and antigenic epitopes, where high-affinity sequences elicit a heightened immune response. However, not all bacterial pathogens have antigenic epitopes. One hypothesized method to enhance the immunogenicity of antigens is through the incorporation of nitrated nonstandard amino acids (nsAA). To determine preferred incorporation sites, we pair the benefit of efficient nsAA incorporation in bacteria with a compatible yeast display of a human protein to develop a screening platform for MHC Class II epitope binding.
The objective of this project is to establish an effective screening platform where S. cerevisiae expresses MHC Class II and E. coli expresses epitopes that bind to MHC Class II. We cultured E. coli carrying epitopes of different binding affinities and induced their expression with arabinose. Similarly, yeast cells expressing MHC Class II were inoculated and cultured in dextrose media and transferred to galactose-induced media. Both strains were labeled with tag-specific antibodies and analyzed using flow cytometry to confirm protein display. To assess binding interactions, a co-incubation assay was performed where bacterial and yeast cells were mixed and incubated. Yeast cells were stained with Calcofluor white while bacterial cells constitutively expressed green-fluorescent protein (GFP). The co-incubation mixtures were observed using microscopy and flow cytometry to detect potential binding.
We show successful expression and display of epitopes in E. coli cells and the HLA-DR401 protein in yeast. Despite confirming protein expression in both organisms, we have not yet achieved binding between the expressed epitopes and MHC Class II. Ongoing optimization of the co-incubation procedures is required to facilitate binding. Upon completion of this project, this platform has the potential for large-scale screening of epitope-MHC Class II interactions, which could significantly contribute to the development of vaccines for antigens with low immunogenicity.