Poster abstracts
Poster number 41 submitted by Olabode Dawodu
Engineering Aminoacyl-tRNA Synthetases (aaRS)/tRNA Pairs to Recognize Cysteine-Reactive Unnatural Amino Acids to Generate Bacterial-Displayed Cyclic Peptides
Olabode Dawodu (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Caitlin Specht (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Alejandro Tapia (Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana), Jennifer Flora (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Jeffery M. Tharp (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana)
Abstract:
Bacteria surface display is a direct evolution technique that allows screening of large peptide libraries to identify novel peptides that bind to a protein of interest. Unlike traditional peptide screening, the peptides are genetically encoded, allowing for the ease of peptide hit determination via sequencing. To form the cyclic peptide, we genetically encoded an unnatural amino acid (uAA) in our peptide that will react with cysteine to form the cyclized peptide. We use an engineered amino-acyl tRNA synthetase (aaRS) that will encode the uAA at TAG stop codons. To confirm installment of the unnatural amino acid at the TAG codon, we utilize our streptavidin model by constructing a plasmid containing a streptavidin-binding peptide that is suppressed by a TAG stop codon. Installment of the uAA was confirmed by flow cytometry by comparing the two cell populations (+/- uAA) that were labeled with streptavidin-PE. Cells supplemented with the uAA, had an increased PE signal compared to cells that were not supplemented with the uAA. These findings gave us support that we can develop a cyclic peptide library to our target protein, programmed cell death ligand 1 (PD-L1); an immune checkpoint protein overexpressed in many cancer cells.
As a starting point, we developed a cyclic peptide library based on a previously reported anti-PD-L1 linear peptide, CLP003. Using fluorescently-activated cell sorting (FACS), we identified four peptides hits that bind to fluorescently-labeled PD-L1. Our results suggest that engineered aaRS/tRNAs can be used to generate and screen novel cyclic peptides to PD-L1.
References:
1.Claudio Zambaldo, Xiaozhou Luo, Angad P. Mehta, and Peter G. Schultz. Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids. Journal of the American Chemical Society 2017;139 (34), 11646-11649.
2.Liu H, Zhao Z, Zhang L, Li Y, Jain A, Barve A, Jin W, Liu Y, Fetse J, Cheng K. Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy. J Immunother Cancer. 2019 Oct 22;7(1):270. doi: 10.1186/s40425-019-0705-y.
3.Tejas Navaratna, Lydia Atangcho, Mukesh Mahajan, Vivekanandan Subramanian, Marshall Case, Andrew Min, Daniel Tresnak, and Greg M. Thurber. Directed Evolution Using Stabilized Bacterial Peptide Display. Journal of the American Chemical Society 2020;142 (4), 1882-1894
Keywords: Aminoacyl-tRNA synthetases, Bacteria display, Cyclic