Abstract
This thesis aims to analyze the tolerability of peptides and mRNA-peptide covalent fusion complexes to undergo cell-free post-translational modifications to expand the scope of biological therapeutics and mRNA display with the aim of obtaining native-like HIV epitopes that tightly bind to HIV broadly neutralizing antibodies (bnAbs). To test this tolerability, we sought to modify selection-winning peptides against bnAb 2G12 by incorporating new purification systems via the attachment of an N-terminal biotin. Likewise, the tolerability of new biotin-mediated purification methods of mRNA display fusions, in which peptides are covalently conjugated to their mRNA via a puromycin linkage, was developed. Achieving these new purification strategies would expand peptide modifications by immobilizing the peptides allowing for simple separation from modification reagents. Unfortunately, poor yields in the translation and purification of both peptides and fusions, as well as failure of chemoenzymatic modifications, limited this study to troubleshooting the technical methods for modifications. Reported here are the efforts to optimize translation and post-translational modification conditions.