Bioconjugation and Protein Engineering for the Development of a Peptide-Protein Conjugate Vaccine and Characterization of an N-terminal Modification Reaction

Post-translational protein modification accounts for a significant amount of biodiversity and is essential for many cellular processes. The development of techniques to mimic native biomolecule modification have evolved into the field of modern bioconjugation. The complementary use of both genetic and chemical methods has provided a large toolbox for an endless possibility of potential bioconjugate constructs, using a wide-variety of synthetic and biologically-derived materials. To this end, reproducing these natural modifications of biomolecules provides researchers a way to interrogate and elucidate the intricate functions within biological systems./nWithin this bioconjugation toolbox, there exist a large number of different chemical reactions for protein modification. The site-specific covalent link between a protein and synthetic moiety, such as a drug or fluorophore, enables the creation of hybrid material that capitalizes on the properties of both individual components. Thus, bioconjugate materials have a wide variety of applications, such as the study of proteins in a biological context, the elucidation of a multi-protein quartenary structure, creating unique protein-based materials, the development of improved therapeutics, and many more./nOne application of site-specific chemical modification of proteins is the development of conjugate vaccines, as described herein. Synthetic vaccines offer great promise as useful therapeutics; however, often individual moieties suffer from poor delivery or weak immunogenicity. Conjugation to a carrier protein can circumvent this issue. Work presented describes the use of cross-reactive material 197 (CRM197) as a carrier protein for the presentation of therapeutic peptide cargo. Heterobifunctional linkers, comprised of orthogonal lysine-reactive and cysteine-reactive handles, were used to modify lysine residues of CRM197 to attach cysteine-containing peptide therapeutics. Ultimately, the bioconjugation strategies explored led to a structurally heterogeneous conjugate material. As structure-immunogenicity relationships exist, we turned to protein engineering of CRM197 to facilitate the creation of structurally homogeneous conjugate material. Work is ongoing to prepare and characterize the resulting peptide-protein conjugates./nIn creating synthetic vaccines, multiple sites of protein modification are often necessary to enable proper immune response. However, often it is desirable to have a site-specific, single modification on a protein of interest. There remains a need for the development of more chemoselective chemical modification of proteins that are mild, efficient, and robust. As a result, novel protein modification techniques target uniquely reactive sites, such as the N-terminal amine, due to its unique environment and pKa. Our group recently reported a single-step N-terminal modification with 2-pyridinecarboxaldehyde (2PCA), which proceeds under physiological conditions. However, certain N-terminal residues were found to have different reactivity and stability toward 2PCA modification. Thus we set out to characterize the reaction mechanism in order to understand this relationship, combining computational analysis, NMR of 2PCA modified peptides, and mass spectrometry on 2PCA modified proteins. With this multifaceted approach, several N-terminal residues were found to strongly promote stable 2PCA modification. Further, we are exploring the key attributes promoting product formation in order to create second generation 2PCA derivatives that will control reaction outcome.

Advisor: Francis, Matthew B