Protein engineering to enable site-specific bioconjugation through catechol-boronate coupling chemistry
| Author(s) | Chattopadhyay, Soumili | |
| Date Accessioned | 2021-11-17T17:09:38Z | |
| Date Available | 2021-11-17T17:09:38Z | |
| Publication Date | 2021 | |
| SWORD Update | 2021-08-11T16:05:35Z | |
| Abstract | Over the past few decades, the customizability provided to biomolecules through protein conjugation has enabled a diverse set of applications, which include protein-based therapeutics, biomaterials, and biosensors. However, current chemical modification techniques suffer from lack of specificity that results in heterogeneous mixtures, limited time stability in vivo, and perturbations to the protein function. Synthetic biology researchers have overcome some of the challenges by utilizing genetic code expansion technology to enable precise formation of protein conjugates. ☐ In this project, we seek to develop a bio-orthogonal protein conjugation platform in Escherichia coli (E. coli) based on catechol-boronate coupling chemistry. This chemistry proceeds in aqueous and mild conditions and can be tuned to be reversible or irreversible according to the end application. In order to perform catechol-boronate conjugation in proteins, we have site-specifically inserted catechol amino acids into proteins using non-standard amino acid (nsAA) incorporation technique. Although a catechol amino acid: L-3,4- dihydroxyphenylalanine (DOPA) is a proven candidate for ribosomal incorporation, an unaddressed challenge is the replacement of tyrosine with DOPA due to the structural similarity. We have investigated in this thesis whether a DOPA analogue having extended side chain (C5-DOPA) with lower resemblance to tyrosine can be site-specifically incorporated into proteins. We have screened a library of published xii OTSs from different families for specificity and activity on C5-DOPA. Based on our results top performing candidates will be engineered as next step for improved specificity to incorporate C5-DOPA at multiple instances in a reporter protein. | en_US |
| Advisor | Kunjapur, Aditya M., | |
| Degree | M.Ch.E. | |
| Department | University of Delaware, Department of Chemical and Biomolecular Engineering | |
| DOI | https://doi.org/10.58088/v4sm-8b19 | |
| Unique Identifier | 1285523842 | |
| URL | https://udspace.udel.edu/handle/19716/29397 | |
| Language | en | |
| Publisher | University of Delaware | en_US |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/protein-engineering-enable-site-specific/docview/2572604829/se-2?accountid=10457 | |
| Keywords | Protein engineering | en_US |
| Keywords | Bioconjugation | en_US |
| Keywords | Catechol-boronate coupling | en_US |
| Title | Protein engineering to enable site-specific bioconjugation through catechol-boronate coupling chemistry | en_US |
| Type | Thesis | en_US |