| Autor: |
Wong DA, Shaver ZM, Cabezas MD, Daniel-Ivad M, Warfel KF, Prasanna DV, Sobol SE, Fernandez R, Nicol R, DeLisa MP, Balskus EP, Karim AS, Jewett MC |
| Jazyk: |
angličtina |
| Zdroj: |
BioRxiv : the preprint server for biology [bioRxiv] 2024 Nov 25. Date of Electronic Publication: 2024 Nov 25. |
| DOI: |
10.1101/2024.03.25.586624 |
| Abstrakt: |
Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTM installing proteins often suffer from low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free protein synthesis (CFPS) with AlphaLISA for the rapid expression and testing of PTM installing proteins. We apply our workflow to two representative classes of peptide and protein therapeutics: ribosomally synthesized and post-translationally modified peptides (RiPPs) and conjugate vaccines. First, we demonstrate how our workflow can be used to characterize the binding activity of RiPP recognition elements, an important first step in RiPP biosynthesis, and be integrated into a biodiscovery pipeline for computationally predicted RiPP products. Then, we adapt our workflow to study and engineer oligosaccharyltransferases (OSTs) involved in conjugate vaccine production, enabling the identification of mutant OSTs and sites within a carrier protein that enable high efficiency production of conjugate vaccines. In total, we expect that our workflow will accelerate design-build-test cycles for engineering PTMs. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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