Development of cell-free platforms for discovering, characterizing, and engineering post-translational modifications

Area of Science:

• Biochemistry
• Protein Engineering
• Synthetic Biology

Background:

• Post-translational modifications (PTMs) are crucial for therapeutic protein and peptide stability and function.
• Existing methods for studying PTM-installing proteins are often low-throughput.
• There is a need for efficient workflows to engineer PTMs for therapeutic applications.

Purpose of the Study:

• To develop a generalizable workflow for rapid expression and testing of PTM-installing proteins.
• To apply this workflow to ribosomally synthesized and post-translationally modified peptides (RiPPs) and conjugate vaccines.
• To accelerate the design-build-test cycles for engineering PTMs.

Main Methods:

• Coupling cell-free protein synthesis (CFPS) with AlphaLISA for protein expression and functional assays.
• Characterizing RiPP recognition element binding activity.
• Studying and engineering oligosaccharyltransferases (OSTs) for conjugate vaccine production.

Main Results:

• Demonstrated workflow for characterizing RiPP recognition elements and integration into biodiscovery pipelines.
• Adapted workflow to identify mutant OSTs and carrier protein sites for efficient conjugate vaccine production.
• Successfully enabled rapid expression and testing of PTM-installing proteins.

Conclusions:

• The developed workflow significantly accelerates the engineering of PTMs.
• This approach facilitates the design and production of novel therapeutic peptides and vaccines.
• The workflow is generalizable across different classes of PTM-installing proteins.