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Updated: Oct 13, 2025

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

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Published on: February 5, 2020

Enzymatic C-Terminal Protein Engineering with Amines.

Fabian B H Rehm¹, Tristan J Tyler¹, Kuok Yap¹

¹Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD 4072, Australia.

Journal of the American Chemical Society

|November 11, 2021

Summary

This study introduces a novel chemoenzymatic method for protein and peptide modification using an engineered transpeptidase. This approach enables efficient, site-specific labeling with diverse amines, overcoming limitations of previous methods.

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Area of Science:

Biochemistry
Chemical Biology
Protein Engineering

Background:

Chemoenzymatic modification enables homogeneous conjugate generation for diverse applications.
Existing transpeptidase methods face limitations including synthetic conjugate preparation, bulky tags, and low substrate turnover.

Purpose of the Study:

To develop a versatile peptide/protein labeling strategy using an engineered transpeptidase.
To overcome limitations of current transpeptidase-mediated ligation methods.

Main Methods:

Engineered a promiscuous transpeptidase for C-terminal asparagine labeling.
Utilized commercially available amines for irreversible incorporation.
Demonstrated applications including protein-drug conjugate preparation, C-to-C protein fusion, and dual-terminal protein labeling.

Main Results:

Achieved irreversible incorporation of diverse amines at C-terminal asparagine.
Successfully prepared a homogeneous protein-drug conjugate.
Generated a genetically inaccessible C-to-C protein fusion.
Demonstrated site-specific, sequential labeling at both protein termini.

Conclusions:

The engineered transpeptidase offers a powerful and versatile platform for chemoenzymatic protein and peptide modification.
This strategy expands the toolkit for creating defined protein conjugates and complex protein architectures.