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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

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Recombinant expression, purification, and characterization of human ATE1 arginyltransferase.

Thilini Abeywansha¹, Abigail Kim², Sahil Bhaskaran¹

¹Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH, United States.

Methods in Enzymology

|August 31, 2025

Summary

This study details a simple method to produce and purify human Arginyl-tRNA-protein transferase 1 (ATE1) enzyme. The optimized process yields highly pure, active ATE1, crucial for understanding protein regulation.

Keywords:

ATE1 Arginylation Arginyltransferase Post-translational modification tRNA

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Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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Published on: August 3, 2017

Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Arginyl-tRNA-protein transferase 1 (ATE1) is a key enzyme involved in protein degradation and regulation.
Understanding ATE1's function requires reliable methods for its expression and purification.
Existing methods may not be efficient for obtaining large quantities of active enzyme.

Purpose of the Study:

To develop a straightforward and efficient method for expressing and purifying recombinant human ATE1 from E. coli.
To enable the production of milligram-scale quantities of highly pure and enzymatically active ATE1.
To establish robust assays for validating ATE1 activity and quantifying its interaction with tRNA.

Main Methods:

Recombinant expression of human ATE1 in E. coli.
Chromatographic purification techniques to achieve high purity (>98%).
Enzymatic arginylation assay coupled with mass spectrometry for activity validation.
Electrophoretic Mobility Shift Assay (EMSA) for quantifying ATE1-tRNA binding affinity.

Main Results:

Successful expression and purification of milligram quantities of recombinant human ATE1.
Achieved over 98% purity for the isolated ATE1 enzyme.
Demonstrated enzymatic activity of the purified ATE1 through arginylation assays.
Quantified ATE1-tRNA binding affinity using EMSA.

Conclusions:

The presented method provides an efficient and reliable way to obtain highly pure, active human ATE1.
This protocol facilitates further research into ATE1's role in protein regulation and turnover.
The validated assays are essential tools for characterizing ATE1 function and interactions.