Related Experiment Video
Updated: May 5, 2026
Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins
Published on: May 30, 2025
A novel quantitative kinase assay using bacterial surface display and flow cytometry
Sónia Troeira Henriques1, Louise Thorstholm, Yen-Hua Huang
1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
Abstract:
The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.
Insights
A new Bacterial Kinase (BacKin) assay uses bacteria to display substrates for cost-effective, high-throughput screening of tyrosine kinase inhibitors. This method offers a simpler, non-radioactive alternative for cancer drug discovery.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Tyrosine kinase inhibition is a validated strategy for cancer treatment.
- Developing novel kinase inhibitors requires efficient screening assays.
- Existing assays often lack cost-effectiveness, simplicity, or broad applicability.
Purpose of the Study:
- To develop a novel, cost-effective, high-throughput screening assay for kinase activity.
- To establish a general, non-radioactive method adaptable for various applications.
- To utilize Bcr-Abl tyrosine kinase as a model for assay development and validation.
Main Methods:
- The Bacterial Kinase (BacKin) assay employs peptide substrates displayed on the surface of *Escherichia coli*.
- Kinase activity is measured by quantifying substrate phosphorylation.
- Flow cytometry is used for examining and quantifying phosphorylation events.
Main Results:
- The BacKin assay provides a cost-effective and high-throughput method for kinase screening.
- Bacteria serve as a self-renewing solid support, eliminating complex chemical strategies.
- The assay is suitable for kinetic studies, mechanistic investigations, and inhibitor characterization.
Conclusions:
- The BacKin assay is a versatile and advantageous platform for identifying small-molecule and peptide-based kinase inhibitors.
- This approach facilitates cancer drug development by offering a simplified and economical screening tool.
- The method demonstrates potential for broad application in kinase research and pharmaceutical laboratories.
