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Issue 2, 2016
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Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

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Abstract

In E. coli and other prokaryotes such as Staphylococcus aureus, and Mycobacterium tuberculosis, biotin protein ligase (BirA) is an emerging drug target as it is the sole enzyme capable of biotin transfer onto the BCCP subunit of ACC. There is currently a gap in simple yet efficient assays for rapidly identifying and characterising inhibitors targeting BirA. We present for the first time the development and validation of a simple and reliable DSF-GTP assay for the high-throughput screening of BirA:ligand interactions using a new GFP-tagged BirA of E. coli. In addition, we developed a new GFP-based biotinylation activity assay taking advantage of a GFP tethered with an AviTag. The data obtained with these assays revealed new insights into how the binding of individual or combinations of ligands affect the overall thermal stability and affinity of BirA. The DSF-GTP assay has a Z′ value of 0.785 that makes it an excellent tool for future high-throughput screening of inhibitory compounds.

Graphical abstract: Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

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Supplementary files

Article information


Submitted
24 Nov 2015
Accepted
25 Nov 2015
First published
02 Dec 2015

This article is Open Access

Anal. Methods, 2016,8, 418-424
Article type
Paper

Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

S. P. Askin, T. E. H. Bond and P. M. Schaeffer, Anal. Methods, 2016, 8, 418 DOI: 10.1039/C5AY03064A

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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