Jump to main content
Jump to site search
Access to RSC content Close the message box

Continue to access RSC content when you are not at your institution. Follow our step-by-step guide.


Issue 2, 2016
Previous Article Next Article

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

Author affiliations

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

Back to tab navigation

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. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material.

Reproduced material should be attributed as follows:

  • For reproduction of material from NJC:
    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
  • For reproduction of material from PCCP:
    [Original citation] - Published by the PCCP Owner Societies.
  • For reproduction of material from PPS:
    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
  • For reproduction of material from all other RSC journals:
    [Original citation] - Published by The Royal Society of Chemistry.

Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.


Social activity

Search articles by author

Spotlight

Advertisements