Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis

Bignon, Christophe, Gruet, Antoine, Longhi, Sonia (October 2022) Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis. International Journal of Molecular Sciences, 23 (21). p. 13167. ISSN 1422-0067

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Abstract

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results.

Item Type:	Paper
Subjects:	bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics Investigative techniques and equipment bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification Investigative techniques and equipment > assays organism description > bacteria organism description > bacteria > escherichia coli bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > green fluorescent protein
CSHL Authors:	Gruet, Antoine
Communities:	CSHL labs > Wigler lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	29 October 2022
Date Deposited:	28 Nov 2022 18:10
Last Modified:	11 Jan 2024 18:54
PMCID:	PMC9658207
URI:	https://repository.cshl.edu/id/eprint/40763

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