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

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Interaction Analysis

Background:

• The split-Green Fluorescent Protein (GFP) reassembly assay is a key method for studying protein-protein interactions (PPIs).
• This technique relies on reconstituting a functional GFP from two non-fluorescent fragments fused to interacting proteins.
• Previous applications have focused on mechanistic insights rather than optimizing the assay itself.

Purpose of the Study:

• To systematically investigate and optimize the split-Green Fluorescent Protein (GFP) reassembly assay for studying PPIs.
• To identify critical methodological parameters that influence the reliability of PPI data, particularly for intrinsically disordered proteins (IDPs).
• To provide a refined protocol for enhanced accuracy in PPI studies using split-GFP.

Main Methods:

• Revisiting and modifying the original split-GFP reassembly assay.
• Co-expression of proteins fused to split-GFP fragments in *E. coli*.
• Systematic testing and analysis of various technical parameters impacting assay performance.

Main Results:

• Identification of several critical parameters that significantly affect the reliability of the split-GFP assay.
• Demonstration of how modifications and parameter choices can avoid common pitfalls.
• Unveiling key factors for obtaining robust and reproducible PPI data.

Conclusions:

• The split-GFP reassembly assay can be significantly improved by careful consideration of specific technical parameters.
• Optimizing these parameters is crucial for accurate PPI studies, especially those involving intrinsically disordered proteins.
• This work provides a foundation for selecting the best bipartite reporter and protocol for specific PPI research needs.