Straightforward Protein-Protein Interaction Interface Mapping via Random Mutagenesis and Mammalian Protein Protein Interaction Trap (MAPPIT).

Laurens Vyncke,Jan Tavernier,Delphine Masschaele,Frank Peelman

doi:10.3390/ijms20092058

Laurens Vyncke, Jan Tavernier + Show 2 more

Open Access

https://doi.org/10.3390/ijms20092058

Copy DOI

Abstract

The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.

Highlights

The expansion of the protein interactomics field is driven by a wide variety of techniques for the detection of protein-protein interactions (PPIs)
Our laboratory developed the mammalian protein-protein interaction trap (MAPPIT), a method combining many of these traits
Transfection conditions are optimized by varying the concentration of bait and prey plasmids and by switching conditions are optimized by varying the concentration of bait and prey plasmids and by switching the bait and prey proteins. (B) The target protein is randomly mutated via error-prone polymerase the baitchain and prey proteins. (B) The target protein is randomly mutated via error-prone polymerase reaction (PCR)

Summary

Introduction

The expansion of the protein interactomics field is driven by a wide variety of techniques for the detection of protein-protein interactions (PPIs). Affinity-purification coupled to mass spectrometry [2], have been complemented with new and improved approaches These enable the study of PPIs at their subcellular location [3,4] or in a physiological context between weak and transient interactions [5,6,7], full-length integral membrane proteins [8] and post-translationally modified proteins [9]. Bait-prey interaction leads to phosphorylation and the recruitment of STAT3 to the gp130 fragment, complementing the JAK-STAT signalling pathway, Int. J. MAPPIT can detect up to one third of all protein interactions in high-throughput screening modus but this number can probably be increased by switching bait and prey, using different bait constructs or using domains instead of full-length proteins [11,12,13]

Methods

Results

Conclusion