Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition

Zhong Yao,Farzaneh Aboualizadeh,Mike Boxem,Igor Jurisica,Igor Stagljar,Jason Kroll,Priscilla Tang,Max Kotlyar,Anna Lyakisheva,Jamie Snider,Indira Akula

doi:10.1038/s41467-020-16299-1

Abstract

Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.

Highlights

To overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL)
A prey protein is fused at its N-terminus with a FLAG tag and an intein C-terminal fragment (IC)
We evaluated the SIMPL system using a benchmarking approach with unbiased PPI reference sets, which has been widely accepted for assessing the overall performance of a PPI method

Summary

Introduction

To overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. PPI detection and analysis are essential for understanding molecular mechanisms of biological processes, elucidating mechanistic details of disease occurrence and progression, as well as for developing new treatments and diagnostics

Methods

Results

Conclusion