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

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.

A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies

“…Protein complementation assays (PCAs) are widely used to detect protein–protein interactions (PPIs) 15 – 19 . In these approaches, a “sensor” protein is split into two fragments, which are then fused to two candidate interacting proteins of interest.…”

Met-HER3 crosstalk supports proliferation via MPZL3 in MET-amplified cancer cells

“…HER3 coimmunoprecipitated with MPZL3-V5 in KatoII cells, but this interaction was reduced by Met inhibition (37.9% of DMSO control, +/-11.2% [SEM.]). This supports the hypothesis that MPZL3 interacts with HER3, and that the HER3-MPZL3 interaction can be modulated by Met activity in MET-amplified cells To test whether MPZL3 could interact directly with HER3, we employed the newly developed Split Intein-Mediated Protein Ligation (SIMPL) technique (Yao et al, 2020). SIMPL employs N-and C-terminal fragments of a self-splicing intein domain fused to bait and prey proteins.…”

“…To test whether MPZL3 could interact directly with HER3, we employed the newly developed Split Intein-Mediated Protein Ligation (SIMPL) technique (Yao et al, 2020). SIMPL employs N- and C-terminal fragments of a self-splicing intein domain fused to bait and prey proteins.…”

Rational Design of Peptide-Based Inhibitors Disrupting Protein-Protein Interactions

“…Importantly, the rapid advances in structural biology and computational biology have enabled the in-depth exploration into the sophisticated conformational ensembles of PPI systems (Qiu et al, 2020 ; Wang et al, 2021 ). With the help of techniques such as molecular dynamics (MD) simulations (Yang et al, 2019 ), the transient intermediate PPI interface conformations can be probed, based on which further structure-based rational inhibitor design can be more easily carried out (Tavakoli and Ganjalikhany, 2019 ; Yao et al, 2020 ). Generally, rational design of peptide-based inhibitors toward PPIs depends heavily on reported crystal structures, which can specifically reveal the principles of protein-protein binding modes.…”