Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Jordan F. Hastings; Jeremy Z.R. Han; Robert F. Shearer; Sean P. Kennedy; Mary Iconomou; Darren N. Saunders; David R. Croucher

doi:10.3791/57109

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Method Article

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

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DOI:

10.3791/57109

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June 15th, 2018

Jordan F. Hastings¹ , Jeremy Z.R. Han¹ , Robert F. Shearer¹^,² , Sean P. Kennedy¹^,³ , Mary Iconomou¹^,⁴ , Darren N. Saunders⁵ , David R. Croucher¹^,⁶^,⁷

¹The Kinghorn Cancer Centre, Garvan Institute of Medical Research, ²Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, ³RCSI Molecular Medicine, Royal College of Surgeons in Ireland, ⁴Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, ⁵School of Medical Sciences, University of New South Wales, ⁶St Vincent's Hospital Clinical School, University of New South Wales, ⁷School of Medicine and Medical Science, University College Dublin

Summary

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Abstract

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.

Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners.

The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Introduction

Protein complex assembly is a key process in maintaining the spatiotemporal specificity of many signalling pathways¹^,². While the critical nature of this regulatory role is widely recognized, there is a lack of experimental techniques available to scrutinize these complexes. Most interactomics studies focus upon interactions with individual proteins, or the sequential enrichment of individual complex components. Here we present a technique for the isolation of a specific protein dimer while excluding the individual moieties of the component proteins as well as complexes formed with competing binding partners