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An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins   

Edited by
Jihyun Kim
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Original research article

A brief version of this protocol appeared in:
Open Biology
May 2017

Abstract

We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain (Sha et al., 2013; Fridy et al., 2014; Schmidt et al., 2016). In this protocol, we describe detailed methodology involved in generating AdPROM constructs and their application in human cell lines for target protein destruction. AdPROM allows functional characterisation of the POI and its efficiency of target protein destruction overcomes many limitations of RNA-interference approaches, which necessitate long treatments and are associated with off-target effects, and CRISPR/Cas9 gene editing, which is not always feasible.

Keywords: AdPROM, Proteolysis, VHL, Cullin2, Ubiquitination, Nanobody, Monobody, CRISPR/Cas9

Background

This protocol enables one to design, build and express AdPROM VHL-nano/monobody constructs in mammalian cell lines to achieve the proteolytic destruction of the endogenous POI. In the original entries, we demonstrated the near-complete destruction of specific target proteins, by using nanobodies that recognise either green fluorescent protein (GFP) (Fridy et al., 2014) or the inflammasomal protein ASC (Schmidt et al., 2016) and two distinct monobodies that recognize the protein tyrosine phosphatase SHP2 (Sha et al., 2013) as target probes, in a number of human cancer cell lines (Fulcher et al., 2016 and 2017). This protocol provides details for the generation of AdPROM constructs, their expression in cells, and monitoring of target protein degradation and can be adapted for use with any nanobody and monobody, both for constitutive and inducible degradation of the POI. The focus of this protocol is not on the generation of nano/monobodies against POIs or CRISPR/Cas9 genome editing (to knockin GFP tags on POIs) but rather the latter steps to facilitate target protein destruction with the AdPROM system.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Macartney, T. J., Sapkota, G. P. and Fulcher, L. J. (2017). An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins. Bio-protocol 7(22): e2614. DOI: 10.21769/BioProtoc.2614.
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