Poster
22

Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag® technologies

Authors

L Wickens1
1 Promega UK Ltd, UK

Abstract

To understand a protein's function inside the cell, studies are often done to remove it using CRISPR-mediated knockout or RNAi knockdown methods. However, these approaches have challenges that include obtaining efficient loss of the targeted protein, or cell death if the protein is found to be essential for cell growth. To overcome these hurdles, we have employed a highly precise and temporally controlled target protein degradation strategy utilizing HaloPROTAC3, a HaloTag® proteolysis targeting chimera small molecule which specifically degrades HaloTag fusion proteins in live cells. HaloPROTAC3, developed by Prof. Craig Crews at Yale University, recruits HaloTag® fusion proteins to VHL E3 ligase complexes, resulting in ubiquitination and degradation of the HaloTag® fusion via the ubiquitin-proteasomal pathway. Endogenous HaloTag® fusion proteins are developed via CRISPR/Cas9 gene editing into either the N- or C-terminal loci of any target protein. Additionally, we've appended the 11 amino acid HiBiT to the HaloTag® fusion protein, allowing for highly quantitative kinetic monitoring of degradation in live cells with the use of luminescence instead of antibodies. We demonstrate how these technologies can be used to elicit robust degradation of target proteins, with control over the protein level as well as the time frame for protein degradation. Also, we provide new opportunities for phenotypic studies in order to investigate the function of essential proteins without the need of protein specific PROTACs.


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ELRIG
The European Laboratory Research & Innovation Group Our Vision : To provide outstanding, leading edge knowledge to the life sciences community on an open access basis
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