EndoBind detects endogenous protein-protein interactions in real time

Anke Bill; Sheryll Espinola; Daniel Guthy; Jacob R Haling; Mylene Lanter; Min Lu; Anthony Marelli; Angelica Mendiola; Loren Miraglia; Brandon L Taylor; Leonardo Vargas; Anthony P Orth; Frederick J King

doi:10.1038/s42003-021-02600-5

EndoBind detects endogenous protein-protein interactions in real time

Commun Biol. 2021 Sep 15;4(1):1085. doi: 10.1038/s42003-021-02600-5.

Authors

Affiliations

¹ Novartis Institute for Biochemical Research, Oncology, Cambridge, MA, USA. anke.bill@novartis.com.
² Genomics Institute of the Novartis Research Foundation, Assay Development and High Throughput Screening, San Diego, CA, USA.
³ Novartis Institute for Biochemical Research, Oncology, Basel, Switzerland.
⁴ Mirati Therapeutics, Inc., Research, San Diego, CA, USA.
⁵ Genomics Institute of the Novartis Research Foundation, Assay Development and High Throughput Screening, San Diego, CA, USA. fred.king@novartis.com.

Abstract

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.

MeSH terms

HEK293 Cells
High-Throughput Screening Assays / methods*
Humans
Kelch-Like ECH-Associated Protein 1 / chemistry*
NF-E2-Related Factor 2 / chemistry*
Protein Binding
Proto-Oncogene Proteins p21(ras) / chemistry*

Substances

KEAP1 protein, human
KRAS protein, human
Kelch-Like ECH-Associated Protein 1
NF-E2-Related Factor 2
NFE2L2 protein, human
Proto-Oncogene Proteins p21(ras)