A sensitive, semi-quantitative mammalian two-hybrid assay

BioTechniques
Elisabeth RiegelThomas Czerny

Abstract

Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system. This inducibility, combined with additional improvements of the iM2H components, results in an up to 100-fold increase in sensitivity compared with conventional M2H approaches. In addition, we include a number of reference interactions in our iM2H approach, which enable semiquantitative assessment of protein interactions. Using Groucho/Tle proteins and their binding partners, we demonstrate the applicability of our iM2H to established protein networks. Finally, to test the applicability of our system for drug screening, the interference of a small-molecule inhibitor on a known protein-protein interaction was te...Continue Reading

References

Dec 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·H A VasavadaS M Weissman
Feb 1, 1991·Molecular and Cellular Biology·C V DangE R Fearon
Oct 5, 1989·Journal of Molecular Biology·M CareyM Ptashne
Oct 10, 1995·Proceedings of the National Academy of Sciences of the United States of America·J L PomerantzP A Sharp
Jul 1, 1995·Experimental Cell Research·Q Ye, H J Worman
Apr 11, 1995·Nucleic Acids Research·M YangS Fields
Jun 25, 1996·Biochemical and Biophysical Research Communications·D Grbavec, S Stifani
Nov 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·S NatesanM Gilman
Nov 21, 2002·Development·Javier López-RíosPaola Bovolenta
Dec 3, 2002·Current Opinion in Biotechnology·Roy Pollock, Tim Clackson
Jan 6, 2004·Science·Lyubomir T VassilevEmily A Liu
Oct 1, 1993·Current Biology : CB·E K O'SheaP S Kim
Oct 16, 2004·BMC Biotechnology·Ajith R VanchaRafael P Ballestero
Oct 25, 2006·Molecular and Cellular Biology·Thomas HeimbucherThomas Czerny
Oct 28, 2006·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Maria FinkThomas Czerny
Sep 30, 2009·Trends in Biochemical Sciences·Sam LievensJan Tavernier
May 24, 2012·The EMBO Journal·Tomas ValentaKonrad Basler
Aug 25, 2012·Methods : a Companion to Methods in Enzymology·Sam LievensJan Tavernier
Oct 1, 2006·Drug Discovery Today. Technologies·Sam LievensJan Tavernier
Apr 7, 2016·Nature Reviews. Drug Discovery·Duncan E ScottJohn Skidmore

❮ Previous
Next ❯

Citations

Mar 11, 2018·Molecular Biotechnology·Matthias VonbrüllThomas Czerny
Aug 1, 2019·Molecular Biology Reports·Elisabeth MertlThomas Czerny
Feb 23, 2020·Biochemistry. Biokhimii︠a︡·E V PoverennayaE A Ponomarenko
Dec 20, 2018·PloS One·Christoph SteurerElisabeth Riegel
Aug 31, 2019·Nature Communications·Soon Gang ChoiYves Jacob

❮ Previous
Next ❯

Methods Mentioned

BETA
two-hybrid
PCR
transfection

Software Mentioned

Rluc
Velcro
Excel

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.