Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation

Amino Acids
Meng-Jun HuZhan-Yun Guo

Abstract

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitat...Continue Reading

References

Dec 14, 2001·The Journal of Biological Chemistry·Neal K WilliamsNicholas E Dixon
Jan 26, 2002·Science·Sheau Yu HsuAaron J W Hsueh
Jul 13, 2002·The Journal of Biological Chemistry·Jin KumagaiAaron J W Hsueh
Oct 3, 2003·The Journal of Biological Chemistry·Changlu LiuTimothy W Lovenberg
Oct 3, 2003·The Journal of Biological Chemistry·Changlu LiuTimothy W Lovenberg
Nov 5, 2004·The Journal of Biological Chemistry·Changlu LiuTimothy W Lovenberg
Jan 11, 2013·Physiological Reviews·R A D BathgateR J Summers
Mar 26, 2013·Journal of Peptide Science : an Official Publication of the European Peptide Society·Wei-Jie ZhangZhan-Yun Guo
Jun 30, 2016·Archives of Biochemistry and Biophysics·Yu LiuZhan-Yun Guo
Oct 25, 2016·British Journal of Pharmacology·Sherie MaAndrew L Gundlach
Dec 7, 2016·British Journal of Pharmacology·Nitin A PatilMohammed Akhter Hossain
Dec 10, 2016·British Journal of Pharmacology·Richard IvellRavinder Anand-Ivell
Jan 25, 2017·Analytical Biochemistry·Christopher R BodleDavid L Roman
Mar 10, 2017·Archives of Biochemistry and Biophysics·Meng-Jun HuZhan-Yun Guo
Jun 14, 2017·Scientific Reports·Dian WeiZhan-Yun Guo
Sep 12, 2017·ACS Chemical Biology·Marie K SchwinnKeith V Wood

❮ Previous
Next ❯

Citations

Jan 23, 2019·The FEBS Journal·Jia-Hui WangZhan-Yun Guo
Jan 24, 2020·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Xiaohan SongNan Wang
Apr 12, 2019·Frontiers in Bioengineering and Biotechnology·Natasha C DaleKevin D G Pfleger

❮ Previous
Next ❯

Methods Mentioned

BETA
bioluminescence resonance
PCR
electrophoresis
transfection
competition binding
saturation binding
competition
BRET

Software Mentioned

SigmaPlot

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.