Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor

He, Shengxiang; Ge, Song; Shi, Jia-Ping; Guo, Yuqi; Guo, Zhan‐Yun

doi:10.1016/j.biochi.2014.08.012

Cited by 21 publications

(14 citation statements)

References 30 publications

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“…The expression construct of the engineered Luc-Cys was generated by insertion of a chemically synthesized DNA linker into the previously generated pNLuc vector (He et al 2014) that was pretreated with restriction enzymes EcoRI and NotI, resulting in the expression construct pET/ Luc-Cys. The coding region of Luc-Cys was confirmed by DNA sequencing.…”

Section: Overexpression and Purification Of Luc-cysmentioning

confidence: 99%

“…Use of the radioactive tracers has drawbacks, such as their short shelf lives and radioactive hazards to operators and environments. In recent studies, we established novel non-radioactive receptor-binding assays for some recombinant protein hormones based on the ultrasensitive bioluminescence of a nanoluciferase (NanoLuc) reporter (He et al 2014;Zhang et al 2013). NanoLuc is a newly developed luciferase reporter with several advantages, such as the brightest bioluminescence reported to date, small size (171 amino acid, 19 kDa), and high stability (Hall et al 2012).…”

Section: Introductionmentioning

confidence: 99%

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Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model

et al. 2015

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Peptide hormones perform important biological functions by binding specific cell membrane receptors. For hormone-receptor interaction studies, receptor-binding assays are widely used. However, conventional receptor-binding assays rely on radioactive tracers that have drawbacks. In recent studies, we established novel non-radioactive receptor-binding assays for some recombinant protein hormones based on the ultrasensitive bioluminescence of a newly developed nanoluciferase (NanoLuc) reporter. In the present work, we extended the novel bioluminescent receptor-binding assay to peptide hormones that have small size and can be conveniently prepared by chemical synthesis. Human ghrelin, a 28-amino acid peptide hormone carrying a special O-fatty acid modification, was used as a model. To prepare a bioluminescent ghrelin tracer, a chemically synthesized ghrelin analog with a unique cysteine residue at the C-terminus was site-specifically conjugated with an engineered NanoLuc with a unique exposed cysteine residue at the C-terminus via a reversible disulfide linkage. The NanoLuc-conjugated ghrelin retained high binding affinity with the ghrelin receptor GHSR1a (K d = 1.14 ± 0.13 nM, n = 3) and was able to sensitively monitor the receptor-binding of various GHSR1a ligands. The novel bioluminescent receptor-binding assay will facilitate the interaction studies of ghrelin with its receptor. We also proposed general procedures for convenient conjugation of other peptide hormones with NanoLuc for novel bioluminescent receptor-binding assays.

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Section: Overexpression and Purification Of Luc-cysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model

et al. 2015

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“…However, the use of radioactive materials is inconvenient for most laboratories. In recent studies, we developed novel bioluminescence ligand-receptor binding assays through chemical conjugation or genetic fusion of a small NanoLuc luciferase to the target protein/peptide hormone [14][15][16][17][18][19][20][21]. As an example, we measured the binding potencies of various ligands with the receptor GHSR1a using the NanoLuc-conjugated ghrelin (ghrelin-Luc) as a non- radioactive tracer [21].…”

Section: Introductionmentioning

confidence: 99%

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Wang

Shao

et al. 2019

The FEBS Journal

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Liver‐expressed antimicrobial peptide 2 (LEAP2) is a highly conserved secretory peptide first isolated in 2003. However, its exact biological functions remained elusive until a recent study identified it as an endogenous antagonist for the growth hormone secretagogue receptor (GHSR1a), a G protein‐coupled receptor for which the gastric peptide ghrelin is the endogenous agonist. By tuning the ghrelin–GHSR1a system, LEAP2 has an important function in energy metabolism. In the present study, we first demonstrated that LEAP2 and ghrelin actually bound to GHSR1a in a competitive manner, rather than in a non‐competitive manner as previously reported, by binding assays and activation assays. Subsequently, we demonstrated that the antagonistic function of LEAP2 was drastically affected by the manner of its addition. LEAP2 primarily affected the maximal activation effect when added before ghrelin, whereas it primarily affected half‐maximal effective concentration when added at the same time as ghrelin. Thus, LEAP2 behaved as a competitive antagonist if added at the same time as the agonist and a non‐competitive antagonist if added before the agonist. This unusual property of LEAP2 might be caused by its slow dissociation from receptor GHSR1a. We also found that the N‐terminal fragment of LEAP2 was important for receptor binding. Our present study revealed an antagonistic mechanism for LEAP2, and will facilitate the design of novel antagonists for receptor GHSR1a in future studies.

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“…In our previous and present studies, we validated the novel bioluminescent binding assay using a variety of protein/peptide hormones with different size and distinct receptors, including relaxin (receptor RXFP1) [ 1 ], INSL3 (receptor RXFP2) [ 5 ], chimeric relaxin family peptide R3/I5 (receptor RXFP3 and RXFP4) [ 35 ], ghrelin (receptor GSHR1a) [ 3 ], leukemia inhibitory factor (receptor LIFR/gp130) [ 4 ], erythropoietin (receptor EPOR) [ 2 ], and FGF2 (receptor FGFR1–4). Although the NanoLuc reporter is large, its attachment did not negatively affect the receptor-binding of a wide range of protein/peptide hormones, provided it was attached to an appropriate position using an appropriate linkeFor wide application of the novel binding assay, we developed two methods for convenient preparation of the bioluminescent ligands, that is, the chemical conjugation method and the genetic fusion method [ 7 ].…”

Section: Resultsmentioning

confidence: 99%

Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

Shao

et al. 2016

PLoS ONE

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We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies.

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Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor

Cited by 21 publications

References 30 publications

Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model

Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

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