Dataset Information

Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase.

ABSTRACT: Extracellular protein interactions mediated by cell surface receptors are essential for intercellular communication in multicellular organisms. Assays to detect extracellular interactions must account for their often weak binding affinities and also the biochemical challenges in solubilising membrane-embedded receptors in an active form. Methods based on detecting direct binding of soluble recombinant receptor ectodomains have been successful, but genome-scale screening is limited by the usual requirement of producing sufficient amounts of each protein in two different forms, usually a "bait" and "prey". Here, we show that oligomeric receptor ectodomains coupled to concatenated units of the light-generating Gaussia luciferase enzyme robustly detected low affinity interactions and reduced the amount of protein required by several orders of magnitude compared to other reporter enzymes. Importantly, we discovered that this flash-type luciferase exhibited a reaction-induced inhibition that permitted the use of a single protein preparation as both bait and prey thereby halving the number of expression plasmids and recombinant proteins required for screening. This approach was tested against a benchmarked set of quantified extracellular interactions and shown to detect extremely weak interactions (KDs????M). This method will facilitate large-scale receptor interaction screening and contribute to the goal of mapping networks of cellular communication.

SUBMITTER: Galaway F

PROVIDER: S-EPMC7324543 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase.

Galaway Francis F Wright Gavin J GJ

Scientific reports 20200629 1

Extracellular protein interactions mediated by cell surface receptors are essential for intercellular communication in multicellular organisms. Assays to detect extracellular interactions must account for their often weak binding affinities and also the biochemical challenges in solubilising membrane-embedded receptors in an active form. Methods based on detecting direct binding of soluble recombinant receptor ectodomains have been successful, but genome-scale screening is limited by the usual r ...[more]

PMID: 32601498

Similar Datasets

Project description:As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3'UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense-antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples.

Dataset Information

Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase.

Publications

Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets