Leishmania tarentolae cell-free based approach for rapid anitbody–antigen interaction analysis

Moradi, Shayli Varasteh; Johnston, Wayne A.; Parker, Jake; Alexandrov, Kirill

doi:10.1016/bs.mie.2021.06.017

Cited by 2 publications

(2 citation statements)

References 11 publications

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“…The reaction was topped up with Ultra-Pure TM water (Invitrogen TM , AUS) to the final volume of 10 μL and incubated at 25 0 C for 5h. LTE lysate production and protein expression set up is described in detail elsewhere (Varasteh Moradi et al, 2021, Johnston et al, 2019.The quality of co-expressed proteins were evaluated through resolving the protein bands on SDS-PAGE gel (Bolt 4-12%, Invitrogen TM ) Following the completion of protein expression, the mixture was diluted 25× with buffer A (25 mM HEPES, 50 mM NaCl, 0.1% BSA and 0.01% Nonidet P-40). The AlphaLISA assay was carried out in an Optiplate-384 Plus plate, using anti-GFP AlphaLISA Acceptor and Streptavidin Donor beads.…”

Section: Hsper2_rmentioning

confidence: 99%

A role for caveolar proteins in regulation of the circadian clock

Fonseka

Weger

et al. 2022

Preprint

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Caveolae are specialized invaginations of the plasma membrane that are formed by the co-assembly of caveolin integral membrane proteins and a cytoplasmic cavin coat complex. Previous work has proposed an interaction of the cavin coat protein, CAVIN3, with the key circadian clock protein, PER2. Here we show that cavin proteins can play a role in the regulation of the circadian clock by external stimuli. Loss of Cavin1 in mice caused a shortening of the free-running period of locomotor activity. CAVIN1 and CAVIN3 were found to play a central role in core clock dynamics with either cavin protein directly interacting with PER2 and their perturbation leading to significant disruption in core clock mRNA expression and CRY1 protein oscillation. In cells, association of cavins and PER2 was increased upon caveola disassembly caused by oxidative stress or by calcium influx, stimuli linked to circadian clock regulation. We thus propose that the caveola system can play a modulatory role in circadian regulation through the cavin proteins.

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Section: Hsper2_rmentioning

confidence: 99%

A role for caveolar proteins in regulation of the circadian clock

Fonseka

Weger

et al. 2022

Preprint

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“…Multiplexing capability, where the analysis can be carried out in 384-well plates, makes this method particularly suitable for the analysis of protein interaction networks (Figure 1B). 24,25 As a proof of concept, we previously performed mapping of protein−protein interactions within the ZIKA virus proteome. 26 Here, we report the reconstitution and analysis of 54 putative protein−protein interactions between the SARS-CoV-2 virus and human proteins previously identified by mass spectrometry and proposed to be druggable targets.…”

Section: ■ Introductionmentioning

confidence: 99%

In Vitro Reconstitution and Analysis of SARS-CoV-2/Host Protein–Protein Interactions

Moradi

Walden

et al. 2023

ACS Omega

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The emergence of viral threats such as Ebola, ZIKA, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires a rapid and efficient approach for elucidating mechanisms of pathogenesis and development of therapeutics. In this context, cell-free protein synthesis (CFPS) holds a promise to resolve the bottlenecks of multiplexed protein production and interaction analysis among host and pathogen proteins. Here, we applied a eukaryotic CFPS system based on Leishmania tarentolae extract (LTE) protein expression in combination with AlphaLISA proximity-based protein interaction technology to identify intraviral and viral-human protein interactions of SARS-CoV-2 virus that can potentially be targeted by the existing or novel antiviral therapeutics. We produced and tested 54 putative human-viral protein pairs in vitro and identified 45 direct binary protein interactions. As a casing example of the assay's suitability for drug development applications, we analyzed the effect of a putative biologic on the human angiotensin-converting enzyme 2/receptor-binding domain (hACE2/RBD) interaction. This suggests that the presented pathogen characterization platform can facilitate the development of new therapeutic agents.

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Leishmania tarentolae cell-free based approach for rapid anitbody–antigen interaction analysis

Cited by 2 publications

References 11 publications

A role for caveolar proteins in regulation of the circadian clock

A role for caveolar proteins in regulation of the circadian clock

In Vitro Reconstitution and Analysis of SARS-CoV-2/Host Protein–Protein Interactions

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