Obelin mutants as reporters in bioluminescent dual-analyte binding assay

Гончарова

Бирюков

et al. 2020

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Bioluminescent solid‐phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer–target complexes during design and development of the specific aptamers. The assay involves Ca2+‐regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.

Section: Methodsmentioning

confidence: 99%

The Ca²⁺‐Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation

Гончарова

Бирюков

et al. 2020

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“…Conjugates Y138F‐anti‐FITC IgG and W92F,H22E‐(dT) 30 were obtained according to the methods described in and , correspondingly.…”

Section: Methodsmentioning

confidence: 99%

Mutants of Ca²⁺‐regulated Photoprotein Obelin for Site‐specific Conjugation

Burakova

Komarova

et al. 2017

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Color variants of Ca 2+ -regulated photoprotein obelin were shown to be an important tool for dual-analyte binding assay. To provide site-directed conjugation with biospecific molecules, several obelin color mutants carrying unique cysteine residues were obtained and characterized for their novel properties. A pair of obelins Y138F,A5C and W92F,H22E,D12C was found to be most suitable (in terms of high bioluminescent activity and stability) as reporters in simultaneous assay of two targets in a sample. Availability of SH-groups, accessible for chemical modification, essentially simplifies the synthesis of biospecific conjugates, increases their yield and conserves obelins' bioluminescence activity. Conjugates with immunoglobulin and oligonucleotide were produced and successfully applied in single nucleotide polymorphism genotyping.

“…Conjugate bovine serum albumin with oligothymidine (BSA‐dT 30 ) was synthesized similarly to the obelin‐dT 30 conjugate with some modification. BSA was incubated with a 40‐fold excess of 2‐iminothiolan in 50 m m BICINE pH 8.5, 5 m m EDTA for 40 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Hybrid Minimal Core Streptavidin–Obelin as a Versatile Reporter for Bioluminescence‐based Bioassay

Bashmakova

Kudryavtsev

et al. 2016

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Ca -regulated photoprotein obelin was genetically fused with a minimum-sized core streptavidin. Hybrid protein (SAV-OL) was produced by bacterial expression and applied as a specific bioluminescent probe in diverse solid-phase assays. The obtained results clearly demonstrate specific activity of each domain indicating its proper folding with favorable space orientation. SAV-OL has been shown to be a much more sensitive label than the chemical conjugate of a full-length streptavidin with obelin.