A high throughput assay of lichenase activity with Congo red dye in plants

Tyurin, A. A.; Suhorukova, Aleksandra V.; Deineko, I. V.; Pavlenko, O. S.; Fridman, Viktoriia A.; Goldenkova-Pavlova, I. V.

doi:10.1186/s13007-021-00801-x

Cited by 5 publications

(3 citation statements)

References 31 publications

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“…The blank samples containing (i) CR (dye background) and (ii) lichenan (125 µg/mL) + CR (substrate background) were used in all measurements. For quantitative estimation, the values of fluorescence intensity were calculated net of the dye and substrate backgrounds using numerical analysis [ 39 ].…”

Section: Methodsmentioning

confidence: 99%

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Tyurin

Mustafaev

Suhorukova

et al. 2022

Plants

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A broad and amazingly intricate network of mechanisms underlying the decoding of a plant genome into the proteome forces the researcher to design new strategies to enhance both the accumulation of recombinant proteins and their purification from plants and to improve the available relevant strategies. In this paper, we propose new approaches to optimize a codon composition of target genes (case study of interferon-αA) and to search for regulatory sequences (case study of 5′UTR), and we demonstrated their effectiveness in increasing the synthesis of recombinant proteins in plant systems. In addition, we convincingly show that the approach utilizing stabilization of the protein product according to the N-end rule or a new protein-stabilizing partner (thermostable lichenase) is sufficiently effective and results in a significant increase in the protein yield manufactured in a plant system. Moreover, it is validly demonstrated that thermostable lichenase as a protein-stabilizing partner not only has no negative effect on the target protein activity (interferon-αA) integrated in its sequence, but rather enhances the accumulation of the target protein product in plant cells. In addition, the retention of lichenase enzyme activity and interferon biological activity after the incubation of plant protein lysates at 65°C and precipitation of nontarget proteins with ethanol is applicable to a rapid and inexpensive purification of fusion proteins, thereby confirming the utility of thermostable lichenase as a protein-stabilizing partner for plant systems.

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Section: Methodsmentioning

confidence: 99%

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Tyurin

Mustafaev

Suhorukova

et al. 2022

Plants

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“…For this purpose, lichenan at a concentration of 125 μg/mL (if not stated otherwise) and Congo red solution at a final concentration of 0.005% were used. The fluorescence was assessed in a Synergy H1 (BioTek, United States) multimode microplate reader using 96-well microtiter plates [9].…”

Section: Quantification Of Licbm3 Lichenasementioning

confidence: 99%

Development of dual reporter vector system for estimating translational activity of regulatory elements

et al. 2022

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Background For the needs of modern biotechnology, a quantitative approach to the control of regulatory elements at all stages of gene expression has long become indispensable. Such a control regime is impossible without a quantitative analysis of the role of each regulatory element or pattern used. Therefore, it seems important to modify and develop the accuracy, reproducibility, and availability of methods for quantifying the contribution of each regulatory code to the implementation of genetic information. Results A new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coliβ-glucuronidase) under the control of different promoters. Herewith we also propose a new method for quantification of the effect of tested regulatory elements on expression, which relies on assessment of the enzyme activities of reporter proteins taking into account the transcription of their genes. Conclusions In our view, this approach makes it possible to precisely determine the amounts of reporter proteins and their transcripts at all stages of expression. The efficiency of the proposed system has been validated by the analysis of the roles of known translation enhancers at the stages of transcription and translation.

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“…For this purpose, lichenan at a concentration of 125 µg/mL (if not stated otherwise) and Congo red solution at a final concentration of 0.005% were used. The fluorescence was assessed in a Synergy H1 (BioTek, United States) multimode microplate reader using 96-well microtiter plates [12].…”

Section: Quantification Of Licbm3 Lichenasementioning

confidence: 99%

A Bireporter Vector System for Assaying Translational Activity of Regulatory Elements

Suhorukova¹,

Tyurin²,

Pavlenko³

et al. 2021

Preprint

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BackgroundMolecular biology has always shown some similarities with computer science. So, considering transient expression, one can see an analogy with a DDoS attack on a computing system. Like the DDoS attack, transient expression can carry a payload. In particular, analysis of the structure of cell mechanisms and signal amplification in the study of very subtle mechanisms of regulation.ResultsA new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coli

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A high throughput assay of lichenase activity with Congo red dye in plants

Cited by 5 publications

References 31 publications

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Development of dual reporter vector system for estimating translational activity of regulatory elements

A Bireporter Vector System for Assaying Translational Activity of Regulatory Elements

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