Background Since the beginning of the use of reporter proteins for expression analysis, a variety of approaches have been developed and proposed; both qualitative and quantitative. The lack of simple methods for direct observation of gene expression in living organisms makes it necessary to continue to propose new methods. In this work, we consider a method for the quantitative analysis of the expression of thermostable lichenase from Clostridium thermocellum used as a sensitive reporter protein. Results In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusions The specific interaction between the dye Congo red and $$\beta$$ β -d-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

Background : To elucidate the functional role of regulatory sequences and contexts identified and predicted during omics studies in the complex mechanisms of gene expression regulation, experimental verification methods in a plant cell are required. For this, the method of transient expression of reporter genes fused with the tested sequences is widely used. Several reporter systems are available that have shown good performance in the studies including the thermostable lichenase Clostridium thermocellum . However, each reporter system has its own limitations with respect to the quantification of the protein product of a reporter gene and, in particular, for the use of high-throughput approaches. Results: In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusion: The specific interaction between the dye Congo red and β -D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

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.

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.

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