Biotechnologia Acta

13-23

Authors: O. Obodovych, V. Sydorenko

The use of plant biomass as a primary source of energy is currently unacceptable both from an economic and environmental point of view. The experience of a number of industries, in particular hydrolysis production, enables to solve the problem of profitability of organic biomass treatment by its deep complex processing with the resulting components whose cost exceeds the cost of organic raw materials as fuel. Currently, the main results of complex processing of organic raw materials are still energy-intensive products ̶ bioethanol and hydrolyzed lignin, which energy characteristics are commensurate with fossil fuels. Bioethanol production from starch-containing, sugar-containing or lignocellulosic raw materials requires the use of different technological stages and, accordingly, the cost of bioethanol for each type of raw material is different. Compared to bioethanol produced from sugar and starch raw materials, bioethanol manufactured from lignocellulosic raw materials is more expensive. Bioethanol obtained from lignocellulosic raw materials is more expensive compared to bioethanol from sugar and starch raw materials. The most energy-intensive in the technology of bioethanol obtaining from lignocellulosic raw materials is the stage of pretreatment of raw materials for hydrolysis, because the process of preliminary preparation and hydrolysis with dilute acids occurs at high temperatures and pressures. During enzymatic hydrolysis, the process temperature is maintained for a long time (up to several days). To ensure deep integrated processing of plant raw materials, as well as to reduce overall costs, it was proposed to improve the technology and equipment, which allow increasing the degree of conversion of raw materials into basic and by-products.

30-40

Authors: E. G. Fomina, E. E. Grigorieva, V. V. Zverko, A. S. Vladyko

A heterologous host has got a unique expression ability of each gene. Differences between the synonymous sequences play an important role in regulation of protein expression in organisms from Escherichia coli to human, and many details of this process remain unclear. The work was aimed to study the composition of codons, its distribution over the sequence and the effect of rare codons on the expression of viral nucleocapsid proteins and their fragments in the heterologous system of E.coli. The plasmid vector pJC 40 and the BL 21 (DE 3) E. coli strain were used for protein expression. The codon composition analysis was performed using the online resource (www.biologicscorp.com). 10 recombinant polypeptides were obtained encoding the complete nucleotide sequence of nucleocapsid proteins (West Nile and hepatitis C viruses) and the fragments including antigenic determinants (Lassa virus, Marburg, Ebola, Crimean-Congo hemorrhagic fever (CCHF), Puumaravala, Hantaan, and lymphocytic choriomeningitis (LHM)). Hybrid plasmid DNAs provide efficient production of these proteins in the prokaryotic system with the recombinant protein yield varying by a factor of 8: from 5 to 40 mg per 1 liter of bacterial culture. No correlation was found between the level of protein expression and the frequency of occurrence of rare codons in the cloned sequence: the maximum frequency of occurrence of rare codons per cloned sequence was observed for the West Nile virus (14.6%), the minimum was for the CCHF virus (6.6%), whereas the expression level for these proteins was 30 and 5 mg/L culture, respectively. The codon adaptation index (CAI) values, calculated on the basis of the codon composition in E. coli, for the cloned viral sequences were in the range from 0.50 to 0.58, which corresponded to the average expressed proteins. The analysis of the distribution profiles of CAI in the cloned sequences indicated the absence of clusters of rare codons that could create difficulties in translation. A statistically significant difference between the frequencies of the distribution of amino acids in the cloned sequences and their content in E. coli was observed for the nucleocapsid proteins of the Marburg, Ebola, West Nile, and hepatitis C viruses.

50-57

Authors: T. A. Yatsenko, S. M. Kharchenko

The plasminogen/plasmin system plays a crucial role in fibrinolysis and regulation of cell functions in a wide range of normal and pathological processes. Investigation of plasminogen/plasmin functions requires the availability of well-characterized and effective molecular tools, such as antibodies. In the present work, the isolation and characterization of rabbit polyclonal antibodies against human plasminogen are described and approaches for the identification of plasminogen and its fragments using the purified antibodies are demonstrated. For the antibodies isolation, standard animal immunization and blood collection procedures, serum isolation, protein salting out and affinity chromatography were performed. For the antibodies characterization and application, the following methods were used: enzyme linked immunoassay (ELISA), Western blotting, FITC-protein conjugation, flow cytometry and spectrofluorometry. The obtained polyclonal rabbit anti-human plasminogen antibodies interacted with human Glu- and Lys-plasminogen, kringles 1-3 and 1-4 of plasminogen, mini-plasminogen, the heavy and light chain of plasmin. We propose the application of anti-plasminogen antibodies for the direct ELISA, Western blot analysis, and for flow cytometry and spectrofluorometric analysis of plasminogen binding with cells. The obtained anti-plasminogen antibodies are promising tools for the investigation of plasminogen/plasmin system functions, either fibrinolytic or signaling.