Fusion with an albumin‐binding domain (ABD) of streptococcal protein G represents a popular approach for half‐life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvβ3‐integrin‐binding protein (JCL) based on the 10th human fibronectin type III domain ( 10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel‐affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD‐L 15 ‐JCL and JCL‐L 14 ‐ABD with HSA was confirmed by analytical size exclusion chromatography and pull‐down assays. Surprisingly, the thermal stability of ABD‐L 15 ‐JCL was dramatically decreased in comparison with JCL and JCL‐L 14 ‐ABD proteins. Pharmacokinetic studies revealed that JCL‐L 14 ‐ABD circulated in murine blood about 10 times longer than ABD‐L 15 ‐JCL and 960 times longer than JCL. Biodistribution studies of JCL‐L 14 ‐ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half‐life extension of the binding proteins based on 10 Fn3.

Endothelial cells play a major role in the development of inflammation and neoangiogenesis in cancer and chronic inflammatory diseases. In 3D cultures, cells are under conditions that closely resemble those existing in healthy and disease-stricken human organs and tissues. Therefore, the development of a 3D model based on the Ea.hy926 endothelial cell line is an urgent need in molecular and cellular biology. Cell cultivation on an anti-adhesive substrate under static conditions was shown to lead to the formation of spheroids (3D cultures). Expression of ICAM-1 and VEGFR-2 and production of cytokines were screened in 2D and 3D cultures in the presence of TNF and VEGF. According to flow cytometry and confocal microscopy data, TNF significantly increased the expression of the cell adhesion molecule ICAM-1 in both 2D and 3D cultures but did not affect the expression level of VEGFR-2. Increased production of pro-inflammatory (IL-8, IL-6, IP-10) and anti-inflammatory (IL-10, TGF-β 1-3) factors was observed in spontaneous 3D cultures but not in 2D cultures, which was confirmed by flow cytometry and qPCR. TNF-induced secretion of IL-10, GM-CSF, and IL-6 was 11-, 4.7-, and 1.6-fold higher, respectively, in 3D cultures compared to 2D cultures. Thus, the use of a Ea.hy926 3D cell culture is a promising approach in studying the effects of anti- and pro-inflammatory agents on endothelial cells.

Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (10Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5T and to construct new artificial TNF-binding proteins. We obtained a 10Fn3 gene combinatorial library and screened it using the bacterial display method. After expression of the selected 10Fn3 variants in Escherichia coli cells and analysis of their TNF-binding activity, we identified proteins that display high affinity for TNF and characterized their properties.

AIM:To investigate the ability of the neuroprotector dimebon to prevent alterations in brain lipid metabolism caused byTNF-α.MATERIAL AND METHODS:The ability of dimebon (2,8-Dimethyl-5-[2-(6-methyl-3-pyridinyl)ethyl]-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole hydrochloride) to prevent alterations in brain lipid metabolism caused byTNF-α was studied in 65 male mice (20+2g weight). TNF-α (10 mkg/mouse), dimebon (0.2 mg/kg) and their combination were injected intraperitoneally. Thirty min, 2, 4 and 24 h after injection, lipid level alterations in total fractions and molecular species of phospholipids (phosphatidylcholine, lysophosphatidylcholine, sphingomyelin and phosphatidylethanolamine) were measured with mass-spectrometry in the hippocampus, cortex and cerebellum.RESULTS AND CONCLUSION:After injection of TNF-α into mice, there are significant changes in the level of all tested phospholipids. Dimebon at a dose of 0.2 mg/kg alone does not cause any changes in the content of all tested phospholipids, but injected together with TNF-α prevents cytokine induced alterations in the lipid content. The selectivity of TNF-α and dimebon influence on certain molecular species of various phospholipids in different parts of mouse brain is found. The presented data suggest protective properties of dimebon preventing the development of proinflammatory syndrome induced by TNF-α in the animal brain.

Cell surface display is a popular approach for the construction of whole-cell biocatalysts, live vaccines, and screening of combinatorial libraries. To develop a novel surface display system for the popular scaffold protein 10th human fibronectin type III domain (10Fn3) in Escherichia coli cells, we have used an α-helical linker and a C-terminal translocator domain from previously characterized autotransporter from Psychrobacter cryohalolentis K5T. The level of 10Fn3 passenger exposure at the cell surface provided by the hybrid autotransporter Fn877 and its C-terminal variants was low. To improve it, the fusion proteins containing 10Fn3 and the native autotransporter passenger Est877 or the cold-active esterase EstPc in different orientations were constructed and expressed as passenger domains. Using the whole-cell ELISA and activity assays, we have demonstrated that N-terminal position of EstPc in the passenger significantly improves the efficiency of the surface display of 10Fn3 in E. coli cells.

Since 1983, Dimebon (Dimebolin) is used clinically in Russia as an antihistamine drug. Recent interest in Dimebolin is associated with its therapeutic effect in patients with Alzheimer’s disease. Animal studies have shown that Dimebon activity is realized via multiple mechanisms. Our experiments performed on the fibroblast cell culture L929 and C57Bl mice have been shown that Dimebon may block cytotoxic signals induced by the proinflammatory cytokines, tumor necrosis factor α (TNFα). Dimebon (10 μg/mL) protected mouse fibroblast cells L929 against toxic action of TNFα. Pretreatment of mice with Dimebon prevented development of changes in molecular species of sphingomyelins and galactosylceramides induced by a single dose administration of TNAα. Dimebon itself did not induce changes in sphingolipids of the investigated brain structures.

В работе представлен новый тип гибридных молекул, включающих красный флуоресцентный белок mCherry и 10-й домен фибронектина человека типа III (10Fn3) – один из альтернативных каркасных белков, на основе которого могут быть получены рекомбинантные аналоги антител различной специфичности. Сконструированы различные варианты гена, кодирующего гибридный флуоресцентный белок, и изучена их экспрессия в клетках Escherichia coli. Установлено, что расположение mCherry на N-конце гибридного белка и предложенная нами модификация его N-концевой аминокислотной последовательности способствуют увеличению выхода продукта экспрессии в растворимой форме. На основе предложенной конструкции получен гибридный флуоресцентный белок ChIBF, содержащий -интегринсвязывающий вариант 10Fn3, и продемонстрирована возможность его использования для визуализации -интегрина на поверхности эпителиальных клеток MDCK методом конфокальной микроскопии.

In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.

Hybrid molecules of a new type bearing a red fluorescent protein mCherry and one of the alternative scaffold proteins, the 10th human fibronectin type III domain ( 10 Fn3), which can be used for the construction of antibody mimics with various binding specificity, were obtained. Different variants of the gene encoding the hybrid fluorescent protein were constructed and their expression in Escherichia coli cells was studied. The mCherry N-terminal position and the modification of its N-terminal amino acid sequence proposed were shown to promote the efficient bacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construct a hybrid fluorescent protein ChIBF containing an α V β 3 -integrin binding variant of 10 Fn3 was obtained, and its use for the visualization of α V β 3 -integrin at the surface of MDCK epithelial cells was demonstrated by confocal microscopy.

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin ((10)Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC(50) of TNF.

Hepatitis C is related to the most important socially significant human infectious diseases. However, there is no vaccine for the hepatitis C virus. The nonstructural protein NS3 of the hepatitis C virus (HCV), which is synthesyzed in the infected cells and it displays protease, NTPase, and helicase enzymatic activities, is one of the possible components of the vaccine. The connection between the effectiveness of the T-cell response to NS3 epitopes and the spontaneous resolution of acute hepatitis C has been shown. The purpose of this work was to compare the immune response of mice to the inoculation of the nucleotide and amino acid sequences of HCV NS3 and their combination, as well as to evaluate the adjuvant activity of the DNA encoding of granulocyte macrophage colony-stimulating factor (GM-CSF) and the influence of regulatory T cells on the effectiveness of the immune response. The maximum anti-HCV NS3 antibody level in the serum (up to 1: 640000) induced the recombinant rNS3 protein introduced with aluminum hydroxide. The most intensive cellular immune response was observed after the simultaneous administration of rNS3 and DNAs encoding full-size NS3 and GM-CSF. A high level of lymphocyte proliferation, accumulation of IFN-γ-secreting cells, and IFN-γ/IL-2 release in response to the stimulators (NS3 antigens of different compositions) were observed in this group of mice. It has been established that the in vitro suppression of regulatory T cells leads to a statistically significant increase in the secretion of IFN-γ. Thus, the simultaneous application of rNS3, along with the DNAs encoding full-size NS3 and GM-CSF, is a promising approach to the development of hepatitis C vaccine. The expediency of adding regulatory T-cell inhibitors in the vaccine composition will be clear after special studies.

Six unique phage antibodies against human tumor necrosis factor (TNF) were selected from the earlier constructed naïve combinatorial library of single-chain antibodies by affinity selection. The TNF binding of these antibodies was examined by enzyme immunoassay and Western blot analysis. The specificity of the selected antibodies was determined from their binding to interferons alpha and gamma, bovine serum albumin, ovalbumin, and ubiquitin. Two antibodies (sA1 and sB3) were converted into a soluble single-chain antibodies as individual molecules. Their affinity proved to be 2.5 and 13.7 nM, respectively.

This review is devoted to the challenging direction of modern molecular biology and bioengineering—the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP binding molecules are a combination of conservative protein cores and hypervariable regions that provide the function of specific binding of the ligand. Structural classification of ASPs includes several types that differ in their molecular targets and potential applications. Construction of artificial binding proteins on the basis of ASPs includes a combinatorial library design with subsequent selection of high-affinity variants by phage display or the more modern cell-free systems. Binding molecules on the basis of ASPs are widely used in various fields of biotechnology and molecular medicine.

Обзор посвящен актуальному направлению современной молекулярной биологии и биоинженерии – свойствам альтернативных каркасных белков (АКБ) и методам получения связывающих молекул на их основе. Молекулы искусственных связывающих белков на основе АКБ представляют собой сочетание консервативного белкового каркаса и гипервариабельных участков, обеспечивающих функцию специфического связывания с лигандом. АКБ, в соответствии со структурными особенностями, можно разделить на несколько классов, которые различаются также спектрами молекулярных мишеней и возможными областями использования. Получение искусственных связывающих белков на основе АКБ включает в себя конструирование комбинаторной библиотеки с последующим отбором высокоаффинных вариантов при помощи фагового дисплея или более современных бесклеточных систем. Связывающие молекулы на основе АКБ находят широкое применение в различных областях биотехнологии и молекулярной медицины.

Из сконструированной ранее неиммунной комбинаторной библиотеки одноцепочечных антител человека методом аффинной селекции были отобраны шесть уникальных фаговых антител против TNF человека. Связывание отобранных фаговых антител с TNF исследовали методами ИФА и Вестерн-блот-анализа. Специфичность отобранных антител определяли по связыванию с интерферонами альфа и гамма, бычьим сывороточным альбумином, овальбумином и убиквитином. Два антитела, sA1 и sВ3, переведены в формат растворимых одноцепочечных антител в виде индивидуальных молекул; их аффинность составила 2.5 и 13.7 нМ соответственно.

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K+ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~ 80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of 15N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.