Vol 88, No 9 (2023)

Antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein (RBD of S-protein) contribute significantly to the humoral immune response during coronavirus infection (COVID-19) and after vaccination. The main focus of the study of the epitope composition of RBD is concentrated on the epitopes recognized by viral neutralizing antibodies. The role of antibodies that bind to RBD but are unable to neutralize the virus in the formation of the immune response remains unclear. In this investigation, the immunochemical properties of two mouse monoclonal antibodies RS17 and S11 against RBD were examined. Both antibodies were shown to have high affinity, but they did not neutralize the virus. The epitopes of these antibodies were localized using phage display: the epitope recognized by the RS17 antibody is located at the N-terminal site of RBD (348-SVYAVNRKRIS-358); the epitope recognized by the S11 antibody is inside the receptor-binding motif of RBD (452-YRLFRKSN-459). Three groups of sera were tested for antibodies competing with non-neutralizing antibodies S11 and RS17: 1) from unvaccinated volunteers, who did not suffer from COVID-19 previously; 2) from people who had had a mild form of COVID-19; 3) from people who had had a severe form of COVID-19. Antibodies competing with the S11 antibody were shown to occur with equal frequency in each of the serum groups studied. At the same time, the presence of antibodies competing with antibody RS17 in the sera was associated with the severity of COVID-19 and was significantly more frequent in the group of sera obtained from patients with severe COVID-19. In conclusion, despite the clear significance of anti-RBD antibodies for the formation of an effective immune response against SARS-CoV-2, it is important to analyze their viral neutralizing activity and to confirm the absence of negative features of obtained anti-RBD antibodies after vaccination.

Extensive application of technologies like phage display in screening peptide and protein combinatorial libraries has not only facilitated creation of new recombinant antibodies but has also significantly enriched repertoire of the protein binders that have polypeptide scaffolds without homology to immunoglobulins. These innovative synthetic binding protein (SBP) platforms have grown in number and now encompass monobodies/adnectins, DARPins, lipocalins/anticalins, and a variety of miniproteins such as affibodies and knottins, among others. They serve as versatile modules for developing complex affinity tools that hold promise in both diagnostic and therapeutic settings. An optimal scaffold typically has low molecular weight, minimal immunogenicity, and demonstrates resistance against various challenging conditions, including proteolysis - making it potentially suitable for peroral administration. Retaining functionality under reducing intracellular milieu is also advantageous. However, paramount to its functionality is the scaffold’s ability to tolerate mutations across numerous positions, allowing for the formation of a sufficiently large target binding region. This is achieved through the library construction, screening, and subsequent expression in an appropriate system. Scaffolds that exhibit high thermodynamic stability are especially coveted by the developers of new SBPs. These are steadily making their way into clinical settings, notably as antagonists of oncoproteins in signaling pathways. This review surveys the diverse landscape of SBPs, placing particular emphasis on the inhibitors targeting the oncoprotein KRAS, and highlights groundbreaking opportunities for SBPs in oncology.

The solute carrier organic anion transporter family member OATP1B1 is one of the most important transporter proteins which mediate the penetration of many endogenous substances and xenobiotics into hepatocytes. A model system providing the expression of the functional protein is needed to assess the interaction of OATP1B1 with various substances. Based on HEK293 cells, we obtained the HEK293-OATP1B1 cell line, permanently expressing the SLCO1B1 gene encoding the OATP1B1 transporter. Expression of the SLCO1B1 gene was confirmed by real-time PCR analysis and immunochemically. The functionality of the transporter was assessed by the transport of atorvastatin, which is a substrate of OATP1B1. Cells of the resulting cell line, which selectively express the functionally active recombinant OATP1B1 transporter, can be used to study its function and to test drugs for belonging to substrates, inducers, and inhibitors of OATP1B1 to assess the risks of drug-drug interactions.

The structure of the Siberian roe deer (Capreolus pygargus) chymosin gene has been established for the first time and its exon/intron organization has been determined. The coding part of the C. pygargus chymosin gene was reconstructed and obtained as a DNA clone using the Golden Gate method. Comparative analysis of the sequences of prochymosins of roe deer, cow and single-humped camel revealed a number of amino acid substitutions in the sites forming the substrate-binding cavity of the enzyme and affecting the specificity subsites S4 and S1′ + S3′. The recombinant plasmid pIP1-Cap was constructed using the integration vector pIP1 for the expression of the roe deer prochymosin gene in CHO-K1 cells. A polyclone of CHO-K1-CYM-Cap cells was obtained, providing synthesis and secretion of recombinant prochymosin into the culture fluid of the producer. As a result of zymogen activation, a recombinant roe deer chymosin preparation with a total milk-clotting activity of 468.4 ± 11.1 IMCU/ml was obtained. The yield of recombinant roe deer chymosin was 500 mg/liter or ≈ 468,000 IMCU/liter, which exceeds the yield of genetically engineered chymosins in most of the expression systems used. The main biochemical properties of the obtained enzyme were compared with commercial preparations of recombinant chymosins of single-humped camel (Camelus dromedarius) and cow (Bos taurus). The specific milk-clotting activity of recombinant C. pygargus chymosin was 938 ± 22 IMCU/mg of protein and was comparable with the indicators of comparison enzymes. The nonspecific proteolytic activity of recombinant roe deer chymosin was 1.4-4.5 times higher than that of cow and camel enzymes. In terms of coagulation specificity, the recombinant C. pygargus chymosin occupied an intermediate position between the genetically engineered analogues of B. taurus and C. dromedarius chymosins. The threshold of thermal stability of recombinant roe deer chymosin was equal to 55°C. At 60°C, the enzyme retained <1% of the initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and restricted access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin β-subunit). In the current work, we have developed a genetic system for the production of recombinant CtxB in E. coli cells and studied the conditions for the synthesis and purification of the target product at the laboratory culture level. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow an E. coli culture in a synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium through Ni²⁺-ions affinity chromatography techniques. Forty-eight hours following CtxB expression induction, the concentration of the target product can be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure while expressing and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level synthesis of recombinant CtxB for medical and research purposes.

The genetic toxin-antitoxin element hok/sok from the natural Escherichiacoli R1 plasmid ensures the segregation stability of the plasmids. Bacterial cells that have lost all copies of the plasmid encoding the short-lived antitoxin die under the action of the long-lived toxin. The hok/sok element in vector plasmids for bacterial expression can increase the productive time of biosynthesis of recombinant proteins, slowing down the accumulation of non-producing cells lacking the target plasmid in the population. In this work, we studied various variants of the position and orientation of the hok/sok element in the standard plasmid pET28a with the inducible T7lac promoter and the kanamycin resistance gene. It was found that the hok/sok element retained functional activity regardless of location on the plasmid and orientation, bacterial cells retained hok/sok plasmids after four days of cultivation without antibiotics and lost the control plasmid without this element. Using the example of three target proteins - E. coli type II asparginase, human growth hormone, and the SARS-CoV-2 virus nucleoprotein, it was demonstrated that for cytoplasmic target proteins, the maximum productivity of bacteria is maintained only when the hok/sok element is located on the plasmid upstream of the target gene promoter. In the case of periplasmic localization of the protein, the productivity of bacteria decreases for all variants of the hok/sok location during cultivation with an antibiotic, and in the case of periodic cultivation of bacteria without an antibiotic, productivity is also better preserved when the hok/sok element is located upstream of the target gene promoter. This variant of the pEHU vector plasmid makes it possible to more than double the biosynthesis of human growth hormone, which is insoluble in the cytoplasm of bacteria, when bacteria are cultivated without antibiotics, and also to maintain asparaginase biosynthesis during periodic cultivation without antibiotics for four days at a level of at least 10 mg/liter. The developed segregation-stabilized plasmid vector can be used to obtain various recombinant proteins in E. coli cells without the use of antibiotics.

Cytochrome CYP102A1 (P450 BM3) from Priestia megaterium (bas. Bacillus megaterium) has a number of specific features making it an ideal target for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme was obtained providing 7β-hydroxylation of steroid substrates of the androstane series with the formation of products possessing anti-inflammatory and neuroprotective activity. In this study, the synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant variant was expressed in Mycolicibacterium smegmatis cells as part of mono- and bicistronic operons together with the synthetic gdh or zwf2 genes encoding glucose dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PD), respectively. The functional activity of the recombinant enzymes was shown in vivo by the example of hydroxylation of androst-4-ene-3,17-dione (AD) to 7β-OH-AD in growing cultures of mycolicibacteria. Biocatalytic activity was doubled by increasing the CYP102A1-LG23 protein solubility in the cell and organizing the cofactor regeneration additional system by introducing GDH and G6PD. The maximum level of 7β-OH-AD amounting 37,68 mol % was achieved by co-expression the cyp102A1-LG23 and gdh genes in M. smegmatis. The results evidence to the perspective of using synthetic genes to obtain recombinant enzymes, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes and can be demand for the methods of microbiological production of 7β-hydroxylated steroids by genetically modified mycolicibacteria.

Methylotrophic yeast Komagataella phaffii are widely used in biotechnology as a host for recombinant protein production. Due to the practical significance of this yeast, it is extremely important to properly select the cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on K. phaffii gene expression was investigated at transcriptomic level. It was demonstrated, that K. phaffii cell response to biotin deficiency strongly depends on the carbon source in the medium. In media containing glycerol, biotin deficiency led to activation of genes involved in biotin metabolism, glyoxylate cycle and synthesis of acetyl-CoA in cytoplasm, as well as repression of genes, involved in lipo- and gluconeogenesis. In methanol containing media, biotin deficiency primarily led to repression of genes, involved in protein synthesis, and activation of cell response to oxidative stress.

Hepatic encephalopathy (HE), a medical term that deals with a neuropsychiatric disorder seen in patients with severe hepatic dysfunction, has been known for more than a century, however, pathogenetic mechanisms underlying cerebral dysfunction during conditions of liver disease are still not entirely understood. There is a consensus that an accumulation of ammonia in brain as a result of impaired detoxification capacity of the liver or the appearance of a portosystemic shunt is a primary cause of HE. Current evidence suggests that ammonia toxicity is mediated by hyperactivation of glutamate receptors, mainly N-methyl-D-aspartate receptors (NMDA-R) and affects many processes of aerobic metabolism that provides energy for a myriad of specific functions and viability of nerve cells. Recent reports on the presence of functional NMDA receptors in erythrocytes and deviations from normal ranges in many blood test parameters that was indicative of impaired hemodynamics and reduced carrying capacity for oxygen in erythrocytes in most patients with HE implicate the relationship between “erythrocyte damage” and cerebral dysfunction. In order to understand how, during hyperammonemia, disturbances in energy metabolism in brain that needs a constant supply of a high level of oxygen in the blood lead to encephalopathy, it is necessary to reveal ammonia-induced disorders in energy metabolism and antioxidant defense system of erythrocytes and explore the potential role of ammonia in reduced brain oxygenation. To detect the said missing link, the activities of erythrocyte antioxidant enzymes and concentrations of GSH, GSSG and H₂O₂ were measured in animals with hyperammonemia by using MK-801, a powerful noncompetitive NMDA receptor antagonist. It was found that the accumulation of ammonia in the blood of animals with hyperammonemia occurs within rat erythrocytes in large amounts and makes these cells which do not contain enzymes for the removal of ammonia, more susceptible to a prooxidant environment of ammonia created during oxidative stress that was completely or partially inhibited with the help of MK-801. Data obtained provide a support to identification of extra risk factors in cognitive disorders and in the prediction of unfavorable outcome with hypoperfusion in patients with elevated concentrations of ammonia in the blood.