Immunoliposomes As a Promising Antiviral Agent against SARS-CoV-2.

According to the World Health Organization, as of January 3, 2020 to September 13, 2023, there were approximately 23 million confirmed cases of COVID-19 reported in the Russian Federation, about 400 thousand of which were fatal. Considering the high rate of mutation of the RNA-containing virus genome, which inevitably leads to the emergence of new infectious strains (Eris and Pyrola), the search for medicinal antiviral agents remains an urgent task. Moreover, taking into account the actively mutating receptor-binding domain, this task requires fundamentally new solutions. This study proposes a candidate immunoliposomal drug that targets the S protein of SARS-CoV-2 by the monoclonal neutralizing antibody P4A1 and ensures the penetration of a highly active ribonuclease into the virus-infected cell, which degrades, among cellular RNA, viral RNA too. We demonstrate a more than 40-fold increase in the neutralizing activity of the developed drug compared to the free monoclonal neutralizing antibody.

Polymer/magnetite carriers functionalized by HER2-DARPin: Avoiding lysosomes during internalization and controlled toxicity of doxorubicin by focused ultrasound induced release.

Nanomedicine has revolutionized the available treatment options during the last decade, but poor selectivity of targeted drug delivery and release is still poses a challenge. In this study, doxorubicin (DOX) and magnetite nanoparticles were encapsulated by freezing-induced loading, coated with polymeric shell bearing two bi-layers of polyarginine/dextran sulphate and finally modified with HER2-specific DARPin proteins. We demonstrated that the enhanced cellular uptake of these nanocarriers predominantly occurs by SKOV-3 (HER2+) cells, in comparison to CHO (HER2-) cells, together with the controlled DOX release using low intensity focused ultrasound (LIFU). In addition, a good ability of DARPin+ capsules to accumulate in the tumor and the possibility of combination therapy with LIFU were demonstrated. A relatively high sensitivity of the obtained nanocarriers to LIFU and their preferential interactions with mitochondria in cancer cells make these carriers promising candidates for cancer treatment, including novel approaches to overcome drug resistance.

Removal of the Translocation Domain and the Furin Cleavage Site Decreases the Relative Hepatotoxicity of the Targeted Antitumor Toxins.

Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatotoxicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.

Synthesis of Magnetic Nanoparticles Stabilized by Magnetite-Binding Protein for Targeted Delivery to Cancer Cells.

A new method for obtaining biomodified magnetite nanoparticles for targeted delivery to cells was developed. The method is based on the use of the C-terminal fragment of the Mms6 protein, which is involved in the magnetite biomineralization during the synthesis of magnetosomes in magnetotactic bacteria Magnetospirillum magneticum AMB-1, and the barnase*barstar high-affinity protein pair. The Mms6 protein fragment is required for stabilizing magnetite, and the barnase*barstar pair mediates the interaction between nanoparticles and the component for modification. The efficiency of this method was confirmed in the synthesis of magnetite nanoparticles recognizing the HER2/neu tumor marker and in the selective labeling of HER2/neu with these nanoparticles on the surface of cancer cells.

Data of self-made Taq DNA polymerase prepared for screening purposes.

DNA analysis is a key procedure in genetic engineering. Nowadays the analysis is often done by PCR with Taq DNA polymerase. Although the last enzyme price is quite low, demand for numerous analyses results in much money expenditure which are not affordable for many laboratories. In a meanwhile, many screening tasks do not require the highly purified enzyme. Taking into account the enzyme unique properties it makes possible to marginally simplify its production without resorting to costly or lengthy techniques such as column chromatography and/or dialysis. Here the data of routine usage of Taq DNA polymerase prepared according to the protocol developed in our laboratory is presented. The protocol takes only several hours to realize and does not need qualified personnel or expensive equipment. Yet it gives the enzyme preparation suitable for most screening purposes. The isolated Taq DNA polymerase stock can be stored as ammonium sulfate suspension in a refrigerator for prolonged period, not less than 6 months. The working enzyme solution is prepared from the stock suspension on demand, not more than once in a month and can be stored also in a refrigerator.

Construction of the plasmid-free strain for human growth hormone production.

The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant.

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

The structure of substrate-free microbial ribonuclease binase and of its complexes with 3'GMP and sulfate ions.

The structures of Bacillus intermedius ribonuclease (binase), an extracellular 109-residue enzyme, and its complexes with 3'GMP and sulfate ions were solved at 1.65 and 2.0 A, respectively. The structures were refined using REFMAC. The crystal of free binase belongs to the space group C2, whereas the crystals of complexes belong to the space group P2(1)2(1)2(1). In both crystal lattices the asymmetric unit contains two molecules which form an identical dimer. The structure of the dimer is such that only one of its subunits can bind the nucleotide in the 3'GMP-binase complex, where the guanyl base is located in the recognition loop of the enzyme. In both binase complex structures the phosphate group of 3'GMP or one of the sulfate ions make an electrostatic interaction with the binase molecule at the catalytic site. A second phosphate-binding site was found in the structures of the complexes at the cleft formed by the loop 34-39, the main chain of Arg82 and the side chain of Trp34. Comparison of the complex and unliganded enzyme crystal structures shows that there are some small but distinct differences in the specificity loop (56-62) and in the loops 34-39 and 99-104 associated with the binding of the nucleotide and sulfate ions.

Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar.

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.