Heterologous signal peptides enable increasing titers of recombinant proteins in mammalian cell culture. Four human heterodimeric glycoprotein hormones (follicle-stimulating hormone, FSH; luteinizing hormone, LH; chorionic gonadotropin, CG; and thyroid-stimulating hormone, TSH) were expressed in stably transfected CHO cells when varying signal peptides of their β-subunits. The signal peptide of human serum albumin proved to be the most effective for the glycoprotein hormone family. The cell specific productivity was increased for LH (2.5 pg/cell, 4-fold increase), TSH (1.6 pg/cell, 13-fold increase), and CG (1.0 pg/cell, 60%-increase). According to the Western blotting and quantitative PCR data, the productivity increase is associated with an increase in the efficiency of translation and translocation of β-subunits of hormones in the endoplasmic reticulum due to their coupling with the heterologous signal peptides.

DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3 '-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5 '-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding. MEZ is a 31-mer aptamer that is highly specific to the RBD from the SARS-CoV-2 Wuhan-Hu-1 strain with Kd = 6.5 nM.

Background The Chinese hamster ovary (CHO) cell line is the main host for the high-titer production of therapeutic and diagnostic proteins in the biopharmaceutical industry. In most cases, plasmids for efficient protein expression in CHO cells are based on the cytomegalovirus (CMV) promoter. The autologous Chinese hamster eukaryotic translation elongation factor 1α (EEF1A1) promoter is a viable alternative to the CMV promoter in industrial applications. The EEF1A1 promoter and its surrounding DNA regions proved to be effective at maintaining high-level and stable expression of recombinant proteins in CHO cells. EEF1A1-based plasmids’ large size can lead to low transfection efficiency and hamper target gene amplification. We hypothesized that an efficient EEF1A1-based expression vector with a long terminal repeat fragment from the Epstein-Barr virus (EBVTR) could be truncated without affecting promoter strength or the long-term stability of target gene expression. Methods We made a series of deletions in the downstream flanking region of the EEF1A1 gene, and then in its upstream flanking region. The resulting plasmids, which coded for the enhanced green fluorescent protein (eGFP), were tested for the level of eGFP expression in the populations of stably transfected CHO DG44 cells and the stability of eGFP expression in the long-term culture in the absence of selection agents. Results It was shown that in the presence of the EBVTR fragment, the entire downstream flanking region of the EEF1A1 gene could be excluded from the plasmid vector. Shortening of the upstream flanking region of the EEF1A1 gene to a length of 2.5 kbp also had no significant effect on the level of eGFP expression or long-term stability. The EBVTR fragment significantly increased expression stability for both the CMV and EEF1A1 promoter-based plasmids, and the expression level drop during the two-month culture was more significant for both CMV promoter-based plasmids. Conclusion Target protein expression stability for the truncated plasmid, based on the EEF1A1 gene and EBVTR fragment, is sufficient for common biopharmaceutical applications, making these plasmid vectors a viable alternative to conventional CMV promoter-based vectors.

We studied the influence of heterologous signal peptides in the β-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When the natural signal peptides of the β-chains were replaced with the heterologous signal peptide of human serum albumin, cell productivity was increased 2–2.5 times for human luteinizing hormone, human chorionic gonadotropin, and human thyroid-stimulating hormone, but not for human follicle-stimulating hormone. No significant increase in cell productivity was observed for human azurocidin signal peptide and human glycoprotein hormone α-chain signal peptide. The used approach allows quick assessing the effect of heterologous signal peptides on the biosynthesis of heterodimeric proteins of various classes.

Determining the presence of antibodies to the SARS-CoV-2 antigens is the best way to identify infected people, regardless of the development of symptoms of COVID-19. The nucleoprotein (NP) of the SARS-CoV-2 is an immunodominant antigen of the virus; anti-NP antibodies are detected in persons previously infected with the virus with the highest titers. Many test systems for detecting antibodies to SARS-CoV-2 contain NP or its fragments as antigen. The sensitivity and specificity of such test systems differ significantly, which can be explained by variations in the antigenic properties of NP caused by differences in the methods of its cultivation, isolation and purification. We investigated this effect for the Escherichia coli-derived SARS-CoV-2 NP, obtained from the cytoplasm in the soluble form. We hypothesized that co-purified nucleic acids that form a strong complex with NP might negatively affect NP’s antigenic properties. Therefore, we have established the NP purification method, which completely eliminates the RNA in the NP preparation. Two stages of RNA removal were used: treatment of the crude lysate of E. coli with RNase A and subsequent selective RNA elution with 2 M NaCl solution. The resulting NP without RNA has a significantly better signal-to-noise ratio when used as an ELISA antigen and tested with a control panel of serum samples with antibodies to SARS-CoV-2; therefore, it is preferable for in vitro diagnostic use. The same increase of the signal-to-noise ratio was detected for the free N-terminal domain of the NP. Complete removal of RNA complexed with NP during purification will significantly improve its antigenic properties, and the absence of RNA in NP preparations should be controlled during the production of this antigen.

The humoral response to the SARS-CoV-2 S protein determines the development of protective immunity against this infection. The standard neutralizing antibodies detection method is a live virus neutralization test. It can be replaced with an ELISA-based surrogate virus neutralization test (sVNT), measuring the ability of serum antibodies to inhibit complex formation between the receptor-binding domain (RBD) of the S protein and the cellular ACE2 receptor. There are conflicting research data on the sVNT methodology and the reliability of its results. We show that the performance of sVNT dramatically improves when the intact RBD from the Wuhan-Hu-1 virus variant is used as the plate coating reagent, and the HRP-conjugated soluble ACE2 is used as the detection reagent. This design omits the pre-incubation step in separate tubes or separate microplate and allows the simple quantification of the results using the linear regression, utilizing only 3–4 test sample dilutions. When this sVNT was performed for 73 convalescent plasma samples, its results showed a very strong correlation with VNT (Spearman’s Rho 0.83). For the RBD, bearing three amino acid substitutions and corresponding to the SARS-CoV-2 beta variant, the inhibitory strength was diminished for 18 out of 20 randomly chosen serum samples, and the magnitude of this decrease was not similar to the change in overall anti-RBD IgG level. The sVNT assay design with the ACE2-HRP is preferable over the assay with the RBD-HRP reagent and is suitable for mass screening of neutralizing antibodies titers.

Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese hamster ovary cell culture broth. We developed a purification process for r-hFSH centered on immunoaffinity chromatography with single-domain recombinant camelid antibodies. The resulting downstream process is simple and devoid of ultrafiltration operations. Studies on chromatography resin resource and ligand leakage showed that the immunoaffinity matrix employed was suitable for industrial use and stable for at least 40 full chromatography cycles, and the leaked single-domain antibody ligand was completely removed by subsequent purification steps. All chromatography resins employed withstood the same 40 cycles of use without significant changes in separation efficiency and product binding capacity. The resulting industrial purification process yielded batches of r-hFSH with consistent levels of purity and bioactivity.

DNA aptamers are oligonucleotides specifically bound to target molecules that can serve as antibodies of nucleic acid nature. For diagnosing the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), methods using antibodies specific to antigens on the virus are broadly used. We generated by classical SELEX a number of aptamers, interacting with the receptor-binding domain of SARS-CoV2 spike protein (SARS-CoV2 Spike RBD) from Wuhan-Hu-1 strain. The sequence identification was performed using a novel methodology based on the nanopore sequencing. For sequence identification of selected aptamers, we created the novel protocol for aptamer identification based on nanopore sequencing. We identified the best aptamer candidate named MEZ. It was chemically synthesized and tested for binding with SARS CoV2 Spike RBD domain of the S-protein from different strains. Kd of the complex is 6.5 nM being comparable with known aptamers. Virus neutralization tests demonstrate similar results for already known and MEZ aptamers. We identified differences for aptamers binding to SARS-CoV-2 Spike RBD from Wuhan-Hu-1 and Omicron strains. MD simulations reveal that the number of hydrogen bonds between the protein and aptamer is higher for the more stable complex. Moreover, dynamic network analysis show that the motions of the aptamer and protein are correlated to a higher extent in a more stable complex. Based on the experimental data and computational results we can conclude that the authentic RBD-aptamer complex has two specific points for interaction and the 3'-end of aptamer is responsible for strain identification. Therefore, the selected aptamer based on experimental data can be an alternative biological element for the development of SARS-CoV-2 diagnostic testing with strain specificity and cost efficiency due to the short length of aptamer being 31 nucleotides.

Variants of genetic constructions are discussed which encode a receptor-binding domain (RBD) of coronavirus SARS-CoV-2 spike protein fused with a monomeric Fc-fragment of human immunoglobulins. A cell line producing RBD-Fc is established. High titers of anti-RBD antibodies and virus-neutralizing antibodies are obtained upon mice immunization with the purified RBD-Fc.

The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. Simultaneous editing of multiple genes makes it possible to obtain a cell line with the required genotype faster than several consecutive rounds of genomic knockout, but the probability of success is lower. Simultaneous editing of the dhfr, glul, bak1, and bax genes in the CHO S cells genome yielded 24 clones with signs of auxotrophy for thymidine and glutamine. Five of them turned out to be dhfr+/–, all five contained a knockout of one or two glul alleles. In one clone, 7 out of 8 target alleles were inactivated by a frameshift, and the second dhfr allele was partially inactivated by insertion of the GAA triplet, which reduced the enzyme activity 2.5 times. The probability of simultaneous knockout of both dhfr alleles increased to 50% when the genome was edited with a pair of guide RNAs directed to one exon of the dhfr gene.

The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.