Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins.

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.

Dimeric ACE2-FC Is Equivalent to Monomeric ACE2 in the Surrogate Virus Neutralization Test.

Angiotensin-converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of protective humoral immune response to a viral infection or vaccine using the surrogate virus neutralization test (sVNT). Soluble ACE2 is also considered as an option for antiviral therapy potentially insensitive to the changes in the SARS-CoV-2 spike protein. Extensive testing of the samples of patient's serum by the sVNT method requires using preparations of ACE2 or ACE2 conjugates with constant properties. We have previously obtained a cell line that is a producer of a soluble monomeric ACE2 and showed that this ACE2 variant can be used in sVNT, preferably as a conjugate with horseradish peroxidase. A cell line that generates an ACE2-Fc fusion protein with high productivity, more than 150 mg/liter of the target protein when cultured in a stirred flask, was obtained for producing a stable and universally applicable form of soluble ACE2. The affinity-purified ACE2-Fc fusion contains a mixture of dimeric and tetrameric forms, but allows obtaining linear response curves for inhibition of binding with the receptor-binding domain of the SARS-CoV-2 spike protein by antibodies. The ACE2-Fc-HRP-based sVNT testing system can be used for practical measurements of the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

Location and Orientation of the Genetic Toxin-Antitoxin Element hok/sok in the Plasmid Affect Expression of Pharmaceutically Significant Proteins in Bacterial Cells.

Genetic toxin-antitoxin element hok/sok from the natural Escherichia coli R1 plasmid ensures segregational stability of plasmids. Bacterial cells that have lost all copies of the plasmid encoding the short-lived antitoxin are killed by the stable toxin. When introduced into bacterial expression vectors, the hok/sok element can increase the productive time of recombinant protein biosynthesis by slowing down accumulation of non-producing cells lacking the expression plasmid. In this work, we studied the effects of position and orientation of the hok/sok element in the standard pET28a plasmid with the inducible T7lac promoter and kanamycin resistance gene. It was found that the hok/sok element retained its functional activity regardless of its location and orientation in the plasmid. Bacterial cells retained the hok/sok-containing plasmids after four days of cultivation without antibiotics, while the control plasmid without this element was lost. Using three target proteins - E. coli type II asparaginase (ASN), human growth hormone (HGH), and SARS-CoV-2 virus nucleoprotein (NP) - it was demonstrated that the maximum productivity of bacteria for the cytoplasmic proteins (HGH and NP) was observed only when the hok/sok element was placed upstream of the target gene promoter. In the case of periplasmic protein localization (ASN), the productivity of bacteria during cultivation with the antibiotic decreased for all variants of the hok/sok location. When the bacteria were cultivated without the antibiotic, the productivity was better preserved when the hok/sok element was located upstream of the target gene promoter. The use of the pEHU vector with the upstream location of the hok/sok element allowed to more than double the yield of HGH (produced as inclusion bodies) in the absence of antibiotic and to maintain ASN biosynthesis at the level of at least 10 mg/liter for four days during cultivation without antibiotics. The developed segregation-stabilized plasmid vectors can be used to obtain various recombinant proteins in E. coli cells without the use of antibiotics.

Drug Transport System Based on Phospholipid Nanoparticles: Production Technology and Characteristics.

One of the current trends in modern pharmaceuticals is the supply of drugs by transport systems. The use of delivery systems allows to increase the therapeutic efficacy, tolerability, and safety of drug therapy. Liposomes, polymer nanoparticles, carbon nanoparticles, blood cells, metal nanoparticles, oxides, etc., are used as transport systems. This work is aimed at obtaining a finished technological product based on soy phospholipids with particle size in the nanometer range and reproducible characteristics (size, charge). For this purpose, we carried out investigations to select the optimal conditions of technological process. The developed technology makes it possible to obtain phospholipid nanoparticles without the use of any solubilizers and/or surfactants, which increases its practical relevance for further work. The versatility of the technology is demonstrated by the example of incorporation of drugs of various chemical nature and pharmacotherapeutic groups.

Chlorin e6 Phospholipid Delivery System Featuring APN/CD13 Targeting Peptides: Cell Death Pathways, Cell Localization, In Vivo Biodistribution.

We have previously designed a phospholipid delivery system for chlorin e6 to increase the efficacy of photodynamic therapy involving a second-generation photosensitizer. Further research into the matter led to double modification of the obtained nanoparticles with ligands exhibiting targeting and cell-penetrating effects: an NGR-containing peptide and heptaarginine (R7), respectively. This study investigated the cell death pathway on HT-1080 tumor cells after treatment with the proposed compositions: the chlorin e6 phospholipid composition and the two-peptide chlorin e6 phospholipid composition. It was demonstrated that most of the cells died by apoptosis. Colocalization analysis of chlorin e6 in the phospholipid composition with two peptides showed mitochondria are one of the targets of the photosensitizer. An HT-1080 tumor-bearing mouse model was used to evaluate the biodistribution of the drug in tumor, liver, and kidney tissues after administration of the study compositions in comparison with free chlorin e6. The photosensitizer mostly accumulated in the tumor tissue of mice administered the phospholipid compositions, and accumulation was increased 2-fold with the peptide-containing composition and approximately 1.5-fold with the unenhanced composition, as compared with free chlorin e6. The enhancement of the chlorin e6 phospholipid composition with targeting and cell-penetrating peptides was found to be effective both in vitro and in vivo.

Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD.

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.

Antigenic properties of the SARS-CoV-2 nucleoprotein are altered by the RNA admixture.

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.

High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector.

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.

High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors.

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the ß-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH.

Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system.

BACKGROUND: Recombinant factor VIII (FVIII), used for haemophilia A therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. Deletion variant of FVIII, in which the entire domain B is replaced by a short linker peptide, was approved for medical use. Efficacy and safety of this FVIII deletion variant are similar to full-length FVIII preparations while the level of production in CHO cells is substantially higher. Typical levels of productivity for CHO cell lines producing deletion variant FVIII-BDD SQ, described elsewhere, are 0.5-2 IU/ml, corresponding to the concentration of FVIII of about 0.2 µg/ml. Using standard vectors based on the cytomegalovirus promoter (CMV) and the dihydrofolate reductase cDNA we have previously obtained the cell line secreting 0.5 IU/ml of FVIII-BDD, which roughly corresponds to the previously published data. RESULTS: An expression system based on CHO genomic sequences including CHO-EEF1A promoter and Epstein-Barr virus terminal repeat fragment allowed us to achieve 80-fold increase in the production level as compared with the conventional expression system based on the CMV promoter. Immediately after the primary selection FVIII -producing cells secreted 5-10 IU/ml of FVIII-BDD, and after multi-stage methotrexate-driven amplification a stable clonal line 11A4H was selected, secreting 39 IU/ml of FVIII-BDD in the simple batch culturing conditions, which considerably exceeds known indicators for industrial producers of this protein. In contrast to other FVIII-BDD producing lines 11A4H accumulates low proportion of the secreted FVIII on the membrane. Its productivity may be further increased approximately two-fold by adding sodium butyrate and butylated hydroxyanisol to the culture medium. A five-stage purification process for the factor VIII was employed. It allowed isolation of the intact FVIII-BDD as was confirmed by mass spectrometry. Purified FVIII-BDD has a specific activity of 11,000 IU/mg, similar to known recombinant FVIII drugs. CONCLUSIONS: The recombinant FVIII-BDD was produced in CHO cells without addition of any animal-derived materials, purified and characterized. Novel genetic constructions for the expression of heterologous proteins combined with optimized cultivation method allowed to obtain the secretion level of biologically active recombinant FVIII increased by almost ten times as compared with the previously published analogues.

New Infestin-4 Mutants with Increased Selectivity against Factor XIIa.

Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 µM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.

Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

BACKGROUND: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure. RESULTS: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative. CONCLUSIONS: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments.

BACKGROUND: Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites. FINDINGS: The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps. CONCLUSIONS: Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.