Expression of the gene encoding blood coagulation factor VIII without domain B in E. coli bacterial expression system.

In this article, we have demonstrated the feasibility of generating an active form of recombinant blood coagulation factor VIII using an E. coli bacterial expression system as a potential treatment for hemophilia type A. Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. So far, all available recombinant FVIII formulations have been produced using eukaryotic expression systems. Mammalian cells can produce catalytically active proteins with all the necessary posttranslational modifications. However, cultivating such cells is time-consuming and highly expensive, and the amount of the obtained product is usually low. In contrast to eukaryotic cells, bacterial culture is inexpensive and allows the acquisition of large quantities of recombinant proteins in a short time. With this study, we aimed to obtain recombinant blood coagulation factor VIII using the E. coli bacterial expression system, a method not previously explored for this purpose. Our research encompasses the synthesis of blood coagulation factor VIII and its expression in a prokaryotic system. To achieve this, we constructed a prokaryotic expression vector containing a synthetic factor VIII gene, which was then used for the transformation of an E. coli bacterial strain. The protein expression was confirmed by mass spectrometry, and we assessed the stability of the gene construct while determining the optimal growth conditions. The production of blood coagulation factor VIII by the E. coli bacterial strain was carried out on a quarter-technical scale. We established the conditions for isolation, denaturation, and renaturation of the protein, and subsequently confirmed the activity of FVIII.

Mast Cells as a Target-A Comprehensive Review of Recent Therapeutic Approaches.

Mast cells (MCs) are the immune cells distributed throughout nearly all tissues, mainly in the skin, near blood vessels and lymph vessels, nerves, lungs, and the intestines. Although MCs are essential to the healthy immune response, their overactivity and pathological states can lead to numerous health hazards. The side effect of mast cell activity is usually caused by degranulation. It can be triggered by immunological factors, such as immunoglobulins, lymphocytes, or antigen-antibody complexes, and non-immune factors, such as radiation and pathogens. An intensive reaction of mast cells can even lead to anaphylaxis, one of the most life-threatening allergic reactions. What is more, mast cells play a role in the tumor microenvironment by modulating various events of tumor biology, such as cell proliferation and survival, angiogenesis, invasiveness, and metastasis. The mechanisms of the mast cell actions are still poorly understood, making it difficult to develop therapies for their pathological condition. This review focuses on the possible therapies targeting mast cell degranulation, anaphylaxis, and MC-derived tumors.

Antimicrobial Peptides: Challenging Journey to the Pharmaceutical, Biomedical, and Cosmeceutical Use.

Antimicrobial peptides (AMPs), or host defence peptides, are short proteins in various life forms. Here we discuss AMPs, which may become a promising substitute or adjuvant in pharmaceutical, biomedical, and cosmeceutical uses. Their pharmacological potential has been investigated intensively, especially as antibacterial and antifungal drugs and as promising antiviral and anticancer agents. AMPs exhibit many properties, and some of these have attracted the attention of the cosmetic industry. AMPs are being developed as novel antibiotics to combat multidrug-resistant pathogens and as potential treatments for various diseases, including cancer, inflammatory disorders, and viral infections. In biomedicine, AMPs are being developed as wound-healing agents because they promote cell growth and tissue repair. The immunomodulatory effects of AMPs could be helpful in the treatment of autoimmune diseases. In the cosmeceutical industry, AMPs are being investigated as potential ingredients in skincare products due to their antioxidant properties (anti-ageing effects) and antibacterial activity, which allows the killing of bacteria that contribute to acne and other skin conditions. The promising benefits of AMPs make them a thrilling area of research, and studies are underway to overcome obstacles and fully harness their therapeutic potential. This review presents the structure, mechanisms of action, possible applications, production methods, and market for AMPs.

Portable Surface Plasmon Resonance Detector for COVID-19 Infection.

Methods based on nucleic acid detection are currently the most commonly used technique in COVID-19 diagnostics. Although generally considered adequate, these methods are characterised by quite a long time-to-result and the necessity to prepare the material taken from the examined person-RNA isolation. For this reason, new detection methods are being sought, especially those characterised by the high speed of the analysis process from the moment of sampling to the result. Currently, serological methods of detecting antibodies against the virus in the patient's blood plasma have attracted much attention. Although they are less precise in determining the current infection, such methods shorten the analysis time to several minutes, making it possible to consider them a promising method for screening tests in people with suspected infection. The described study investigated the feasibility of a surface plasmon resonance (SPR)-based detection system for on-site COVID-19 diagnostics. A simple-to-use portable device was proposed for the fast detection of anti-SARS-CoV-2 antibodies in human plasma. SARS-CoV-2-positive and -negative patient blood plasma samples were investigated and compared with the ELISA test. The receptor-binding domain (RBD) of spike protein from SARS-CoV-2 was selected as a binding molecule for the study. Then, the process of antibody detection using this peptide was examined under laboratory conditions on a commercially available SPR device. The portable device was prepared and tested on plasma samples from humans. The results were compared with those obtained in the same patients using the reference diagnostic method. The detection system is effective in the detection of anti-SARS-CoV-2 with the detection limit of 40 ng/mL. It was shown that it is a portable device that can correctly examine human plasma samples within a 10 min timeframe.

In vitro immune evaluation of adenoviral vector-based platform for infectious diseases.

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions.

BACKGROUND: Gene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called "trivial overlapping", there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome's novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family. RESULTS: A single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR' protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR' have opposing functions - RepR is crucial for initiation of pIGRK replication, while RepR' is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression. CONCLUSIONS: Regulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

The factor VIII protein and its function.

Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. Human factor VIII is a single chain of about 300 kDa consisting of domains described as A1-A2-B-A3-C1-C2. The protein undergoes processing prior to secretion into blood resulting in a heavy chain of 200 kDa (A1-A2-B) and a light chain of 80 kDa (A3-C1-C2) linked by metal ions. The role of factor VIII is to increase the catalytic efficiency of factor IXa in the activation of factor X. Variants of these factors lead frequently also to severe bleeding disorders.

Expression of yeast deubiquitination enzyme UBP1 analogues in E. coli.

BACKGROUND: It has been shown that proteins fused to ubiquitin undergo greater expression in E. coli and are easier to purify and renaturate than nonhybrid foreign proteins. However, there is no commercial source of large quantities of specific deubiquitinating proteases. This is the reason why hybrid proteins containing ubiquitin at their N-end cannot be used in large scale biotechnological processes. RESULTS AND CONCLUSION: We have described the synthesis of the yeast deubiquination enzyme UBP1 muteins in E. coli. We have shown that an efficient overproduction of the enzyme in E. coli may be achieved after the introduction of several changes in the nucleotide sequence encoding UBP1. One of the conditions of an effective synthesis of the UBP1 muteins is the removal of the 5'-end sequence encoding the transmembrane region of the enzyme. The obtained variants of the enzyme may be successfully used for processing large amounts of hybrid proteins comprising ubiquitin or tagged ubiquitin at their N-ends.