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

Expression of the gene encoding blood coagulation factor VIII without domain B in <i>E. coli</i> 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

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

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