NAD+-Dependent Formate Dehydrogenase from Themotolerant Yeast Ogataea parapolymorpha: Properties and Protein Engineering of the N-Terminal Sequence
AbstractPreviously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeastOgataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained inEscherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties ...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Recent Developments in Bioprocessing of Recombinant Antibody Fragments
AbstractBiotechnological and biomedical applications of antibodies have been on a steady rise since the 1980s. As unique and highly specific bioreagents, monoclonal antibodies (mAbs) have been widely exploited and approved as therapeutic agents. However, the use of mAbs has limitations for therapeutic applications. Antibody fragments (AbFs) with preserved antigen-binding sites have a significant potential to overcome the disadvantages of conventional mAbs, such as hete rogeneous tissue distribution after systemic administration, especially in solid tumors, and Fc-mediated bystander activation of the immune system. AbFs ...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Heterologous Expression of Recombinant Proteins and Their Derivatives Used as Carriers for Conjugate Vaccines
AbstractCarrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in the disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification, and conjugation of already known and well-established proteins. The use of recombinant proteins has a number of advantages over isolation from natura...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Reconstitution of Calcium Channel Protein Orai3 into Liposomes for Functional Studies
AbstractStore-operated calcium entry (SOCE) is the main mechanism for the Ca2+ influx in non-excitable cells. The two major components of SOCE are stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. SOCE requires interaction between STIM1 and Orai. Mammals have three Orai homologs: Orai1, Orai2, and Orai3. Although Orai1 has been widely studied and proven to essential for numerous cellular processes, Orai3 has also attracted a significant attention recently. The gating and activation mechanisms of Orai3 have yet to be fully elucid...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Location and Orientation of the Genetic Toxin –Antitoxin Element hok/sok in the Plasmid Affect Expression of Pharmaceutically Significant Proteins in Bacterial Cells
AbstractGenetic toxin –antitoxin elementhok/sok from the naturalEscherichia 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, thehok/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 thehok/sok element in the standard pET28a plasmid with the inducibleT7lac ...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Novel B-Cell Epitopes of Non-Neutralizing Antibodies in the Receptor-Binding Domain of the SARS-CoV-2 S-Protein with Different Effects on the Severity of COVID-19
In conclusion, despite the clear significance of anti -RBD antibodies in the effective immune response against SARS-CoV-2, it is important to analyze their virus-neutralizing activity and to confirm absence of the antibody-mediated enhancement of infection by the anti-RBD antibodies. (Source: Biochemistry (Moscow))
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Non-Immunoglobulin Synthetic Binding Proteins for Oncology
AbstractExtensive application of technologies like phage display in screening peptide and protein combinatorial libraries has not only facilitated creation of new recombinant antibodies but has also significantly enriched repertoire of the protein binders that have polypeptide scaffolds without homology to immunoglobulins. These innovative synthetic binding protein (SBP) platforms have grown in number and now encompass monobodies/adnectins, DARPins, lipocalins/anticalins, and a variety of miniproteins such as affibodies and knottins, among others. They serve as versatile modules for developing complex affinity tools that h...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Analysis of Some Biochemical Properties of Recombinant Siberian Roe Deer (Capreolus pygargus) Chymosin Obtained in the Mammalian Cell Culture (CHO-K1)
AbstractStructure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene ofC. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1 ′ + S3′ specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to expres...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells
AbstractRecombinant proteins produced inEscherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in theE. coli genome. The second approach, which has not been previously used, was to increase expression ofE. coli protein YciM. According to the published data, elevated expression of YciM leads ...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
State-of-the-Art Approaches to Heterologous Expression of Bispecific Antibodies Targeting Solid Tumors
AbstractBispecific antibodies (bsAbs) are some of the most promising biotherapeutics due to the versatility provided by their structure and functional features. bsAbs simultaneously bind two antigens or two epitopes on the same antigen. Moreover, they are capable of directing immune effector cells to cancer cells and delivering various compounds (radionuclides, toxins, and immunologic agents) to the target cells, thus offering a broad spectrum of clinical applications. Current review is focused on the technologies used in bsAb engineering, current progress and prospects of these antibodies, and selection of various heterol...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii
AbstractEntomopathogenic bacteria of the genusPhotorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in thePhotorhabdus genomes, immediately after theprtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor fromPhotorhabdus laumondii subsp.laumondii TT01 was cloned and expressed inEscherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Impaired Enzymatic Antioxidant Defense in Erythrocytes of Rats with Ammonia-Induced Encephalopathy: Role of NMDA Receptors
AbstractHepatic encephalopathy (HE), a neuropsychiatric disorder developing in patients with severe hepatic dysfunction, has been known for more than a century. However, pathogenetic mechanisms of cerebral dysfunction associated with liver disease are still poorly understood. There is a consensus that the primary cause of HE is accumulation of ammonia in the brain as a result of impaired liver detoxification capacity or the portosystemic shunt. Current evidence suggests that ammonia toxicity is mediated by hyperactivation of glutamate receptors, mainly N-methyl-D-aspartate receptors (NMDARs), and affects brain aerobic meta...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Dimeric ACE2-FC Is Equivalent to Monomeric ACE2 in the Surrogate Virus Neutralization Test
AbstractAngiotensin-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 pre...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Effect of Biotin Starvation on Gene Expression in Komagataella phaffii Cells
In this study the effect of biotin starvation on gene expression inK. phaffii at transcriptome level was investigated. It was demonstrated, that the response ofK. phaffii cell to biotin deficiency strongly depends on the carbon source in the medium. In the media containing glycerol, biotin deficiency led to activation of the genes involved in biotin metabolism, glyoxylate cycle, and synthesis of acetyl-CoA in cytoplasm, as well as repression of the genes, involved in lipo- and gluconeogenesis. In the methanol-containing media, biotin deficiency primarily led to repression of the genes, involved in protein synthesis, an...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin β-Subunit Production in E. coli
AbstractCholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin β-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB inE. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to growE. coli culture in the...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
