Features of the Mechanism of Proton Transport in ESR, Retinal Protein from Exiguobacterium sibiricum
AbstractRetinal-containing light-sensitive proteins – rhodopsins – are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein ofExiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond...
Source: Biochemistry (Moscow) - October 1, 2023 Category: Biochemistry Source Type: research
Mitochondrial Network: Electric Cable and More
AbstractMitochondria in a cell can unite and organize complex, extended structures that occupy the entire cellular volume, providing an equal supply with energy in the form of ATP synthesized in mitochondria. In accordance with the chemiosmotic concept, the oxidation energy of respiratory substrates is largely stored in the form of an electrical potential difference on the inner membrane of mitochondria. The theory of the functioning of extended mitochondrial structures as intracellular electrical wires suggests that mitochondria provide the fastest delivery of electrical energy through the cellular volume, followed by the...
Source: Biochemistry (Moscow) - October 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
Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis
In this study, syntheticcyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing thegdh (glucose dehydrogenase, GDH) orzwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene inMycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7 β-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7β-OH-A...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Ultra-Fast Mass Spectrometry in Plant Biochemistry: Response of Winter Wheat Proteomics to Pre-Sowing Treatment with Iron Compounds
AbstractIn recent years, ultrafast liquid chromatography/mass spectrometry methods have been extensively developed for the use in proteome profiling in biochemical studies. These methods are intended for express monitoring of cell response to biotic stimuli and elucidation of correlation of molecular changes with biological processes and phenotypical changes. New technologies, including the use of nanomaterials, are actively introduced to increase agricultural production. However, this requires complex approbation of new fertilizers and investigation of mechanisms underlying the biotic effects on the germination, growth, a...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
Generation of a Cell Line Selectively Producing Functionally Active OATP1B1 Transporter
AbstractThe solute carrier organic anion transporter family member, OATP1B1, is one of the most important transporter proteins, which mediate penetration of many endogenous substances and xenobiotics into hepatocytes. A model system providing expression of the functional protein is needed to assess interaction of OATP1B1 with various substances. Based on the HEK293 cells, we obtained the HEK293-OATP1B1 cell line, constitutively expressing theSLCO1B1 gene encoding the OATP1B1 transporter. Expression of theSLCO1B1 gene was confirmed by real-time PCR analysis and Western blotting. Functionality of the transporter was assessed...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
CRISPR/Cas9-Mediated Genome Editing of the Komagataella phaffii to Obtain a Phytase-Producer Markerless Strain
AbstractUsing CRISPR/Cas9 system, the recipient strainsK. phaffii VKPM Y-5013 (His– phenotype) andK. phaffii VKPM Y-5014 (Leu– phenotype) were derived from theK. phaffii VKPM Y-4287 strain, which has a high expression potential. Based on the developed recipient strains, markerless producers of heterologous proteins could be obtained. Efficiency of the gene inactivation with different variants of sgRNA ranged from 65 to 98% and from 15 to 72% for theHIS4 andLEU2 genes, respectively. The recipient strains retained growth characteristics of the parent strain and exhibited high expression potential, as estimated by t...
Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research
