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
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
Effect of Hippocampal Overexpression of Dopamine Neurotrophic Factor (CDNF) on Behavior of Mice with Genetic Predisposition to Depressive-Like Behavior
AbstractCerebral dopamine neurotrophic factor (CDNF) is a promising agent for Parkinson ’s disease treatment. However, its role in regulation of non-motor behavior including various psychopathologies remains unclear. In this regard, the aim of the present work was to study effect of CDNF overexpression in hippocampus on behavior of the ASC mice (Antidepressant Sensitive Cataleptics) with genetic predisposition to depressive-like behavior. CDNF overexpression in the mouse hippocampal neurons was induced using an adeno-associated viral vector. Four weeks after stereotaxic injection of the AAV-CDNF construct into the dorsal...
Source: Biochemistry (Moscow) - August 1, 2023 Category: Biochemistry Source Type: research
Long-Term Experimental Hyperglycemia Does Not Impair Macrovascular Endothelial Barrier Integrity and Function in vitro
AbstractHyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Manyin vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observedin vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC)in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, ...
Source: Biochemistry (Moscow) - August 1, 2023 Category: Biochemistry Source Type: research
Complexes and Supramolecular Associates of Dodecyl-Containing Oligonucleotides with Serum Albumin
AbstractSerum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiolo...
Source: Biochemistry (Moscow) - August 1, 2023 Category: Biochemistry Source Type: research
Constitutive Androstane Receptor Agonist Initiates Metabolic Activity Required for Hepatocyte Proliferation
AbstractActivation of the constitutive androstane receptor (CAR, NR1I3) by chemical compounds induces liver hyperplasia in rodents. 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a mouse CAR agonist, is most often used to study chemically induced liver hyperplasia and hepatocyte proliferationin vivo. TCPOBOP is a potent murine liver chemical mitogen, which induces rapid liver hyperplasia in mice independently of liver injury. In recent years, great amount of data has been accumulated on the transcription program that characterizes the TCPOBOP-induced hepatocyte proliferation. However, there are only few data abou...
Source: Biochemistry (Moscow) - August 1, 2023 Category: Biochemistry Source Type: research
Hybrid Implants Based on Calcium-Magnesium Silicate Ceramic Diopside as a Carrier of Recombinant BMP-2 and Demineralized Bone Matrix as a Scaffold: Ectopic Osteogenesis in Intramuscular Implantation in Mice
AbstractHigh efficiency of hybrid implants based on calcium-magnesium silicate ceramic, diopside, as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously using the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue usingin vivo bioreactor principle in the patient ’s own body, effectiveness of ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At the dose of 7 μg of BMP-2 per implant, dense agg...
Source: Biochemistry (Moscow) - August 1, 2023 Category: Biochemistry Source Type: research
