Heterologous Expression of Human IFNγ and Anti-IL17 Antibody in Leishmania tarentolae Promastigote.

BACKGROUND: Leishmania is an intracellular flagellate protozoan parasite that causes a wide range of clinical diseases in humans. The basis of immunological resistance against leishmaniasis depends on Thl reactions and is within the time period of cytokine function. METHODS: In this study, human anti-IL17 antibody and IFNγ-producing promastigote were produced to be used in leishmanization. A sequence of light and heavy chains' gene of anti-IL17 antibody and human IFNγ (hIFNγ) was obtained from the NCBI database and synthesized in the ECORV reaction site in the plasmid pGH, which it's called pGH-hIFNγ-antiIL17. The synthesized part using the restriction enzyme ECORV was extracted from the plasmid and after purification by electroporation was transferred to Iranian lizard Leishmania (I.L.L). Evaluation of structural presence in the I.L.L genome at the level of DNA and mRNA was assessed. The expressions of hIFNγ and anti-IL17 were evaluated and confirmed using ELISA and western blot analysis. The hIFNγ secreted from the culture medium was collected at high concentrations of 124.36 ± 6.47 pg/mL. RESULTS: Targeted gene replacement into the I.L.L genome was successfully performed for the first time using the pGH-hIFNγ-antiIL17 plasmid in an identical replacement process. Stabilized recombinant DNA contains a target gene that has no toxicity to the parasite. CONCLUSIONS: The effective achievement of producing a recombinant gene was done for the first time by replacing the I.L.L-CPC gene with plasmid pGH-hIFNγ-antiIL17 by targeted gene replacement. This cab can regulate the production of hIFNγ and anti-IL17. This makes it a viable choice for eliminating leishmania.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

PURPOSE: Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. METHODS: After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. RESULTS: The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. CONCLUSION: The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

Enzymatic and antibacterial activity of the recombinant endolysin PVP-SE1gp146 expressed in Hansenula polymorpha.

Antibiotic resistance, a major global concern, highlights the need for discovering alternative therapies. Recently, endolysins have garnered attention as antibacterial tools with a lower resistance development rate compared to conventional antibiotics, and their production in various expression hosts holds significance. Given its generally recognized as safe (GRAS) status and other advantages, Hansenula polymorpha offers a promising host for endolysin production. PVP-SE1gp146 originates from the Salmonella Enteritidis-specific phage PVP-SE1, which has been previously characterized. We inserted the PVP-SE1gp146 coding gene into the H. polymorpha expression vector pHIPX4. The resulting recombinant, pHIPX4-PVP-SE1gp146, was then introduced into H. polymorpha NCYC495 to facilitate the production of the endolysin PVP-SE1gp146. The expression level of the PVP-SE1gp146 protein was assessed, and it was determined to be approximately 43 mg/l of yeast culture medium. The enzymatic (muralytic) activity of this endolysin was also evaluated, corresponding to the version produced by the E. coli Bl21 strain. The endolysin exhibited admissible antibacterial activity against several gram-negative species, including P. aeruginosa, E. coli, and A. baumannii, while showing an almost negligible impact on K. pneumoniae. Endolysin production within GRAS-approved hosts holds potential for combating antibiotic-resistant bacteria. Challenges involve optimizing concentrations, targeting gram-negative species and improving attachment to bacterial cell walls. Addressing these issues requires dedicated research in endolysin engineering and a comprehensive evaluation of their production in diverse expression hosts.

Production of a Ribosome-Displayed Mouse scFv Antibody Against CD133, Analysis of Its Molecular Docking, and Molecular Dynamic Simulations of Their Interactions.

The pentaspan transmembrane glycoprotein CD133, prominin-1, is expressed in cancer stem cells in many tumors and is promising as a novel target for the delivery of cytotoxic drugs to cancer-initiating cells. In this study, we prepared a mouse library of single-chain variable fragment (scFv) antibodies using mRNAs isolated from mice immunized with the third extracellular domain of a recombinant CD133 (D-EC3). First, the scFvs were directly exposed to D-EC3 to select a new specific scFv with high affinity against CD133 using the ribosome display method. Then, the selected scFv was characterized by the indirect enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), and in silico analyses included molecular docking and molecular dynamics simulations. Based on ELISA results, scFv 2 had a higher affinity for recombinant CD133, and it was considered for further analysis. Next, the immunocytochemistry and flow cytometry experiments confirmed that the obtained scFv could bind to the CD133 expressing HT-29 cells. Furthermore, the results of in silico analysis verified the ability of the scFv 2 antibody to bind and detect the D-EC3 antigen through key residues employed in antigen-antibody interactions. Our results suggest that ribosome display could be applied as a rapid and valid method for isolation of scFv with high affinity and specificity. Also, studying the mechanism of interaction between CD133's scFv and D-EC3 with two approaches of experimental and in silico analysis has potential importance for the design and development of antibody with improved properties.

Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains.

BACKGROUND: Genome manipulation of Leishmania species and the creation of modified strains are widely employed strategies for various purposes, including gene function studies, the development of live attenuated vaccines, and the engineering of host cells for protein production. OBJECTIVE: Despite the introduction of novel manipulation approaches like CRISPR/Cas9 technology with significant advancements in recent years, the development of a reliable protocol for efficiently and precisely altering the genes of Leishmania strains remains a challenging endeavor. Following the successful adaptation of the CRISPR/Cas9 system for higher eukaryotic cells, several research groups have endeavored to apply this system to manipulate the genome of Leishmania. RESULTS: Despite the substantial differences between Leishmania and higher eukaryotes, the CRISPR/Cas9 system has been effectively tested and applied in Leishmania.  CONCLUSION: This comprehensive review summarizes all the CRISPR/Cas9 systems that have been employed in Leishmania, providing details on their methods and the expression systems for Cas9 and gRNA. The review also explores the various applications of the CRISPR system in Leishmania, including the deletion of multicopy gene families, the development of the Leishmania vaccine, complete gene deletions, investigations into chromosomal translocations, protein tagging, gene replacement, large-scale gene knockout, genome editing through cytosine base replacement, and its innovative use in the detection of Leishmania. In addition, the review offers an up-to-date overview of all double-strand break repair mechanisms in Leishmania.

Preliminary Information of Iranian Lizard Leishmania Promastigote Transcriptome Sequencing by Next Generation Sequencing (NGS) Method.

Background: A lizard Leishmania has been isolated from a lizard (Agama agilis) in Iran. Its genome sequence has not been determined, so far. Methods: The study was done at Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2017-2023. Leishmania promastigotes were cultured in RPMI1640 culture medium and collected at logarithmic phase by centrigugation. Parasite RNA was extracted by the Qiagene standard kit and its quantity and quality was determined and sequenced by NGS method with Illumina PE machine at BGI Company (China). Results: The number of 8316 mRNA, 83 tRNA, 63 rRNA, 83 ncRNA, 5 snRNA, 1039 snoRNA, 36 region, and 3 repeat regions, 8343 CDS, 9597 Exon and 9292 Genes were identified in promastigote of Iranian lizard Leishmania. Conclusion: Genomic elements of Iranian lizards Leishmania (with unique characteristics) were determined and identified by NGS system.

Ectopic expression of insulin in a type 1 diabetic rat model by injection of manipulated mesenchymal stem cells with an insulin construct driven by a glucose-sensitive promoter in the port vein.

The treatment of type 1 diabetes through islet cell transplantation is a complex process, facing challenges such as allograft rejections and a limited supply of donors. One potential solution is to utilize the liver as an alternative for natural insulin production, as hepatocytes can secrete proteins and respond to glucose levels. Recent research has shown promising results in using mesenchymal stem cells as a potential cure for diabetes. The study utilized a diabetic rat model, confirmed through blood sugar measurement. A plasmid vector was designed with specific genetic components, synthesized by biotech company, and then Inserted vector into a plasmid with resistance genes and bacterial origin. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured and transfected with the plasmid using Lipofectamine 3000. Polymerase chain reaction was employed to confirm successful transfection using specific primers. For the animal study, 30 male Wistar rats were divided into six groups, each comprising five rats. The control group did not receive any treatment, while the second group received MSCs via Portal Vein Injection. The third group received MSCs transfected with a specific construct via Portal Vein Injection. The fourth group was induced to develop diabetes through streptozotocin (STZ) injection, the fifth group developed diabetes and received untransfected MSCs via Portal Vein Injection, and the sixth group received MSCs transfected with the specific construct via Portal Vein Injection. To manage Pain, appropriate pain control was administered to the rats for 3 days after the surgery. Fixed liver tissues obtained from the euthanized rats were utilized for immunohistochemistry. In this study, immunohistochemical techniques were used to examine insulin expression in different groups of rats. The control groups showed high levels of insulin expression, while the diabetic groups exhibited lower expression. However, there was a significant difference between the diabetic groups treated with MSC and transgenic MSC cells. All groups had similar baseline glucose levels, but the diabetic groups showed a significant increase after STZ injection, whereas the control and MSC groups did not. Postintervention, both the control and MSC groups had similar glucose levels to the post-STZ levels. However, diabetes-induced groups experienced a significant decrease in glucose levels, with the transfected MSCs showing a greater decrease than the untransfected MSCs. The study suggested that treatment with MSCs, especially transfected ones, can effectively reduce glucose levels in rats with diabetes. In this research, rat BM-MSCs were utilized to create insulin-producing mesenchymal cells with glucose-sensitive insulin expression. The cells were transferred to the liver of diabetic rats via portal vein injection, leading to an increase in insulin expression. This study proposes a novel approach for cell therapy and delivery in the treatment of type 1 diabetes.

Serum Level of Antibodies Against Novel Acinetobacter Baumannii OmpA-selected Peptides in ICU Staff: Promise for the Future of Vaccine Development.

Extensively drug-resistant Acinetobacter baumannii is considered one of the most dangerous threats to global health, requiring novel therapeutic interventions. The outer membrane protein A (OmpA) is an immunogenic agent that triggers immune responses. The current study evaluated serum antibody levels against previously determined immunogenic OmpA peptides from A baumannii in ICU staff. Serum samples were collected from 62 ICU staff members (representing the exposed group), healthy controls (representing the nonexposed group), and patients with systemic lupus erythematosus (SLE) (as controls for nonspecific antibody reactions). After excluding the cross-reactive antibodies via Escherichia coli lysate pretreatment, all the samples were assessed in the vicinity of A baumannii lysate by enzyme-linked immunosorbent assay (ELISA). All the positive samples were assessed for interaction with previously designed and selected peptides using ELISA. The protective potential of positive serum antibodies was surveyed in vitro using an opsonophagocytic study. The most antibody positive samples against one of the dominant peptides were determined in the ICU personnel (75%).  SLE serum samples did not react with candidate peptides. The strongest positive reaction was observed in serum treatment with one of the OmpA peptides (No. 5) with significant differences compared to other designed peptides. Our findings showed that ICU samples have substantially higher antibody levels than the nonexposed group; Positive samples show strong results in the opsonophagocytosiis assay. This study demonstrates A baumannii colonization at human mucosal surfaces, especially in exposed healthy workers. Novel OmpA-derived peptides could be used to identify immunogenic vaccine candidates. Therefore, more studies are needed  before this peptide and antibody levels are used in diagnosis, prevention, or treatment.

Modified Phenol/Chloroform-Free DNA Isolation from Yeast and Other Fungi by Non-Enzymatic Salting Out Method.

The most commonly used fungal DNA extraction techniques require enzymatic digestion or hazardous chemicals. Here, we describe a quick non-enzymatic salt-out method that we developed for isolating high-quality DNA from various fungal species. DNA was extracted from four species of typical fungi, including Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, and Aspergillus flavus. Results that we obtained from agarose gel electrophoresis and spectrophotometry verified the integrity, high quality, and purity of the extracted DNAs. Also, PCR amplification and restriction enzyme digestion confirmed the efficiency of the extracted DNAs in molecular biology applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Yeast and fungal cultivation for DNA extraction Basic Protocol 2: Modified salting out DNA extraction protocol Support Protocol 1: PCR amplification of extracted genomes Support Protocol 2: Digestion with restriction enzyme.

In vitro Gluten Degradation Using Recombinant Eurygaster Integriceps Prolyl Endoprotease: Implications for Celiac Disease.

Background: Celiac disease (CD) is a gluten-sensitive chronic autoimmune enteropathy. A strict life-long gluten-free diet is the only efficient and accepted treatment until now. However, maintaining a truly gluten-free status is both difficult and costly, often resulting in a social burden for the person. Moreover, 2 to 5 percent of patients fail to improve clinically and histologically upon elimination of dietary gluten. Therefore, novel therapeutic approaches, including gluten degrading enzymes, are an unmet need of celiac patients. Objectives: To evaluate the function of sunn pest prolyl endoprotease for gluten and gliadin hydrolysis in vitro. Materials and Methods: The spPEP was expressed as a recombinant protein in E. coli BL21 (DE3), and its catalytic activity was assessed by SDS-PAGE and RP-HPLC analyses. Results: Production of a 100-kDa spPEP protein was confirmed by SDS-PAGE and western blot analysis. Also, we demonstrate that spPEP efficiently degrades gluten and α-gliadin (30-40 kDa) in vitro under conditions similar to the GI and is resistant to pepsin and trypsin. Conclusion: The gathered data demonstrated that spPEP might be a novel candidate for Oral Enzymatic Therapy (OET) in CD and other gluten-related disorders.

Polycistronic Expression of Multi-Subunit Complexes in the Eukaryotic Environment: A Narrative Review.

Protein complexes are involved in many vital biological processes. Therefore, researchers need these protein complexes for biochemical and biophysical studies. Several methods exist for expressing multi-subunit proteins in eukaryotic cells, such as 2A sequences, IRES, or intein. Nevertheless, each of these elements has several disadvantages that limit their usage. In this article, we suggest a new system for expressing multi-subunit proteins, which have several advantages over existing methods meanwhile it, lacks most of their disadvantages. Leishmania is a unicellular eukaryote and member of the Trypanosomatidae family. In the expression system of Leishmania, pre-long RNAs that contain several protein sequences transcribe. Then these long RNAs separate into mature mRNAs in the process named trans splicing. For producing multi-subunit protein, Leishmania transformed with a vector containing the sequences of all subunits. Therefore, those subunits translate and form the complex under eukaryotic cell conditions. The sequence of each protein must separate by the spatial sequence needed for trans splicing. Based on a Leishmania expression pattern, not only is it possible to produce the complexes with the correct structures and post-translational modifications, but also it is possible to overcome previous method problems.

Effects of human chorionic gonadotropin-producing peripheral blood mononuclear cells on the endometrial receptivity and implantation sites of the mouse uterus.

OBJECTIVE: This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. METHODS: In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. RESULTS: DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. CONCLUSION: Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.

Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies.

Fibroblasts are the main cells of connective tissue and have pivotal roles in the proliferative and maturation phases of wound healing. These cells can secrete various cytokines, growth factors, and collagen. Vascular endothelial growth factor (VEGF) is a unique factor in the migration process of fibroblast cells through induces wound healing cascade components such as angiogenesis, collagen deposition, and epithelialization. This study aimed to create VEGF165 overexpressing fibroblast cells to evaluate angiogenesis function in wound healing. In vitro, a novel recombinant expression vector, pcDNA3.1(-)-VEGF, was produced and transfected into the fibroblast cells. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying and qualifying methods. Mechanical, physical, and survival properties of polyurethane-cellulose acetate (PU-CA) scaffold were investigated. Finally, in vivo, the angiogenic potential was evaluated in four groups containing control, PU-CA, PU-CA with fibroblast cells, and VEGF-expressing cells on days 0, 2, 5, 12 and 15. Wound biopsies were harvested and the healing process was histopathologically evaluated on different days. qRT-PCR showed VEGF overexpression (sevenfold) in genetically-manipulated cells compared to fibroblast cells. Recombinant VEGF expression was also confirmed by western blotting. Manipulated fibroblast cells represented more angiogenesis than other groups on the second day after surgery, which was also confirmed by the antiCD31 antibody. The percentage of wound closure area on day 5 in genetically-manipulated Hu02 and Hu02 groups showed a significant reduction of wound area compared to other groups. These findings indicate that overexpression of VEGF165 in fibroblast cells results in enhanced angiogenesis and formation of granulated tissue in the early stage of the healing process, which can show its therapeutic potential in patients with impaired wound healing and also provide functional support for gene therapy.

Production of CFTR Mutant Gene Model by Homologous Recombination System.

OBJECTIVE: The most common mutation in cystic fibrosis (CF), (ΔF508-CFTR), results in impaired protein maturation, folding and transportation to the surface of the cell. As a consequence of impaired protein maturation and/or transport from the extracellular matrix to the cell, different systems are influenced, including gastrointestinal system and glandular system, reproductive system and respiratory systems. CF models are essential tools to provide further knowledge of CF pathophysiology. With this aim, we designed a transgenic CF model based on the homologous recombination (HR) system. MATERIALS AND METHODS: In this experimental study, a specifically designed construct containing the CFTR gene with F508del was cloned into a PTZ57R cloning vector and then the construct was transformed into the male pronucleus by microinjection after in vitro fertilization (IVF). Then the rates of blastocyst formation and embryonic development at 72 hours after IVF, were evaluated using the inverted microscope and the insertion of the construct was approved by polymerase chain reaction (PCR) method. RESULTS: The CFTR gene was successfully cloned into the PTZ57R cloning vector and overall, from 22 injected cells, 5 blastocysts were observed after pronuclear injection of the CFTR gene construct. PCR verification of the blastocyst with CFTR-specific primers represented complete recombination of CFTR into the mouse genome. CONCLUSION: For the first time we designed a unique genome construction that can be detected using a simple PCR method. The pronuclear injection was performed for the transformation of the genome construct into the male pronuclei using microinjection and the development of zygote to the blastocyst stage has been observed following transgenesis.

Cloning, Expression and Purification of Full-length Recombinant Ecarin and Comparing Its Expression and Function with Its Truncated Form.

Ecarin is a metalloproteinase found in snake venom (SVMP) with an important role in coagulation and control of hemostasis. It can specifically produce active-thrombin from prethrombin-2 and does not differentiate between normal and abnormal prothrombin. It is used in diagnostic tests and to evaluate the treatment process of many diseases. There are many drawbacks associated with separating these compounds from snake venom. Therefore, in this study, full-length recombinant Ecarin (r-Ecarin) was cloned, expressed, and purified in eukaryotic host cells. To determine the most effective form of the enzyme, r-Ecarin was compared with the recombinant truncated form, which has only the metalloprotease domain of the protein (r-Ecamet) in terms of function and expression. Briefly, A DNA construct composed of sequence-encoding Ecarin was designed and cloned into pCAGGS expression vector and, subsequently, expressed in Chinese Hamster Ovary (CHO) cells. To identify the enzymatic activity of expressed protein, a bioactivity assay was performed. Blood coagulation time and expression levels of r-Ecarin and r-Ecamet proteins were compared. Also, a histopathological assessment was carried out on the liver of mice treated with these proteins. Comparison of r-Ecarin and r-Ecamet expression pattern demonstrated that full-length Ecarin expression has at least 2-fold higher expression in eukaryotic cells. Determination of r-Ecarin function proved that this protein is capable of prothrombin cleavage and producing thrombin. Comparison of PT test results between the r-Ecarin and r-Ecamet showed that there is a significant difference in the activity of the two enzymes and the full-length protein coagulates the blood in less time.

Purslane (Portulaca oleracea L.) as a novel green-bioreactor for expression of human serum albumin (HSA) gene.

Transgenic plants showed high potential to become a valuable and safe source of bio-compounds that can be used as therapeutics without any require for pooled human blood products. Human serum albumin (HSA) is one of the best-selling pharmaceuticals in the world because it is utilized for treating several acute illnesses, including hypovolemia, burns, and hemorrhage. This work was aimed to investigate the production of recombinant HSA (rHSA) protein in a plant-based expression platform. For this, we used in-planta and tissue culture-based Agrobacterium-mediated transformation (TCBAT) procedures to insert HSA gene into purslane (Portulaca oleracea L.) genome. The purslane seeds and leaves were infected with A. tumefaciens strain LBA4404 containing the HSA gene on pBI121 plasmid, and then regenerated into transgenic plant on MS medium. The qRT-PCR, southern hybridization, western blotting, and ELISA analysis were accomplished to corroborate the insertion and expression of HSA gene in transgenic plantlets. The molecular asses indicated that HSA gene was successfully transferred and expressed in purslane plants using in-planta and TCBAT methods. The first attempt to express rHSA in purslane resulted in a low-level accumulation of the protein in the transgenic plant shoots. Therefore, we used a synthetic 5'UTR (synJ) to enhance HSA transcript stability and translation efficiency. The results suggested that the synJ caused pronounced enhancement of rHSA expression rate. The highest amount of rHSA protein was recorded in transgenic purslane generated by TCBAT method (33.92 ± 4.31 µg/g FW).

The OmpA of commensal Escherichia coli of CRC patients affects apoptosis of the HCT116 colon cancer cell line.

BACKGROUND: Colorectal cancer ranks third globally among all types of cancers. Dysbiosis of the gut microbiota of people with CRC is one of the effective agents in the tumorigenesis and metastasis in this type of cancer. The population of Escherichia coli strains, a component of gut microbiota, is increased in the gut of people with CRC compared with healthy people. So, E.coli strains isolated from these patients may have a role in tumorigenesis. Because the most isolated strains belong to the B2 phylogenuetic group, there seems to be a linkage between the bacterium components and malignancy. MATERIAL AND METHODS: In this study, the proteomic comparison between isolated Ecoli from CRC patients and healthy people was assayed. The isolated spot was studied by Two-dimensional gel electrophoresis (2DE) and Liquid chromatography-mass spectrometry (LC-MS). The results showed that the expression of Outer membrane protein A (OmpA) protein increased in the commensal E.coli B2 phylogenetic group isolated from CRC patients. Additionally, we analyzed the effect of the OmpA protein on the expression of the four genes related to apoptosis in the HCT116 colon cancer cell line. RESULTS: This study identified that OmpA protein was overexpressed in the commensal E.coli B2 phylogenetic group isolated from CRC patients compared to the E.coli from the control group. This protein significantly decreased the expression of Bax and Bak, pro-apoptotic genes, as well as the expression of P53 in the HCT116 Cell Line, P < 0.0001. LC-MS and protein bioinformatics results confirmed that this protein is outer membrane protein A, which can bind to nucleic acid and some of the organelle proteins on the eukaryotic cell surface. CONCLUSIONS: According to our invitro and insilico investigations, OmpA of gut E.coli strains that belong to the B2 phylogenetic group can affect the eukaryotic cell cycle.

Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

BACKGROUND: CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. OBJECTIVE: As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. METHODS: CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. RESULTS: When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 µM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 µM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 µg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. CONCLUSION: These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote.

Background: Leishmania is a eukaryotic protozoan parasite belonging to the Trypanosomatidae family. The Iranian Lizard Leishmania (I.L.L.), which is nonpathogenic to mammals, shows great promise to be used as an expression system for recombinant protein production. Unlike other Leishmania strains, the ideal culture medium for I.L.L. has not been established, although it is commonly cultured in the RPMI1640 medium. Methods: We investigated the growth rate of the wild and recombinant I.L.L. in BHI, RPMI1640, LB, and M199 media with and without FBS, hemin, or lyophilized rabbit serum. Subsequently, the expression rate of the recombinant protein in these media was compared. Results: The growth rate of I.L.L. in RPMI1640 medium and LB broth was similar and supplementation with 10% FBS did not affect the growth rate. The amount of protein expression in the LB medium was higher than in the other three media. Conclusion: The LB broth is an appropriate medium for I.L.L. culture and recombinant protein production.

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.