جستجوی مقالات مرتبط با کلیدواژه "recombinant protein" در نشریات گروه "پزشکی"

Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.

This study aimed to engineer a recombinant chimeric fusion protein by combining a nanobody-targeting domain with an exotoxin effector domain. The chimeric protein was designed to bind surface-expressed GRP78 on cancer cells, facilitating internalization and inducing apoptosis to inhibit proliferation and survival.

Using a flexible linker, we designed two constructs linking VHH nanobody domains to Pseudomonas exotoxin (PE) domains II, III, and Ib. These constructs were then optimized for expression in E. coli BL21 (DE3) using the pET28a vector. Following the expression of the recombinant proteins, we purified them and tested their binding capability, cytotoxicity, and ability to induce apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7, as well as in control cell lines HEK-293 and MDA-MB-468. The binding affinity was measured using a cell-based ELISA, internalization was assessed through Western blotting, cytotoxicity was evaluated by an MTT assay, and apoptosis was determined using flow cytometry with an Annexin V kit.

The immunotoxin specifically bound to cancer cells expressing csGRP78. The results of the cytotoxicity test showed that the cytotoxic effect of two constructs, I and II, depended on concentration and time. With an increase in both components, the effect of recombinant proteins also increased. Both constructs were able to penetrate and induce apoptosis in csGRP78+ cells.

These immunotoxin structures showed therapeutic potential against GRP78-expressing cancers, making them suitable candidates for targeted therapy pending in vivo studies.

Shiga toxin-producing E. coli (STEC) are bacteria causing severe foodborne diseases, with E. coli O157:H7 being a significant public health concern. Infection may occur following exposure to or drinking the contaminated water or the consumption of the contaminated food, especially meat and dairy products. We aimed to optimize a sandwich ELISA method using purified poly-IgG against HI chimera protein for E. coli O157:H7 detection. We induced the recombinant chimeric antigen (HI) in a prokaryotic host and purified it through a Ni-NTA column. After refolding the antigen, mice and rabbits were immunized and the poly-IgGs were purified from sera using a protein G column. Recombinant HI (60 kDa) was expressed in E. coli BL21 (yield: 1.2 mg/L) and purified via the Ni-NTA column. Antibodies were generated in mice and rabbits, serving as detection and capture antibodies. The optimized antibody concentrations were 1.25 μg/ml for capture and 0.312 μg/ml for detection. Our sandwich ELISA demonstrated high sensitivity (limit of detection: 104 CFU/ml) and specificity for E. coli O157:H7, confirmed by testing against different bacteria. Our developed sandwich ELISA has proven to be a highly sensitive method for the detection of E. coli O157:H7, capable of reliably detecting bacterial concentrations as low as 104 CFU/ml.

Botulinum neurotoxin (BoNT), a lethal bacterial toxin causing neuroparalytic disease, necessitates robust detection methods to prevent and manage botulism outbreaks. The receptor-binding domain of the toxin's heavy chain (Hc) has been extensively explored as a potential BoNT vaccine candidate. This study's primary goal is the swift detection of Botulinum neurotoxin type A (BoNT/A) using a sandwich ELISA method employing polyclonal antibodies. The recombinant BoNT/A-285HcC was induced with one mM IPTG at 25°C for 18 hours to reduce inclusion bodies and purified using Ni-NTA under non-denaturing conditions. Immunization of animals followed a specific regimen using purified BoNT/A-285HcC recombinant antigen and Freund's adjuvant. IgG antibodies from immunized mice serum were isolated using protein G resin. The purified antibodies' reactivity with recombinant BoNT/A-285HcC protein was assessed through western blotting. Efficient protein expression was achieved, yielding 50 mg/L. The recombinant BoNT/A-285HcC, with a molecular weight of 46 kDa, was purified with a near 90% purity level. ELISA results demonstrated a significant rise in anti-BoNT/A antibody titers following three doses. Western blot analysis confirmed the specific binding capability of the purified anti-BoNT/A IgG. Ultimately, the sandwich ELISA developed in this study exhibited the ability to detect 100 pg/ml of BoNT/A, utilizing 1.25 µg/ml of mice antibody as the capture and 0.3 µg/ml of rabbit antibody as the detection antibody. Purified polyclonal antibodies against recombinant BoNT/A-285HcC can be effectively employed in diagnostic serological tests for BoNT/A detection, with a limit of detection (LOD) of 100 pg/ml, significantly enhancing our ability to combat BoNT-related threats and ensuring the safety of medical applications.

 Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active.

 Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells.

 Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4±7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8±2.5 IU/0.1 mL).

 A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

Hepatitis A virus (HAV) is a causative agent of acute hepatitis in humans, infecting more than one million individuals every year, including both symptomatic and asymptomatic infections. The currently available preventive vaccines for HAV are based on either wild-type or live-attenuated virus strains, which can contribute to the costliness of the vaccination process. Therefore, it may be worthwhile to explore the potential of subunit vaccines that utilize immunogenic viral products.

This study presents the results of a novel recombinant protein production study that employed the native structures of HAV-VP1 and HBs-Ag. The fusion protein underwent comprehensive characterization to evaluate its potential applications in diagnostics and immunization. The truncated recombinant protein, HAV-VP1 (position 99-259 aa) -HBs-Ag, was successfully expressed in the Escherichia coli BL21-DE3 system.

The recombinant protein, with a molecular weight of 46 kDa, was evaluated using SDS-PAGE gel electrophoresis and confirmed by western blotting. The fusion protein was successfully detected in serum samples positive for HBV or HAV using anti-HBs and anti-VP1 antibodies. Additionally, it elicited a potent humoral response in BALB/c mice.

The novel recombinant protein described in this study has the potential to serve as a bivalent vaccine against HAV and HBV infections. The next step involves evaluating the immunogenicity and safety profile of the protein.

Rotaviruses (RV) and hepatitis A virus (HAV) are pathogens responsible for more than 2 million hospitalizations, especially in developing countries, due to transmission through the fecal-oral route. Currently, there are several FDA-approved RV and HAV vaccines available which are based on killed or attenuated viruses. However, these vaccines often have side effects and low efficacy in eliciting specific immunity. Therefore, the design of a vaccine based on a recombinant protein, composed of RV and HAV antigens seems essential.

We used bioinformatics tools to design and analyze the properties, predict the structure and evaluate the function, immunogenicity, antigenicity, and truncated sequences of HAV VP1 and RV VP8 as a dual vaccine platform. The predicted epitopes were expressed as a recombinant protein in Escherichia coli BL21 where half of the VP1 protein was fused with the Rota protein VP8 using  pET24a expression vector,.

The expressed protein was confirmed by SDS-PAGE and Western blotting. Subsequently, high-scale expression, purification, refolding and determination of the protein concentration (~2.4 µg/µl) were obtained.

Upon completion of the future immunogenicity evaluation through injection into mice, the present fusion protein can potentially serve as a candidate for a recombinant vaccine against both RV and HAV infections.

Botulinum neurotoxin is one of the most potent toxins. This neurotoxin is a biological weapon due to its high lethality and simplicity of preparation. The receptor-binding domain of botulinum neurotoxin is a promising vaccine candidate. In this study, the immunogenicity of the C-terminal domain of botulinum neurotoxin type A binding domain was investigated.

The synthetic gene encoding 285 C-terminal amino acids of BoNT/A binding domain fused to trxA gene, was constructed. The sequence was optimized codon usage for expression in E. coli and subcloned into pET-17b expression vector. The recombinant protein was expressed using 1 mM IPTG and purified by affinity chromatography on a column of Ni-NTA. The recombinant protein was confirmed by Western blotting and was used to immunize mice. The indirect ELISA and t-test were used to assess and compare antibody titers against recombinant protein.

The codon adaptation index of the contract was altered from 0.62 to 0.90 after optimization. The minimum energy of the predicted mRNA structure was (-308.39) kcal/mol. SDS-PAGE and Western blotting confirmed the 44/6 kDa recombinant protein. Following immunization, mice elicited significant IgG antibodies in serum compared to control mice (p<0.05).

The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice. Future studies may develop the recombinant antigen as a potential immunogenic candidate against botulinum neurotoxin type A.

The treatment of Staphylococcus aureus infections has become a public health crisis due to the extensive development of antimicrobial resistance. Antimicrobial peptides (AMPs) have been introduced as promising naturally-derived antimicrobial alternatives to antibiotics. LL-37 and oncorhyncin II are 2 AMPs with notable proven antibacterial effects.

This study aimed to produce recombinant LL-37 and oncorhyncin II and investigate their synergistic effects on S. aureus (ATCC25923).

The synthetic genes of LL-37 and oncorhyncin II were individually ligated into the pET32a expression vector. Transformed pET32a was introduced into Escherichia coli BL21 as an expression host. The protein expression and purification steps were optimized, and the biological effectiveness of the peptides was evaluated by assessing the minimum inhibitory concentration (MIC), time-kill, and growth kinetic tests against S. aureus.

The MIC assay confirmed the effective antibacterial performances of LL-37 and oncorhyncin II against S. aureus at 30.6 and 47.93 g/mL, respectively. The peptides’ synergistic activity was validated by the checkerboard method. A combination of LL-37 and oncorhyncin II at 2  MIC showed a sharp decline of the viable cells with over 3-time reductions in log 10 colony-forming units (CFU)/mL within the first 5 hours. The growth kinetic results confirmed the high effectiveness of the peptides’ combination in eliminating the bacterial inoculum turbidity by 50% reduction during the first hour of exposure.

The produced recombinant LL-37 and oncorhyncin II showed effective antimicrobial function against S. aureus. The synergistic performance of the peptides was repeatedly confirmed through checkerboard, time-kill, and growth kinetic assays.

Inosine 5-monophosphate dehydrogenase 1 (IMPDH1) is the rate-limiting enzyme in the de novo purine nucleotide biosynthesis. IMPDH1 catalyzes IMP-oxidation to XMP, which in continue is converted to guanine nucleotides. Like mammals, the mouse IMPDH1 (mH1) has retinal-specific isoforms named H1 (546) and H1 (603). Mutations in the IMPDH1 gene are believed to cause retinal degenerative disease, retinitis pigmentosa, mediated by retinal-specific gene variants. After RNA extraction from the mouse retina, RT-PCR was done using NdeI and XhoI harboring primers. Tree mH1 isoform genes were amplified and cloned into a pET26b+ vector separately. The recombinant expression vectors were then transformed in E. coli BL21 (DE3) strain, expressed under IPTG-induced conditions and purified with Ni-NTA agarose resin. Activity assay of recombinant proteins was done by using spectrophotometric methods. Here, we cloned and optimized the expression and the purification of recombinant mH1 canonical and retinal isoforms in E. coli to gain soluble and highly active protein for further functional assays. Recombinant protein production in prokaryotic hosts, especially E. coli, is the most common method in large-scale of protein production for functional and structural studies. However, maximal yield and activity of recombinant proteins require optimal conditions for expression and purification, which is what we showed in the present study for the mouse IMPDH1 recombinant isoforms.

 Multidrug-resistant (MDR) Acinetobacter baumannii is one of the most common nosocomial pathogens. Antimicrobial peptides (AMPs) have been introduced as a viable alternative to antibiotics in the treatment of MDR pathogens.

 This study was designed to assess the in vitro pharmacokinetics of the combination of two potent AMPs, LL-37 and oncorhyncin II, against A. baumannii (ATCC19606).

 The synthesized genes of oncorhyncin II and LL-37 were introduced into Escherichia coli BL21 as the expression host. The minimum inhibitory concentration (MIC), time-kills, and growth kinetics of these peptides were used to evaluate their antimicrobial efficiencies against A. baumannii (ATCC19606).

 LL-37 and oncorhyncin II recombinant peptides showed MIC of 30.6 and 95.87 µg/mL against A. baumannii, respectively. Additive action was confirmed by combining the generated AMPs at the checkerboard approach. The combination of LL-37 and oncorhyncin II at 2 × MIC resulted in a rapid drop in log10 CFU/mL of A. baumannii in the time-kill and growth kinetic findings studies.

 The combination of the produced LL-37 and oncorhyncin II synergizes the bioactivity of the individual peptides. Therefore, these peptides or their combinations might function as novel antibiotics and be used to develop and produce new antimicrobial drugs for the treatment of infections caused by A. baumannii.

Escherichia coli is one of the most commonly used organisms for producing recombinant protein. The recognized cell genome and well-known cell factory of the bacteria make it an ideal heterologous system of preference for the production of recombinant proteins. Over recent years, this cell system has been modified to improve the production of therapeutic proteins, and several methodologies are now available. One of the scientists' most preferred strategies is maintaining cell survival by optimizing the culture conditions. This review summarises some experiments related to those culture conditions and discusses how they affect recombinant protein expression in E. coli.

Homodimericbone morphogenetic protein-7 (BMP-7) plays a key role in bone metabolism. The functionality of human BMP-7 protein is dependent on its disulfide bond formation and proper folding. Therefore, the expression of soluble recombinant BMP-7 using Escherichia colicells as the host remains a challenge. Given the need for these disulfide-bonded proteins for stabilized native conformation, the cytoplasm of SHuffle®T7 Express as an E. coliengineered strain can effectively fold disulfide-bonded proteins with a needfor proper oxidative folding. These cellular features turn the SHuffle®expression system into an efficient host for the recombinant production of human BMP-7 protein. A soluble dimeric form of recombinant human BMP-7 (rhBMP-7) which has a wide range of applications in medicine and can be used in the treatment of bone defects was produced using the SHuffle®strain as the expression system. This study demonstrated the production of rhBMP-7 using E. coliSHuffle®T7 Express strain. Also, an effective protocol was proposed for the expression and purification of soluble human BMP-7. In addition, it was found that the genetically engineered SHuffle®strain can efficiently enhance the solubility of recombinant human BMP-7 as a therapeutic target.

The use of chimeric proteins that selectively interact with various immune cell receptors to treat oncology patients has increased. One effective way to obtain recombinant proteins is to use the E. coli expression system. However, in eukaryotic protein production in E. coli, severaldifficulties arise that can be solved by fusing the target protein with thioredoxin. Thioredoxin can enhance solubility, but its large size can lead to an erroneous assessment of protein solubility, folding, and activity. The present study examined the ligand-binding activity of PD-L1, and CTLA-4 receptors
fused with thioredoxin.

The de novo synthesized genes of the extracellular domains of the PD-L1 and CTLA-4 were cloned into the pET28 and pET32 expression plasmids and used to transform E. coli BL21 cells. Purified recombinant proteins were characterized by western blotting, LC-MS/MS spectrometry, and
ELISA.

Amino acid sequence comparisons of the recombinant proteins obtained by LC-MS/MS with the SwissProt database resulted in the highest comparison scores from 4950 to 13396. The binding efficiencies of recombinant human B7-1 Fc to rCTLA-4 and rTrx-CTLA-4 proteins in ELISA did not
differ significantly. Similar results were obtained with recombinant rhesus monkey PD-1 hFc against rPD-L1 and rTrx-PD-L1.

Recombinant proteins specifically reacted with recombinant human B7-1 Fc and recombinant rhesus monkey PD-1 hFc. The fusion of thioredoxin with recombinant proteins through linkers slightly affected the activity of the extracellular domains of CTLA-4 and PD-L1.

Avian infectious bronchitis (IB) is an economically important, highly contagious, acute, upper-respiratory-tract disease of chickens and other bird species, caused by the avian coronavirus infectious bronchitis virus (IBV). The aim of this study was the expression of the S1 gene protein genotype variant-2 in pET28a. In vitro protein expression is an important method for obtaining many viral proteins to investigate their protective and biological properties. Since the S1 glycoprotein of the infectious bronchitis virus induces virus-neutralizing antibodies, it is an appropriate candidate for producing a recombinant vaccine against infectious bronchitis disease.

In this study, the S1 gene fragment of infectious bronchitis virus genotype IS/1494/06 was amplified using Reverse Transcriptase-Polymerase Chain Reaction (RT_PCR) and was inserted into the pTG19-T vector. The construct was subcloned into pET28a and inserted into E.coli BL21 competent cells.

The insertion was proved by PCR analysis, and isolation of the S1 gene from the construct was carried out. Finally, it was sequenced. The SDS-PAGE assay showed that a protein about 60 kDa was expressed, but the western blot assay did not confirm the expression of the S1 protein.

S1 protein did not express well. To obtain properly expressed proteins, we suggest trying different expression vectors or choosing smaller important portions of the S1 gene.

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.

 Overexpression of programmed cell death ligand 1 (PD-L1) in tumor cells and subsequent interaction with the programmed cell death protein 1 (PD-1) in tumor-infiltrating T cells cause an immune evasion of the tumor from cytotoxic T-cells. Therefore, inhibiting such interaction by a recombinant PD-1 can hinder tumor growth and extend the survival rate.

 The mouse extracellular domain of PD-1 (mPD-1) was expressed in BL21 (DE3) E. coli strain and purified using nickel affinity chromatography. The binding ability of the purified protein to human PD-L1 was studied using ELISA. Finally, the tumor-bearing mice were used to evaluate the potential antitumor effect.

 The recombinant mPD-1 showed a significant binding capacity to human PD-L1 at the molecular level. The tumor size significantly decreased in the tumor-bearing mice after the intra-tumoral injections of mPD-1. Moreover, the survival rate increased significantly after eight weeks of monitoring. The histopathology revealed the necrosis in the tumor tissue of the control group compared to the mPD-1 received mice.

 Our outcomes propose that interaction blockade between PD-1 and PD-L1 is a promising approach for targeted tumor therapy.

Although, conventional methods for the expression of polyomaviruses and herpesviruses recombinant proteins for serological  assays  and  vaccine  developments  in baculoviruses are well established, the manipulations are laborious and time consuming.

A new expression system based on plasmid was used to express two polyomaviruses major capsid protein VP1 (JCV VP1 and BKV VP1), and two herpesviruses glycoproteins (HSV-1 gD and VZV gE) in insect cells. A ligation independent cloning (LIC) was applied to generate the recombinant plasmids. Transfection of Sf9 insect cells were performed using the recombinants. The produced proteins were analysed using SDS-PAGE, immunofluorescence, and immunoblotting.

JCV-VP1, BKV-VP1, VZV-gE and HSV-1gD were successfully expressed in the insect cells, 48 h post-infection and detected in cytoplasm and cell membranes with immunoreactivity. This plasmid based expression system took 5 days to express the protein.

The plasmid based expression system in insect cells was highly efficient and would be ideal for rapid expression of polyomaviruses and herpesviruses proteins in insect cells to be potentially used in applications such as vaccine components and serological assays.

Shigellosis is a form of acute intestinal infection and one of the global health problems that occur in human by pathogenic species of Shigella. Producing a cost-effective and protective vaccine against the pathogenic strains of these bacteria will have a significant effect on the improvement of public health. The purpose of this research was to design, express and purify a multiepitope protein as a candidate vaccine against Shigella pathogenic species.

The multi-epitope protein-encoding Ipas, Omps and IcsA genes was designed based on previous bioinformatics assessments and synthesized in pET28a (+) expression vector. Since no detectable expression was observed, the gene was subcloned into pET32a (+). The pET32a (+) recombinant vector containing the desired gene was transferred into Escherichia coli BL21 (DE3) and the expression of the recombinant protein was induced using IPTG. The protein was purified using a nickel column. Finally, the Western blotting method was used to confirm the expression of the recombinant protein.

The sub-cloning of the gene was confirmed using PCR reaction. Gene expression analysis showed that the desired protein had a suitable expression. Western blotting analysis confirmed the expression of the recombinant protein.

The expressed and purified multi-epitope recombinant protein, containing the main epitopes of the common antigens of pathogenic Shigella species could be achieved as the first step to design a multiepitope vaccine candidate against shigellosis.