Investigating the effect of cGRP78 vaccine against different cancer cells and its role in reducing melanoma metastasis.

Background and purpose: Treatment of malignancies with chemotherapy and surgery is often associated with disease recurrence and metastasis. Immunotherapy improves cancer treatment by creating an active response against tumor antigens. Various cancer cells express a large amount of glucose-regulated protein 78 (GRP78) protein on their surface. Stimulating the immune system against this antigen can expose cancer cells to the immune system. Herein, we investigated the effectiveness of a cGRP78-based vaccine against different cancer cells. Experimental approach: BALB/c mice were immunized with the cGRP78. The humoral immune response against different cancer cells was assessed by Cell-ELISA. The cellular immunity response was determined by splenocyte proliferation assay with different cancer antigens. The effect of vaccination on metastasis was investigated in vaccinated mice by injecting melanoma cancer cells into the tail of mice. Findings/Results: These results indicated that the cGRP78 has acceptable antigenicity and stimulates the immune system to produce antibodies. After three injections, the amount of produced antibody was significantly different from the control group. Compared to the other three cell types, Hela and HepG2 showed the highest reaction to the serum of vaccinated mice. Cellular immunity against the B16F10 cell line had the best results compared to other cells. The metastasis results showed that after 30 days, the growth of B16F10 melanoma cancer cells was not noticeable in the lung tissue of vaccinated mice. Conclusion and implications: Considering the resistance of vaccinated mice to metastasis, this vaccine offers a promising prospect for cancer treatment by inhibiting the spread of cancer cells.

Introduction of a new recombinant vaccine based on GRP78 for breast cancer immunotherapy and evaluation in a mouse model.

Introduction: Breast cancer is one of the most prevalent malignancies in women. Several treatment options are available today, including surgery, chemotherapy, and radiotherapy. Immunotherapy, as a highly specific therapy, involves adaptive immune responses and immunological memory. In our present research, we used the recombinant C-terminal domain of the GRP78 (glucose- regulated protein 78) protein to induce an immune response and investigate its therapeutic impact in the 4T1 breast cancer model. Methods: BALB/c mice were immunized with the cGRP78 protein. The humoral immune response was assessed by ELISA. Then, BALB/c mice were injected subcutaneously with 1×106 4T1 tumor cells. Subsequently, tumor size and survival rate measurements, MTT, and cytokine assays were performed. Results: The animals receiving the cGRP78 vaccine showed significantly more favorable survival and slower tumor growth rates compared with unvaccinated tumor-bearing mice as the negative control mice. Circulating levels of tumoricidal cytokines such as IFNγ were higher, whereas tolerogenic cytokines such as IL-2, 6, and 10 either did not increase or had a decreasing trend in mice receiving cGRP78. Conclusion: cGRP78 vaccines generated potent immunotherapeutic effects in a breast cancer mouse model. This novel strategy of targeting the GRP78 protein can promote the development of cancer vaccines and immunotherapies for breast cancer malignancies.

A novel approach to enhance the performance of kallikrein 6 enzyme using Pichia pastoris GS115 as a host.

Background and purpose: Enzyme engineering is the process of raising enzyme efficiency and activity by altering amino acid sequences. Kallikrein 6 (KLK6) enzyme is a secreted serine protease involved in a variety of physiological and pathological activities. The increased expression of KLK6 plays a key role in various diseases. Instability and spontaneous activation and deactivation are major challenges in the study of this enzyme. This study aimed to create a stable pro-KLK6 enzyme by enzyme engineering, designing a specific cleavage site for enterokinase, and using Pichia pastoris GS115 as a host cell. Then, recombinant pro-KLK6 was used to introduce a novel inhibitor for it. Experimental approach: An engineered pro-KLK6 gene was cloned into the pPICZα A expression vector. Then, it was expressed in P. pastoris GS115 and purified by Ni-NTA chromatography. An inactive engineered pro-KLK6 gene was cleaved by enterokinase and converted to an active KLK6. The KLK6 enzyme activity and its kinetic parameters were measured using N-benzoyl-L-arginine ethyl ester (BAEE) substrates. Findings/Results: The secretory form of the pro-KLK6 was expressed at about 11 mg/L in P. pastoris (GS115). Before activation with enterokinase, pro-KLK6 was inactive and did not activate spontaneously. The kinetic parameters, including Km and Vmax, were estimated at 113.59 µM and 0.432 µM/s, respectively. Conclusion and implications: A stable pro-KLK6 enzyme was produced using P. pastoris (GS115) as the host cell and a specific cleavage site for enterokinase. Additionally, this study assessed the kinetic parameters of the KLK6 enzyme using the BAEE substrate for the first time.

Anti-EGFR bioengineered bacterial outer membrane vesicles as targeted immunotherapy candidate in triple-negative breast tumor murine model.

Cancer immunotherapy employing checkpoint inhibitors holds great promise across diverse cancers; nonetheless, a substantial proportion of patients (ranging from 55 to 87%) remain unresponsive to this treatment. To amplify therapeutic efficiency, we propose a synergistic therapeutic strategy that entails the deployment of targeted nano-sized particles carrying Toll-like receptor (TLR) agonists to the tumor site. This innovative approach seeks to activate intratumoral antigen-presenting cells using bioengineered outer membrane vesicles (OMVs) derived from gram-negative bacteria. These OMVs possess inherent attributes of surface-exposed immune stimulators and TLR-activating components, rendering them intriguing candidates for investigation. These OMVs were meticulously designed to selectively target cancer cells exhibiting an overexpression of epidermal growth factor receptor (EGFR). To gauge the precision of this targeting, the conducted affinity-based assays aimed at determining the equilibrium dissociation constant of the single-chain variable fragment employed for this purpose. In vitro experiments confirmed the OMVs' proficiency in adhering to EGFR-overexpressed cancer cells. Moreover, the evaluation extended to an in vivo context, where the therapeutic effect of nanovesicles was appraised within the tumor microenvironment of the triple-negative breast cancer mouse model. Notably, both intraperitoneal and intratumoral administrations of nanovesicles exhibited the ability to activate natural killer cells and skew M2 macrophage towards an M1 phenotype. The combined scrutiny of in vitro and in vivo findings underscores the potential efficiency of OMVs as a promising strategy for future anti-tumor endeavors.

Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1).

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10-3 (S-1), and kcat/Km 62.77 (M-1 S-1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

Covid-19 prevention and treatment by targeting Fc-fusion proteins: An experience to fight emerging diseases.

The coronavirus disease 2019 (Covid-19) pandemic has been considered a major threat to human health. Effective therapeutic approaches are urgently required. Spike protein and the Angiotensin-converting enzyme 2 (ACE2) receptors have critical roles in SARS-CoV-2 infection. As a result, these two proteins are considered potential targets for the development of a wide variety of biotherapeutics and vaccines for controlling Covid-19. The fusion proteins have desirable medicinal properties, including high serum half-life, stability, and solubility in the body. Moreover, other Fc-fusion proteins used to treat other diseases have no known side effects. These Fc-fusion proteins are valuable biopharmaceuticals and have been proposed as therapeutic candidates for the treatment and prevention of Covid-19 owing to their potential therapeutic benefits.

Synthesis, Biological Activity Evaluation, Docking and Molecular Dynamics Studies of New Triazole-Tetrahydropyrimidinone(thione) Hybrid Scaffolds as Urease Inhibitors.

New series of triazole-tetrahydropyrimidinone(thione) hybrids (9a-g) were synthesized. FT-IR, 1 H-NMR, 13 C-NMR, elemental analysis and mass spectroscopic studies characterized the structures of the synthesized compounds. Then, the synthesized compounds were screened to determine the urease inhibitory activity. Methyl 4-(4-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9c) exhibited the highest urease inhibitory activity (IC50 =25.02 µM) among the compounds which was almost similar to thiourea as standard (IC50 =22.32 µM). The docking study of the screened compounds demonstrated that these compounds fit well in the urease active site. Based on the docking study, compound 9c with the highest urease inhibitory activity showed chelates with both Ni2+ ions of the urease active site. Moreover, the molecular dynamic study of the most potent compounds showed that they created important interactions with the active site flap residues, His322, Cys321, and Met317.

Synthesis, biological assessment, and computational investigations of nifedipine and monastrol analogues as anti-leishmanial major and anti-microbial agents.

Leishmaniasis includes a range of parasitic diseases caused by numerous types of the protozoan kinetoplastid parasite. Fungal and bacterial pathogens have led to infectious illnesses causing some main public health problem in current years. A series of dihydropyridine and tetrahydropyrimidine derivatives having fluoro, bromo, and nitro substituents at para-phenyl ring on C4 of dihydropyridine and tetrahydropyrimidine rings were synthesized. Then, anti-leishmanial and antimicrobial potencies of compounds were assessed. All compounds were synthesized via Hantzsch and Biginelli reactions. All derivatives were evaluated for their anti-leishmanial and antimicrobial activities. Moreover, docking and molecular dynamics simulation calculations of the compounds in PRT1 binding site were performed to report the results of anti-leishmanial and antimicrobial activities. Compounds 4a and 4b showed the highest anti-amastigote and anti-promastigote activities. Compound 4a revealed the highest antimicrobial activity against E. coli, P. aeruginosa, and C. albicans strains. In addition, compound 4c showed the highest activity against S. aureus. The fluoro, bromo, and nitro substituents in para-position of phenyl group at C4 of dihydropyridine and tetrahydropyrimidine moieties as well as the bulk and length of the chain linking to the ester moieties are essential for anti-leishmanial and anti-microbial activities of these derivatives. Low cytotoxicity was shown by most of derivatives against macrophages. The molecular docking studies were in agreement with in vitro assay. Moreover, hydrogen binds, RMSF, RMSD, and Rg, strongly showed the steady binding of 4a and 4b compounds in PRT1 active site.

Nanobodies; new molecular instruments with special specifications for targeting, diagnosis and treatment of triple-negative breast cancer.

Breast cancer is the most common type of cancer in women and the second leading cause of cancer death in female. Triple-negative breast cancer has a more aggressive proliferation and a poorer clinical diagnosis than other breast cancers. The most common treatments for TNBC are chemotherapy, surgical removal, and radiation therapy, which impose many side effects and costs on patients. Nanobodies have superior advantages, which makes them attractive for use in therapeutic agents and diagnostic kits. There are numerous techniques suggested by investigators for early detection of breast cancer. Nevertheless, there are fewer molecular diagnostic methods in the case of TNBC due to the lack of expression of famous breast cancer antigens in TNBC. Although conventional antibodies have a high ability to detect tumor cell markers, their large size, instability, and costly production cause a lot of problems. Since the HER-2 do not express in TNBC diagnosis, the production of nanobodies for the diagnosis and treatment of cancer cells should be performed against other antigens expressed in TNBC. In this review, nanobodies which developed against triple negative breast cancer, were classified based on type of antigen.

Design, synthesis, and in silico studies of tetrahydropyrimidine analogs as urease enzyme inhibitors.

The urease enzyme, a metalloenzyme having Ni2+ ions, is recognized in some bacteria, fungi, and plants. Particularly, it is vital to the progress of infections induced by pathogenic microbes, such as Proteus mirabilis and Helicobacter pylori. Herein, we reported the synthesis of a series of tetrahydropyrimidine derivatives and evaluated their antiurease activity. Finally, quantitative and qualitative analyses of the derivatives were performed via in silico studies. Urease inhibitory activity was determined as the reaction of H. pylori urease with different concentrations of compounds, and thiourea was used as a standard compound. Docking and dynamics methodologies were applied to study the interactions of the best compounds with the amino acids in the active site. All compounds showed good to excellent antiurease activity. The potent compounds were not cytotoxic against the HUVEC normal cell line. Based on the docking study, compound 4e with the highest urease inhibitory activity (IC50 = 6.81 ± 1.42 µM) showed chelates with both Ni2+ ions of the urease active site. Further, compound 4f displayed a very good inhibitory activity (IC50 = 8.45 ± 1.64 µM) in comparison to thiourea (IC50 = 22.03 ± 1.24 µM). The molecular docking and dynamics simulation results were correlated with the in vitro assay results. Moreover, the derivatives 4a-n followed Lipinski's rule-of-five and had drug-likeness properties.

In silico and in vitro studies of thiosemicarbazone-indole hybrid compounds as potent α-glycosidase inhibitors.

It is essential to study α-glucosidase enzyme (EC 3.2.1.20) inhibitors because of their physiological role as well as their clinical relevance. In previous research, a novel series of thiosemicarbazone-indole hybrid compounds were synthesized and reported. In the current research, α-glucosidase inhibitory activity of the derivatives was evaluated and then in silico studies were carried out on screened compounds. All derivatives exhibited a magnificent α-glucosidase inhibitory activity (IC50 = 27.0 ± 1.0-97.4 ± 1.5 µM) toward the acarbose as reference drug (IC50 = 750.0 ± 1.5 µM). Compound 1i having phenyl ring at the thiosemicarbazone moiety and the trimethoxymethyl substituent at phenyl moiety of C2 position of indole ring was the most potent compound (IC50 = 27.0 ± 1.0 µM) among other compounds. A kinetic study of 1i revealed that is a competitive inhibitor against α-glucosidase. Moreover, the molecular docking studies established that screened derivatives interacted with the essential amino acids in the active site. Finally, based on the molecular dynamics simulations and free binding energy calculations, complexes 1d, 1i and 1k with α-glucosidase showed a good stability in the active site. Van der Waals and electrostatic interactions also exhibited the most contributions to the stability of these complexes. Moreover, all the screened compounds showed agreeable ADME properties for oral bio-availability, and good drug-likeness.

Outlook of therapeutic and diagnostic competency of nanobodies against SARS-CoV-2: A systematic review.

PURPOSE: The newly emerged coronavirus (SARS-CoV-2) continues to infect humans, and no completely efficient treatment has yet been found. Antibody therapy is one way to control infection caused by COVID-19, but the use of classical antibodies has many disadvantages. Heavy chain antibodies (HCAbs) are single-domain antibodies derived from the Camelidae family. The variable part of these antibodies (Nanobodies or VHH) has interesting properties such as small size, identify criptic epitopes, stability in harsh conditions, good tissue permeability and cost-effective production causing nanobodies have become a good candidate in the treatment and diagnosis of viral infections. METHODS: Totally 157 records (up to November 10, 2021), were recognized to be reviewed in this study. 62 studies were removed after first step screening due to their deviation from inclusion criteria. The remaining 95 studies were reviewed in details. After removing articles that were not in the study area, 45 remaining studies met the inclusion criteria and were qualified to be included in the systematic review. RESULTS: In this systematic review, the application of nanobodies in the treatment and detection of COVID-19 infection was reviewed. The results of this study showed that extensive and sufficient studies have been performed in the field of production of nanobodies against SARS-CoV-2 virus and the obtained nanobodies have a great potential for use in patients infected with SARS-CoV-2 virus. CONCLUSION: According to the obtained results, it was found that nanobodies can be used effectively in the treatment and diagnosis of SARS-CoV-2 virus.

Nanobodies: a tool to open new horizons in diagnosis and treatment of prostate cancer.

BACKGROUND: Prostate cancer is one of the most common cancers in men and its incidence has increased dramatically in the last decade. This increase in the detection of this type of cancer is based more on the detection of PSA or PSMA antigens as the most important specific antigens of this cancer, and this early detection has greatly helped in the more optimal treatment of patients. MAIN BODY: Many methods have been proposed by researchers for early detection of prostate cancer, but most of the methods used today to detect this type of cancer have been using classical antibodies. Although classical antibodies are able to detect tumor cell markers, but instability, large size, costly and laborious production, and random immobility characteristics, causes many problems. Nanobodies or VHHs, which are derived from camel heavy chain antibodies, have special advantages and have eliminated the disadvantages of classical antibodies which makes them attractive to use in biosensors and cancer diagnostic kits. The research that has been done so far shows that the introduced nanobodies are created for the purpose of targeting, detecting and sensing prostate cancer cells with two main purposes. The first is the efficient identification of prostate cancer and the second is the elimination of cancer cells. CONCLUSION: Research shows the use of specific nanobodies against prostate cancer antigens in the design of biosensors and target therapy will be very interesting. In this review article, these nanobodies are introduced and categorized based on their performance.

Emerging Technologies for the Treatment of COVID-19.

The new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), turned into a pandemic affecting more than 200 countries. Due to the high rate of transmission and mortality, finding specific and effective treatment options for this infection is currently of urgent importance. Emerging technologies have created a promising platform for developing novel treatment options for various viral diseases such as the SARS-CoV-2 virus. Here, we have described potential novel therapeutic options based on the structure and pathophysiological mechanism of the SARS-CoV-2 virus, as well as the results of previous studies on similar viruses such as SARS and MERS. Many of these approaches can be used for controlling viral infection by reducing the viral damage or by increasing the potency of the host response. Owing to their high sensitivity, specificity, and reproducibility, siRNAs, aptamers, nanobodies, neutralizing antibodies, and different types of peptides can be used for interference with viral replication or for blocking internalization. Receptor agonists and interferon-inducing agents are also potential options to balance and enhance the innate immune response against SARS-CoV-2. Solid evidence on the efficacy and safety of such novel technologies is yet to be established although many well-designed clinical trials are underway to address these issues.

Isolation and characterization of a novel nanobody for detection of GRP78 expressing cancer cells.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy-chain antibodies also known as "nanobody." The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c-terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high-affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy.

Nanobodies, the potent agents to detect and treat the Coronavirus infections: A systematic review.

The newly emerged coronavirus (SARS-CoV-2) continues to infect humans, and no effective treatment has yet been found. Antibody therapy is one way to control infection caused by COVID-19. However, the use of classical antibodies raises complex issues. Heavy chain antibodies (HCAbs) are single-domain antibodies derived from the Camelidae family. The variable part of these antibodies (Nanobodies or VHH) has interesting properties such as small size, cost-effective production, and good tissue permeability, causing VHH to be regarded as an antiviral therapeutics. However, the small size of nanobodies may lead to low antigen binding affinity and rapid renal clearance. In this systematic review, the application of nanobodies in the treatment of COVID-19 infection and other similar infections (MERS and SARS) was reviewed.

Expression of recombinant G-CSF receptor domains and their inhibitory role on G-CSF function.

BACKGROUND AND PURPOSE: Granulocyte colony-stimulating factor (G-CSF) is routinely used in combination with chemotherapy to battle neutropenia. However, studies suggest that this chemokine may increase the risk of metastasis and malignancy in many cancers. To counteract the adverse effects of G-CSF in cancer, antibodies have been used to block its action. However, antibodies are large and complex molecules which makes their production expensive. Thus in this study, we aim to construct different structure variants of the G-CSF receptor containing different domains and select the best variant that prevents the adverse actions of this chemokine. These novel structures are smaller than antibodies and easier to produce. EXPERIMENTAL APPROACH: Different domains of the G-CSF receptor were designed and cloned into the pET28a expression vector. These recombinant receptor subunits were then expressed in Escherichia coli and purified using standard affinity chromatography techniques. Interaction of recombinant receptor subunits with G-CSF was assessed using enzyme-linked immunosorbent assay and NFS60 cells. FINDINGS / RESULTS: Two recombinant receptor subunits containing D1 + D2 + D3 domains and D2 domain showed the strongest inhibitory activity to G-CSF. CONCLUSION AND IMPLICATIONS: These novel recombinant receptor variants could be candidates for further studies in the development of novel therapeutics.

Engineering an anti-granulocyte colony stimulating factor receptor nanobody for improved affinity.

AIMS: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. MAIN METHODS: We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SIGNIFICANCE: Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.

Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.

Structure prediction, expression, and antigenicity of c-terminal of GRP78.

Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient deprivation in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-ß-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches.