Computational analysis of collagenase from different Vibrio, Clostridium and Bacillus strains to find new enzyme sources

Collagenase is one the important enzyme, which is applied in varied fields ranging from tannery, food and cosmetic industries to clinical therapies. Currently, the commercially available collagenase enzyme has been produced by Clostridium histolyticum bacteria. In our study, in order to find new sources of collagenase producer, 30 collagenases from different species of Clostridium, Vibrio and Bacillus were evaluated from the view of phylogenetic relation, domain architecture and Physiochemical features. Totally our results indicate that the non-pathogenic C. novyi (NT) with the aliphatic index (80.68), instability index (27), pI (6.54), Mw (112.838 kDa) and two PPC domain could be suggested as potent bacteria for industrial production of collagenase

Computational analysis of collagenase from different Vibrio, Clostridium and Bacillus strains to find new enzyme sources

Collagenase is one the important enzyme, which is applied in varied fields ranging from tannery, food and cosmetic industries to clinical therapies. Currently, the commercially available collagenase enzyme has been produced by Clostridium histolyticum bacteria. In our study, in order to find new sources of collagenase producer, 30 collagenases from different species of Clostridium, Vibrio and Bacillus were evaluated from the view of phylogenetic relation, domain architecture and physiochemical features. Totally our results indicate that the non-pathogenic C. novyi (NT) with the aliphatic index (80.68), instability index (27), pI (6.54), Mw (112.838 kDa) and two PPC domain could be suggested as a potent bacteria for industrial production of collagenase

In-silico selection of appropriate signal peptides for romiplostim secretory production in Escherichia coli

Background: Thrombocytopenia is a prevalent condition caused by a reduction in platelet production, increased platelet depletion, or a combination of both processes. Romiplostim, the most potent drug and the only thrombopoietin (TPO) receptor agonist, consists of four TPO receptor–activating peptides fused as a fusion protein to the human IgG1 heavy chain segment. Romiplostim could be produced by recombinant DNA technology in Escherichia coli. Extracellular and periplasmic secretion using signal peptides (SPs) of the recombinant proteins could have several advantages in purification and protein recovery. SPs are short amino-acid sequences used in protein translocation through various membranes. The structure of the different SPs identified in various bacteria and their compatibility with the protein could affect the transfer success and efficacy. Objective: This study aimed to identify the best SP for extracellular secretion of romiplostim to facilitate its purification and reduce production costs in E. coli. Methods: The amino acid sequences of the SPs and the position of their cleavage sites in the proteins were evaluated using the Signal 5.0 server. The solubility of the proteins produced in E. coli was predicted by protein-sol. The physicochemical parameters of the SPs were analyzed by ProtParam using the ExPASy server. Results/Conclusion: We found that SfmC is the most suitable candidate, followed by TorT and bla. However, this result should be confirmed by further experimental assessment. Furthermore, for the successful secretion of a recombinant protein, the optimal growth conditions should be controlled in addition to a suitable SP

Designing an HCV diagnostic kit for common genotypes of the virus in Iran based on conserved regions of core, NS3-protease, NS4A/B, and NS5A/B antigens: an in silico approach

Hepatitis C virus (HCV) is a life-threatening virus that causes liver infection. If it is not detected in an early phase, the virus can lead to severe liver damages, including hepatic fibrosis, liver cirrhosis, and hepatocellular carcinoma. Today, computational design of the HCV diagnostic kit is employed to increase the specificity and sensitivity of the ELISA (enzyme-linked immunosorbent assay) diagnosis method according to the specific genotypes of the virus in each geographical region as well as to reduce costs in developing and low-income countries. The aim of this study was to design a multi-epitope protein from common HCV genotypes in Iran (1a, 1b, and 3a). For this purpose, potential immunodominant epitopes and highly antigenic regions were identified for six antigenic proteins and all of the segments were joined using a proper linker. The physico-chemical characteristics of the designed multi-epitope protein were evaluated and tertiary structures of the construct were modeled. Then, the models were evaluated and the best one was determined. Finally, the sequence of the protein was reverse-translated and optimized for high expression in E. coli expression host. The findings of the present study indicated that the designed construct could detect the common HCV genotypes in Iran with high sensitivity and specificity

Production of a novel multi-epitope vaccine based on outer membrane proteins of Klebsiella pneumoniae

Klebsiella pneumoniae is a hospital-acquired pathogen that leads to various infections. Hence, efforts to develop an effective vaccine against that pathogen are well documented. Our interest is the production of the previously designed multi-epitope vaccine construct against the K. pneumoniae in a prokaryotic host. Therefore, a new construct containing the nucleotide sequence of the novel vaccine was successfully expressed in Escherichia coli and then purified by Ni-NTA spin column. The purified recombinant protein can be considered as potential vaccine candidate for wet-laboratory analysis aiming to fight K pneumoniae

In Silico Analysis of Glutaminase from Different Species of Escherichia and Bacillus

Background: Glutaminase (EC 3.5.1.2) catalyzes the hydrolytic degradation of L-glutamine to L-glutamic acid and has been introduced for cancer therapy in recent years. The present study was an in silico analysis of glutaminase to further elucidate its structure and physicochemical properties. Methods: Forty glutaminase protein sequences from different species of Escherichia and Bacillus obtained from the UniProt Protein Database were characterized for homology search, physiochemical properties, phylogenetic tree construction, motif, superfamily search, and multiple sequence alignment. Results: The sequence level homology was obtained among different groups of glutaminase enzymes, which belonged to superfamily serine-dependent β-lactamases and penicillin-binding proteins. The phylogenetic tree constructed indicated 2 main clusters for the glutaminases. The distribution of common β-lactamase motifs was also observed; however, various non-common motifs were also observed. Conclusion: Our results showed that the existence of a conserved motif with a signature amino-acid sequence of β-lactamases could be considered for the genetic engineering of glutaminases in view of their potential application in cancer therapy. Nonetheless, further research is needed to improve the stability of glutaminases and decrease their immunogenicity in both medical and food industrial applications

Efficacy of co-immunization with the DNA and peptide vaccines containing SYCP1 and ACRBP epitopes in a murine triple-negative breast cancer model

Multiepitope cancer vaccines have gained lots of attention for prophylactic and therapeutic purposes in cancer patients. In our previous study, multiepitope DNA and peptide cancer vaccines consisted of the most immunodominant epitopes of ACRBP and SYCP1 antigens were designed by bioinformatic tools. In this study, the effect of prophylactic co-immunization with these DNA and peptide cancer vaccines in the 4T1 breast cancer animal model was assessed. Serum levels of the peptide-specific IgG total, IgG2a and IgG1 were measured by enzyme-linked immunosorbent assay (ELISA). Also, the efficacy of the immunized mice splenocytes’ for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) was evaluated. The co-immunization caused a significant (P < .05) increase in the serum levels of IgG1 and IgG2a. The co-immunized mice splenocytes exhibited significantly enhanced IL-4 (6.6-fold) and IFN-γ (19-fold) production. Also, their lymphocytes exhibited higher proliferation rate (3-fold) and granzyme B production (6.5-fold) in comparison with the control. The prophylactic co-immunization significantly decreased the breast tumors’ volume (78%) and increased the tumor-bearing mice survival time (37.5%) in comparison with the control. Taking together, prophylactic co-immunization with these multiepitope DNA and peptide cancer vaccines can activate the immune system against breast cancer. However, further experiments are needed to evaluate their efficacy from different angles

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

BackgroundThe sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis.Methodology/principal findingsThe coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively.Conclusions/significanceThe preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required

Deep analysis of N-cadherin/ADH-1 interaction: a computational survey

<p>Due to the considerable role of N-cadherin in cancer metastasis, tumor growth, and progression, inhibition of this protein has been highly regarded in recent years. Although ADH-1 has been known as an appropriate inhibitor of N-cadherin in clinical trials, its chemical nature and binding mode with N-cadherin have not been precisely specified yet. Accordingly, in this study, quantum mechanics calculations were used to investigate the chemical nature of ADH-1. These calculations clarify the molecular properties of ADH-1 and determine its reactive sites. Based on the results, the oxygen atoms are suitable for electrophilic reactivity, while the hydrogen atoms that are connected to nitrogen atoms are the favorite sites for nucleophilic reactivity. The higher electronegativity of the oxygen atoms makes them the most reactive portions in this molecule. Molecular docking and molecular dynamics (MD) simulation have also been applied to specify the binding mode of ADH-1 with N-cadherin and determine the important residues of N-cadherin involving in the interaction with ADH-1. Moreover, the verified model by MD simulation has been studied to extract the free energy value and find driving forces. These calculations and molecular electrostatic potential map of ADH-1 indicated that hydrophobic and electrostatic interactions are almost equally involved in the implantation of ADH-1 in the N-cadherin binding site. The presented results not only enable a closer examination of N-cadherin in complex with ADH-1 molecule, but also are very beneficial in designing new inhibitors for N-cadherin and can help to save time and cost in this field.</p

Designing a Humanized Immunotoxin Based on HER2 Specific scFv and DFF40 Toxin Against Breast Cancer: An In-Silico Study

Breast cancer is the most frequent cancer in women worldwide. Human epidermal growth factor receptor 2 (HER2) is a receptor which is overexpressed in breast cancer cells. Targeting this receptor could be a key factor for treatment of breast cancer patients. Herceptin is an antibody which can bind to HER2 receptor, in addition Herceptin derived single chain fragment V (scFv) can be used in designing immunotoxin for targeting HER2 positive cancer cells. DFF40 is a nuclease activated by caspase-3 and responsible for genomic DNA fragmentation during apoptosis. In this study, we used bioinformatics tools to design an immunotoxin containing HER2 specific scFv and DFF40 toxin. An immunotoxin construct was designed by linking scFv and DFF40 amino acids sequence via a peptide linker. The secondary structure, physicochemical features, solubility, and allergenicity of construct were predicted. The tertiary structure was built, refined, and evaluated. Protein-protein docking, and molecular dynamics studies were carried out for evaluation of immunotoxin-receptor binding, and the stability of the immunotoxin, respectively. The results indicated that the designed construct could be a stable protein with appropriate solubility, which is not an allergen and has a suitable structure which can bind to HER2 appropriately. Finally, this construct could be a promising candidate for producing an HER2 targeting immunotoxin. However, different in vitro and in vivo immunological assays should be performed to confirm the efficacy of the designed construct