In silico design of a trivalent multi-epitope global-coverage vaccine-candidate protein against influenza viruses: evaluation by molecular dynamics and immune system simulation.

Hemagglutinin (HA), a variable viral surface protein, is essential for influenza vaccine development. Annually, traditional trivalent vaccines containing influenza A/H1N1, A/H3N2 and B viruses are administered globally, which are not very effective for the mutations in HA protein. The aim of this study was to design a multi-epitope vaccine containing epitopes of the HA protein of H1N1, H3N2 and B viruses using immunoinformatics methods. The HA protein epitope prediction was performed using Immune Epitope Database. Toxicity, antigenicity and conservancy of the epitopes were evaluated using ToxinPred, VaxiJen and Epitope Conservancy Analysis tools, respectively. Then, nontoxic, antigenic and high conserved epitopes with high prediction scores were selected. Their binding affinity was evaluated against human and mouse MHC class I and II molecules using the HPEPDOCK tool. Physicochemical properties and post-translational modifications were evaluated using ProtParam, SOLpro and MusiteDeep tools, respectively. Top selected epitopes were joined using linkers to produce the best effective recombinant trivalent vaccine candidate to elicit cellular and humoral immune responses in mouse and human host models. These sequences were modeled and verified. By evaluating the results of various analyses of all models and the most similarity to the native HA protein, model 5 was selected as the best model. Finally, in silico cloning of this model as vaccine candidate was performed in pET21. This study was a computer-aided analysis for a multi-epitope trivalent recombinant vaccine candidate against influenza viruses. The efficiency of our best model of vaccine candidates should be validated using in vitro and in vivo studies.Communicated by Ramaswamy H. Sarma.

Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis.

INTRODUCTION: L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes. METHOD: In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties. RESULT: Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase's asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme. CONCLUSION: In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.

Computational peptide engineering approach for selection of the new C05 antibody-driven peptide with potency to blocking influenza a virus attachment; from in silico to in vivo.

Antiviral drugs are currently used to prevent or treat viral infections like influenza A Virus (IAV). Nonetheless, annual genetic mutations of influenza viruses make them resistant to efficient treatment by current medications. Antiviral peptides have recently attracted researchers' attention and can potentially supplant the current medications. This study aimed to design peptides against IAV propagation. For this purpose, P2 and P3 peptides were computationally designed based on the HCDR3 region of the C05 antibody (a monoclonal antibody that neutralizes influenza HA protein and inhibits the virus attachment). The synthesized peptides were tested against the influenza A virus (A/Puerto Rico/8/34 (H1N1)) in vitro, and the most efficient peptide was selected for in vivo experiments. It was shown that the designed peptide shows much more prophylactic and therapeutic effects against the virus. These findings demonstrated that the designed peptide can control the virus infection without any cytotoxicity effect. Antiviral peptide design is acknowledged as a critical tactic to manage viral infections by preventing viral binding to the host cells.Communicated by Ramaswamy H. Sarma.

Crucial role of non-hydrophobic residues in H-region signal peptide on secretory production of l-asparaginase II in Escherichia coli.

Protein secretion into the periplasmic space requires several interactions between the amino-terminal signal sequence of the protein and secretion machinery components. Therefore, modification of the components of amino-terminal sequence can be used as a powerful strategy to improve secretion efficiency. The hydrophobic region is an important domain for signal peptide function due to interaction with different components of the secretion apparatus. In this study, to evaluate the effect of hydrophobicity level and secondary structure of the h-domain signal peptide on the secretion efficiency, a series of missense mutations were constructed in the hydrophobic domain of the l-asparaginase II signal peptide. The h-region hydrophobicity level of mutants G8L, T16L, and G8L/T16L was increased compared with the wild-type. In addition, the amino acid glycine as a helix-breaker residue was substituted with leucine (G8L), forming a stable and extended α-helix structure in the h-domain. The effect of introducing an aromatic residue in this region was also investigated by mutant G8F. Our mutagenesis studies showed that increasing the hydrophobicity levels, extending the α-helical conformation, and the introduction of an aromatic residue within the h-region signal sequence reduced the secretion level of asparaginase. These results imply a vital role of non-hydrophobic residues in the H-region.

Optimization of IL-1RA structure to achieve a smaller protein with a higher affinity to its receptor.

Interleukine-1 family cytokines are key orchestrators of innate and adaptive immunity. In particular, up-regulation of IL-1R1 via its agonistic ligands consisting of IL-1ß and IL-1α is implicated in a variety of human diseases, such as rheumatoid arthritis, psoriasis, type I diabetes, amyotrophic lateral sclerosis, and dry-eye disease. Until now, there are no small-molecule inhibitors of the IL-1R1 with increased antagonistic potency to be used for the treatment of peripheral inflammation. The objective of this study was to engineer a low-molecular-weight version of IL-1RA with increased affinity and enhanced antagonistic activity for potential therapeutic use. To develop a smaller protein-ligand with a better affinity to IL-1R, we used bioinformatics studies and in silico simulations to anticipate non-binding areas on IL-1RA. In this study, we have identified a 41aa (F57-F98) non-binding site of IL-1RA. Overall RMSF of the Truncated complex (1.5 nm) was lower than the Native complex (2 nm), which could prove higher stability of the Truncated complex. The free binding energy of the T-IL-1RA (- 1087.037 kJ/mol) was significantly lower than the IL-1RA (- 836.819 kJ/mol) which could demonstrate a higher binding affinity of the truncated ligand with its receptor as a result of new important interactions. These findings have demonstrated a higher binding affinity of the T-IL-1RA with its receptor than the native protein. These results should: have an impact on the development of new treatments that block IL-1 signaling, although more research is needed in vitro and in vivo.

TNFα priming through its interaction with TNFR2 enhances endothelial progenitor cell immunosuppressive effect: new hope for their widespread clinical application.

BACKGROUND: Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. METHODS: EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. RESULTS: Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. CONCLUSIONS: We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs' longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration. Video Abstract.

Simulation-based protein engineering of R. erythropolis FMN oxidoreductase (DszD).

The sulfur contents of fossil fuels have negative impacts on the environment and human health. The bio-catalytic desulfurization strategies and the biological refinement of fossil fuels are a cost-effective process compared to classical chemistry desulfurization. Rhodococcus erythropolis IGTS8 is able to metabolize the organic sulfur compound by the unique genes cluster (i.e. DszA, B, C and D genes) in the 4S metabolic pathway. The dszD gene codes a key enzyme for sulfur reduction in the gene cluster. In this study, the structure of the DszD enzyme was predicted and then the key residues toward FMN binding were identified which were Thr62, Ser63, Asn77, and Ala79. To investigate the effect of manipulation in key residues on the enzymatic activity of the DszD, different mutations were performed on key residues. The molecular docking simulation showed that A79I and A79N mutants have the lowest binding free energies compared to the wild-type enzyme in binding with FMN substrate. A 50 ns molecular dynamics (MD) simulation performed using GROMACS software. The RMSD and RMSF analysis showed that two mutants are more stable than the wild-type enzyme during MD simulation. The binding free energies between FMN substrate and complexes were calculated and analyzed by the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) method. The experimental results showed that the enzyme activity for the oxidoreductase process toward biodesulfurization increased 1.9 and 2.3 fold for A79I and A79N mutants, respectively.

Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide.

Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl ß-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.

BACKGROUND: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. OBJECTIVES: We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. MATERIALS AND METHODS: Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. RESULTS: In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. CONCLUSIONS: By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.

Cloning and expression of alkane hydroxylase-1 from Alcanivorax borkumensis in Escherichia coli.

Enzymes with hydroxylating activity on alkanes have potential application as biotransformation catalysts in chemical and pharmaceutical industry. Genome of Alcanivorax borkumensis, a marine bacterium with hydrocarbon dissimilation activity, contains at least two P450 monooxygenases and two nonheme monooxygenases, AlkB1 and AlkB2, respectively. Presumably, all these enzymes possess alkane hydroxylating activity. Both AlkB1 and AlkB2 are membrane proteins. Two accessory proteins, rubredoxin and rubredoxin reductase, supply the reducing equivalent from nicotinamide adenine dinucleotide phosphate reduced (NADPH to hydroxylases. Rubredoxin reductase catalyses the reduction of rubredoxin by oxidation of NADPH, and rubredoxin transfers the electrons to the alkane hydroxylase to complete the hydroxylation reaction. Here, we sought to investigate the expression of alkB1 gene in Escherichia coli. Therefore, we amplified alkB1 gene from A. borkumensis genome by polymerase chain reaction and cloned it in the expression vector pET26 upstream of His-tag sequence. Predisposed BL21 (DE3) cells were transformed by the recombinant vector. At last, expression of recombinant enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Regarding the potential ability of this enzyme in hydroxylation of long-chained alkanes, the application of it would be studied in petroleum downstream industries.

Rubredoxin reductase from Alcanivorax borkumensis: expression and characterization.

Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems.

Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of Rhodococcus erythropolis.

Microbial desulfurization is potentially an alternative process to chemical desulfurization of fossil fuels and their refined products. The dibenzothiophene desulfurizing system of Rhodococcus erythropolis includes DszD which is an NADH-dependent FMN oxidoreductase with 192 residues that is responsible for supplying reducing equivalents in the form of FMNH(2) to monooxygenases, DszA and DszC. We performed amino acid sequence comparisons and structural predictions based on the crystal structure of available pdb files for three flavin reductases PheA2, HpaC(Tt) and HpaC(St) with the closest structural homology to IGTS8 DszD. The Thr62 residue in DszD was substituted with Asn and Ala by site-directed single amino acid mutagenesis. Variants T62N and T62A showed 5 and 7 fold increase in activities based on the recombinant wild type DszD, respectively. This study revealed the critical role of position 62 in enzyme activity. These results represent the first experimental report on flavin reductase mutation in R. erythropolis and will pave the way for further optimization of the biodesulfurization process.

Production of a recombinant alkane hydroxylase (AlkB2) from Alcanivorax borkumensis.

Alcanivorax borkumensis is an oil-degrading marine bacterium. Its genome contains genes coding for three cytochrome P450s and two integral membrane alkane hydroxylases (AlkB1 & AlkB2), all assumed to perform hydroxylation of different linear or branched alkanes. Although, the sequence of alkB2 has been determined, the molecular characterization and the substrate specificity of AlkB2 require more precise investigation. In this study, AlkB2 from A. borkumensis SK2 was expressed in Escherichia coli to examine the functionality of AlkB2 as a hydroxylating enzyme. Furthermore, the activity of the enzyme in the presence of the accessory proteins, rubredoxin (RubA) and rubredoxin reductase (RubB), produced in E. coli BL21(DE3)plysS cells, was determined. Recombinant AlkB2 is produced in an active form and rubredoxin is the intermediate electron donor to AlkB2 and can replace AlkG function, when NADH is the prime electron donor.

Cloning and expression of simian rotavirus spike protein (VP4) in insect cells by baculovirus expression system.

BACKGROUND: VP4 protein is as spikes on rotavirus outer capsid shell which is responsible for virus attachment to the host. VP4 induces production of neutralizing antibodies which could be used for serotyping of different isolates. METHODS: Simian rotavirus SA11 gene 4 cDNA was cloned into a cloning plasmid pDONRTM by recombination reaction using clonase II enzyme mix. The resulting clone was called VP4-entry clone. In the second recombination reaction, cloned gene was inserted into the linear DNA of the Baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) downstream of the strong polyhedrin promoter. The recombinant AcNPV-VP4 DNA was transfected by lipofection into the insect cell line, Spodoptera frugiperda (Sf9) cells. Expression of VP4 in the Sf9 cells was confirmed by the immunofluorescence test using rabbit polyclonal anti-rotavirus and anti-rabbit FTIC-conjugated antibodies by Western immuno-blotting technique. The antigenicity of the expressed protein was determined by immunizing rabbits and testing the sera by Western-blotting and neutralization method. RESULTS: The cloned VP4 gene was obtained and expressed in baculovirus system. The specificity of the expressed protein was confirmed by its reactivity with anti-rotavirus antibody. Antibody produced against the expressed protein showed neutralizing activity for rotavirus indicating that the protein was biologically active and could induce natural antibody response. CONCLUSION: The expressed protein from rotavirus VP4 gene has a potential for development of rotavirus vaccine.

Characterization of the heme environment in Arabidopsis thaliana fatty acid alpha-dioxygenase-1.

Plant alpha-dioxygenases (PADOX) are hemoproteins in the myeloperoxidase family. We have used a variety of spectroscopic, mutagenic, and kinetic approaches to characterize the heme environment in Arabidopsis thaliana PADOX-1. Recombinant PADOX-1 purified to homogeneity contained 1 mol of heme bound tightly but noncovalently per protein monomer. Electronic absorbance, electron paramagnetic resonance, and magnetic circular dichroism spectra showed a high spin ferric heme that could be reduced to the ferrous state by dithionite. Cyanide bound relatively weakly in the ferric PADOX-1 heme vicinity (K(d) approximately 10 mm) but did not shift the heme to the low spin state. Cyanide was a very strong inhibitor of the fatty acid oxygenase activity (K(i) approximately 5 microm) and increased the K(m) value for oxygen but not that for fatty acid. Spectroscopic analyses indicated that carbon monoxide, azide, imidazole, and a variety of substituted imidazoles did not bind appreciably in the ferric PADOX-1 heme vicinity. Substitution of His-163 and His-389 with cysteine, glutamine, tyrosine, or methionine resulted in variable degrees of perturbation of the heme absorbance spectrum and oxygenase activity, consistent with His-389 serving as the proximal heme ligand and indicating that the heme has a functional role in catalysis. Overall, A. thaliana PADOX-1 resembles a b-type cytochrome, although with much more restricted access to the distal face of the heme than seen in most other myeloperoxidase family members, explaining the previously puzzling lack of peroxidase activity in the plant protein. PADOX-1 is unusual in that it has a high affinity, inhibitory cyanide-binding site distinct from the distal heme face and the fatty acid site.

Role of Asn-382 and Thr-383 in activation and inactivation of human prostaglandin H synthase cyclooxygenase catalysis.

Cyclooxygenase catalysis by prostaglandin H synthase-1 and -2 (PGHS-1 and -2) requires activation of the normally latent enzyme by peroxide-dependent generation of a free radical at Tyr-385 (PGHS-1 numbering) in the cyclooxygenase active site; the Tyr-385 radical has also been linked to self-inactivation processes that impose an ultimate limit on cyclooxygenase catalysis. Cyclooxygenase activation is more resistant to suppression by cytosolic glutathione peroxidase in PGHS-2 than in PGHS-1. This differential response to peroxide scavenging enzymes provides a basis for the differential catalytic regulation of the two PGHS isoforms observed in vivo. We sought to identify structural differences between the isoforms, which could account for the differential cyclooxygenase activation, and used site-directed mutagenesis of recombinant human PGHS-2 to focus on one heme-vicinity residue that diverges between the two isoforms, Thr-383, and an adjacent residue that is conserved between the isoforms, Asn-382. Substitutions of Thr-383 (histidine in most PGHS-1) with histidine or aspartate decreased cyclooxygenase activation efficiency by about 40%, with little effect on cyclooxygenase specific activity or self-inactivation. Substitutions of Asn-382 with alanine, aspartate, or leucine had little effect on the cyclooxygenase specific activity or activation efficiency but almost doubled the cyclooxygenase catalytic output before self-inactivation. Asn-382 and Thr-383 mutations did not appreciably alter the Km value for arachidonate, the cyclooxygenase product profile, or the Tyr-385 radical spectroscopic characteristics, confirming the structural integrity of the cyclooxygenase site. The side chain structures of Asn-382 and Thr-383 in PGHS-2 thus selectively influence two important aspects of cyclooxygenase catalytic regulation: activation by peroxide and self-inactivation.