Expasy ProtParam Research Articles

Homology modeling and insilico approach of cleome gynandra - an indigenous medicinal plant

Cleome gynandra is a widespread medicinal plant belonging to the family Capparaceae. In Ayurvedic medicine C. gynandra is a main component in Narayana Churna. It has numerous properties like Anthelmintic, in ear diseases, pruritis and several other diseases like gastro intestinal disorders and gastrointestinal infections etc. This is an effort to gather and document evidence on different features of C. gynnadra and highlight the need for survey and development. In this current study, nine proteins of C. gynandra were identify by using of bioinformatics tools. The bioinformatic study of the characterization of proteins of C.gynandra were using Expasy Protparam server, 3D structure was done using SWISS MODEL. Plants ofdifferent family show uniqueness 98% and above were particular and its sequences retrieved, aligned using Clustal Omega. Secondary Structure prediction exhibited that α – helix, random coil, β – turn and long strand leads. Phylogenetic analysis of Glyceraldehyde 3 PO4 of C. gynandra exposes that the Capparaceae families are closely related. Insilco sequence analysis of C. gynandra showed that these proteins taken from different organisms linked organized evolutionarily as they possess conserved regions in their protein sequences.These results will be helpful to further study on C. gynandra protein functions at molecular or structural levels and also valuable in homology modelling and insilico approach.

Delineating the Immuno-Dominant Antigenic Vaccine Peptides Against gacS-Sensor Kinase in Acinetobacter baumannii: An in silico Investigational Approach

ObjectivesTo predict the novel vaccine peptide candidates against gacS protein involved with the citrate utilization in the two-component system of A. baumannii-associated virulence as an alternative strategy to combat the multi-drug resistant strains using an immuno-informatic approach.MethodsThe study is designed as an observational in silico study design with the application of BepiPred, AlgPred, VaxiJen, AntigenPro, SolPro, Expasy ProtParam server, IEDB database, and MHC cluster analytical tools and servers to predict the immuno-dominant B-cell and T-cell epitopes from gacS FASTA sequences retrieved from UNIPROT database. Further peptide interactions with TLR-4 was assessed based on the number of hydrogen bonds.ResultsNine peptides (20aa) with the highest score of 1 were selected from the 137 epitopes, and five were predicted as antigenic epitopes (E1–E5). E3 was selected as the potent antigen (score: 0.939537) and E1 as the best vaccine candidate (score: 0.9803) under AntigenPro and Vaxijen server, respectively. SolPro predicted all epitopes as soluble peptides. ProtParam predictions showed E3 and E5 as stable proteins with a shelf life of 3.5 and 1.9 h and possessed negative GRAVY values. PsortB server predicted a final localization score of 7.88 for the gacS protein sequence as a cytoplasmic membrane protein. IEDB conservancy analysis showed 100% conserved sequences within the gacS sequence, and class I conservancy yielded positive values for all epitopes. Cluster analysis showed strong interactions, and the protein-peptide interactions with TLR-2 finally detected E5 as the best interacting peptide (H bonds = 14) followed by E3 (H bonds = 12).ConclusionThe study suggests five antigenic peptides as promiscuous vaccine candidates to target the gacS of A. baumannii using immuno-informatic approach toward the peptide synthesis and in vitro analysis. However, the study recommends further experimental validation for immunological response and memory through in vivo studies.

In silico analysis and homology modeling of strictosidine synthase involved in alkaloid biosynthesis in catharanthus roseus

BackgroundIn this study, strictosidine synthase (STR) from Catharanthus roseus that plays an important role in alkaloid biosynthesis was selected. The purpose of this work was to perform in silico analysis and to predict the three-dimensional structure of this protein that is not available. ResultsPhysicochemical characterization was performed by Expasy’s Protparam server. The computed theoretical isoelectric point (pI) found to be less than 7 indicates the acidic nature of this protein. The aliphatic index 73.04 indicates the thermal stability of the protein. Grand average hydropathy (GRAVY) was predicted to be − 285; this lower value of GRAVY shows the possibility of better interaction of this protein with water. Functional analysis of these proteins was performed by SOSUI server which predicted the transmembrane helix. Secondary structure analysis was carried out by SOPMA that revealed that Alpha helix dominated among secondary structure elements followed by random coil, extended strand, and beta turns. The modeling of the three-dimensional structure of the STR was performed by Swiss model. The model was validated using protein structure checking tools PROCHECK and PROVE. ConclusionsThis study reveals in silico analysis by Expasy Protparam server, SOPMA, and SOSUI server. Homology modeling of STR was performed by Swiss model.

In-silico analysis and homology modelling of antioxidant proteins present in Pisum sativum

Garden Peas are edible seeds that have an essential nutrient required for human diet. Peas contain phytochemicals that shows antioxidant activities. A computational approach was adopted to analyse the characteristic properties and structure of the Peas antioxidant proteins. Superoxidase dismutase (SOD), catalase, phospholipid hydroperoxide g lutathione p eroxidase-like p rotein (PHGPX) and ascorbate peroxidase (APX) are the antioxidant proteins which have been used in this study. Physiochemical properties such as isoelectric point, molecular weight, aliphatic index etc. were analysed by using the Expasy's Prot Param server. Secondary structure of these proteins was analysed to study the functional characteristic of the protein. The crystal structure of the ascorbate peroxidase was available, but the crystal structures of other proteins were not available in any of the protein structure database. Hence the threedimensional structures of these proteins were generated using Swiss Modeller and Geno3D followed by validation through SAVES server. This analysis will provide additional help for the analysis of crystal structure for further experimentation.

In silico structural homology modelling of EST073 motif coding protein of tea Camellia sinensis (L)

BackgroundTea (Camellia sinensis (L). O. Kuntze) is known as the oldest, mild stimulating caffeine containing non-alcoholic beverage. One of the major threats in south Asian tea industry is the blister blight leaf disease (BB), caused by the fungus Exobasidium vexans Masse. SSR DNA marker EST SSR 073 is used as a molecular marker to tag blister blight disease resistance trait of tea. The amino acid sequences were derived from cDNA sequences related to EST SSR 073 of BB susceptible (TRI 2023) and BB resistant (TRI 2043) cultivars. An attempt has been made to understand the structural characteristics and variations of EST SSR 073 locus that may reveal the factors influencing the BB resistance of tea with multiple bioinformatics tools such as ORF finder, ExPasy ProtParam tools, modeler V 9.17, Rampage server, UCSF-Chimera, and HADDOCK docking server. ResultsThe primary, secondary, and tertiary structures of EST SSR 073 coding protein were analyzed using the amino acid sequences of both BB resistant TRI 2043 and BB susceptible TRI 2023 tea cultivars. The coding amino acid sequences of both the cultivars were homologous to photosystem I subunit protein (PsaD I) of Pisum sativum. The predicted 3D structures of proteins were validated and considered as an acceptable overall stereochemical quality. The BB resistant protein showed CT repeat extension and did not involve in topology of the PsaD I subunit. The C terminal truncation of BB resistance caused the formation of hydrogen bonds interacting with PsaD I and other subunits of photosystem I in the modeled three-dimensional protein structure. ConclusionsCamellia sinensis EST 073 SSR motif coding protein was identified as the PsaD I subunit of photosystem I. The exact mechanism of PsaD I conferring the resistance for blister blight in tea needs to be further investigated.

In silico structural modeling and analysis of physicochemical properties of curcumin synthase (CURS1, CURS2, and CURS3) proteins of Curcuma longa

BackgroundPharmaceutically important curcuminoid synthesis in C. longa is controlled by CURS1, CURS2, and CURS3 genes. The present study detected the physicochemical properties and structural characteristics including the secondary and 3D structure of CURS proteins. The primary, secondary, and tertiary structure of the CURS proteins were modeled and characterized using multiple bioinformatics tools such as ExPasy ProtParam tools, self-optimized prediction method with alignment (SOPMA), PSIPRED, and SWISS-MODEL. The predicted secondary structure of curcumin synthase provided an α-helix and random coil as the major components. The reliability of the modeled structure was confirmed using PROCHECK and QMEAN programs. ResultsThe molecular weight of CURS1 is 21093.19 Da, theoretical pI as 4.93, and an aliphatic index of 99.19. Molecular weight of CURS2 and CURS3 proteins are 20266.13 Da and 20629.52 Da, theoretical pI as 5.28 and 4.96, and an aliphatic index of 89.30 and 86.37, respectively. In the predicted secondary structure of CURS proteins, alpha helices and random coils of CURS1, CUR2, and CURS3 were 42.72, 41.38, and 44.74% and 24.87, 31.03, and 17.89, respectively. The extended strands were 16.24, 19.40, and 17.89. QMEAN Z-score is − 0.83, − 0.89, and − 1.09 for CURS1, CURS2, and CURS3, respectively. ConclusionPrediction of the 3D model of a protein by in silico analysis is a highly challenging aspect to confirm the NMR or X-ray crystallographic data. This report can contribute to the understanding of the structure, physicochemical properties, structural motifs, and protein-protein interaction of CURS1, CUR2, and CURS3.

In silico functional and structural characterization of hepatitis B virus PreS/S-gene in Iranian patients infected with chronic hepatitis B virus genotype D

ObjectiveChronic hepatitis B (CHB) virus infection is the most prevalent chronic liver disease and has become a serious threat to human health. In this study, we attempted to specify and predict several properties including physicochemical, mutation sites, B-cell epitopes, phosphorylation sites, N-link, O-link glycosylation sites, and protein structures of S protein isolated from Ahvaz. Materials and methodsInitially, hepatitis B virus DNA (HBV DNA) was extracted from five sera samples of untreated chronic hepatitis B patients. The full-length HBV genomes were amplified and then cloned in pTZ57 R/T vector. The full sequences of HBV were registered in the GenBank with accessions numbers (MK355500), (MK355501) and (MK693107-9). PROTSCALE, Expasy's ProtParam, immuneepitope, ABCpred, BcePred, Bepipred, Algpred, VaxiJen, SCRATCH, DiANNA, plus a number of online analytical processing tools were used to analyse and predict the preS/S gene of genotype D sequences. The present study is the first analytical research on samples obtained from Ahvaz. ResultsWe found major hydrophilic region (MHR) mutations at “a” determining region that included K122R, N131T, F134Y, P142L, and T126N mutations. Moreover, Ahvaz sequences revealed four sites (4, 112, 166, and 309) in the preS/S gene for N-glycosylation that could possibly be a potential target for anti-HBV therapy. ConclusionIn the present study, mutations were identified at positions T113S and N131T within the MHR region of S protein; these mutations can potentially decrease the effect of hepatitis B vaccination in vaccine recipients.

Comparative analysis of structural and functional aspects of phytoene synthase from Meiothermus taiwanensis strain RP

PurposeThe aim of this study is the in silico characterization of the structure and function of the phytoene synthase (PSY) of a red carotenoid producing thermophile Meiothermus taiwanensis strain RP with a comparative approach.MethodsPSYs from M. taiwanensis strain RP and other groups of thermophilic, mesophilic and psychrophilic bacteria, plants, protozoa, and algae were analyzed by ExPASy ProtParam, NCBI Conserved Domain Search, SOPMA, PSIPRED, Robetta server, ProQ, and QMEAN, with the superposition of 3-D structures in PyMOL.ResultsRP PSY shows the highest (97.5 %) similarity with M. ruber and the lowest with the psychrophile Gelidibacter algens (36.7 %). The amino acid sequence of RP PSY is one of the shortest, with 275 residues. The instability index of RP PSY is much lower compared with plant sequences. Alanine, arginine, glycine, and leucine residues are the highest in Meiothermus sp., and they have a high amount of alpha-helix. Most of the 32 active site residues are conserved in all the sequences. However, some residues are more prone to substitutions in other PSYs except M. ruber. The three-dimensional structures of M. taiwanensis strain RP, Gelidibacter algens, Thermus thermophiles, Meiothermus ruber, and Brassica napus PSYs were homology modeled, validated, and submitted to Protein Model Databank. The superposition of the 3-D structures shows that their active site region structure is identical.ConclusionRP PSY is one of the most stable PSYs and knowledge of its individual properties, similarities, and dissimilarities with other PSYs may be useful for genetic engineering and purification of the protein for improved carotenoid production.

Antiproliferative and apoptotic effects of proteins from black seeds (Nigella sativa) on human breast MCF-7 cancer cell line

BackgroundNigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line.MethodsNS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools.ResultsGel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC50 values of 14.25 ± 0.84 and 8.05 ± 0.22 μg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range.ConclusionOur findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.

LEA Genlerinin Dişbudak (Fraxinus excelsior) Genomunda Tanımlanması ve Karakterizasyonu

Aim of study: LEA proteins have a critical role in the abiotic stress response of plants. Ash belongs to the olive family and its genome sequence is complete. The LEA genes has not been extensively analyzed, although there are annotations in the ash genome. Therefore, it was aimed to perform genome-wide analysis of LEA genes in ash genome using bioinformatic tools in this study. 
 Materials and methods: Ash and LEA protein sequences were obtained from the Ash Tree Genome and LEAP database respectively. Homologous LEA peptides in ash were found using CLC Genomic Workbench 11. Properties of ash LEA proteins were determined with Expasy PROTPARAM. MEGA7 was used to construct the phylogenetic tree. Functional analysis of ash LEA proteins was carried out via Blast2GO. miRNAs targeting transcripts of ash LEA proteins were detected with psRNATarget. The three-dimensional structures of ash LEA proteins were predicted using PHYRE2.
 Main result: 118 LEA genes (FexLEA) were identified in ash genome. FexLEA were divided into 5 distinct clusters according to phylogenetic analysis. The major molecular function of FexLEA was found as the binding activity. miR838 was the most common miRNA targeting FexLEA transcripts. 
 Highlights: This study will provide the basis for further functional analysis of LEA proteins in ash.

Comparative modeling and mutual docking of structurally uncharacterized heat shock protein 70 and heat shock factor-1 proteins in water buffalo

Aim:In this study, a wide range of in silico investigation of Bubalus bubalis (BB) heat shock protein 70 (HSP70) and heat shock factor-1 (HSF1) has been performed, ranging from sequence evaluation among species to homology modeling along with their docking studies to decipher the interacting residues of both molecules.Materials and Methods:Protein sequences of BB HSP70 and HSF1 were retrieved from NCBI database in FASTA format. Primary and secondary structure prediction were computed using Expasy ProtParam server and Phyre2 server, respectively. TMHMM server was used to identify the transmembrane regions in HSP70. Multiple sequence alignment and comparative analysis of the protein was carried out using MAFFT and visualization was created using ESPript 3.0. Phylogenetic analysis was accomplished by COBALT. Interactions of HSP70 with other proteins were studied using STRING database. Modeller 9.18, RaptorX, Swiss-Modeller, Phyre2, and I-TASSER were utilized to design the three-dimensional structure of these proteins followed by refinement; energy minimization was accomplished using ModRefiner and SPDBV program. Stereochemical quality along with the accuracy of the predicted models and their visualization was observed by PROCHECK program of PDBsum and UCSF Chimera, respectively. ClusPro 2.0 server was accessed for the docking of the receptor protein with the ligand.Results:The lower value of Grand Average of Hydropathy indicates the more hydrophilic nature of HSP70 protein. Value of the instability index (II) classified the protein as stable. No transmembrane region was reported for HSP70 by TMHMM server. Phylogenetic analysis based on multiple sequence alignments (MSAs) by COBALT indicated more evolutionarily closeness of Bos indicus (BI) with Bos taurus as compared to BI and BB. STRING database clearly indicates the HSF1 as one of the interacting molecules among 10 interacting partners with HSP 70. The best hit of 3D model of HSP70 protein and HSF1 was retrieved from I-TASSER and Phyre2, respectively. Interacting residues and type of bonding between both the molecules which were docked by ClusPro 2.0 were decoded by PIC server. Hydrophobic interactions, protein-protein main-chain-side-chain hydrogen bonds, and protein-protein side-chain-side-chain hydrogen bonds were delineated in this study.Conclusion:This is the first-ever study on in silico interaction of HSP70 and HSF1 proteins in BB. Several bioinformatics web tools were utilized to study secondary structure along with comparative modeling, physicochemical properties, and protein-protein interaction. The various interacting amino acid residues of both proteins have been indicated in this study.

Designing and Modeling of Multi-epitope Proteins for Diagnosis of Toxocara canis Infection

Serological investigation is the main method to achieve satisfactory results in Toxocara canis diagnosis. The accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize. Therefore significant efforts have been made to find alternative reagents as capture antigens. Multi-epitope peptides are potential diagnostic markers to improve the accuracy of diagnostic kits. The main aim of this study is the prediction and design of a novel synthetic protein consisting of multiple immunodominant B cell epitopes by Use of three proteins TES-120, TES-30, and TES-26 of T. canis. Primary, secondary and tertiary structures of the proteins were analyzed by using several various online software (ExPasy, IEDB, ABCpred, SVMTriP). Then, B cell construct was assessed by machine learning and Physico-chemical approaches and finally, homology modeling of 3D structure of protein was evaluated. The results of in silico analyses indicated that regions with high immunogenicity for TES-120 protein are located at between residues 97–167, for TES-30 protein are in the residues 52–102, 172–207 and for TES-26 are in the residues 33–83, 130–180. These regions could have good potential features for designing the Multi-epitopes. Finally, selected epitopes were linked to each other by linkers. The average length of the constructs was 342 bp. Also, the high proportion of random coils and extended strands in construct suggest that the protein form antigenic epitopes. Expasy ProtParam classified the constructs with moderate stability and 56.2% residues of constructs were located in favored regions of the Ramachandran plot. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide can laid a theoretical basis to develop an appropriate diagnostic kit for human toxocariasis.

Structural and functional analysis of ferritin heavy chain subunit in Oryziaslatipes

In North East region of India, iron toxicity is one of the major problems in culture fisheries. To overcome this challenge, it is necessary to identify the role of ferritin protein as an iron detoxificant and store in fishes. The Heavy chain in a ferritin protein possess di- Fe binding site in the fourth helix that interacts with oxygen. In this paper attempt has been made to study the structure and function of ferritin heavy chain subunit of Oryzias latipes from amino acid sequence. Physicochemical characterization by Expasy ProtParam form tools reveals that the protein is acidic, unstable and hydrophilic. A hydropathy scale showed two peak with significant score above the threshold value (0 to + value) but TMHMM conclude that there were one transmembrane domain within protein. The secondary structures contain alpha helix (56.50%), extended strands (10.73%) and coiled region (32.77%). The query sequence shows homology to the selected template (structure of mouse heavy chain modified ferritin by X-ray diffraction technique) with maximum % identity. To analyse the phylogenetic relationship, ML tree was constructed between Oryzias latipes and Oryzias melastigma for the ferritin heavy sub unit along with Cyprinus carpio as out crossed. Two distinct clads formation was observed between same genus species and the out crossed species. In protein-protein interaction analysis via STRING 10.0 tool, two enriched pathways of KEGG, six Inter Pro domains, one PFAM protein domain, one Uniprot keywords and eleven functional parameters of network analysis were identified in Oryzias latipes. The overall investigation reveals the structural features and their association in detoxification and iron homeostasis. Keywords: Ferritin Heavy Chain; Oryzias latipes; Physicochemical Characterization; Homology Modeling

CLONING OF DNA POLYMERASE-ǀ GENE FROM THERMOPHILIC Bacillus licheniformis STRAIN NWMF1 INTO AN E.coli EXPRESSION SYSTEM

DNA polymerase, catalyze template directed synthesis of DNA from nucleotide triphosphate. Thermostable DNA polymerase-ǀ (DNAP-ǀ) has been a common reagent in molecular biology because of its use in DNA amplification and DNA sequencing by PCR. DNAP-ǀ produced in moderate thermophiles such as Bacillus species may not be suitable for PCR, However, moderately thermophilic DNAP-ǀ from Bacillus has been used in molecular biology techniques such as loop mediated isothermal amplification. It is a low cost alternative to detect certain infectious diseases such as tuberculosis, malaria and can be applied in low/middle income countries. The objective of the study was isolation and cloning of DNAP-ǀ gene from native thermophilic bacterium, Bacillus licheniformis strain NWMF1 and over-expression by using expression host E. coli BL21(DE3)pLysS. A gram +ve endospore forming thermophilic bacterium was isolated from soil near the hot-water springs at Polonnaruwa, Sri Lanka. The identification of Bacillus licheniformis strain NWMF1 was carried out using morphological tests and 16s r.RNA gene sequence analysis. Initially the gene was cloned into pGEMT-easy vector and transformed into E. coli JM109 followed by sequence confirmation and protein blast analysis by NCBI. Thereafter the DNAP-ǀ gene re-cloned into PET28a+ vector and transformed into E. coli BL21(DE3)pLysS expression host. Recombinant E. coli clones were confirmed by colony PCR. Sequence analysis confirmed the presence of the complete gene (2640bp) including start and stop codons. The complete protein sequence consists 879 amino acids. SDS-PAGE and analysis by EXPASy–ProtParam indicated the molecular weight of DNAP-ǀ as ~92 kDa. Polymerase activity of His-tag purified DNAP-ǀ was demonstrated by PCR methodology.

Pemodelan Homologi Protein Receptor Orphan Receptor-1 (ROR-1) Sebagai Target Terapi Chronic Lymphocytic Leukemia (CLL

Receptor Orphan Reseptor-1 (ROR-1) is a trans-membrane protein consists of 937 amino acid residues located on 1p31.3 chomosome. ROR-1 has an important role in leukemogenesis of CLL cells. Co-expressions of ROR-1 and TCL enhance leukemogenesis and caused AKT’s phosphorylation, cell’s proliferations, and resistances to apoptosis. This study had done physicochemical characteristics analysis, secondary structure and tertiary structure prediction of ROR-1 Protein using few types of bioinformation tools. The physicochemical characteristics analysis was done by using Expasy Protparam server and the prediction of secondary structure was using Sopma and Psipred. The prediction of ROR-1’s tertiary structure was done by using Modeller 9.19 software and resulting five ROR-1’s tertiary structures predicted model. The accuration of the results then evaluated using Ramachandran’s plot analysis and it was showed Model 3 is the best models wich percentage is 82%.Keywords: ROR-1, Choronic Lymphocytic Leukemia (CLL), homology modeling, Modeller, Ramachandran plot

In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants.

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N2 fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.

Insilico analysis of Myostatin protein of Labeo calbasu

Myostatin is a protein released in autocrine fashion which exerts negative effect for skeletal muscle growth during myoblast proliferation and differentiation in teleost fishes. Myostatin is a member of the TGF-β family encoded by MSTN gene, also known as growth differentiation factor 8(GDF-8). MSTN gene knock-out exhibited hypertrophy and hyperplasia of skeletal muscle, but over expression or systemic administration of myostatin causes muscle atrophy. Blocking of myostatin activity helps in generating higher muscle growth rate and mutation in MSTN gene creates double-muscle phenotype. Therefore, insilico analysis of Myostatin in an Indian major carp species Labeo calbasu has carried out in the present study for understanding structural organization of the concerned protein. Amino acid sequence heaving the accession number AEN75195.1 was retrieved from NCBI. The sequence was subjected to Expasy ProtParam, PSIPRED and GORIV, PDB sum and SWISSMODEL/ Workspace for finding out the physicochemical properties, secondary structure prediction and template selection procedure respectively. The predicted 3D structure of Myostatin by Swiss model server was validated through RAMPAGE server and then submitted to Protein Model Database (PMDB) heaving the ID No: PM0081037. The extracellular location was also analysed by Hum-mPLoc 2.0. The structural examination of Myostatin will help in development of antagonistic protein or inhibitor, which can shed a new direction for the growth promotion of piscine species during various periods of life cycle.

In silico sequence analysis of predicted beta-amylase 7-like protein in Juglans regia L.

Walnut ( Juglans regia L.) is a deciduous tree of the Juglandaceae family. Beta-amylase (β-amylase, EC 3.2.1.2) is an enzyme that catalyses hydrolysis of glycosidic bonds in polysaccharides. In this study; sequence, physicochemical, and three-dimensional analyses of predicted β-amylase 7-like protein in Juglans regia using various bioinformatic tools were conducted. The physicochemical properties of the predict β-amylase 7-like protein were analyzed by using ExPASy ProtParam tool that revealed the molecular weight (MW), Isoelectric Points ( pI ), total number of negatively charged residues (Asp + Glu), total number of positively charged residues (Arg + Lys), instability index, aliphatic index, and GRAVY (Grand Average of Hydropathy) values. Subcellular localization using CELLO v.2.5, putative phosphorylation sites using NetPhos 3.1 server, domain analysis using Pfam, and secondary structure prediction using SOPMA were accom-plished. To predict the 3D structure of the predict β-amylase 7-like protein, homology models were applied using PSIPRED, RAMPAGE, and PyMOL programs. The results of our study provide insight into fundamental characteristics of the predicted β-amylase 7-like protein in Juglans regia.

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7–L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7–L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis.

In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.