Small-sized Peptides Research Articles

The utility of proteases in proteomics, from sequence profiling to structure and function analysis.

In mass spectrometry (MS)-based bottom-up proteomics, protease digestion plays an essential role in profiling both proteome sequences and post-translational modifications (PTMs). Trypsin is the gold standard in digesting intact proteins into small-size peptides, which are more suitable for high-performance liquid chromatography (HPLC) separation and tandem MS (MS/MS) characterization. However, protein sequences lacking Lys and Arg cannot be cleaved by trypsin and may be missed in conventional proteomic analysis. Proteases with cleavage sites complementary to trypsin are widely applied in proteomic analysis to greatly improve the coverage of proteome sequences and PTM sites. In this review, we survey the common and newly emerging proteases used in proteomics analysis mainly in the last 5 years, focusing on their unique cleavage features and specific proteomics applications such as missing protein characterization, new PTM discovery, and de novo sequencing. In addition, we summarize the applications of proteases in structural proteomics and protein function analysis in recent years. Finally, we discuss the future development directions of new proteases and applications in proteomics.

Probing the Spatiotemporal Dynamics of Oxytocin in the Brain Tissue Using a Simple Peptide Alkyne-Tagging Approach.

The dynamics of oxytocin and its site of action in the brain are poorly understood due to the lack of appropriate tools, despite the interest in the central action of oxytocin signaling. Here, we develop and apply an oxytocin analogue probe by conjugating it with an alkyne via a widely applicable simple coupling reaction. Alkyne-tagged oxytocin behaves similarly to endogenous oxytocin while allowing specific and highly sensitive detection of extracellularly applied oxytocin. Using this probe, we find the existence of high-affinity specific binding sites of oxytocin in the hippocampus. Furthermore, characterization of oxytocin dynamics reveals the cellular basis of its volume transmission in the brain tissue. Finally, we show the wide applicability of this technique for other centrally acting peptides. Thus, the alkyne tagging strategy provides a unique opportunity to characterize the spatiotemporal dynamics of oxytocin and other small-sized peptides in the brain tissue.

Simple and rapid pipeline for the production of cyclic and linear small-sized peptides in E. coli

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

Amyloid-beta peptide (25\u201335) triggers a reorganization of lipid membranes driven by temperature changes

The amyloid-beta peptide (Aβ) is considered a key factor in Alzheimer's disease (AD) ever since the discovery of the disease. The understanding of its damaging influence has however shifted recently from large fibrils observed in the inter-cellular environment to the small oligomers interacting with a cell membrane. We studied the effect of temperature on the latter interactions by evaluating the structural characteristics of zwitterionic phosphatidylcholine (PC) membranes with incorporated Aβ25–35 peptide. By means of small angle neutron scattering (SANS), we have observed for the first time a spontaneous reformation of extruded unilamellar vesicles (EULVs) to discoidal bicelle-like structures (BLSs) and small unilamellar vesicles (SULVs). These changes in the membrane self-organization happen during the thermodynamic phase transitions of lipids and only in the presence of the peptide. We interpret the dramatic changes in the membrane's overall shape with parallel changes in its thickness as the Aβ25–35 triggered membrane damage and a consequent reorganization of its structure. The suggested process is consistent with an action of separate peptides or small size peptide oligomers rather than the result of large Aβ fibrils.

New short cationic antibacterial peptides. Synthesis, biological activity and mechanism of action

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.

Antibacterial Properties of Small-Size Peptide Derived from Penetratin against Oral Streptococci.

Periodontitis, an infectious disease originating from dental biofilms that causes the irreversible loss of alveolar bone, is accompanied by gradual biofilm formation and the continuous progression of inflammation. A small peptide derived from penetratin, Arg-Gln-Ile-Arg-Arg-Trp-Trp-Gln-Arg-NH2 (RR9), appears to have antibacterial properties against selected strains associated with periodontitis. The purpose of this research is to assess the antibacterial activity and mechanism of RR9 against the initial oral colonizers Streptococci oralis, Streptococci gordonii, and Streptococci sanguinis and to investigate the cytotoxicity of RR9 on human gingival fibroblasts in vitro. The effects of RR9 on the initial oral settlers of planktonic and biofilm states were evaluated by measuring the MIC, MBC, bactericidal kinetics, and antibiofilm activity. Visual evidence and antibacterial mechanisms were identified, and the anti-inflammatory activity and cytotoxicity were measured. The results demonstrated that RR9 can inhibit the growth of streptococci in the planktonic state and during biofilm formation in vitro while keeping a low toxicity against eukaryotic cells. The antibacterial mechanism was proven to be related to the lower expression of sspA in streptococci. RR9 may be used as a potential antimicrobial and anti-infective agent for periodontal disease.

Bacteria do it better! Proteomics suggests the molecular basis for improved digestibility of sourdough products

The aim of this study was to evaluate the dynamics of proteolysis during dough fermentation started with different lactic acid bacteria species, through the identification of intermediate and small-sized peptides generated during fermentation. Single-strain cultures of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Companilactobacillus alimentarius, and Leuconostoc pseudomesenteroides were assayed as sourdough starters. Assays were carried out at lab-scale for 48 h of fermentation, using both unstarted and yeast-leavened dough as controls. Physicochemical and microbiological analyses were combined with peptidomic and proteomic profiling, identifying several hundreds of peptides mainly released from the water-soluble wheat proteins, including β-amylase, triticin, and serpins. Both α- and γ-gliadins were hydrolyzed, though only at the N-terminal domain, while the central protein region – encrypting celiac disease epitopes– remained unaffected. The bacterial-mediated consumption of sugars and the concomitant hydrolysis of starch degrading β-amylase could underlie improved digestibility and several nutritionally beneficial effects of sourdough baked products.

Fractionation of Tilapia By-Product Protein Hydrolysate Using Multilayer Configuration of Ultrafiltration Membrane

Production of small-sized peptides is significant because of their health benefits. Ultrafiltration (UF) membrane provides an effective fractionation of small-sized peptides on a large scale. Thus, the present study was aimed to evaluate the performance of multilayer UF membrane in fractionating tilapia fish by-product (TB) protein hydrolysate by observing the permeate flux, peptide transmission, and peptide distribution under different stirring speed, pH of feed solution, and salt concentration (NaCl). The fractionation process was carried out using a dead-end UF membrane system that consists of a stack of two membrane sheets with different (10/5 kDa) and similar (5/5 kDa) pore sizes in one device. The highest permeate flux (10/5 kDa–39.5 to 47.3 L/m2.h; 5/5 kDa– 15.8 to 20.3 L/m2.h) and peptide transmission (10/5 kDa–51.8 to 61.0%; 5/5 kDa–18.3 to 23.3%) for both multilayer membrane configurations were obtained at 3.0 bar, 600 rpm, pH 8, and without the addition of salt. It was also found that the permeates were enriched with small-size peptides (<500 Da) with a concentration of 0.58 g/L (10/5 kDa) and 0.65 g/L (5/5 kDa) as compared to large-sized peptides (500–1500 Da) with concentration of 0.56 g/L (10/5 kDa) and 0.36 g/L (5/5 kDa). This might indicate the enrichment of small-size peptides through the multilayer membrane which could potentially enhance the biological activity of the protein hydrolysate fraction.

Quantitatively mapping the interaction of HER2 and EGFR on cell membranes with peptide probes.

Human epidermal growth factor receptor-2 (HER2) is a member of the epidermal growth factor receptor (HER) family that is involved in various biological processes such as cell proliferation, survival, differentiation, migration and invasion. It generally functions in the form of homo-/hetero-dimers or oligomers with other HER family members. Although its essential roles in cellular activities have been widely recognized, questions concerning the spatial distribution of HER2 on the membranes and the interactions between it and other ErbB family members remain obscure. Here, we obtained a high-quality dSTORM image of HER2 nanoscale clusters recognized by peptide probes, and found that HER2 forms clusters containing different numbers of molecules on cell membranes. Moreover, we found that HER2 and EGFR formed hetero-oligomers on non-stimulated cell membranes, whereas EGF stimulation reduced the degree of heteromerization, suggesting that HER2 and EGFR hetero-oligomers may inhibit the activation of EGFR. Collectively, our work revealed the clustered distribution of HER2 and quantified the changes of the interaction between HER2 and EGFR in the resting and active states at the single molecular level, which promotes a deeper understanding of the protein-protein interaction on cell membranes.

EFFECT OF GERMINATION ON FREE RADICAL SCAVENGING ACTIVITIES AND ANGIOTENSIN I-CONVERTING ENZYME INHIBITORY OF MELINJO (Gnetum gnemon L) SEED PROTEINS

The effect of germination on free radical scavenging activities and Angiotensin I-Converting Enzyme (ACE-I) inhibitory of melinjo (Gnetum gnemon) seed proteins was studied to determine its potential use as a nutraceutical ingredient. Investigations into the protein profiles, amino acid composition, free radical scavenging activities and ACE-I-inhibitory of proteins extracted from melinjo seed during germination (0 to 21 days) were carried out. The concentration of amino acids detected in the melinjo seed samples ranged from 0.30 to 12.75 g/100 g protein and the degree of hydrolysis during germination at 21st days was significantly highest than that of either (0-14 days). Furthermore, with all samples a large small-sized peptides degree (

Fermentation of soybean meal improves growth performance and immune response of abruptly weaned Holstein calves during cold weather

We studied the effect of different levels of soybean meal (SBM) and fermented soybean meal (FSBM) on the growth performance, blood parameters and immune response of abruptly-weaned calves in cold weather. Thirty-nine Holstein calves (body weight = 39.5 ± 3.3 kg) were randomly assigned to one of three dietary treatments: 27% SBM + 0% FSBM (FSBM0, control, n = 13); 18% SBM + 9% FSBM (FSBM9, n = 13); and 13.5% SBM + 13.5% FSBM (FSBM13, n = 13) from October 2017 to January 2018. The average weekly temperature was 8.5 ± 2.3 °C (95%, confidence interval) over a period of 9 weeks (wk) of the experiment. All the calves were weaned abruptly by removing the milk meal at the age of 59 days, but remained in individual hutches until the end of the experiment. The FSBM used in our experiment had a higher crude protein content, a higher percentage of rumen-undegradable protein, and a higher percentage of small-sized peptides (consisting of 2 to 3 amino acids) than SBM. A treatment × wk interaction was detected for the average daily gain (ADG) in which FSBM13-fed calves gained the least at wk 2 of the experiment, but gained the most at wk 6, after which they gained as FSBM9-fed calves. Calves fed FSBM0 initially had the highest ADG but at the end of wk 8 and 9, gained the lowest. In addition, starter intake also showed a significant treatment × wk interaction, with FSBM13- and FSBM9-fed calves showing a higher starter intake between wk 8 and 9 than FSBM0-fed calves. Calves fed FSBM13 had a higher withers and hip height than those fed other diets. The percentage of neutrophils (NEU), neutrophil-to-lymphocyte ratio (NEU:LYM), and serum glucose concentration were higher in FSBM0-fed calves, 1 and 3 days post-weaning (DPW, 0 DPW = 59 days), but they had a lower percentage of LYM compared to FSBM13-fed calves. The white blood cells (WBCs) count also showed a treatment × wk interaction, with FSBM9-fed calves having higher WBCs between -2 and 3 DPW. The insulin concentration also exhibited a treatment × wk interaction. In this way, the FSBM13-fed calves had the lowest level of insulin, but those fed FSBM0 significantly increased their insulin level between -2 and 3 DPW. The concentrations of interleukin 1B and serum amyloid A were lower in FSBM13-fed calves than in those fed other diets. In conclusion, in this study, feeding FSBM increased calf growth performance while reducing the weaning stress via a pronounced reduction in the concentration of pro-inflammatory mediators.

Peptides in cosmeceuticals

Skin aging is part of human global aging which becomes evident along the years following different ways in organs, tissues and cells. Aging of the internal organs is masked whilst the skin is the first organ displaying visible signs of passing time. As life expectancy is growing everywhere at a fast pace, many people are on a quest for the fountain of youth, and age usually factors into the perception of beauty.Peptides feature molecules constituted by chains of two or more amino acids connected by peptide bonds. There are a lot of physiological peptides present in the organism, most of them having a signalling function. Due to the potent activity of these physiological peptides, it was tempting using their model and mimic them in order to get focused therapeutic activities. This trend was initiated in the beginning of twenty-first century, and many peptides were synthesized and are used as active ingredients in cosmeceuticals. Most of these peptides are oriented towards the treatment of skin aging, mainly for the attenuation of fine lines and wrinkles. Others are positioned in the management of hyperpigmentation or may also be antimicrobial peptides. Finally, peptides also exist which can improve hair growth. Formulation of peptides in cosmeceuticals is relatively easy, however attention must be paid to their transcutaneous penetration. For small-sized peptides there is usually no problem, whilst for bigger-sized peptides various artefacts like encapsulation or branching with fatty acids must be used. Another concern about peptides is their eventual toxicity, and there is currently insufficient responses from the industry about this matter.The majority of research studies are focused on the development of anti-aging actives, and there is still room for research to be carried out to evaluate other functions of these actives and create new actives for other indications. Understanding of cellular mechanisms, gene regulation, receptor activity and metabolic interactions increases constantly and many more peptides will probably appear and have applications in cosmeceuticals.

Effect of Egg White Hydrolysates on the Shrinkage, Cooking Loss and Texture of Pork Meat Slices

The potential of using hydrolyzed egg white (hEW) as a food additive to decrease shrinkage, cooking loss of pork meat slices was investigated. Functional characteristics including solubility, Water-Holding Capacity (WHC) and Oil-Binding Capacity (OBC) of three hEWs by using different kinds of enzymes, a commercial small size peptide S and native egg white (NEw) were evaluated. Pork meat slices (8 cm×6 cm) with thickness of 4 mm were soaked in the same weight of solution contained hEW, small peptide S or NEw at a protein concentration of 10% (w/v) at 4°C for 24 h respectively. Results showed that, cooking loss (%) and shrinkage rate (%) decreased when raw meat was immersed in the solution which contained hEWs or NEw. Meat treated by hEW obtained by using enzyme-Thermoase PC10F® showed an excellent WHC and resulted in the least shrinkage rate (3.2%) compared with the meat soaked in the same portion of water (shrinkage rate of 22.7%). Moreover, by using a scanning electron microscopy, relative larger holes and a grainy appearance of endomysium sheaths were also observed in the meat treated by this hEW. Treated by hEWs and NEw contributed to the decrease of elasticity and toughness of cooked meat. Thus, treated by hEWs may permit reduction in cooking loss as and shrinkage rate of pork meat slices.

Digestion differently affects the ability of native and thermally aggregated ovalbumin to trigger basophil activation

Ovalbumin (OVA), a major allergen from hen's egg albumen, tends to aggregate when heated. Depending on the balance of attractive and repulsive interactions, heat-induced OVA aggregates have various morphologies, which differ in digestibility. In the context of food allergy to egg, we investigated the ability of native and thermally aggregated OVA as well as their digests to induce the degranulation of a humanized rat basophil leukemia (RBL) cell line, which was sensitized with a pool of sera from egg-allergic children. Native and two thermally aggregated OVA forms were digested in vitro using a gastrointestinal digestion model based on the INFOGEST harmonized protocol including a final degradation with jejunal brush border membranes (BBM) enzymes. The course of digestion was monitored by the OPA method and by RP-HPLC. Digestibility was OVA small aggregates>OVA large aggregates>>native OVA and BBM peptidases only significantly hydrolyzed small-sized peptides from gastro-duodenal digests of the aggregates. The degranulation ability of the native OVA slightly changed during the gastric phase but mostly decreased during the duodenal digestion with no further change with BBM digestion. The degranulation ability of aggregates, which was significantly lower than the ability of native OVA, was not significantly affected by digestion. Digestibility and ability to induce basophil degranulation can thus not be straightforward linked.

Evaluation on performance of dead-end ultrafiltration membrane in fractionating tilapia by-product protein hydrolysate

Conversion of tilapia by-products through enzymatic hydrolysis is a promising alternative to produce a fish protein hydrolysate (FPH). Tilapia by-product (TB) protein hydrolysate consists of peptide mixtures in various sizes. Recovery of small-sized peptides is increasingly becoming a priority due to their special characteristics, which can provide an excellent physiological function. Ultrafiltration (UF) membrane is an effective and suitable tool for fractionating small size peptides in a large scale. The objective of this study was to evaluate the membrane performance based on the permeate flux and peptide transmission. The fractionation of TB protein hydrolysate was performed through a dead-end UF membrane (10 and 5 kDa) with the effect of stirring speed (0–600 rpm), pH (3, 5, 7, 8 and 9) and salt concentration (NaCl; 0 M, 0.2 M, 0.4 M, and 0.6 M) at varies pressure (1.0, 1.5, 2.0, 2.5 and 3.0 bar). It was found that the transmembrane pressure of 2.5 bar, stirring speed of 600 rpm, pH 8 and without addition of NaCl turned out to be the best parameters which gave permeate flux of 53 L/m2h (10 kDa) and 27 L/m2h (5 kDa), while for the peptide transmission, the value of 87.33% (10 kDa) and 36.11% (5kDa) was obtained. This study indicates that by adding salt, it has lowered the permeate flux and peptide transmission. Finally, through a well-controlled of operating and physicochemical parameters, it was possible to produce peptides with sizes lower than 1000 Da.

Cucurbitaceae Seed Protein Hydrolysates as a Potential Source of Bioactive Peptides with Functional Properties.

Seeds from Cucurbitaceae plants (squashes, pumpkins, melons, etc.) have been used both as protein-rich food ingredients and nutraceutical agents by many indigenous cultures for millennia. However, relatively little is known about the bioactive components (e.g., peptides) of the Cucurbitaceae seed proteins (CSP) and their specific effects on human health. Therefore, this paper aims to provide a comprehensive review of latest research on bioactive and functional properties of CSP isolates and hydrolysates. Enzymatic hydrolysis can introduce a series of changes to the CSP structure and improve its bioactive and functional properties, including the enhanced protein solubility over a wide range of pH values. Small-sized peptides in CSP hydrolysates seem to enhance their bioactive properties but adversely affect their functional properties. Therefore, medium degrees of hydrolysis seem to benefit the overall improvement of bioactive and functional properties of CSP hydrolysates. Among the reported bioactive properties of CSP isolates and hydrolysates, their antioxidant, antihypertensive, and antihyperglycaemic activities stand out. Therefore, they could potentially substitute synthetic antioxidants and drugs which might have adverse secondary effects on human health. CSP isolates and hydrolysates could also be implemented as functional food ingredients, thanks to their favorable amino acid composition and good emulsifying and foaming properties.

Soy Pulp Extract Inhibits Angiotensin I-Converting Enzyme (ACE) Activity In Vitro: Evidence for Its Potential Hypertension-Improving Action

ABSTRACTSoy pulp, called “okara” in Japanese, is known as a by-product of the production of bean curd (tofu), and expected to contain a variety of biologically active substances derived from soybean. However, the biological activities of okara ingredients have not yet been fully understood, and the effectiveness of okara as a functional food seems necessary to be further evaluated. Then the effect of okara extract on angiotensin I-converting enzyme (ACE) activity was examined in vitro, and the extract was shown to cause the inhibition of ACE activity in a manner depending on its concentration. Kinetic analysis indicated that this enzyme inhibition was accompanied by an increase in the Km value without any change in Vmax. Further studies suggested that putative inhibitory substances contained in the extract might be heat stable and dialyzable, and recovered mostly in the peptide fraction obtained by a spin-column separation and a high performance liquid chromatography (HPLC) fractionation. Therefore, the extract was speculated to contain small-size peptides responsible for the inhibitory effect of okara extract on ACE activity, and could be expected to improve the hypertensive conditions by reducing the production of hypertensive peptide.

Small Peptides Derived from Penetratin as Antibacterial Agents.

The synthesis, in vitro evaluation and conformational study of several small-size peptides acting as antibacterial agents are reported. Among the compounds evaluated, the peptides Arg-Gln-Ile-Lys-Ile-Trp-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2 , Arg-Gln-Ile-Lys-Ile-Arg-Arg-Met-Lys-Trp-Arg-NH2 , and Arg-Gln-Ile-Trp-Trp-Trp-Trp-Gln-Arg-NH2 exhibited significant antibacterial activity. These were found to be very active antibacterial compounds, considering their small molecular size. In order to better understand the antibacterial activity obtained for these peptides, an exhaustive conformational analysis was performed, using both theoretical calculations and experimental measurements. Molecular dynamics simulations using two different media (water and trifluoroethanol/water) were employed. The results of these theoretical calculations were corroborated by experimental circular dichroism measurements. A brief discussion on the possible mechanism of action of these peptides at molecular level is also presented. Some of the peptides reported here constitute very interesting structures to be used as starting compounds for the design of new small-size peptides possessing antibacterial activity.

New small-size peptides modulators of the exosite of BACE1 obtained from a structure-based design

We report here two new small-size peptides acting as modulators of the β-site APP cleaving enzyme 1 (BACE1) exosite. Ac-YPYFDPL-NH2 and Ac-YPYDIPL-NH2 displayed a moderate but significant inhibitory effect on BACE1. These peptides were obtained from a molecular modeling study. By combining MD simulations with ab initio and DFT calculations, a simple and generally applicable procedure to evaluate the binding energies of small-size peptides interacting with the exosite of the BACE1 is reported here. The structural aspects obtained for the different complexes were analyzed providing a clear picture about the binding interactions of these peptides. These interactions have been investigated within the framework of the density functional theory and the quantum theory of atoms in molecules using a reduced model. Although the approach used here was traditionally applied to the study of noncovalent interactions in small molecules complexes in gas phase, we show, through in this work, that this methodology is also a very powerful tool for the study of biomolecular complexes, providing a very detailed description of the binding event of peptides modulators at the exosite of BACE1.

MHC binding peptides for designing of vaccines against Japanese encephalitis virus: A computational approach.

Japanese encephalitis (JE), a viral disease has seen a drastic and fatal enlargement in the northern states of India in the current decade. The better and exact cure for the disease is still in waiting. For the cause an in silico strategy in the development of the peptide vaccine has been taken here for the study. A computational approach to find out the Major Histocompatibility Complex (MHC) binding peptide has been implemented. The prediction analysis identified MHC class I (using propred I) and MHC class II (using propred) binding peptides at an expectable percent predicted IC (50) threshold values. These predicted Human leukocyte antigen [HLA] allele binding peptides were further analyzed for potential conserved region using an Immune Epitope Database and Analysis Resource (IEDB). This analysis shows that HLA-DRB1*0101, HLA-DRB3*0101, HLA-DRB1*0401, HLA-DRB1*0102 and HLA-DRB1*07:01% of class II (in genotype 2) and HLA-A*0101, HLA-A*02, HLA-A*0301, HLA-A*2402, HLA-B*0702 and HLA-B*4402% of HLA I (in genotype 3) bound peptides are conserved. The predicted peptides MHC class I are ILDSNGDIIGLY, FVMDEAHFTDPA, KTRKILPQIIK, RLMSPNRVPNYNLF, APTRVVAAEMAEAL, YENVFHTLW and MHC class II molecule are TTGVYRIMARGILGT, NYNLFVMDEAHFTDP, AAAIFMTATPPGTTD, GDTTTGVYRIMARGI and FGEVGAVSL found to be top ranking with potential super antigenic property by binding to all HLA. Out of these the predicted peptide FVMDEAHFTDPA for allele HLA-A*02:01 in MHC class I and NYNLFVMDEAHFTDP for allele HLA-DRB3*01:01 in MHC class II was observed to be most potent and can be further proposed as a significant vaccine in the process. The reported results revealed that the immune-informatics techniques implemented in the development of small size peptide is useful in the development of vaccines against the Japanese encephalitis virus (JEV).