Phage display was first described by George P. Smith when it was shown that virus particles were capable of presenting foreign proteins on their surface. The technology has paved the way for the evolution of various biomolecules presentation and diverse selection strategies. This unique feature has been applied as a versatile platform for numerous applications in drug discovery, protein engineering, diagnostics, and vaccine development. Over the decades, the limits of biomolecules displayed on phage particles have expanded from peptides to proteomes and even alternative scaffolds. This has allowed phage display to be viewed as a versatile display platform to accommodate various biomolecules ranging from small peptides to larger proteomes which has significantly impacted advancements in the biomedical industry. This review will explore the vast array of biomolecules that have been successfully employed in phage display technology in biomedical research.
Antibiotic resistance is a growing concern that is affecting public health globally. The search for alternative antimicrobial agents has become increasingly important. Antimicrobial peptides (AMPs) produced by Bacillus spp. have emerged as a promising alternative to antibiotics, due to their broad-spectrum antimicrobial activity against resistant pathogens. In this review, we provide an overview of Bacillus-derived AMPs, including their classification into ribosomal (bacteriocins) and non-ribosomal peptides (lipopeptides and polyketides). Additionally, we delve into the molecular mechanisms of AMP production and describe the key biosynthetic gene clusters involved. Despite their potential, the low yield of AMPs produced under normal laboratory conditions remains a challenge to large-scale production. This review thus concludes with a comprehensive summary of recent studies aimed at enhancing the productivity of Bacillus-derived AMPs. In addition to medium optimization and genetic manipulation, various molecular strategies have been explored to increase the production of recombinant antimicrobial peptides (AMPs). These include the selection of appropriate expression systems, the engineering of expression promoters, and metabolic engineering. Bacillus-derived AMPs offer great potential as alternative antimicrobial agents, and this review provides valuable insights on the strategies to enhance their production yield, which may have significant implications for combating antibiotic resistance. KEY POINTS: • Bacillus-derived AMP is a potential alternative therapy for resistant pathogens • Bacillus produces two main classes of AMPs: ribosomal and non-ribosomal peptides • AMP yield can be enhanced using culture optimization and molecular approaches.
Bioactive peptides, as products of hydrolysis of diverse food proteins, are the focus of current research. They exert various biological roles, one of the most crucial of which is the antioxidant activity. Reverse relationship between antioxidant intake and diseases has been approved through plenty of studies. Antioxidant activity of bioactive peptides can be attributed to their radical scavenging, inhibition of lipid peroxidation and metal ion chelation properties of peptides. It also has been proposed that peptide structure and its amino acid sequence can affect its antioxidative properties. This paper reviews bioactive peptides from food sources concerning their antioxidant activities. Additionally, specific characteristics of antioxidative bioactive peptides, enzymatic production, methods to evaluate antioxidant capacity, bioavailability, and safety concerns of peptides are reviewed.
Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of the PRH1 locus. Gene frequencies of the At productive allele (PRH1(6)) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n = 746). These three proteins were not found in 106 Indian subjects.
Matched MeSH terms: Peptides/genetics*; Salivary Proteins and Peptides/genetics*
Three human saliva genetic markers, namely, salivary peroxidase (SAPX), Pm, and Ph proteins, were investigated in the three major ethnic groups of Malaysia: Malays, Chinese, and Indians. For Pm, the allelic frequencies of Pm+ for Malays, Chinese, and Indians are 0.385 +/- 0.030, 0.282 +/- 0.026, and 0.289 +/- 0.026 respectively. For Ph, the allelic frequencies of Ph+ are 0.082 +/- 0.016 for Malays, 0.109 +/- 0.017 for Chinese, and 0.062 +/- 0.013 for Indians. For SAPX, the allelic frequencies of SAPX1 in Malays, Chinese, and Indians are 0.762 +/- 0.027, 0.755 +/- 0.027, and 0.723 +/- 0.026 respectively.
Matched MeSH terms: Salivary Proteins and Peptides/genetics*
Spider venoms are vast natural pharmacopoeias selected by evolution. The venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. We tested six of them (latarcins 1, 2a, 3a, 4b, 5, and cytoinsectotoxin 1a) for their ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the genes of the selected peptides. Controlled expression of the transgenes led to a significant decrease of C. trachomatis viability inside the infected cells.
As anticancer peptides (ACPs) have attracted great interest for cancer treatment, several approaches based on machine learning have been proposed for ACP identification. Although existing methods have afforded high prediction accuracies, however such models are using a large number of descriptors together with complex ensemble approaches that consequently leads to low interpretability and thus poses a challenge for biologists and biochemists. Therefore, it is desirable to develop a simple, interpretable and efficient predictor for accurate ACP identification as well as providing the means for the rational design of new anticancer peptides with promising potential for clinical application. Herein, we propose a novel flexible scoring card method (FSCM) making use of propensity scores of local and global sequential information for the development of a sequence-based ACP predictor (named iACP-FSCM) for improving the prediction accuracy and model interpretability. To the best of our knowledge, iACP-FSCM represents the first sequence-based ACP predictor for rationalizing an in-depth understanding into the molecular basis for the enhancement of anticancer activities of peptides via the use of FSCM-derived propensity scores. The independent testing results showed that the iACP-FSCM provided accuracies of 0.825 and 0.910 as evaluated on the main and alternative datasets, respectively. Results from comparative benchmarking demonstrated that iACP-FSCM could outperform seven other existing ACP predictors with marked improvements of 7% and 17% for accuracy and MCC, respectively, on the main dataset. Furthermore, the iACP-FSCM (0.910) achieved very comparable results to that of the state-of-the-art ensemble model AntiCP2.0 (0.920) as evaluated on the alternative dataset. Comparative results demonstrated that iACP-FSCM was the most suitable choice for ACP identification and characterization considering its simplicity, interpretability and generalizability. It is highly anticipated that the iACP-FSCM may be a robust tool for the rapid screening and identification of promising ACPs for clinical use.
In this study, we reported a complete molecular characterization including bioinformatics features, gene expression, peptide synthesis and its antimicrobial activities of an anti-lipopolysaccharide (LPS) factor (ALF) cDNA identified from the established cDNA library of freshwater prawn Macrobrachium rosenbergii (named as MrALF). The mature protein has an estimated molecular weight of 11.240 kDa with an isoelectric point of 9.46. The bioinformatics analysis showed that the MrALF contains an antimicrobial peptide (AMP) region between T54 and P77 with two conserved cysteine residues (Cys55 and Cys76) which have an anti-parallel β-sheet confirmation. The β-sheet is predicted as cationic with hydrophobic nature containing a net charge of +5. The depicted AMP region is determined to be amphipathic with a predicted hydrophobic face 'FPVFI'. A highest MrALF gene expression was observed in hemocytes and is up-regulated with virus [white spot syndrome baculovirus (WSBV)], bacteria (Aeromonas hydrophila) and Escherichia coli LPS at various time points. The LPS binding region of MrALF peptide was synthesized to study the antimicrobial property, bactericidal efficiency and hemolytic capacity. The peptide showed antimicrobial activity against both the Gram-negative and Gram-positive bacteria. The bactericidal assay showed that the peptide recognized the LPS of bacterial cell walls and binding on its substrate and thereby efficiently distinguishing the pathogens. The hemolytic activity of MrALF peptide is functioning in a concentration dependant manner. In summary, the comprehensive analysis of MrALF showed it to be an effective antimicrobial peptide and thus it plays a crucial role in the defense mechanism of M. rosenbergii.
Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.
Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical-pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour samples of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators. Using peptide matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), we have identified m/z values which can classify 88% of all tumours correctly. The top discriminative m/z values were identified using a combination of in situ sequencing and LC-MS/MS from digested tumour samples. Two of the proteins identified, plectin and α-Actin-2, were used for validation studies using LC-MS/MS data independent analysis (DIA) and immunohistochemistry. In summary, MALDI-MSI has the potential to identify discriminators of metastasis using primary tumour samples.
Extracellular senile plaques and intracellular neurofibrillary tangles are the neuropathological findings of the Alzheimer's disease (AD). Based on the amyloid cascade hypothesis, the main component of senile plaques, the amyloid-beta (Aβ) peptide, and its derivative called amyloid precursor protein (APP) both have been found to place their central roles in AD development for years. However, the recent therapeutics have yet to reverse or halt this disease. Previous evidence demonstrates that the accumulation of Aβ peptides and APP can exert neurotoxicity and ultimately neuronal cell death. Hence, we discuss the mechanisms of excessive production of Aβ peptides and APP serving as pathophysiologic stimuli for the initiation of various cell signalling pathways including apoptosis, necrosis, necroptosis and autophagy which lead to neuronal cell death. Conversely, the activation of such pathways could also result in the abnormal generation of APP and Aβ peptides. An elucidation of actions of APP and its metabolite, Aβ, could be vital in suggesting novel therapeutic opportunities.
Ultraviolet (UV) radiation causes damage in all living organisms, including DNA damage that leads to cell death. Herein, we provide a new technique for UV radiation protection through intracellular short peptide expression. The late embryogenesis abundant (LEA) peptide, which functions as a shield that protects macromolecules from various abiotic stress, was obtained from the Polypedilum vanderplanki group 3 LEA protein. Recombinant Escherichia coli BL21 (DE3) expressing functional LEA short peptide in vivo were exposed to UVA and UVC radiation for 4, 6, and 8 h. E. coli transformants expressing the LEA peptide showed higher cell viability under both UVA and UVC treatment at all time points as compared with that of the control. Furthermore, the cells expressing LEA peptide showed a higher number of colony-forming units per dilution under UVA and UVC treatment. These results suggested that expression of the short peptide could be useful for the development of genetically modified organisms and in applications that require resilience of organisms to UV radiation.
Several bioactive molecules isolated from the saliva of blood-sucking arthropods, such as mosquitoes, have been shown to exhibit potential anticoagulant function. We have previously identified a 30kDa allergen named Aegyptin-like protein (alALP), which is highly homologous to Aegyptin, from the salivary glands of female Aedes albopictus (Asian tiger mosquito). In this study, we identified the conserved functional domain of alALP by using bioinformatic tools, and expressed the His-tagged alALP recombinant protein in sf9 insect cells by generation and transfection of a baculoviral expression plasmid carrying the fulllength cDNA of alALP. We purified this recombinant protein and examined its function on the inhibition of blood coagulation. The results showed that the purified His-alALP prolonged the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) in vitro as well as the Bleeding Time (BT) in vivo, which suggest that alALP could be a novel anticoagulant.
Matched MeSH terms: Salivary Proteins and Peptides/genetics
Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the hallmark pathologies of amyloid-beta plaques and neurofibrillary tangles. Symptoms of this devastating disease include behavioral changes and deterioration of higher cognitive functions. Impairment of neurogenesis has also been shown to occur in AD, which adversely impacts new neuronal cell growth, differentiation, and survival. This impairment possibly results from the cumulative effects of the various pathologies of AD. Preclinical studies have suggested that the administration of melatonin-the pineal hormone primarily responsible for the regulation of the circadian rhythm-targets the effects of AD pathologies and improves cognitive impairment. It is postulated that by mitigating the effect of these pathologies, melatonin can also rescue neurogenesis impairment. This review aims to explore the effect of AD pathologies on neurogenesis, as well as the mechanisms by which melatonin is able to ameliorate AD pathologies to potentially promote neurogenesis.
The objective of this study was to screen and identify α-amylase inhibitor peptides from Pinto bean. Five Pinto bean bioactive peptides were successfully identified: PPHMLP (P1), PLPWGAGF (P3), PPHMGGP (P6), PLPLHMLP (P7) and LSSLEMGSLGALFVCM (P9). Based on ELISA results, their promising optical density values were 1.27; 3.71, 1.67, 3.20 and 1.03, respectively, which indicated the binding interaction between the peptide and α-amylase occurred. The highest inhibitory activity (66.72%) of the chemically synthesized peptide was shown in SyP9 followed by SyP1 (48.86%), SyP3 (31.17%), SyP7 (27.88%) and SyP6 (23.96%). The IC50 values were 1.97, 8.96, 14.63, 18.45 and 20.56mgml(-1), respectively. Structure activity relationship study revealed that α-amylase was inhibited due to its residues of Ala230, Asp229, Asp326, Tyr54, Met195, Leu194 and His233 were bound. On the other hand, the residues of PBBP (i.e. histidine, proline and methionine) were found to have the highest potency in the binding interaction.
Chitinases play a vital role during the pathogenic invasion and immunosuppression in various organisms including invertebrates and vertebrates. In this study, we have investigated the participation of MrChit-3 (Macrobrachium rosenbergii Chitinase-3) during host-pathogenic interaction in freshwater prawn, M. rosenbergii. Quantitative real-time PCR analysis showed that the expression of MrChit-3 was up-regulated during bacterial, viral and laminarin challenge. Moreover, to understand the antimicrobial role of the GH18 domain, a putative membrane-targeting antimicrobial peptide (MrVG) was identified from the GH18 domain region of the protein and it was chemically synthesized. Physico-chemical features of the GH18 derived antimicrobial peptide (AMP) was assessed by various in silico tools and the antimicrobial property of the peptide was confirmed from in vitro studies. The membrane targeting mechanism of the peptide was determined by flow cytometry (FACS) and scanning electron microscope (SEM) analysis. Interestingly, the peptide was able to inhibit the growth of a chitinolytic fungal pathogen, Aspergillus niger, which was isolated from the shells of M. rosenbergii. The toxicity studies such as hemolysis activity on human blood erythrocytes and cell viability assay with primary kidney cells, HEK293 of MrVG revealed that the peptide was not involved in inducing any toxicity.
Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
A plasmid construct was designed in which the gene of antimicrobial peptide melittin is controlled by the tetracycline-responsive promoter of human cytomegalovirus, aided by a constitutively expressed trans-activator protein gene. Its vaginal administration and induction of melittin gene transcription with doxycycline markedly suppressed subsequent genital tract infection of mice by Mycoplasma hominis and Chlamydia trachomatis. At least half of the melittin-protected animals proved free of either pathogen within 3-4 weeks. Recombinant plasmids expressing genes of antimicrobial peptides hold much promise as agents for prevention and control of urogenital latent infections.
Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.