Peptides and proteins with antifungal activity from medicinal plants

Introduction: Peptides and proteins are bio-macromolecules with diverse interesting bioactivities, formed of amino acids joined by peptidic bounds. Their resources include animals, plants, fungi, bacteria and insects. Various biological effects from these agents including antioxidant, antimicrobial, anti-inflammatory, anti-hypertensive and opioid activity have been reported. Antimicrobial peptides have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have demonstrated activities towards phytopathogens, as well as against human pathogenic organisms such as viruses, bacteria, fungi, protozoa and parasites. Antifungal proteins have captured the attention of a large number of investigators regarding to their economic implications. Plants are one of the attractive sources of antifungal peptides and proteins. Methods and Results: Searching keywords of bioactive peptides and proteins, antimicrobial, antifungal and plant in different search engines resulted in numerous published articles. According to the litritures, herbal antifungal proteins and peptides can be classified, based on their structures and/or functions, into groups including chitinases, glucanases, thaumatin-like proteins, thionins, and cyclophilin-like proteins. These proteins may demonstrate different fungal specificities. The mechanisms of antifungal actions of only several antifungal proteins including thaumatin-like proteins and chitinases have been elucidated. Some of the chitinase-like proteins have been reported from plants for instance: Dolichin, an antifungal protein from field beans (Dolichos lablab) with strong antifungal activity against Rhizoctonia solani, a 28 kDa, protein from cowpea (Vigna unguiculata) seeds, pinto bean (Phaseolus vulgaris cv. pinto) seeds, and ricebean (Delandia umbellata) seeds, another protein from Panax notoginseng (sanchi ginseng) roots with potent antifungal activity against F. oxysporum and Pananotin from sanchi ginseng roots. Conclusions: From this survey it can be concluded that different proteins and peptides of diverse structures are elaborated by a diversity of plants with wide antifungal activities. Some of these antifungal proteins have also exhibited antiviral inhibitory activitis toward the enzymes of human immunodeficiency virus

Use of Green Fluorescent Protein (GFP) Vector in Classical Restriction Enzyme-based Cloning Methods of Gateway Cloning System

Generating of an expression clone that can produce the pharmaceutical proteins in an efficient and soluble form at high levels is considered as an important step in pharmaceutical industry. Recombination-based cloning could be a quick and efficient way for generating expression vectors. Thus, both efficient and robust subcloning is vital for the construction of gene expression vectors. In this study, we used the traditional restriction enzyme-based cloning methods for generation of expression-ready clones by the most well-known commercial cloning technologies, Gateway

The Effect of Arginine as an Anti-Aggregation Excipient on Recombinant Human Growth Hormone

Aggregation is one of the main physical instabilities of proteins, which might occur during all steps of the manufacturing and storage of products. The presence of protein aggregates may result in the reduction of activity, induce immunologic responses and failure of therapeutic efficiency. Therefore, using additives in drug formulations is one of the essential approaches to prevent protein aggregation. The main objective of this study was to evaluate the inhibitory influence of arginine or glycine as excipients on the aggregation behavior of recombinant human growth hormone (rhGH). Two types of mechanical and thermal stresses including freeze-thaw and vortex-agitation were applied to the 1 mg/mL protein solution in PBS buffer (25 mM, pH = 7) in the presence and absence of arginine and glycine. The influence of arginine or glycine at the concentration of 320 mM on reduction of rhGH thermal/mechanical-induced aggregation was evaluated using SE-HPLC and turbidity measurement. The results of this study revealed that the monomer concentration decreased linearly; and therefore, aggregate formation was intensified with the increase in the number of freeze-thaw cycles. Moreover, it was found that a significant amount of rhGH (> 80%) was rapidly adsorbed at the walls of the vessels or converted to insoluble aggregates. Arginine decreased the insoluble aggregate formed during the freeze-thaw cycling more effectively than glycine. In addition, following the vortex-agitation stress, arginine had the optimum preventive effect in aggregate formation in contrast to glycine, which increased the formation of insoluble aggregates. The findings revealed that arginine may be a potential additive in preserving rhGH against thermal/mechanical-induced aggregation.Highlights Aggregation is one of the main physical instabilities of proteins.Protein aggregation may result in a compromise of safety and efficacy of biopharmaceutical products.Arginine at the concentration of 320 mM reduced rhGH thermal/mechanical-induced aggregation. Arginine, in contrast to glycine, optimally decreased the formation of insoluble aggregates

Concerns in the Design and Development of Novel Antimicrobial Peptides

Peptide and protein based therapeutics are the most promising approaches in today medicine. Bioactive peptides can be valuable drugs in the treatment of various illnesses, such as cardiovascular and neurodegenerative diseases. Cell toxic peptides can be considered for cancer or infection therapy. Antimicrobial peptides (AMPs) are one of the most interesting antibiotic groups in this regard, especially in drug resistance infections. Numerous AMPs have been discovered from the natural source; however, artificial synthetic ones have been also developed based on rational design or bioinformatics modeling. Physicochemical features of AMPs are highly important in their antibacterial activity as well as their toxicity. The best AMP is the one that has selective potent antimicrobial bioactivity and no or least hemolytic and cytotoxic effect. In this review, various structural factors affecting the AMPs bioactivity, such as AMPs size, charge, amphipathicity, and amino acid sequence are illustrated considering the most recently published articles. Finally, the trends in AMP design and development are discussed. HIGHLIGHTS•Antimicrobial peptides are highly interesting antibiotics in multi-drug resistance infections.•Antimicrobial peptides are short peptides with less than 50 amino acids and overall positive charge.•First and second structural features of AMPs are important factors in their bioactivities.•Cyclization and branching of AMPs could affect their pharmacokinetic and pharmacodynamics

The Relationship Between Janus Kinase Pathways and MicroRNAs

Janus Kinase (JAK) family is a group of four signaling enzymes composing of four distinct domains and involved in the intracellular pathways of cytokine downstream signaling. There are two kinase domains at C-terminal of protein, one of which is regulatory and the other has the main functionality in phosphorylation of target proteins. JAKs involve in the critical physiological processes, including immune response, growth, and differentiation. Mutations or malfunction of JAKs gene can result in pathological conditions like immuno-inflammatory diseases and malignancies. Targeting of JAK enzymes has been considered as effective therapeutic approaches in immuno-inflammatory disorders and different types of hematopoietic cancers or solid tumors. Rather than cytokines that are the natural modulators and the small chemical inhibitors developed as the therapeutic modulators of JAK enzymes, miRNAs can exert regulatory activity on JAKs. miRNAs are valuable biomarkers and regulatory elements of different pathophysiological conditions, particularly cancers. The relationships between JAK enzymes and miRNA are bi-directional, as the JAKs activity through JAK-STAT pathway as well as some other non-STAT pathways, control the expressions of various genes. These connections help scientists to design and develop novel therapeutic agents and predict the prognosis of disease following therapeutic regimens, based on these two critical components of cell biology. HIGHLIGHTS•Janus kinase family consists of four signaling enzymes involved in cytokine signaling pathways.•Modifications of JAK enzymes resulted in various pathological conditions.•JAK2 modification is reported in several types of cancers.•JAK modulators have been approved by FDA for treatment of several immunological and neoplastic disorders

Development and stability comparison of targeted therapeutic nanomolecules of aptamer-miRNA conjugates using two methods of conjugation

Introduction: An important issue in cancer therapy is the achievement of desired therapeutic response with the least adverse effects. To achieve this goal, targeted drug delivery systems were developed. Aptamers, mainly DNA/RNA aptamers, are the attractive affinity ligands for the cancer cell surface specific antigens. Besides, microRNAs are another type of therapeutic and diagnostic oligonucleotides that have been recently studied in various cancers. miRNAs are small double stranded RNAs with important roles in cell regulatory pathways. Profile changes of miRNAs can result in cancer development. External addition of miRNAs or their elimination using antagomiR can lead to the efficient treatment of related disease. Targeted delivery of therapeutic agents to the site of action with less adverse effects is the most challenging issue in anticancer chemotherapeutic agents as well as miRNA therapy. In addition, miRNAs stability in biological systems can be improved by targeting strategy. In this study, a cancer specific aptamer (anti-nucleolin aptamer) and a functional miRNA in cell growth and proliferation (miRNAlet-7d) were used in the development of targeted nano-molecules as an efficient anti-proliferative agent for cancer cells.   Methods and Results: Sequences of A1411 aptamer and miRNA let-7d were extracted from related databanks and were chemically synthesized with amine and thiol modification in the 3' terminals or with a 17 nucleotides sticky extension at 3' terminal.  The sequences were conjugated covalently using SM(PEG)2 hetero-bi-functional cross-linker or un-covalently by annealing the sticky ends. Conjugation was confirmed using polyacrylamide gel electrophoresis 15%. The serum stability of these two types of conjugates were evaluated using up to 48 h incubation of conjugates in human serum (AB+). Stability of covalent conjugate using SM(PEG)2 linker was at least two hours more than the un-covalent one.   Conclusions: Remarkable advantages of this nano-molecule were targeted and relative stable delivery of miRNA as the therapeutic agent with probable synergistic effect of two oligonucleotides of miRNA and aptamer in the proliferation inhibition of cancer cells

Direct Immobilization of Coagulation Factor VIII on Au/Fe3O4 Shell/Core Magnetic Nanoparticles for Analytical Application

Protein-coated nanoparticles have diverse applications in biomedical science. The protein hydrophobic domains or surface electrostatic charge conducts adsorption of proteins to different surfaces. This property can be customized to immobilize specific molecules on solid supports for experimental screenings or purification processes. To develop highly selective affinity ligands—such as aptamers—against specific protein targets, protein-coated magnetic particles have been successfully applied. This approach could be highly efficient in affinity ligand development against coagulation factor VIII.In this study, magnetic nanoparticles were prepared by co-precipitation method and, then, a gold coating was run on the MNPs’ surface. The gold coating could add some attractive specifications to the protein immobilized nanoparticles during the aptamer selection process, such as simultaneous affinity determination of aptameric oligonucleotides by fluorescence-based methods. The gold surface has been indicated as a specific feature for covalent binding to the sulphur functional groups of various molecules. In proteins, sulphur units of cysteine or methionine might be bound covalently to the gold surface. In addition, nonspecific and non-covalent attachment of proteins to the gold particles may be performed. Therefore, a series of samples containing different mass ratios of protein to gold magnetic nanoparticles (GMNPs) were evaluated to find the best conditions for coagulation factor VIII immobilization. The results showed that the best condition for high coating efficiency was 48 h incubation at 4 ºC of protein and GMNPs with a mass ratio of 0.5% in PBS 25mM, with pH=7 as binding buffer.Highlights:Magnetic nanoparticles are the most attractive nanostructures in biomedical and bio-analytical fields.The protein coating on MNPs has been found to have wide clinical and analytical applications.Coagulation factor VIII (FVIII) is a valuable therapeutic human protein in the market.Attachment of a large protein like F VIII to GMNPs is affected by various environmental factors

Optimization of Aflatoxin B1 Aptasensing

Combination of aptamers with DNAzymes attracted intense attention for development of DNA-based biosensors for detection of mycotoxins. In the present study a combination of aflatoxin B1 specific aptamer and HRP- (horseradish peroxidase-) mimicking DNAzyme was optimized for detecting aflatoxin B1. Detecting approach is based on the binding affinity of aflatoxin B1 to its specific aptamer and conversion of substrate to a detectable colorimetric signal by a linked DNAzyme. Compared to conventional methods for aflatoxin B1 detection, DNA-based assay has the advantages of low cost, long-term stability, and rapid, simple, and user-friendly steps

Author Package, TPPS, Vol. 4 (2019)

The Trends in Peptide and Protein Sciences is a peer-reviewed, online-only (previously print-online), scientific journal owned by Protein Technology Research Center, Shahid Beheshti University of Medical Sciences and documents in all important aspects of the research in peptides and proteins focusing on analytics and impurities, bioinformatics, biopharmaceuticals and vaccines, biotechnology, chemical synthesis, conformational analysis, design and  development of protein therapeutics, determination of structure, enzymology, folding and sequencing,  formulation and stability, function, genetics,  immunology, kinetics, modeling, molecular biology, pharmacokinetics and pharmacodynamics of therapeutic proteins and antibodies, pharmacology,  protein engineering and development, protein-protein interaction, proteomics, purification/expression/production, simulation, thermodynamics and  hydrodynamics and protein biomarkers. The aim of this Journal is to publish high quality original research articles, reviews, short communications and letters and to provide a medium for scientists and researchers to share their findings from the area of peptides and proteins. The Trends in Peptide and Protein Sciences is published in collaboration with Iranian Association of Pharmaceutical Scientists. From volume 3 (2018) of TPPS, articles are continuously published online only, as soon as the review process is completed