Peptide synthesis: chemical or enzymatic

Peptides are molecules of paramount importance in the fields of health care and nutrition. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant. The present review pretends to establish a non-biased appreciation of the advantages, potentials, drawbacks and limitations of both technologies. Chemical synthesis is thoroughly reviewed and their potentials and limitations assessed, focusing on the different strategies and challenges for large-scale synthesis. Then, the enzymatic synthesis of peptides with proteolytic enzymes is reviewed considering medium, biocatalyst and substrate engineering, and recent advances and challenges in the field are analyzed. Even though chemical synthesis is the most mature technology for peptide synthesis, lack of specificity and environmental burden are severe drawbacks that can in principle be successfully overcame by enzyme biocatalysis. However, productivity of enzymatic synthesis is lower, costs of biocatalysts are usually high and no protocols exist for its validation and scale-up, representing challenges that are being actively confronted by intense research and development in this area. The combination of chemical and enzymatic synthesis is probably the way to go, since the good properties of each technology can be synergistically used in the context of one process objective

Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout ( Oncorhynchus mykiss )

Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays

Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids

Background: Tumour necrosis factor-alpha (TNF-\u3b1) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-\u3b1 has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-\u3b1 detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-\u3b1 antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-\u3b1 of salmonid fish. TNF-\u3b1 epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-\u3b1, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-\u3b1 positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-\u3b1 function

Anti-peptide antibodies: A tool for detecting IL-8 in salmonids

Background: Interleukin 8 is a chemokine that is produced by several types of cells, like macrophages and has chemotactic activity in particular on neutrophils, playing a key role during the inflammatory process. It has been demonstrated at the molecular level that this molecule is present and conserved in several vertebrate groups, pointing its importance. Analysis of the amino acid sequence of IL-8, projected from cDNA of Salmo salar , presents homology with the sequences of mammals, poultry and lamprey, indicating the presence of a homologous molecule in higher fish. However, there is no information at protein level, which allows characterizing the regulatory role of this molecule during the immune response in fish. Results: In this work, we designed and synthesized an epitope peptide of 10 residues with a purity of 95% and mass of 1158.7 kDa, which showed a random coil structure. From this peptide it was able to generate a polyclonal mono-specific antibody which was capable of detecting the whole molecule of IL-8 in tissue and cellular model of salmonids. Conclusions: The resulting antibody is a versatile tool for detecting IL-8 by different immune techniques such as ELISA, dot blot, western blotting and immunocytofluorescence. Analysis of IL-8 at proteomic level is a useful method for characterizing immune properties of this molecule in fish