ABSTRACT
APHC3 is an analgesic polypeptide that was found in the sea anemone (Heteractis crispa), and contains 56 amino acid residues. This polypeptide is of interest for the development of medications for diseases, associated with inflammatory or neuropathological processes, as well as its use as an analgesic. This work presents an innovative biotechnological method for APHC3 production. We have constructed a recombinant plasmid intended for biosynthesizing the fusion protein consisting of a chitin-binding domain, DnaB mini-intein from Synechocystis sp. capable of undergoing pH-dependent self-cleavage, and the target peptide. In the process of biosynthesis the fusion protein aggregates and forms the inclusion bodies that are welcomed since APHC3 is a cytotoxic peptide. The target peptide recovery process developed by us involves 3 chromatographic steps. The method developed by us enables to produce 940â¯mg of the recombinant APHC3 from 100â¯g of the inclusion bodies. The method is straightforward to implement and scale up. The recombinant APHC3 activity and effectiveness as an analgesic was proved by animal testing.
Subject(s)
Chromatography/methods , Cnidarian Venoms/isolation & purification , Gene Expression , Inteins , Peptides/isolation & purification , Sea Anemones/metabolism , Animals , Cloning, Molecular , Cnidarian Venoms/genetics , Escherichia coli/genetics , Intercellular Signaling Peptides and Proteins , Peptides/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Method of highly sensitive registration of magnetic nanoparticles by their nonlinear magnetization is used in a novel sandwich-type immunoassay for detection of staphylococcal toxins in complex media of virtually any volume, with increasing sensitivity at higher sample volume. The signal is read out from the entire volume of a nontransparent 3D fiber structure employed as a solid phase, which provides large reaction surface, quick reagent mixing, as well as antigen immunofiltration directly in the course of the assay. The method has demonstrated near-linear dose-response curves within a wide range of ~3 decades, while detection of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST) in neat milk without sample preparation. The limits of detection (LOD) as low as 4 and 10 pg/mL for TSST and SEA, respectively, were obtained in 2-h format using 30-mL samples. The second, 25-min format, showed the LOD of 0.1 and 0.3 ng/mL for the same toxins in a 150 µL sample. The developed immunoassay can be applied in food safety control, in vitro diagnostics, and veterinary for a variety of research from express tests in the field to highly sensitive laboratory tests.
Subject(s)
Enterotoxins/analysis , Immunoassay , Magnetite Nanoparticles/chemistry , Animals , Enterotoxins/genetics , Mice , Mice, Inbred BALB CABSTRACT
A method for generation of highly specific miniantibodies within the phage particle has been developed, and used to produce antibodies against Staphylococcus enterotoxin type C1. Under successive panning of the non-immune phage miniantibody (scFv) library with enterotoxins SE (types A, B, C1, D, E, G, and I) adsorbed on the plate surface, we generated 11 individual phage clones to Staphylococcus enterotoxin type C1. Five of them interacted specifically only with SEC1 and had no cross-reactions with the other enterotoxins.