Pilot production of the recombinant peptide toxin of Heteractis crispa as a potential analgesic by intein-mediated technology

• Published in: Protein expression and purification 2018-05, Vol.145, p.71-76
• Main Authors: Esipov, Roman S., Makarov, Dmitry A., Stepanenko, Vasily N., Kostromina, Maria A., Muravyova, Tatyana I., Andreev, Yaroslav A., Dyachenko, Igor A., Kozlov, Sergey A., Grishin, Evgeny V.
• Format: Article
• Language: English
• Subjects: Analgesic polypeptide; Animals; Chromatography - methods; Cloning, Molecular; Cnidarian Venoms - genetics; Cnidarian Venoms - isolation & purification; Escherichia coli - genetics; Fusion protein; Gene Expression; Intein; Inteins; Peptides - genetics; Peptides - isolation & purification; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant protein; Sea Anemones - metabolism; Toxin
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2017.12.011

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 940 mg of the recombinant APHC3 from 100 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. •The method developed by us enables to produce 940 mg of the recombinant APHC3 from 100 g of the inclusion bodies.•The fusion protein content in the inclusion bodies was 62%.•The latency of jumping response on the animal models was equal for recombinant and natural APHC3.•The suggested method is straightforward and cost efficient.