Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization

Duleepa Pathiraja, Juan Byun, Saeyoung Lee, In Geol Choi

Research output: Contribution to journalArticle

Abstract

Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development.

Original languageEnglish
Pages (from-to)124-129
Number of pages6
JournalJournal of Biotechnology
Volume308
DOIs
Publication statusPublished - 2020 Jan 20

Fingerprint

Amanitins
Fungal Proteins
Mycotoxins
Cyclization
Escherichia coli
Peptides
Lead compounds
Gene encoding
RNA
Agaricales
Biomass
Fusion reactions
Solubility
prolyl oligopeptidase
Spectroscopy
Proteins
Cyclic Peptides
RNA Polymerase II
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Post Translational Protein Processing

Keywords

  • Cyclic peptide
  • Prolyl oligopeptidase
  • RiPP
  • α-Amanitin

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization. / Pathiraja, Duleepa; Byun, Juan; Lee, Saeyoung; Choi, In Geol.

In: Journal of Biotechnology, Vol. 308, 20.01.2020, p. 124-129.

Research output: Contribution to journalArticle

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