Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization.
J Biotechnol
; 308: 124-129, 2020 Jan 20.
Article
en En
| MEDLINE
| ID: mdl-31837370
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Agaricales
/
Escherichia coli
/
Amanitinas
Idioma:
En
Revista:
J Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2020
Tipo del documento:
Article
Pais de publicación:
HOLANDA
/
HOLLAND
/
NETHERLANDS
/
NL
/
PAISES BAJOS
/
THE NETHERLANDS