TY - JOUR
T1 - Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization
AU - Pathiraja, Duleepa
AU - Byun, Juan
AU - Lee, Saeyoung
AU - Choi, In Geol
PY - 2020/1/20
Y1 - 2020/1/20
N2 - 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.
AB - 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.
KW - Cyclic peptide
KW - Prolyl oligopeptidase
KW - RiPP
KW - α-Amanitin
UR - http://www.scopus.com/inward/record.url?scp=85076700078&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85076700078&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2019.12.004
DO - 10.1016/j.jbiotec.2019.12.004
M3 - Article
C2 - 31837370
AN - SCOPUS:85076700078
VL - 308
SP - 124
EP - 129
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
ER -