title: Kinetic analysis, expression pattern, and production of a recombinant fungal protease inhibitor of tasar silkworm Antheraea mylitta.
abstract: Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K                 (                   i                 )) of 60 nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India.
authors.forename: Sobhan
authors.initials: S
authors.lastname: Roy
authors.forename: Venugopal Rao
authors.initials: VR
authors.lastname: Ravipati
authors.forename: Suvankar
authors.initials: S
authors.lastname: Ghorai
authors.forename: Mrinmay
authors.initials: M
authors.lastname: Chakrabarti
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Das
authors.forename: Ananta Kumar
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D000818:Animals; D001236:Aspergillus oryzae / Q000187:drug effects; D005658:Fungi / Q000201:enzymology*; D015870:Gene Expression; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D009036:Moths / Q000201:enzymology / Q000235:genetics; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: C541006:FPI-1 protease inhibitor, Antheraea mylitta; D019476:Insect Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins