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Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta

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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.

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Acknowledgments

This work was supported by a grant from the Indian Council of Medical Research, Government of India.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Sobhan Roy, Venugopal Rao Ravipati, Suvankar Ghorai, Mrinmay Chakrabarti, Amit Kumar Das & Ananta Kumar Ghosh

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  1. Sobhan Roy
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  2. Venugopal Rao Ravipati
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  3. Suvankar Ghorai
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  5. Amit Kumar Das
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  6. Ananta Kumar Ghosh
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Correspondence to Ananta Kumar Ghosh.

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Roy, S., Ravipati, V.R., Ghorai, S. et al. Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta . Appl Biochem Biotechnol 168, 1076–1085 (2012). https://doi.org/10.1007/s12010-012-9842-1

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