REVISITING RUBISCO AS A PROTEIN SUBSTRATE FOR INSECT MIDGUT PROTEASES

Authors

  • Usha Bhardwaj,

    Corresponding author
    1. Plant-Insect Interactions Group, Department of Botany, Delhi University, Delhi, India
    • Correspondence to: U. Bhardwaj, Plant-Insect Interactions Group, Department of Botany, Delhi University, Delhi 110007, India. E-mail: ushakhatri@gmail.com

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  • Amit Bhardwaj,

    1. Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
    Current affiliation:
    1. International Center of Public Health, New Jersey Medical School, Rutgers, Newark, New Jersey 07103, USA
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  • Rakesh Kumar,

    1. Plant-Insect Interactions Group, Department of Botany, Delhi University, Delhi, India
    Current affiliation:
    1. Center for Environmental Management of Degraded Ecosystems, Delhi University, Delhi, India
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  • Sadhu Leelavathi,

    1. Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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  • Vanga Siva Reddy,

    1. Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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  • Sudeshna Mazumdar-Leighton

    1. Plant-Insect Interactions Group, Department of Botany, Delhi University, Delhi, India
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  • Grant sponsor: Funding from University Grants Commission to Delhi University for strengthening R & D.

Abstract

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.

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