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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL AND BIOTECHNOLOGY APPLICATIONS FOR INSECT PEST MANAGEMENT Title: Characterisation of functional and insecticidal properties of a cathepsin L-like proteinase from flesh fly (Sacrophaga peregrina) involved in differentiation of imaginal discs.

Authors
item Phillip, Judith - DURHAM UNIVERSITY
item Fitches, Elaine - CENTRAL SCIENCE LAB
item Harrison, Robert
item Bonning, Bryony - IOWA STATE UNIV.
item Gatehouse, John - DURHAM UNIVERSITY

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 5, 2007
Publication Date: June 1, 2007
Citation: Phillip, J., Fitches, E., Harrison, R.L., Bonning, B., Gatehouse, J.A. Characterisation of functional and insecticidal properties of a cathepsin L-like proteinase from flesh fly (Sacrophaga peregrina) involved in differentiation of imaginal discs. Insect Biochemistry and Molecular Biology. 37:589-60.

Interpretive Summary: Insect pests cause billions of dollars of damage to crops each year. The use of chemical insecticides to control insect pests can have negative ecological, environmental, and health consequences. Proteins that selectively kill insect pests without harming plants and other animals potentially can be used in place of chemical insecticides to protect crops. In this study, the biochemical properties of an insecticidal protein (a cysteine protease) were examined. The requirements for the insecticidal activity of this protein were determined. The information in this study will help researchers develop strategies for using this particular class of insect protein in crop protection.

Technical Abstract: ScathL is a cathepsin L-like cysteine proteinase from Sacrophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs. The protein has a potential insecticidal activity, since recombinant baculoviruses engineered to express ScathL have an enhanced ability to kill lepidopteran larvae. An expression system based on the methylotrophic yeast Pichia pastoris was used to produce ScathL in quantities sufficient for purification and characterisation. Although the expression construct contained the full proprotein coding sequence for ScathL, the proprotein polypeptide could only be detected in culture supernatant at early stages of expression. Purified recombinant protein, contained only a polypeptide corresponding to mature ScathL as a result of autocatalytic activation. Purified recombinant ScathL was active in the presence of reducing agents,and could be readily inactivated by oxidation. The enzyme hydrolysed a typical cathepsin L substrate, Z-Phe-Arg-AMC, with optimal activity at pH 5.5. ScathL was sensitive to pH, losing activity rapidly at pH values above 6.0, and showed decreasing activity with increasing ionic strength. The enzyme showed limited proteolytic activity towards protein substrates, with digestion resulting in large uncleaved fragments. ScathL was found to be insecticidal towards larvae of the tomato moth, Lacanobia oleracera, following injection into the haemolymph, in a dose-dependent manner. Production of a mutant form of ScathL, ScathLcys-ala, in which the active site cysteine was replaced by alanine, showed that enzyme activity was necessary for the complete proprotein processing observed during production as a recombinant protein, and for insecticidal activity against lepidopteran larvae.

Last Modified: 8/27/2014
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