MOLECULAR CLONING OF A COMPLIMENTARY DNA SEQUENCE ENCODING A CUTICLE DEGRADING PROTEASE PRODUCED BY ENTOMOPATHOGENIC FUNGI
Patent No. 5,962,765
Issued: October 5, 1999
Inventors: Raymond St.Leger, Donald W. Roberts, and Richard C. Staples, Ithaca, NY
We have studied the regulation of the extracellular chymoelastase protease (Pr1) of Metarhizium anisopliae, an enzyme involved in the penetration of insect cuticle by Metarhizium and other entomopathogenic fungi. We report here the isolation and characterization of a Pr1 cDNA clone with a full length insert. Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide and a propeptide and the mature protein is predicted to have a relative molecular mass of 28.6 kDa. The primary structure of Pr1 shares extensive homology (30-60%) with enzymes of the subtilisin subclass of the serine endopeptidases and the serine, histidine and aspartyl components of the active site in subtilisins are preserved. The genes coding for chymoelastase or slightly altered versions thereof, can be used to transform various organisms (i.e. fungi, viruses, plants, bacteria, etc.) such that the transformed organisms are capable of producing chymoelastase in recoverable quantities. Fragments and derviatives of a DNA sequence coding for a chymoelastase could be used to code for a polypeptide having an activity which can: a) bind to insect cuticle; b) enhance signal processing of proteins; c) hydrolyse polypepetides; d) suppress protease expression; or e) be used as a probe to identify homologous genes in organisms. While chymoelastases and Pr1 have been previously isolated, new and novel uses for chymoelastase are disclosed, wherein the chymoelastase is used to selectively degrade protein in the presence of non-protein polymers. A new insecticide insecticide is disclosed which comprises a recombinant virus, microorganism, cell, plant or fungi infects, is eaten by or otherwise taken up by, an insect and expresses the enzyme Pr1 within said insect such that Pr1 activates a prophenoloxidase system within said insect.
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