Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs.
ScathL is a cathepsin L-like cysteine proteinase from Sarcophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs, through proteolysis of components of basement membranes. An expression system based on the methylotrophic yeast Pichia pastoris was used to produce recombinant ScathL. Although the expression construct contained the full proprotein coding sequence for ScathL, the proprotein was only detected in culture supernatant at early stages of expression by Western blotting. The purified recombinant protein contained only a polypeptide similar to mature ScathL, as a result of autocatalytic processing. After activation by reducing agents, the enzyme hydrolysed the cathepsin L substrate Z-Phe-Arg-AMC, with optimal activity at pH 5.5. ScathL showed decreasing activity with increasing ionic strength above 0.3M NaCl, and lost activity irreversibly at pH > or = 7.5. The enzyme showed limited activity towards protein substrates, digesting only to large fragments. ScathL was insecticidal towards larvae of the tomato moth, Lacanobia oleracera, following injection into the haemolymph. It caused melanisation, although no evidence of extensive proteolysis in haemolymph or gut was observed. Production of a inactive mutant form of ScathL showed that enzyme activity was necessary for the complete proprotein processing observed during production as a recombinant protein, and for insecticidal activity.
Philip
Judith M D
JM
School of Biological and Biomedical Sciences, Durham University, South Road, Durham, UK.
Fitches
Elaine
E
Harrison
Robert L
RL
Bonning
Bryony
B
Gatehouse
John A
JA
D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004175:Diptera / Q000201:enzymology* / Q000254:growth & development; D004789:Enzyme Activation; D006458:Hemolymph / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000494:pharmacology; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects; D009154:Mutation; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012492:Salts / Q000378:metabolism
D007306:Insecticides; D011994:Recombinant Proteins; D012492:Salts; D003546:Cysteine Endopeptidases