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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.
Insect Biochem Mol Biol. 2007 Jun; 37(6):589-600.IB

Abstract

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.

Authors+Show Affiliations

School of Biological and Biomedical Sciences, Durham University, South Road, Durham, UK.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

17517336

Citation

Philip, Judith M D., et al. "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." Insect Biochemistry and Molecular Biology, vol. 37, no. 6, 2007, pp. 589-600.
Philip JM, Fitches E, Harrison RL, et al. 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. Insect Biochem Mol Biol. 2007;37(6):589-600.
Philip, J. M., Fitches, E., Harrison, R. L., Bonning, B., & Gatehouse, J. A. (2007). 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. Insect Biochemistry and Molecular Biology, 37(6), 589-600.
Philip JM, et al. 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. Insect Biochem Mol Biol. 2007;37(6):589-600. PubMed PMID: 17517336.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Philip,Judith M D, AU - Fitches,Elaine, AU - Harrison,Robert L, AU - Bonning,Bryony, AU - Gatehouse,John A, Y1 - 2007/03/12/ PY - 2006/12/15/received PY - 2007/03/01/revised PY - 2007/03/05/accepted PY - 2007/5/23/pubmed PY - 2007/7/26/medline PY - 2007/5/23/entrez SP - 589 EP - 600 JF - Insect biochemistry and molecular biology JO - Insect Biochem Mol Biol VL - 37 IS - 6 N2 - 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. SN - 0965-1748 UR - https://www.unboundmedicine.com/medline/citation/17517336/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0965-1748(07)00059-8 DB - PRIME DP - Unbound Medicine ER -