@article {466,
	title = {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},
	volume = {37},
	year = {Submitted},
	note = {08-10-200906-02-2012NormalSchool of Biological and Biomedical Sciences, Durham University, South Road, Durham, UK.enghttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve\&db=pubmed\&dopt=Abstract\&list_uids=17517336},
	pages = {589 - 600},
	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 \&gt; 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.},
	keywords = {Amino Acid Sequence, Animals, Cysteine Endopeptidases/genetics/isolation \&amp, development, Diptera/*enzymology/growth \&amp, Enzyme Activation, Hemolymph/metabolism, Hydrogen-Ion Concentration, Insecticides/pharmacology, Larva/drug effects, Lepidoptera/drug effects, Mutation, Pichia/genetics, purification/*metabolism/pharmacology, purification/metabolism/pharmacology, Recombinant Proteins/genetics/isolation \&amp, Salts/metabolism},
	author = {Philip, J. M. and Fitches, E. and Harrison, R. L. and Bonning, B. and Gatehouse, J. A.}
}
@article {468,
	title = {Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein},
	volume = {37},
	year = {Submitted},
	note = {08-10-200906-02-2012NormalLaboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan.enghttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve\&db=pubmed\&dopt=Abstract\&list_uids=17681235},
	pages = {960 - 968},
	abstract = {A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.},
	keywords = {Amino Acid Sequence, Animals, Cathepsins/drug effects/isolation \&amp, Cloning, Molecular, Conserved Sequence, Cysteine Proteinase Inhibitors/genetics/*isolation \&amp, development, DNA Primers, Escherichia coli/genetics, Hemolymph/*chemistry, Humans, inhibitors, Larva, Manduca/*growth \&amp, Molecular Sequence Data, Papain/antagonists \&amp, Polymerase Chain Reaction, purification/metabolism, purification/pharmacology, Recombinant Proteins/isolation \&amp, Sequence Alignment, Sequence Homology, Amino Acid},
	author = {Miyaji, T. and Kouzuma, Y. and Yaguchi, J. and Matsumoto, R. and Kanost, M. R. and Kramer, K. J. and Yonekura, M.}
}
