cDNA cloning and heterologous expression of a wheat proteinase inhibitor of subtilisin and chymotrypsin (WSCI) that interferes with digestive enzymes of insect pests.
A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production, wsci cDNA was cloned into the E. coli vector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae ( Helicoverpa armigera , Plodia interpunctella and Tenebrio molitor ). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.
Di Gennaro
Simone
S
Department of Agrobiologia e Agrochimica, Universitá della Tuscia, Italy.
Ficca
Anna G
AG
Panichi
Daniela
D
Poerio
Elia
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D019476:Insect Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007313:Insecta / Q000187:drug effects / Q000201:enzymology*; D007814:Larva / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D020860:Subtilisin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013552:Swine; D014908:Triticum / Q000201:enzymology*
D018076:DNA, Complementary; D019476:Insect Proteins; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C504099:WSCI protein, Triticum; D002918:Chymotrypsin; D020860:Subtilisin