WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical—chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made...

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Bibliographic Details
Published in:Planta 2011-10, Vol.234 (4), p.723-735
Main Authors: Di Maro, Antimo, Farisei, Francesca, Panichi, Daniela, Severino, Valeria, Bruni, Natalia, Ficca, Anna Grazia, Ferranti, Pasquale, Capuzzi, Valeria, Tedeschi, Francesca, Poerio, Elia
Format: Article
Language:English
Subjects:
Agriculture
Amino Acid Sequence
Amino acids
Animals
Barley
Base Sequence
Biological and medical sciences
Biomedical and Life Sciences
Cattle
Chymotrypsin - antagonists & inhibitors
Complementary DNA
Consensus Sequence
DNA, Complementary - genetics
Ecology
Enzymes
Escherichia coli - genetics
Escherichia coli - metabolism
Forestry
Fundamental and applied biological sciences. Psychology
Insecta - enzymology
Life Sciences
Models, Molecular
Molecular Sequence Data
Molecular Weight
Original Article
Plant Proteins - chemistry
Plant Proteins - genetics
Plant Proteins - isolation & purification
Plant Proteins - metabolism
Plant Sciences
Protease inhibitors
Protease Inhibitors - chemistry
Protease Inhibitors - isolation & purification
Protease Inhibitors - metabolism
Protein isoforms
Proteins
RNA, Messenger - genetics
RNA, Plant - genetics
Sensitivity and Specificity
Sequence Alignment
Sequence Analysis, Protein
Three dimensional modeling
Triticum - chemistry
Triticum - metabolism
Trypsin inhibitors
Ungulates
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Summary:A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical—chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7—89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.
ISSN:0032-0935
1432-2048
DOI:10.1007/s00425-011-1437-5