title: Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus.
abstract: The Mexican bean weevil, Zabrotes subfasciatus, feeds on several seeds such as Vigna unguiculata, Phaseolus vulgaris, and Pisum sativum, causing severe crop losses. This ability to obtain essential compounds from different diets could possibly be explained due to a wide variability of digestive proteinases present in the weevil's midgut. These may improve digestion of many different dietary proteins. Coleopteran serine-like proteinases have not been thoroughly characterized at the molecular level. In this report, a full-length cDNA encoding a trypsin-like protein, named ZsTRYP, was isolated from Z. subfasciatus larvae using RT-PCR, 5' and 3' RACE techniques. The quantitative real-time PCR analysis strongly correlated the Zstryp transcript accumulation to the major feeding developmental larval stage. Zstryp cDNA was subcloned into pET101 vector and expressed in a Escherichia coli BL21(DE3) strain. Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to purify a 29.0-kDa recombinant enzyme. The purified ZsTRYP was then assayed with several synthetic peptide substrates and also challenged with different inhibitors. The biochemical data allowed us to classify ZsTRYP as a trypsin. Moreover, homology modeling analysis indicated a typical trypsin structural core and a conserved catalytic triad (His(41), Asp(86), and Ser(182)).
authors.affiliation: Embrapa Recursos Genéticos e Biotecnologia, Brasilia-DF, Brazil.
authors.forename: Cláudio P
authors.initials: CP
authors.lastname: Magalhães
authors.forename: Rodrigo R
authors.initials: RR
authors.lastname: Fragoso
authors.forename: Djair S L
authors.initials: DS
authors.lastname: Souza
authors.forename: Aulus E A D
authors.initials: AE
authors.lastname: Barbosa
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Silva
authors.forename: Flávio
authors.initials: F
authors.lastname: Finardi-Filho
authors.forename: Maria C M
authors.initials: MC
authors.lastname: da Silva
authors.forename: Thales L
authors.initials: TL
authors.lastname: Rocha
authors.forename: Octavio L
authors.initials: OL
authors.lastname: Franco
authors.forename: Maria F
authors.initials: MF
authors.lastname: Grossi-de-Sa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D017510:Protein Folding; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D048889:Weevils / Q000201:enzymology* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D012697:Serine Endopeptidases