Molecular and Structural Characterization of a Trypsin Highly Expressed in Larval Stage of Zabrotes subfasciatus

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 (His41, Asp86, and Ser182). Arch. Insect Biochem. Physiol. 66:169–182, 2007. © 2007 Wiley-Liss, Inc.