Molecular Cloning, Gene Structure, Expression, and Enzyme Activity of a Serine Protease from Water Scorpion, Laccotrephes japonensis (Hemiptera: Nepidae)

Abstract

Serine proteases are major insect enzymes involved in the digestion of dietary proteins and in the process of blood meal digestion. In this study, cDNA was constructed using the whole body of Laccotrephes japonensis. The flanking sequences of the 5-and 3-end of this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained a 963-bp ORF encoding 320 amino acids. The deduced amino acid sequence showed 62% identity with the Creontiades dilutus serine protease, 58% with the Lygus lineolaris trypsin precursor, and 54% with the Triatoma infestans salivary trypsin. To assess the expression of the L. japonensis serine protease (JGsp), the JGsp gene was cloned into a baculovirus transfer vector, pBac-1, and expressed in Sf9 cells (Spodoptera frugiperda). SDS-PAGE and western blot analysis have shown that the JGsp recombinant protein was a monomer with a molecular weight of about 32 kDa. Recombinant JGsp has shown activity in the protease enzyme assay using gelatin as a substrate.