Molecular cloning, sequencing and expression of a cDNA encoding an intestinal trehalase from Spodoptera frugiperda (SfTre1).
Silva, M.C.P., Terra, W. & Ferreira, C.
Departamento de Bioquímica, Instituto de Química, USP-SP, São Paulo.
Trehalases are enzymes that play a central role in insect, Nematoda and fungi. Nevertheless, neither their tertiary structure nor their catalytical groups are known.
Total RNA was isolated from larval midgut tissues and reverse transcriptions were performed with an oligo-(dT) primer. Degenerate primers corresponding to conserved regions in insect trehalases were also used to amplify a PCR product corresponding to the SfTre1 cDNA. Using specific primers based on this sequence for PCR, we obtained the complete cDNA sequence. The putative coded enzyme has 587 amino acids, a signal peptide with 23 amino acids and 6 glycosilation sites. The sequence shows highest identity and similarity ( 61 and 76%, respectively), to the soluble digestive trehalase from Bombyx mori.
SfTre1 cDNA was subcloned in pET-28a expression vector (pET-28a-Tre), which was used to transform E. coli BL21 DE3 strain. The cells were grown and treated with 1 mM IPTG for 5 h (37°C or 25°C, 250 r.p.m.) or for 15 h (20°C, 250 r.p.m.). SfTre1 was found mainly in inclusion bodies but the specific activity found in soluble fraction of cells induced at 20 0C for 16h ( 3,905 mU/mg) is high enough to be used in our future studies.
Soluble SfTre1 was purified using a 1 mL HisTrapFF column, showing a single band with 62 kDa after SDS-PAGE. Purified SfTre1 has Km, half-life of thermal inactivation at 50°C and specific activity similar to the values found for the soluble trehalase purified from S. frugiperda midguts.
Supported by FAPESP and CNPq.
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