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Keywords:

  • trehalase;
  • Chironumus riparius;
  • RACE-PCR;
  • touchdown PCR;
  • homology modeling

Trehalase is involved in the control of trehalose concentration, the main blood sugar in insects. Here, we describe the molecular cloning of the cDNA encoding for the soluble form of the trehalase from the midge larvae of Chironomus riparius, a well-known bioindicator of the quality of freshwater environments. Molecular cloning was achieved through multiple alignment of Diptera trehalase sequences, allowing the synthesis of internal homology-based primers; the complete open reading frame(ORF) was subsequently obtained through RACE-PCR(where RACE is rapid amplification of cDNA ends). The cDNA contained the 5′ untranslated region (UTR), the 3′ UTR including a poly(A) tail and the ORF of 1,725 bp consisting of 574 amino acid residues with a predicted molecular mass of 65,778 Da. Recombinant trehalase was successfully expressed in Escherichia coli as a His-tagged protein and purified on Ni-NTA affinity chromatography. Primary structure analysis showed a series of characteristic features shared by all insect trehalases, while three-dimensional structure prediction yielded the typical glucosidase fold, the two key residues involved in the catalytic mechanism being conserved. Production of recombinant insect trehalases opens the way to structural characterizations of the catalytic site, which might represent, among others, an element for reconsidering the enzyme as a target in pest insects’ control.