Expression of functional Anopheles merus α-amylase in the baculovirus/Spodoptera frugiperda system

The Anopheles merus ( Diptera, Nematocera, Culicoidea) alpha-amylase gene (AmerAmy, GenBank Accession Number U01210) was amplified with its own or with the Zabrotes subfasciatus alpha-amylase signal peptide (ZsAmerAmy, GenBank Accession Number AY270183) by PCR, using designed primers. The AmerAmy gene was sequenced from its promotor to the TGA codon. As a positive control, the Z. subfasciatus alpha-amylase gene with its own signal peptide (ZsAmy, GenBank Accession Number AF255722) was also amplified by PCR. These three sequences were inserted into the baculovirus genome using the Bac-to-Bac(TM) system. Recombinant baculovirus preparations were used to infect Sf9 Spodoptera frugiperda insect cells. The A. merus alpha-amylase was successfully expressed as an active enzyme detected mainly in cell culture supernatants.