Expression of functional Anopheles merus alpha-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 subfasciatusalpha-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. subfasciatusalpha-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 trade mark system. Recombinant baculovirus preparations were used to infect Sf9 Spodoptera frugiperda insect cells. The A. merusalpha-amylase was successfully expressed as an active enzyme detected mainly in cell culture supernatants.
Chimoy Effio
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Instituto de Quimica, Universidade de São Paulo, São Paulo, Brazil.
Folgueras-Flatschart
A V
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Montor
W R
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Pernasetti
F M
FM
Pueyo
M T
MT
Sogayar
M C
MC
Effo
P C
PC
D000818:Animals; D000852:Anopheles / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D018929:Cell Culture Techniques; D002845:Chromatography; D001517:Coleoptera / Q000235:genetics*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D008969:Molecular Sequence Data; D018411:Spodoptera / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D000516:alpha-Amylases