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In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti by Alberto A Rascón; Johnathon Gearin; Jun Isoe; Roger L Miesfeld is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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## Background The major Dengue virus vector __Aedes aegypti__ requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of __Ae. aegypti__ midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes. ## Results We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k~cat~/K~M~) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed

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Author

Alberto A Rascón; Johnathon Gearin; Jun Isoe; Roger L Miesfeld

Publisher

BioMed Central; Springer (Biomed Central Ltd.); London: BioMed Central; Springer Science and Business Media LLC (ISSN 1471-2091)

Published

2011

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)