A Biochemical Study of The Autocatalytic Nature of a Serine Protease (Early Trypsin) From The Aedes aegypti Female Mosquito

The female Aedes aegypti mosquito requires specific midgut proteases to digest blood meals in a biphasic manner for completion of the gonotrophic cycle. The anautogenous nature of the female mosquito has led to the mosquito in becoming an efficient vector in transmitting several bloodborne pathogens: Chikungunya, Yellow Fever, Dengue and the Zika virus. With the many flaws associated with current vector control strategies, our research lab is focused on the development of a potential novel vector control strategy by targeting midgut proteases involved in blood meal digestion. The role of one of the four most abundantly expressed proteases, AaET, an early phase trypsin-like serine protease, is the focus of investigation. Characterization of AaET is needed to understand the overall relationship between the mosquito midgut digestion and viral transmission. N-terminal and C-terminal His6-tagged AaET, AaET with a pseudo propeptide (EK), and two mutant AaET genes were cloned and recombinantly expressed in bacterial cells. N-terminal His6-tagged AaET and AaET-EK were purified and tested for activity in vitro. The inactive AaET (WT) zymogen potentially exhibits autocatalytic behavior contributing to purification challenges and low protein yields