In Vitro Activation of Purified Aedes Aegypti Mosquito Early Trypsin (AaET) Wild‐type in Comparison to the Inactive AaET Mutant (Active Site Serine to Alanine)

The female Aedes aegypti mosquito requires specific midgut proteases to degrade blood meals in a biphasic manner for completion of the gonotrophic cycle. Upon acquisition of multiple blood meals, the female anthrophophilic Aedes aegypti mosquito may become infected with several viruses, such as Dengue, Yellow fever, Chikugunya, and Zika viruses. Once infected, the mosquito can transmit these pathogens to its human hosts, making the mosquito an efficient biological vector. Current vector control strategies have not shown to be efficient, therefore studying and potentially inhibiting the required proteases for blood meal digestion could provide a new vector control strategy. This study is focusing on Aedes aegypti Early Trypsin (AaET), an early phase, trypsin‐like serine protease. This enzyme is one of four most abundant midgut proteases involved in blood meal digestion. The specific activity role of AaET wild‐type (WT) in blood meal digestion has yet to be fully elucidated. The enzyme has been solubly expressed using bacteria as a host, which has led to auto‐catalytic activity. Despite this, zymogen AaET (WT) has been successfully purified and activated using a specific buffer exchange approach that removes dithiothreitol (a reducing reagent that was added in order to inhibit auto degradation during the purification process). Proteolytic activity of zymogen and the active form of AaET (WT) were determined using a chromogenic trypsin substrate, BApNA, to compare to published results. To determine if AaET (WT) is truly autocatalytic, an AaET Mutant (MU) was constructed by mutating the Serine residue in the active site to an Alanine, and was subsequently purified to compare to the wild type form. Support or Funding Information SC3 Research Grant from NIH