Publication Date

Summer 2023

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Advisor

Alberto A. Rascón Jr.; Ningkun Wang; Gianmarc Graziolli

Abstract

The female Aedes aegypti mosquito is a primary vector for the transmission of blood–borne viral pathogens such as Chikungunya, Zika, Yellow Fever, and Dengue. The female mosquito will become infected with these pathogens through the uptake of a blood meal from an infected host. The midgut digestive enzymes are required to digest the blood meal to obtain necessary nutrients needed for the completion of the gonotrophic cycle. Blood meal digestion is biphasic and digestive enzymes are expressed at different time points after uptake of a blood meal. The focus of this study is on an early phase protease known as Aedes aegypti Early Trypsin (AaET). Initial attempts at obtaining soluble inactive zymogen led to autoactivation and low abundance of recombinantly expressed protease, making it difficult to isolate enough enzyme for activity assays and structural studies. Thus, an optimized protocol was developed to improve proper folding of the protease as well as to produce an abundance of the inactive zymogen form. The AaET gene was cloned into the pET28a expression vector and transformed into E. coli T7 SHuffle cells. Several growth conditions were tested, such as varying growth temperatures, concentration of the inducer (IPTG), and the presence or absence of a small molecule osmolyte (betaine). From these studies, bacterial growth in TB media at 10℃ with 0.025 mM IPTG and the addition of 1 mM betaine resulted in the best and most successful production of soluble recombinant AaET in the inactive form. Future research will focus on protein purification, enzyme kinetics, proteolytic specificity, and cleavage of blood meal protein substrates.

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