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Recombinant Expression and Purification of Wild‐type JHA15 from Aedes aegypti
Ist Teil von
The FASEB journal, 2020-04, Vol.34 (S1), p.1-1
Erscheinungsjahr
2020
Link zum Volltext
Quelle
Wiley Online Library
Beschreibungen/Notizen
Abstract only The Aedes aegypti mosquito serves as a major vector of the Dengue, yellow fever, and zika viruses, which can be found in tropical regions around the world, as well in certain regions of the United States. The primary method of disease transmission is through the bite of an infected female mosquito taking a blood meal from an uninfected human host, which is a process necessary for the gonotrophic cycle. Once the blood meal is consumed, midgut proteases degrade blood meal proteins into amino acids and peptides that are then used in egg development. The common proteases found in the midgut, which are expressed after a blood meal, consist of chymotrypsins, trypsins, and exo‐peptidases. The focus of this study is on the JHA15 protease, which is believed to exhibit chymotrypsin‐like activity and is constantly expressed immediately after a blood meal. However, the exact role in this process has not been fully elucidated. Therefore, we are working towards understanding the catalytic and specific activity of the protease in vitro , which will help determine the specific role in the mosquito. To initiate these studies, JHA15 with the wild type pro‐peptide region was cloned into the pET28a vector. Once the gene was cloned, soluble protease expression was achieved utilizing Shuffle T7 competent Escherichia coli cells (NEB) grown in TB media at 10°C. The protease was purified with a Ni +2 ‐chelating column and purity analyzed through SDS‐PAGE. Future research will focus on activation and enzyme kinetics of the protease to determine if it is indeed involved in blood meal digestion.