Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Alexia Perryman - San Jose State University
Co-Author(s): Taylor McCann and Alberto A. Rascon, San Jose State University, San Jose, CA
Dengue, chikungunya, and yellow fever viruses are problematic in tropical and subtropical areas. The transmission of these viruses to humans can largely be attributed to female Aedes aegypti mosquitoes. Recently, cases of humans infected with the Dengue and chikungunya viruses have appeared in the United States as a result of the presence of these efficient biological vectors. Transmission occurs when the female Aedes aegypti mosquito acquires a bloodmeal needed to provide nutrients for egg development and maturation. Vital digestive enzymes expressed in the mosquito’s midgut are responsible for cleaving proteins into amino acids necessary for this process. Bloodmeal digestion occurs in a biphasic manner as protease activity can be detected 1 to 6 hours post blood meal, known as the early phase, with higher protease activity observed 12 to 36 hours post blood meal, known as the late phase. Aedes aegypti serine protease V, or AaSPV, is believed to be expressed in the midgut at fairly constant levels throughout the bloodmeal digestion process, yet the potential role it plays in blood meal digestion is not clear. Other serine proteases from the Aedes aegypti midgut contain a His-Ser-Asp catalytic triad; however, in AaSPV, the histidine is replaced by a leucine. The presence of the catalytic triad could be a key component in conferring proteolytic activity. Due to difficulty in obtaining soluble protease expression utilizing various growth experimental conditions, the pMAL bacterial expression vector was employed to produce a maltose binding protein (MBP) fusion with the AaSPV protease. AaSPV was originally cloned into the pET28a vector to yield an N-terminally his6-tagged protease, then subcloned into the pMAL vector in order to keep the N-terminal his6-tag. The MBP-AaSPV fusion was solubly expressed using E. coli and purified through a batch method using wheat starch. Cleavage of the MBP-AaSPV fusion protein with recombinant enterokinase will expose the N-terminal his6-tag and be purified to homogeneity using a Nickel column. This will allow us to study the protease in vitro and determine if it is a true digestive enzyme, playing a role in bloodmeal digestion.
Funder Acknowledgement(s): This work is supported in part by NIH grant number 5T34GM008253-28 to AP.
Faculty Advisor: Alberto Rascon,