Abstract 1456 Expression, Purification, and Enzymology of GAPDH from the Venom of the Parasitoid Wasp Ganaspis hookeri

G. hookeri are obligate parasitoid wasps that inject Drosophila larvae with their eggs and venom. Upon infection by the wasp, the fly initiates an immune response that results in encapsulation of the wasp egg alongside metabolic changes to support an increase in metabolic requirements. The infection results in a switch from oxidative phosphorylation to glycolysis in the hemocytes of the host Drosophila. In insects, gluconeogenesis in the fat body, a liver-like tissue, generates glucose which is then converted to trehalose and transported to the hemolymph where it is available for use to fight off infection. G. hookeri venom contains 168 proteins, eight of which are glycolytic enzymes with only hexokinase and phosphofructokinase being absent. Our goal is to determine the role of these glycolytic enzymes in promoting the wasp infection. This study focuses on glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A vector (pET23a) encoding GAPDH with a hexahistidine tag attached at the carboxyl terminus was expressed in E. coli. SDS-PAGE and immunoblotting were used to confirm that GAPDH was expressed and in a soluble form. The fusion protein was purified using nickel column chromatography and mass spectrometry, both MALDI and LC-MS/MS, were used to validate the purified proteins. Michaelis Menten kinetics will be performed to compare the kinetic constants of the venom GAPDH to that of Drosophila melanogaster GAPDH. This work was carried out as a course-based undergraduate research (CURE) project at Siena College, Oklahoma Christian University, and UNC Pembroke, and externally funded by NIH R25 IPERT grant 1R25GM130528-01A1 to PIs R. Kelly and C. Goller at North Carolina State University and Co-PI M. Santisteban at UNC Pembroke, and NIH R35GM133760 to NTM.