Authors:

Morgan Grace Laskowski

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About Paper:

Cardiovascular disease is the leading cause of death among adults in the United States and worldwide. Phospholipase C (PLC) enzymes such as PLCb are important for healthy cardiovascular funcIon. These enzymes hydrolyze the membrane phospholipid phosphaIdylinositol-4,5-bisphosphate (PIP2) to generate the second messengers diacyclglycerol (DAG) and inositol-1,4,5 triphosphate (IP3), which acIvate PKC and release intracellular calcium stores, respecIvely. DysregulaIon of these second messenger pathways can lead to disease and heart failure. PLCb is regulated by the heterotrimeric G protein subunits Gaq and Gbg, but exactly how PLCb is recruited to and interacts with the plasma membrane requires further study. In this project, we aim to generate a HaloTag-PLCb3 construct for use in future microscopy studies with model membranes. We are using cloning, baculovirus producIon, and protein purificaIon techniques to work toward this goal. A PLCb3 construct in the baculoviral expression vector, pFastBac-HTA was linearized by PCR, and a Halo-7 Tag was added on the N- terminus of the protein via ligaIon-independent cloning. This construct will be used to produce baculovirus to express this fusion protein in insect cells, and purificaIon will be performed via affinity and size-exclusion chromatography. Following purificaIon, we will covalently label the protein with a fluorescent ligand via the HaloTag to further invesIgate how PLCb interacts with membranes in vitro. These findings will give insights into the kineIc behavior of PLCs on model membrane systems and increase our understanding of these criIcal enzymes. Keywords: Phospholipase C (PLC); Cloning; PLCB3

Source:

Purdue University / 2024

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Co-authors:

Morgan Grace Laskowski