Alzheimer’s disease (AD) is a devastating form of dementia with 5 million people suffering from the disease in the United States alone. Thus, there is a need to understand the cause of AD so that therapeutics can be developed to treat it. The greatest risk factor for AD is the APOE gene, which encodes the apolipoprotein Eprotein. Three predominant APOE alleles exist humans, which give rise to three isoforms, called apoE2, E3, and E4. Individuals with apoE4 are ten times more likely to develop AD. In contrast, the apoE2 isoform has a neuroprotective effect and is associated with a 2-fold lower risk of developing AD. A growing body of evidence has demonstrated that apoE plays a major role in AD and cognitive aging in an isoform-specific manner, through either contributing to or counteracting AD pathogenesis. Our lab is interested in studying the molecular mechanisms of apoE in AD, but we first needed to establish a system to produce apoE protein in mammalian cells. Here, I present the culmination of the work I conducted in the Adams Lab establishing a system to evaluate apo isoform-specific effects in mammalian cells, focusing on the establishment of the 293 FreeStyle Expression system, a serum-free, high-density HEK cell culture system, to generate recombinant apoE in mammalian cells. From the generation of apoE expression constructs to the quantification of secreted apoE protein levels, this project establishes the tools necessary to study apoE in the context of AD in mammalian cell culture.
Dr. Kenneth W. Adams, Thesis Advisor
Dr. Heather H. Marella, Committee Member
Dr. Merideth K. Krevosky, Committee Member
Cunningham, Michaela. (2022). Establishing the Freestyle 293 Expression System as a Tool to Generate Recombinant Apolipoprotein E in Mammalian Cells. In BSU Honors Program Theses and Projects. Item 522. Available at: https://vc.bridgew.edu/honors_proj/522
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