Title

Fortilin- An Evaluation of Affinity Tag Viability for the Use in Mass Production of Recombinant Protein

Document Type

Student Presentation

Presentation Date

4-15-2019

College

College of Arts and Sciences

Department

Department of Chemistry & Biochemistry

Faculty Sponsor

Dr. Lisa Warner

Abstract

Fortilin is a novel, antiapoptotic protein found in the nucleus, cytosol, and extracellular space. Fortilin’s anti-apoptotic action can lead to atherosclerosis and subsequent cardiac pathogenesis through a foam cell sparing action. Inhibition of fortilin could lead to a novel treatment for heart disease. Novel inhibitors of fortilin have been developed, however, the mechanistic details are still unknown. To understand the mechanism of action, a structural biology approach will be taken. To proceed, we require large quantities of pure fortilin for experimentation. Thus, the objective of this project was to design and test fortilin constructs with different purification tags and cleavage sites to find optimal conditions to produce recombinant protein in milligram quantities. A total of four plasmids with amino terminal affinity tags were evaluated, and cleavage was evaluated using SDS-gel electrophoresis. Despite high expression, no affinity tags were easily removed by cleavage with specific proteases. Furthermore, NMR spectra have demonstrated that at least one of the tested tags interacts with the fortilin active site which yields a non-viable result. We hypothesize that the cleavage site is not easily accessible on the protein surface. So, we will proceed with a new construct where the tag may not need to be cleaved.

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