Generation of fusion protein constructs to track cellular levels of mutant alpha-1 antitrypsin
OA Version
Citation
Abstract
Alpha-1 antitrypsin deficiency (AATD) in the liver results from misfolding and intracellular aggregation of Alpha-1 antitrypsin (AAT) proteins. A system capable of tracking intracellular AAT levels would be highly useful as a means of identifying the effects of potential therapeutic compounds or genetic modulations. The objective of this study was to apply fusion protein constructs comprising AAT and fluorescent reporters to visualize and quantify intracellular trafficking and levels of wild-type (M) and mutant (Z) AAT in the liver. Initially, fusion protein constructs were synthesized using Gibson Assembly. To study the relevance to hepatic biology lentiviruses expressing these constructs were then produced and used to transduce the construct into HepG2, a liver cell line. After sorting and isolating a mEmerald+ve HepG2 cell population, the cells were characterized through Enzyme Linked Immunosorbent Assay, Fluorescence-activated cell sorting and Immunofluorescence image analyses.
Fusion protein constructs represent an approach that will allow real-time quantification of AAT levels in live cells. Future directions will include the application of the constructs generated in this project to evaluate the effects of potential therapeutic compounds and studying the disease risk modifying genes using high throughput screening on levels of intracellular mutant ZAAT proteins.
Description
License
Attribution-NonCommercial-NoDerivatives 4.0 International