In-Vitro Expression of FET Proteins

Abstract

The FET proteins, FUS, EWSR1, and TAF15 are RNA binding proteins that form amyloid-like plaques through aggregation. Research into these proteins is important due to their presence and implication in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The objective of this project was to clone the FET genes into an in-vitro expression system. Using this method, the need to culture cells to obtain protein for in-vitro experiments testing protein-protein interactions and RNA binding ability would be removed. This project was based around the cloning of each FET gene into a pT7CFE1-CHis plasmid vector using MEGAWHOP protocols. After cloning of a gene, in-vitro translation (IVT) using HeLa lysates was facilitated using the internal ribosomal entry site (IRES) sequence found in the pT7CFE1-CHis plasmid. During this project, in-vitro translation constructs were designed and tested with a pilot in-vitro translation assay performed using the initial FUS in-vitro translation MEGAWHOP product, revealing the unexpected result of a double insertion of FUS into the pT7CFE1-CHis plasmid. After alterations to the in-vitro translation constructs, FUS and TAF15 megaprimers have been reliably produced for proper insertion into the pT7CFE1-CHis plasmid.