Date of Award

12-2025

Degree Type

Honors College Thesis

Academic Program

Chemistry BS

Department

Chemistry and Biochemistry

First Advisor

Dr. Vijay Rangachari

Advisor Department

Chemistry and Biochemistry

Abstract

TAR DNA-binding protein 43 (TDP-43) is a 43 kDa RNA-binding protein linked to several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease. 5-7,28,30,33 Studying full-length TDP-43 has been difficult because of its intrinsic instability and tendency to aggregate.19,25,28,33,36 In this study, we focused on overcoming the difficulties to express full-length TDP-43 in E. coli recombinantly. We focused on expressing full-length TDP-43 as a fusion construct with a maltose-binding protein (MBP) to improve solubility and stability. Our strategy was to purify TDP-43-MBP fusion and make biomolecular condensates with RNA before cleaving the tag with thrombin protease. We successfully expressed the fusion construct and purified it using nickel affinity and size-exclusion chromatography. The protein showed high purity, confirmed by the expected molecular weight by MALDI-ToF mass spectrometry. Turbidity assays indicated the formation of a condensate containing RNA via liquid–liquid phase separation (LLPS).12-15,19,25 However, confocal microscopy did not reveal visible droplets, suggesting that the in vitro conditions may not be optimal. Thrombin digestion efficiently removed the MBP tag, as confirmed by MALDI-ToF spectra showing a 42.8 kDa peak, but a low signal suggested that the cleaved protein aggregated or precipitated, a behavior commonly observed in previous findings where MBP-free TDP-43 is highly unstable.19,25,28,33 Further studies are ongoing to find optimal conditions for tag-free TDP-43 purification. But this study has developed a reproducible approach for expressing and purifying full-length TDP-43.

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