Purification and Structural Characterization of N-Terminal 190 Amino Acid Deleted Essential Mammalian Protein; Transcription Termination Factor 1

Abstract

The mammalian transcription termination factor 1 (TTF1) is an essential protein that plays diverse cellular physiological functions like transcription regulation (both initiation and termination), replication fork blockage, chromatin remodeling, and DNA damage repair. Hence, understanding the structure and mechanism conferred by its variable conformations is important. However, so far, almost nothing is known about the structure of either the full-length protein or any of its domains in isolation. Since the fulllength protein even after multiple attempts could not be purified in soluble form, we have codon optimized, expressed, and purified the N-terminal 190 amino acid deleted TTF1 (ΔN190TTF1) protein. In this study, we characterized this essential protein by studying its homogeneity, molecular size, and secondary structure using tools like dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, Raman spectroscopy, and atomic force microscopy (AFM). By CD spectroscopy and DLS, we confirmed that the purified protein is homogeneous and soluble. CD spectroscopy also revealed that ΔN190TTF1 is a helical protein, which was further established by analysis of Raman spectra and amide I region deconvolution studies. The DLS study estimated the size of a single protein molecule to be 17.2 nm (in aqueous solution). Our structural and biophysical characterization of this essential protein will open avenues toward solving the structure to atomic resolution and will also encourage researchers to investigate the mechanism behind its diverse functions attributed to its various domains.

Novel Function of Transcription Factor TTF1 in UV Mediated DNA Damage Repair in Mammalian Cells

“…Histidine tagged codon optimized mammalian TTF1 was expressed in BL21-DE3 cells and purified as mentioned in Tiwari et. al [12]. This purified protein was then immobilized on Ni-NTA column.…”

“…Overexpression of TTF1 has been reported in various tumours, indicating that TTF1 is required in higher quantities to meet the higher rate of ribosome biogenesis to cope up with hyper-proliferation of tumorous cells. Considering the essential and multifunctional nature of this protein, it becomes important than ever to characterize the mechanism behind its function and regulation [11][12][13].…”