High-yield production in E. coli and characterization of full-length functional p13II protein from human T-cell leukemia virus type 1

Abstract

Human T-cell leukemia virus type 1 is an oncovirus that causes aggressive adult T-cell leukemia but is also responsible for severe neurodegenerative and endocrine disorders. Combatting HTLV-1 infections requires a detailed understanding of the viral mechanisms in the host. Therefore, in vitro studies of important virus-encoded proteins would be critical. Our focus herein is on the HTLV-1-encoded regulatory protein p13^II, which interacts with the inner mitochondrial membrane, increasing its permeability to cations (predominantly potassium, K⁺). Thereby, this protein affects mitochondrial homeostasis. We report on our progress in developing specific protocols for heterologous expression of p13^II in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13^II, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13^II C-terminus was instrumental in increasing the persistently low expression of soluble p13^II in its native form. We subsequently developed approaches for protein spin labeling and a conformation study using double electron-electron resonance (DEER) spectroscopy and a fluorescence-based cation uptake assay for p13^II in liposomes. Our DEER results point to large protein conformation changes occurring upon transition from the soluble to the membrane-bound state. The functional assay on p13^II-assisted transport of thallium (Tl⁺) through the membrane, wherein Tl⁺ substituted for K⁺, suggests transmembrane potential involvement in p13^II function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13^II and would aid in the development of structure-based protein inhibitors and markers.

Keywords: DEER-detected p13(II) self-association; Fluorescence-based p13(II) liposome uptake assay; High-yield protein production; Human T-cell leukemia virus type 1 encoded protein p13(II); Viral protein-mitochondrial membrane association.