High-yield production in E. coli and characterization of full-length functional p13 protein from human T-cell leukemia virus type 1.

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, 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 in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13 C-terminus was instrumental in increasing the persistently low expression of soluble p13 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 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-assisted transport of thallium (Tl) through the membrane, wherein Tl substituted for K, suggests transmembrane potential involvement in p13 function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13 and would aid in the development of structure-based protein inhibitors and markers.