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

Elka R Georgieva, Peter P Borbat, Christina Fanouraki, Jack H Freed

Protein expression and purification 2020 Sep

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Human T-cell leukemia virus type 1 is an oncovirus that causes aggressive adult T-cell leuk emia 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 p13II, 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 p13II in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13II, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13II C-terminus was instrumental in increasing the persistently low expression of soluble p13II 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 p13II 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 p13II-assisted transport of thallium (Tl+) through the membrane, wherein Tl+ substituted for K+, suggests transmembrane potential involvement in p13II function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13II and would aid in the development of structure-based protein inhibitors and markers. Copyright © 2020 Elsevier Inc. All rights reserved.

Citation

Elka R Georgieva, Peter P Borbat, Christina Fanouraki, Jack H Freed. High-yield production in E. coli and characterization of full-length functional p13II protein from human T-cell leukemia virus type 1. Protein expression and purification. 2020 Sep;173:105659