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Technical Challenges of HIV Tat Protein Purification

Wednesday, September 13, 2017 — Poster Session II

3:30 p.m. – 5:00 p.m.
FAES Terrace
NINDS
VIROL-8

Authors

  • UA Santamaria
  • MM Bachani
  • JP Steiner
  • AL Popescu
  • W Li
  • A Nath

Abstract

Background: Despite advancements and developments in antiretroviral therapy for Human Immunodeficiency Virus (HIV) the Trans-Activator of Transcription (Tat) protein remain in HIV infected cells. Furthermore, tat is neurotoxic and elicits immune system responses. Purification of tat is needed for the development of mechanistic and therapeutic studies. Methods: Plasmids with the tat gene were created from the HIV clades A, B, C, and D and were expressed in E. coli with a histidine tag. The protein was then separated from cell debris using nickel based affinity chromatography; the protein is lyophilized and stored at -80° C. Results: Expression of levels of Tat was influenced by a multitude of factors. First, the different HIV clades showed variable amounts of expression with clade B yields being the lowest since it was toxic to the bacteria. Second, Tat gets easily polymerized via cysteine-cysteine bonding. Furthermore, the cysteine and methionine residues also readily oxidize in ambient conditions. Tat can also form intens and autocleave. Finally, Tat can non-specifically bind to plastics, cellular proteins, and bacterial RNA. Multiple precautions need to be taken to prevent and manage protein loss; for example, the use of silicon tubes, prevention of freeze-thaw cycles, and the elimination of binding partners. Conclusions: We have identified multiple challenges in the purification and storage of Tat protein, and have developed protocols to overcome these hurdles.

Category: Virology