Recombinant HIV-1 Tat C Protein Expression and Characterization

HIV-1 Tat C protein has been expressed and purified as per our standardized protocol, described elsewhere in detail [23 (link)]. Immunoconfirmation of recombinant HIV-1 Tat C protein was done by western blot analysis using anti-Tat antibody (NIH AIDS Research and Reference Reagent Program). Endotoxin level of purified recombinant HIV-1 Tat C protein was measured by Limulus Amebocyte Lysate (LAL) assay (Lonza) as per manufacturer’s protocol. The level of endotoxin was found in range of 0.04 EU/μg of purified protein; which was much below the acceptable limit set by international standards. Transcriptional activity of recombinant HIV-1 Tat C protein was checked by transactivation assay as described previously [23 (link)]. CEM-GFP cells are T-cell lines, carrying a stably integrated GFP gene under the control of HIV-1 subtype-B LTR. Purified HIV-1 Tat C protein (5 μg) was transfected in CEM-GFP cells, with proteo-juice protein transfection reagent (Novagen). GFP expressions were visualized with fluorescence microscope (Axio Observer-A1, Carl Zeiss, Germany). All protocols were approved by the Centre for Cellular and Molecular Biology (CCMB), Hyderabad Institutional Biosafety Committee.