Expression and Cellular Internalization of Two Tat-Conjugated Fluorescent Proteins
ARMY MEDICAL RESEARCH INST OF CHEMICAL DEFENSE ABERDEEN PROVING GROUND MD
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Two hybrid vectors were designed for the expression in E. coli of fluorescent fusion proteins containing the protein translocation domain designated as Tat. The Tat domain was introduced to promote the entry of cargo protein, in this case the fluorophore yellow fluorescent protein YFP, into cells. The first construct was made by fusing Tat with YFP. The second Tat fusion protein was constructed to contain YFP and the palmitoylation domain Palm from SNAP-25. The Palm domain was intended to bind the fusion protein to intracellular membranes and trap the fluorophore inside the cells. Intracellular localization of both proteins was demonstrated by laser confocal microscopy. This research serves as proof of the concept that such Tat fusion constructs may be useful in intracellular delivery of proteins and drugs that normally cannot penetrate the cell membrane and that the Tat domain remains functional with an intracellular palmitoylation trapping domain present.
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