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Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells

Division of Life Sciences, Hallym University, Chunchon 200-702, Korea¹
Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702, Korea²

Author for correspondence: Soo Young Choi. Present address: Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea. Fax +82 33 241 1463. e-mail sychoi{at}hallym.ac.kr

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains a high proportion of arginine and lysine residues, is responsible for highly efficient protein transduction through the plasma membrane. To identify the role of the PTD sequence motif in transduction, various deletions and substitutions were introduced into the PTD. Tat–green fluorescent protein (GFP) fusion proteins, containing various lengths of the Tat PTD, were expressed and the extent of their transduction into mammalian cells was analysed by Western blot analysis and fluorescence microscopy. Deletion analysis of PTD mapped to a nine amino acid motif (residues 49–57: RKKRRQRRR) sufficient for transduction. Further deletion of this Tat basic domain either at the N terminus or at the C terminus significantly decreased transduction efficiency. The transduction efficiencies of GFPs fused to nine consecutive lysine (9Lys–GFP) or arginine (9Arg–GFP) residues were similar to that of Tat(49–57)–GFP. The transduced proteins localized to both the nucleus and the cytosol, as assessed by confocal microscopy and Western blot analysis of subcellular fractions from transduced cells. Thus, the availability of recombinant GFP fusion proteins facilitates the simple and specific identification of protein transduction mediated by these peptide sequences. The modified PTD sequences designed in this study may provide useful tools necessary for delivering therapeutic proteins/peptides into cells.

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