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.
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.
Park
Jinseu
J
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Ryu
Jiyoon
J
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Kim
Kyeong-Ae
KA
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Lee
Hak Joo
HJ
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Bahn
Jae Hoon
JH
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Han
Kyuhyung
K
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Choi
Eui Yul
EY
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Lee
Kil Soo
KS
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
Kwon
Hyeok Yil
HY
Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702, Korea2.
Choi
Soo Young
SY
Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1.
D020816:Amino Acid Motifs; D001692:Biological Transport; D015696:Gene Products, tat / Q000737:chemistry* / Q000502:physiology; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins