Enhanced uptake of a heterologous protein with an HIV-1 Tat protein transduction domains (PTD) at both termini.
Poor membrane permeability of proteins is a major limitation of protein therapy. In a previous study, we showed that the minimal sequence required for efficient transduction of Tat-GFP is the basic domain from 49-57 of HIV-1 Tat called the protein transduction domain (PTD. Here we have generated HIV-1 Tat PTD GFP fusion proteins in which HIV-1 Tat PTD is fused with the N- and/or C-termini of GFP. The various GFP fusion proteins were purified from Escherichia coli and characterized for their ability to enter mammalian cells using Western blot analysis, confocal microscopy and flow cytometry. The GFP fusion protein with Tat PTD at its C-terminus was taken up as efficiently as the GFP fusion protein with Tat PTD at its N-terminus. However, the same protein with PTDs at its both termini was taken up even more efficiently. All the GFP fusion proteins were present in both the nucleus and cytosol of the transduced cells. Uptake was lower at 4 degrees C than at 37 degrees C. The availability of the expression vectors developed in this study may help to devise novel strategies in the rational development of protein-based drugs.
Ryu
Jiyoon
J
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea.
Han
Kyuhyung
K
Park
Jinseu
J
Choi
Soo Young
SY
D015696:Gene Products, tat / Q000378:metabolism*; D017930:Genes, Reporter; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus