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. 2007 Mar 20;2(3):167-70.
doi: 10.1021/cb600429k. Epub 2007 Feb 23.

Arginine grafting to endow cell permeability

Stephen M Fuchs, Ronald T Raines

Arginine grafting to endow cell permeability

Stephen M Fuchs et al. ACS Chem Biol. .

Abstract

We report on a means to endow proteins with the ability to permeate mammalian cells without appending an exogenous domain. Our approach is to install a cationic patch on the surface of a target protein by the grafting of arginine residues. Doing so with GFP did not compromise conformational stability but enabled efficient cellular uptake that was dependent on cell-surface glycosaminoglycans. We anticipate that this cell-permeable variant of GFP, which obviates the need for transfection, will be useful for numerous applications in cell biology and that the method of arginine grafting will be broadly applicable.

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Figures

Figure 1
Figure 1
Scheme for arginine grafting to create a cell-permeable variant of GFP (cpGFP). (top) Ribbon model depicting the location of the five anionic residues in GFP that were replaced with arginine to yield a surface comprised of ten cationic residues. The fluorophore is depicted in space-filling mode. (bottom) Space-filling model depicting the effect of the arginine substitutions on the electropotential surface (blue: cationic; red: anionic).
Figure 2
Figure 2
Images of the internalization of GFP variants into living human and rodent cells. HeLa cells were incubated with cpGFP (a, 10 µM; b, 1 µM; c, 0.1 µM) and eGFP (d, 10 µM) for 3 h in Opti-MEM medium at 37 °C. Cells were then placed in fresh medium for 1 h and stained with Hoescht 33342 (blue) and propidium iodide (red) for 15 min prior to visualization by confocal microscopy. (e) CHO-K1 and (f) CHO-745 cells (which are GAG-deficient) were incubated with cpGFP (2 µM) for 3 h at 37 °C in Opti-MEM medium. Cells were then placed in fresh medium for 1 h and stained with Hoescht 33342 (blue) and propidium iodide (red) for 15 min prior to visualization.
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