Polyarginine as a multifunctional fusion tag

Abstract

Fusion to cationic peptides, such as nonaarginine (R(9)), provides a means to deliver molecular cargo into mammalian cells. Here, we provide a thorough analysis of the effect of an R(9) tag on the attributes of a model protein: bovine pancreatic ribonuclease (RNase A). The R(9) tag diminishes the conformational stability of RNase A (DeltaT(m)=-8 degrees C in phosphate-buffered saline). This effect is nearly mitigated by the addition of salt. The tag does not compromise the enzymatic activity of RNase A. An R(9) tag facilitates the purification of RNase A by cation-exchange chromatography and enables the adsorption of RNase A on glass slides and silica resin with the retention of enzymatic activity. The tag can be removed precisely and completely by treatment with carboxypeptidase B. Finally, the R(9) tag increases both the cellular uptake of RNase A and the cytotoxicity of G88R RNase A, a variant that evades the cytosolic ribonuclease inhibitor protein. Thus, we conclude that polyarginine is a versatile protein fusion tag.

Figure 1.

Effect of an R₉ tag on the purification of a protein by cation-exchange chromatography. (A) RNase A-R₉ was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. (B) SDS-PAGE gel of RNase A-R₉ before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.

Figure 2.

Effect of an R₉ tag on the conformational stability of a protein. (Top) Thermal denaturation of RNase A (□) (T_m=63.7 ± 1.0°C) and RNase A-R₉ (▪) (T_m=54.0 ± 1.0°C) in PBS. (Bottom) Conformational stability of RNase A (□) and RNase A-R₉ (▪) in 50 mM sodium phosphate buffer (pH 7.2), containing NaCl (0–1.00 M).

Figure 3.

Effect of an R₉ tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R₉ (10 μM, A) or fluorescein-labeled RNase A (10 μM, B) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.

Figure 4.

Effect of an R₉ tag on the cytotoxicity of wild-type ribonuclease A, its G88R variant, and Onconase. Cell proliferation was determined by incorporation of [methyl-³H]thymidine into cellular DNA after a 44-h incubation with a ribonuclease. Each data point is expressed as a percentage of a PBS control. Variants with R₉ tags are denoted with filled symbols: RNase A-R₉,•; G88R RNase A-R₉, ▪ Variants without R₉ tags are denoted with open symbols: RNase A, ○; G88R RNase A,□; ONC, ▵. IC₅₀ values are listed in Table 1.

Figure 5.

Effect of an R₉ tag on the adsorption of a protein to a glass slide and silica resin. (A) Fluorescent images of fluorescein-labeled RNase A-R₉ and RNase A (10–0.01 μM) adsorbed on to a glass slide. (B) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R₉ or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R₉.