Polyarginine as a multifunctional fusion tag

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 R9 tag on the attributes of a model protein: bovine pancreatic ribonuclease (RNase A). The R9 tag diminishes the conformational stability of RNase A (Delta T-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.