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Polyarginine as a multifunctional fusion tag

¹ Department of Biochemistry and ² Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA

(RECEIVED February 2, 2005; FINAL REVISION March 26, 2005; ACCEPTED March 26, 2005)

Fusion to cationic peptides, such as nonaarginine (R₉), 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 (T_m=–8°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 R₉ 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 R₉ 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.

Keywords: affinity tag; conformational stability; enzymatic activity; protein adsorption; protein transduction domain; ribonuclease A

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051393805.

Reprint requests to: Ronald T. Raines, Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA; e-mail: raines{at}biochem.wisc.edu; fax: (608) 262-3453.

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