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 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.
Fuchs
Stephen M
SM
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA. raines@biochem.wisc.edu.
Raines
Ronald T
RT
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D010455:Peptides; D011993:Recombinant Fusion Proteins; C015462:polyarginine; D012259:Ribonuclease, Pancreatic