Cationic peanut peroxidase: expression and characterization in transgenic tobacco and purification of the histidine-tagged protein

Abstract Cationic peanut ( Arachis hypogaea ) peroxidase (CPRX) is the major isozyme of peanut peroxidases secreted into the culture medium from the cells growing in liquid suspension culture. In this report, CPRX was expressed in transgenic tobacco ( Nicotiana tabacum ) plants and represented about 0.3% of the total soluble proteins. In order to purify the expressed protein, a sequence encoding a C-terminal tag of six consecutive histidine residues was introduced into the corresponding cDNA bearing a native N-terminal signal sequence facilitating secretion of CPRX. The expressed CPRX-his6 was purified under nondenaturing conditions by a single-step affinity chromatography using NTA resin. The purified CPRX-his6 was fully functional as native CPRX. CPRX is a glycoprotein. Monoclonal antibodies specific for epitope occurring at the region where oligosaccharide is linked to the polypeptide backbone recognized CPRX-his6, indicating the protein was also glycosylated properly in tobacco cells. Western blotting analysis on proteins from spent medium confirmed CPRX-his6 was secreted into the liquid medium, indicating the native CPRX signal peptide was sufficient to direct the secretion of CPRX-his6 in tobacco cells.