Selenoprotein P expression, purification, and immunochemical characterization

Most selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon, Selenoprotein P is unique in that its mRNA encodes 10-12 selenocysteine residues, depending on species. In a ddition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in p art due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/s top codons, Herein, we report for the first time the expression of recombin ant rat selenoprotein P in a transiently transfected human epithelial kidne y cell line, as well as the endogenously expressed protein from HepG2 and C hinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified th e recombinant and endogenously expressed proteins using nickel-agarose affi nity chromatography, We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with comm ercial anti-histidine antibodies. The ability to express, purify, and immun ochemically detect the recombinant protein provides a foundation for invest igating the functions and efficiency of expression of this intriguing prote in.