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 addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese 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 the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein.
Tujebajeva
R M
RM
Thyroid Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Harney
J W
JW
Berry
M J
MJ
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D015870:Gene Expression; D006031:Glycosylation; D006639:Histidine / Q000276:immunology; D006801:Humans; D011233:Precipitin Tests; D011506:Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D012644:Selenium Radioisotopes; D017279:Selenocysteine / Q000378:metabolism*; D051149:Selenoprotein P; D051140:Selenoproteins
D000906:Antibodies; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012644:Selenium Radioisotopes; D051149:Selenoprotein P; D051140:Selenoproteins; D017279:Selenocysteine; D006639:Histidine