Importance of the carboxyl terminus of human thyroid peroxidase in the efficient expression of the protein in eukaryotic cells.
Carboxyl terminal truncation of membrane-associated human thyroid peroxidase (hTPO), with the elimination of its single membrane-spanning and short intracytoplasmic regions, generates a soluble, secreted, enzymatically active protein (amino acids 1-848). In order to determine the effects of further carboxyl terminal deletions on the expression of hTPO, Chinese hamster ovary cells were stably transfected with plasmids constructed to express amino acids 1-771, 1-636, 1-539 and 1-382 of the 933 amino acid TPO protein, respectively. Unlike hTPO1-848, the more severely truncated TPO mutant proteins could not be detected in conditioned media by polyclonal anti-TPO antibodies. Using detergent-solubilized microsomal proteins from these cells, very low levels of hTPO1-771 (approximately 90 kDa), but not the more extensive deletion mutations, were detected by these anti-TPO antibodies. Confirmation of the loss of efficient expression of more severely truncated hTPO was obtained using a anti-hTPO monoclonal antibody with an epitope near the amino terminus and which recognizes only the denatured protein. The mRNA for all hTPO mutants was detected in the stably-transfected Chinese hamster ovary cells. In summary, the present study indicates that a largely intact extracellular portion of hTPO is required for expression in eukaryotic cells.
Finke
R
R
Thyroid Molecular Biology Unit, Veterans' Administration Medical Center, San Francisco, CA 94121.
Foti
D
D
Rapoport
B
B
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D006224:Cricetinae; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D007453:Iodide Peroxidase / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D012333:RNA, Messenger; D007453:Iodide Peroxidase