Importance of the carboxyl terminus of human thyroid peroxidase in the efficient expression of the protein in eukaryotic cells

Abstract

Listen

Translate article

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 hTPO 1–848, 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 hTPO 1–771 (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.