Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells.
Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 microgram/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.
Fan
J L
JL
Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston 77555, USA.
Patibandla
S A
SA
Kimura
S
S
Rao
T N
TN
Desai
R K
RK
Seetharamaiah
G S
GS
Kurosky
A
A
Prabhakar
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D013967:Thyroiditis, Autoimmune / Q000097:blood
D000906:Antibodies; D002241:Carbohydrates; D011994:Recombinant Proteins; D007453:Iodide Peroxidase