title: Recombinant human thyroid peroxidase produced in insect cells has similar properties to native human thyroid peroxidase.
abstract: Purified native human thyroid peroxidase (nTPO) isolated from thyroid tissue and recombinant (r)TPO produced in High Five insect cells have been compared. nTPO and rTPO were purified to about 95% homogeneity and showed similar UV and visual spectra and similar 412 nm per 280 nm absorbance ratios (0.4 for nTPO and 0.4 for rTPO). The nTPO and rTPO guaiacol oxidation enzyme activities were about 1,000 guaiacol units per milligram of protein. TPO autoantibody binding characteristics of nTPO and rTPO were analyzed in an assay based on 125I-labeled nTPO and precipitation with protein A. In the assay, the effect of unlabeled nTPO or rTPO on TPO autoantibody binding from 25 patients sera was studied. Unlabeled nTPO or rTPO (from 0 to 160 ng/mL) inhibited the binding of TPO autoantibodies in a dose-dependent manner in the case of each serum studied (from 100% in the absence of unlabeled TPO to 5%-10% in the presence of 160 ng/mL of TPO). The inhibition profile for each serum was essentially identical in the case of both TPO preparations. The effect of TPO autoantibodies on enzyme activity of rTPO was analyzed after incubation of rTPO with TPO autoantibody-positive serum immunoglobulin G (IgG) (n = 12), TPO monoclonal antibodies reactive with two different epitopes on the TPO, IgG (n = 3) from glutamic acid decarboxylase autoantibody positive patient sera, and IgG (n = 3) from healthy blood donors. Effective complexing of TPO by TPO autoantibodies was tested by precipitating the complexes with solid phase protein A and measuring the TPO enzyme activity in the resulting supernatants. These studies showed that the TPO enzyme activity was not affected by incubation with TPO autoantibody-positive IgG or monoclonal antibodies despite effective complexing of the autoantibodies with TPO. Overall, our studies demonstrate that nTPO and rTPO produced in insect cells are very similar in terms of enzyme activity, UV and visible spectra, and reactivity with autoantibodies. Furthermore, in our study, TPO autoantibodies did not appear to inhibit TPO enzyme activity.
authors.affiliation: FIRS Laboratories, RSR Ltd. Parc Ty Glas, Llanishen, Cardiff, United Kingdom.
authors.forename: P
authors.initials: P
authors.lastname: Gut
authors.forename: F
authors.initials: F
authors.lastname: Grennan Jones
authors.forename: A
authors.initials: A
authors.lastname: Sullivan
authors.forename: K
authors.initials: K
authors.lastname: Ziemnicka
authors.forename: S
authors.initials: S
authors.lastname: Smith
authors.forename: D
authors.initials: D
authors.lastname: Jaskólski
authors.forename: J
authors.initials: J
authors.lastname: Furmaniak
authors.forename: B
authors.initials: B
authors.lastname: Rees Smith
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood / Q000378:metabolism; D001327:Autoimmune Diseases / Q000201:enzymology / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006139:Guaiacol / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007313:Insecta / Q000378:metabolism*; D007453:Iodide Peroxidase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013959:Thyroid Diseases / Q000276:immunology
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D006139:Guaiacol; D007453:Iodide Peroxidase