Recombinant human thyroid peroxidase produced in insect cells has similar properties to native human thyroid peroxidase

Purified native human thyroid peroxidase (nTPO) isolated from thyroid tissu e and recombinant (r)TPO produced in High Five insect cells have been compa red. nTPO and rTPO were purified to about 95% homogeneity and showed simila r 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 a ctivities were about 1,000 guaiacol units per milligram of protein. TPO aut oantibody binding characteristics of nTPO and rTPO were analyzed in an assa y based on I-125-labeled nTPO and precipitation with protein A. In the assa y, 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) in hibited 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 (I gG) (n = 12), TPO monoclonal antibodies reactive with two different epitope s on the TPO, IgG (n = 3) from glutamic acid decarboxylase autoantibody pos itive patient sera, and IgG (n = 3) from healthy blood donors. Effective co mplexing of TPO by TPO autoantibodies was tested by precipitating the compl exes with solid phase protein A and measuring the TPO enzyme activity in th e resulting supernatants. These studies showed that the TPO enzyme activity was not affected by incubation with TPO autoantibody-positive IgG or monoc lonal antibodies despite effective complexing of the autoantibodies with TP O. Overall, our studies demonstrate that nTPO and rTPO produced in insect c ells are very similar in terms of enzyme activity, UV and visible spectra, and reactivity with autoantibodies. Furthermore, in our study, TPO autoanti bodies did not appear to inhibit TPO enzyme activity.