ИСТИНА

Anionic tobacco peroxidase (TOP) has many advantages over horseradish peroxidase isoen-zyme C (HRP C) widely used in biosensors and enzyme immune assay (higher storage and operational stability, higher activity in chemiluminescent oxidation of luminol, no need of chemiluminescence en-hancers). Recombinant TOP is expressed in E. coli cells as insoluble inclusion bodies. Refolding pro-cedure was developed (Hushpulian D.M. et al. Biochemistry(Moscow), 2003) but yield of active protein was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved. Conjugates of wild-type rTOP and mutant rTOP Phe140Tyr with goat IgG against mouse IgG we prepared and tested. Catalytic activity of enzyme in both conjugates did not change in reaction of chemiluminescent oxidation of luminol. New conjugates provided higher sensitivity and accuracy in mouse IgG detection compared to conjugate of goat IgG with HRP. This work was supported by the Russian Foundation for Basic Research (project no. 13-04-02013-a).