In Vitro Recombinant Rat Urate Oxidase In The Host Cell Degradation Study Of The Way

Rat UOX cDNA has been subcloned into pTRE, a responsive plasmid in the Tet-off gene expression system, renamed pTRE-rUOX. Cotransfected with pTRE-rUOX and a selectable plasmid pTK-Hyg into the Tet-off cell line CHO-AA8 by Clonfectin? Transfection Reagent. After 14 days of selection, 50 individual clones were isolated and grown separately in the presence of hygromycin (200 μg/ml). Five individual double stable cell lines were selected for further studies, which confirmed that cells showed a high level of UOX enzyme specific activity and lowest background expression. We successfully establish a tetracycline (Tc) controlled gene expression model to regulate expression of rat UOX in vitro. After different time points, UOX level in the cotransfected cells were analyzed by Southern blotting and Western blotting. The location and configuration of UOX were confirmed by transmission and immunoelectron microscopic studies.In the first experiment, Southern blotting showed that the double transfected stable cell lines exhibited rat UOX DNA and revealed a high level of expression. Western blotting revealed recombinant rat UOX from the cells in the absence of Tc or in the early stages of Tc treatment. Several days after treated with Tc, the expression of rat UOX was gradually declined and cannot be observed on 7 day after the Tc treatment by Western blot. No endogenous UOX DNA or protein was detected in CHO-AA8 cells.As a inducer, Tc competitively bind with tetR that inhibit tTA binding with TRE in response plasmid, blocking transcription of UOX gene. After Tc was added into medium, UOX DNA broke down gradually by nuclease. In contrast, the expressed protein wasgradually broke down by the proteo-metabolism system.In the second experiment, immunoelectron microscopic observations revealed numerous gold particles localized on the crystalloid structure present in the cells, indicating that the UOX was significantly targeted in peroxisomes of the cells. In routine transmission electron microscopy, the cells expressing recombinant rat UOX showed numerous high electron dense granules in the cytoplasm in lower magnification. Few lysosomes in the cytoplasm and around the UOX structure can also be observed. When the cells treated with Tc at day 5, the integrated UOX structure can not be observed in the cytoplasm and the number of lysosomes increased in the cytoplasm. When the cells treated with Tc at day 7, the UOX structure can not be observed in the lysosomes or around. An acid phosphatase staining was performed in order to confirm that the autophagosome-like structures have lysosomal enzymes, the structures displayed positive staining. Immunoelectron microscopy revealed no gold particle in phagolysosomes on day 7 after the Tc treatment.These observations suggest the entire organelle rather than a single protein within the peroxisomes is degraded once that rat UOX gene expression was turn off and phagocytic vacuole/lysosome pathway (microautophagic process) may plays an major role in degradation of the recombinant protein under the experimental conditions.