The production of cervical cancer-related HPV16 E7 oncoprotein in rice seeds

Cervical cancer is a leading cause of cancer-related death in women of developing countries and the second most prevalent cancer among women of developed countries. HR-HPV (high-risk human papillomavirus) type 16 is mainly considered as the causative agent of more than half of clinical cervical cancer cases, and the E7 oncoprotein of HPV16 is responsible for causing and maintaining the transformed state of tumor cells. HPV16 E7 is regarded as a tumor-specific antigen because of continual expression in cancer cells, and hence is a promising target for a therapeutic vaccine against cervical cancer. Since the last decade, plant-based expression systems have been promoted for large-scale production of recombinant proteins of pharmaceutical, industrial and agricultural applications. In this study, HPV16 E7 is produced in rice seeds by manipulating the secretory pathway and targeting endomembrane system organelles of rice endosperm cells for better yield and stability. Large numbers of independent transformants of secretory expression cassettes (pE7 and p←E7) and vacuolar expression cassette (pE7CTPP), expressing recombinant E7 under the control of the rice glutelin promoter and its signal peptide were produced by Agrobacterium transformation of Oryza sativa L. cv. Nipponbare. The expression of recombinant E7 was confirmed by Western blot analysis and ELISA (enzyme linked immunosorbent assay). The pE7 and p←E7 transformants, differing with respect to the proximity of glutelin promoter to CaMV 35S promoter (governing expression of selectable marker gene), accumulate recombinant E7 to similar levels. Transformants of pE7CTPP, containing the rice lectin CTPP (carboxyl terminal propeptide) for vacuolar targeting of recombinant E7, accumulate recombinant E7 to a level 3-4 fold more than that in pE7 and p←E7 transformants. Non-cleavage of CTPP from recombinant E7 (E7-CTPP) was observed, and suggests the inappropriateness of utilization of a CTPP for vacuolar targeting of recombinant proteins of pharmaceutical importance. The recombinant E7 was present predominantly in an insoluble form of high-oligomeric order in rice endosperm cells. In the pE7 and p←E7 transformants, the recombinant E7 was engineered to follow the default pathway to the intercellular space of rice endosperm cells. However, fluorescence and electron microscopy showed that recombinant E7 was deposited in PSVs (protein storage vacuoles) and prolamin protein bodies. When rice lectin CTPP was utilized in addition to the glutelin signal peptide, the recombinant E7-CTPP exhibited a similar localization pattern to PSVs (protein storage vacuoles) and prolamin protein bodies. Thus, the specialized nature of storage tissue appears to influence the recombinant protein trafficking and localization.