AbstractBackgroundHuman papillomavirus (HPV) is the causative agent of nearly all forms of cervical cancer, which can arise upon viral integration into the host genome and concurrent loss of viral regulatory gene E2. Gene‐based delivery approaches show that E2 reintroduction reduces proliferative capacity and promotes apoptosis in vitro.AimsThis work explored if our calcium‐dependent protein‐based delivery system, TAT‐CaM, could deliver functional E2 protein directly into cervical cancer cells to limit proliferative capacity and induce cell death.Materials and ResultsTAT‐CaM and the HPV16 E2 protein containing a CaM‐binding sequence (CBS‐E2) were expressed and purified fromEscherichia coli. Calcium‐dependent binding kinetics were verified by biolayer interferometry. Equimolar TAT‐CaM:CBS‐E2 constructs were delivered into the HPV16+SiHa cell line and uptake verified by confocal microscopy. Proliferative capacity was measured by MTS assay and cell death was measured by release of lactate dehydrogenase. As a control, human microvascular cells (HMECs) were used. As expected, TAT‐CaM bound CBS‐E2 with high affinity in the presence of calcium and rapidly disassociated upon its removal. After introduction by TAT‐CaM, fluorescently labeled CBS‐E2 was detected in cellular interiors by orthogonal projections taken at the depth of the nucleus. In dividing cells, E2 relocalized to regions associated with the mitotic spindle. Cells receiving a daily dose of CBS‐E2 for 4 days showed a significant reduction in metabolic activity at low doses and increased cell death at high doses compared to controls. This phenotype was retained for 7 days with no further treatments. When subcultured on day 12, treated cells regained their proliferative capacity.ConclusionsUsing the TAT‐CaM platform, bioactive E2 protein was delivered into living cervical cancer cells, inducing senescence and cell death in a time‐ and dose‐dependent manner. These results suggest that this nucleic acid and virus‐free delivery method could be harnessed to develop novel, effective protein therapeutics.