Researcher(s)
- Kavya Gajjar, , University of Delaware
Faculty Mentor(s)
- Subhasis Biswas, Medical and Molecular Sciences, University of Delaware
Abstract
The E1 replication protein of Human Papillomavirus (HPV) plays a critical role in viral DNA replication, which makes it a key target for understanding HPV-driven oncogenesis. Biochemical characterization of E1 has proven difficult, however, because the wild-type protein is highly insoluble and therefore hard to extract, purify, and study in vitro. To address this problem, the project pursues a two-part strategy that pairs a Δ71 sequence truncation with an in vivo, mammalian-like glycosylation system, aiming to convert E1 from an insoluble protein into a soluble, experimentally workable form.
Using custom primers and PCR, we truncated the region associated with E1 insolubility. The purified PCR products were digested and directionally ligated into a pFastBac vector, which was then transformed into DH5α E. coli. Ten clones were screened by analyzing recombinant plasmids, yielding four at high DNA concentration (approximately 400 ng/µL). These constructs were transformed into EMBacY cells for blue/white colony selection and subsequent bacmid preparation. In parallel, we prepared a SweetBac bacmid system to introduce humanized glycosylation, intended to improve solubility by adding hydrophilic glycan groups. This system was used to transfect Sf9 insect cells and generate a V1 helper virus.
The final insect-cell co-infections are still in progress. We expect that the combined E1 and Δ71 truncation and SweetBac virus-mediated in vivo glycosylation will yield glycosylated soluble E1 within the insect cells. The resulting soluble protein will be purified by column chromatography to provide the yield needed to analyze real-time DNA and protein binding dynamics using Bio-Layer Interferometry (BLI). If successful, engineering a soluble E1 protein would establish a robust platform for directly investigating the strain-specific replication mechanisms that shape HPV virulence.