Expression and detection of recombinant sheep prion protein (RecShPrPC) using human neuroblastoma cells and Western blotting

Scope and method of study. The objectives of the present study was to overexpress the sheep normal prion protein (ShPrP) using a mammalian expression vector. The prion protein gene which was in the zeroblunt vector was PCR amplified and was subcloned into pcDNA6/myc/HisB plasmid. The quality of the PCR reaction and the orientation of the cloned gene within vector were determined by DNA sequencing. Upon confirmation of this, the His-tagged fusion protein gene was transfected into human neuroblastoma cells (SK-N-SH) for overexpression of the protein. The proteins expressed were solubilized in guanidinium chloride and purifications were attempted with different conditions using the Nickel Nitriloactetate chromatographic system.;Findings and conclusions. We developed an expression system for overexpressing the sheep normal prion protein with the human neuroblastoma cells. We also conclude that the prurification of this particular protein using the nickel affinity chromatographic column did not yield significant quantities of the same.