Expression of prion protein (PrP) molecules in a recombinant adeno-associated virus (rAAV). Our initial experiments have been to express PrP in an AAV vector obtained from collaborators at the University of Florida. We have so far cloned into the AAV plasmids three PrP genes made available by Dr. Sue Priola?s group: mouse PrP bearing an epitope tag, denoted as 3F4 (Mo3F4 PrP); Mo3F4 PrP from which the first a-strand structure has been deleted, denoted Aa1 PrP; and hamster PrP, denoted Ha PrP. The 3F4 epitope is normally present on Ha PrP, but when inserted into Mo PrP it offers the potential to discriminate between two mouse PrP molecules in the same cell or animal. We have demonstrated that all of these genes are expressed on the membrane of cells when the constructs are transfected in. The patterns of expression appear to be the same as those seen with other systems being employed in LPVD. We are presently engaged in work to package the PrP constructs in to rAAV. Expression of an anti-PrP single chain antibody in a recombinant AAV (rAAV). Although attempts to demonstrate a protective host response in natural or experimental TSE disease have been unsuccessful, several groups have recently shown that monoclonal antibodies directed against certain regions of the PrP molecule can clear PrPres and infectivity from infected cell cultures, and can attenuate or prevent disease in vivo. A single chain antibody or single chain variable fragment (scFv) based on an anti-PrP monoclonal antibody would be a powerful tool to study PrP pathogenesis. Therefore, we have begun experiments to prepare an scFv based on the monoclonal antibody 3F4 and express it in AAV. The relatively small size of the complete scFv is compatible with AAV expression. The strategy is to build the scFv and verify its expression and function in a prokaryotic system, and then move it into AAV. Using RT-PCR and conserved primer sets, we cloned and sequenced the variable regions from the mouse IgG heavy (VH) and light (VL) chain mRNAs from the 3F4 hybridoma cell line. The sequences contained easily recognizable complement determining regions (CDRs) and framework sequences characteristic of IgG. The VH and VL gene segments were then joined with a flexible linker sequence and adapted for prokaryotic expression as a periplasmic protein with a C-terminal His tag. The scFv-3F4 was expressed at high level, but the protein accumulated in the insoluble fraction of the bacteria. We have tried several protocols to renature the molecules and form the disulfide linkages crucial to optimal function. Preliminary immunofluorescence results on refolded scFv-3F4 suggested that the scFv has activity against cells expressing the 3F4 epitope on cells. Methods:
| Speare, Jonathan O; Rush 3rd, Thomas S; Bloom, Marshall E et al. (2003) The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein. J Biol Chem 278:12522-9 |
