NR AXTL
AU Kav,N.; Shi,Y.; Polymenidou,M.; Aguzzi,A.; Yajima,W.
TI Properties of an Anti-mouse Prion Protein-specific Single-chain Antibody
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.68
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB The abnormal isoform of prion proteins is believed to be the agent responsible for Transmissible Spongiform Encephalopathies (TSE), such as human sporadic Creutzfeldt-Jakob disease. These nervous system disorders are closely associated with conformational alterations of the host-encoded cellular prion proteins (PrPc) into their pathological form, PrPsc. Experiments in mice have shown that anti-PrP antibodies can interfere with prion replication. Single-chain antibodies (ScFv) are fully functional molecules consisting of the variable regions of antibody heavy and light chains and can be efficiently expressed in a number of systems including bacteria and plants. The objective of this study was to generate ScFvs with high affinity and specificity for mouse PrPc and to perform large scale expression and purification of these ScFv antibodies for subsequent experiments. RNA samples from hybridoma cells were prepared and cDNA synthesized using First-Strand cDNA Synthesis Kit (GE Healthcare). ScFvs specific for prion protein were isolated using phage-display. This ScFv was subsequently expressed in a bacterial system, purified to homogeneity and characterized by ELISA. After 3 rounds of panning against the N-terminal region of the mouse PrPc, 10 individual ScFv clones recognizing the peptide epitope were identified and sequenced. The ScFv consisted of 249 amino acids and sequence variations were found in both the complementarity determining regions (CDRs) and the frame regions. Clone pJB-M02-02 produced the highest signal against the peptide epitope and was observed to posses properties similar to the original monoclonal antibody. The induction of the ScFv gene in E.coli resulted in significant accumulation of the ScFv within 3 hours. Although several protein induction conditions were tested, the ScFv was expressed exclusively in inclusion bodies. After purification of the ScFv from the insoluble protein fractions and a protein refolding protocol, the refolded ScFv was characterized by ELISA and found to be functional. We have isolated a ScFv using phage display techniques which recognizes the N-terminal part of the mouse PrPc protein, expressed it in large quantities and purified it to homogeneity. The properties of the ScFv are similar to that of the original monoclonal antibody and provides us with a valuable tool to investigate the structure and function of PrP.
AD K. Nat, Y. Shi, W. Yajima, University of Alberta, Agricultural, Food and Nutritional Science, Canada; M. Polymenidou, A. Aguzzi, University of Zürich, Institute for Neuropathology, Switzerland
SP englisch
PO Schottland