Expression, purification and characterization of ricin vectors used for exogenous antigen delivery into the MHC class I presentation pathway

Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smithet al. J Immunol 2002; 169:99–107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression inE. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterizedin vitro , via anN -glycosidase assay, andin vivo , by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass.