Expression, purification and characterization of ricin vectors used for exogenous antigen delivery into the MHC class I presentation pathway
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Last Update: 2021-03-02
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Source: Internet
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Author: User
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Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith
et 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 in
E. coli
and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized
in vitro
, via an
N
-glycosidase assay, and
in 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.
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