Protein Characterization
Resolution of cfIL-22 protein on 12% SDS-PAGE generated three major bands. The ~20 kDa and 24 kDa (Fig. 2.2C) bands were predicted to be associated with differential post-translational processing of the cfIL-22 monomer and the ~44kDa band corresponded to the predicted size of a cfIL-22 dimer. All three products were excised from a Coomassie-stained gel and confirmed by MS/MS analysis to be cfIL-22. N-terminal sequencing of the 20 and 24kDa bands confirmed that the signal peptide was fully processed in both monomeric forms of cfIL-22 expressed in plants (supporting material Figure S5). PNGase F removal of N-glycans resulted in a single band product at 20kDa (supporting material Figure S6). This confirms the prediction that the 24kDa band corresponds to cfIL-22 protein with a single N-glycan.