FIG. 5.
Amino-terminal sequence analysis of the 12-kDa, carboxyl-terminal MBP-p23/p12 cleavage product. After cleavage of MBP-p13/p12 with recombinant 3CLpro, separation by SDS-polyacrylamide gel electrophoresis, and transfer to a polyvinylidene difluoride membrane, the 12-kDa cleavage product was subjected to Edman degradation. Phenylthiohydantoin-amino acids generated during each reaction cycle were detected by their absorbance at 269 nm (expressed as milli-absorbance units [mAU]) and identified by their characteristic retention times on a reversed-phase HPLC support. (a) Chromatogram of phenylthiohydantoin-amino acid standards; (b to f) chromatograms of phenylthiohydantoin-amino acids from reaction cycles 1 to 5, respectively. Specific peaks of phenylthiohydantoin-amino acids are indicated in the single-letter code.