FIG 5 .
NPTH-dependent autocleavage is not necessary to achieve substrate specificity switching. (A) Illustration of the positions of the introduction of human rhinovirus 3C protease cleavage sites on the crystal structure of the C-terminal domain of SpaS (SpaSc) (PDB accession no. 3C01). (B) The expression and cleavage of SpaSFLAG and 3C protease in the indicated strains were analyzed in whole-cell lysates by SDS-PAGE, Western blotting, and immunodetection of the FLAG epitope. Functionality of substrate specificity switching was profiled by SDS-PAGE of TCA-precipitated culture supernatants, Western blotting, and immunodetection of InvJ, SipB, and SptP. Asterisks indicate the cleaved C-terminal fragments of the engineered SpaS variants. Abbreviations: 3C, human rhinovirus 3C protease; wc, whole cells; sup, culture supernatant.