M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
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