Deletion of the carboxy terminal part of stefin B does not have a major effect for binding to papain.

Determination of crystal structures of chicken cystatin and human stefin B complexed with papain revealed a novel model of protease inhibition and also structural differences between two cysteine proteinase inhibitor (CPI) families. According to the 3D alignment, stefins have an extension of 9 amino acids on their carboxy terminus in comparison with cystatins. The extension was not expected to make a major contribution to interaction with the enzyme. A deletion mutant of stefin B, corresponding in length to the carboxy terminal sequence of chicken cystatin, was constructed by the use of polymerase chain reaction (PCR). This (C3S, delta 89-98) human stefin B, 10 amino acids shorter, inhibited papain with a Ki of 0.012 nM which is comparable to the Ki of 0.03 nM for authentic, nondeleted recombinant stefin B. This finding thus confirms the tertiary structure-based alignment.