Proteases are involved in various biological functions. Thus, inhibition of 
their activities is scientifically interesting and medically important. However, 
there is no systematic method established to date to generate endopeptidase 
inhibitory peptides. Here, we report a general system to identify endopeptidase 
inhibitory peptides based on the use of in vitro evolution. Using this system, 
we generated peptides that inhibit cathepsin E (CE) specifically at a 
submicromolar IC(50). This system generates protease inhibitor peptides 
utilizing techniques of cDNA display, selection-by-function, Y-ligation-based 
block shuffling, and others. We further demonstrated the importance and 
effectiveness of a secondary library for obtaining small-sized and active 
peptides. CE inhibitory peptides generated by this method were characterized by 
a small size (8 to 12 aa) and quite different sequences, suggesting that they 
bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained 
here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of 
pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally 
similar to CE). The general applicability of this system suggests that it may be 
useful to identify inhibitory peptides for various kinds of proteases and that 
it may therefore contribute to protein science and drug discovery. The peptide 
binding to a protein is discussed in comparison with the antibody binding to an 
antigen.