Understanding Protease Resistance Using mRNA Display

Peptides (small proteins, generally less than 30 amino acids) have several therapeutic advantages over common antibodies: they are cheaper, faster and easier to produce and purify, and they generally do not require cold temperatures for storage. However, their current applications have been limited due to their instability in the presence of proteases and peptidases naturally present in the human body. These enzymes can degrade peptides in a matter of minutes, leaving the peptides unable to reach their target to be effective. Understanding the rules governing the cleavage process of proteases and peptidases can significantly advance the field of peptide therapeutics. Previous studies of protease and peptidase activity required each peptide to be individually synthesized, purified, and tested. By combining high-throughput sequencing with mRNA display,1 we were able to simultaneously analyze the stability of millions of peptides against common proteases. We studied the cleavage patterns of chymotrypsin, trypsin, and proteinase K, proteases commonly used in biological research. Our preliminary results show clear cleavage activity bias of each protease, and we are able to identify particularly effective and ineffective substrates for each protease. Furthermore, our model successfully predicts the relative stabilities for peptides found in the literature.