An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions.

The Escherichia coli outer-membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2'. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon-like peptide-1 (7-37) in 4 M urea. The P1-P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The k(cat)/ K(m) ratio increased with basic residues at P4 or P6, mainly due to a lower K(m) rather than an increase in k(cat). On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli-expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.