Engineering of a macromolecular scaffold to develop specific protease inhibitors

Nat Biotechnol. 2003 Sep;21(9):1063-8. doi: 10.1038/nbt860. Epub 2003 Aug 17.


The specific inhibition of serine proteases, which are crucial switches in many physiologically important processes, is of value both for basic research and for therapeutic applications. Ecotin, a potent macromolecular inhibitor of serine proteases of the S1A family, presents an attractive scaffold to engineer specific protease inhibitors because of its large inhibitor-protease interface. Using synthetic shuffling in combination with a restricted tetranomial diversity, we created ecotin libraries that are mutated at all 20 amino acid residues in the binding interface. The efficacy of these libraries was demonstrated against the serine protease plasma kallikrein (Pkal). Competitive phage display selection yielded a Pkal inhibitor with an apparent dissociation equilibrium constant (K(i)*) of 11 pM, whereas K(i)* values for related proteases (such as Factor Xa (FXa), Factor XIa (FXIa), urokinase-type plasminogen activator (uPA), thrombin, and membrane-type serine protease 1 (MT-SP1)) were four to seven orders of magnitude higher. The adaptability of the scaffold was demonstrated by the isolation of inhibitors to two additional serine proteases, MT-SP1/matriptase and Factor XIIa.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Escherichia coli Proteins / biosynthesis*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification
  • Feasibility Studies
  • Macromolecular Substances
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed / physiology
  • Periplasmic Proteins*
  • Plasma Kallikrein / chemistry
  • Plasma Kallikrein / metabolism
  • Protein Conformation
  • Protein Engineering / methods*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / metabolism*
  • Serine Proteinase Inhibitors / chemistry*
  • Serine Proteinase Inhibitors / genetics
  • Serine Proteinase Inhibitors / isolation & purification
  • Serine Proteinase Inhibitors / metabolism*


  • Eco protein, E coli
  • Escherichia coli Proteins
  • Macromolecular Substances
  • Periplasmic Proteins
  • Recombinant Proteins
  • Serine Proteinase Inhibitors
  • Serine Endopeptidases
  • matriptase
  • Plasma Kallikrein