Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching"

Nat Biotechnol. 2002 Sep;20(9):908-13. doi: 10.1038/nbt725. Epub 2002 Aug 5.


We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Publication types

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

MeSH terms

  • Computer Simulation
  • Cytokines / chemistry
  • Cytokines / genetics
  • Cytokines / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genes, Switch
  • Granulocyte Colony-Stimulating Factor / chemical synthesis*
  • Granulocyte Colony-Stimulating Factor / genetics*
  • Granulocyte Colony-Stimulating Factor / metabolism
  • Histidine / chemistry*
  • Histidine / genetics*
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Conformation
  • Protein Engineering / methods*
  • Receptors, Granulocyte Colony-Stimulating Factor / chemistry
  • Receptors, Granulocyte Colony-Stimulating Factor / genetics
  • Receptors, Granulocyte Colony-Stimulating Factor / metabolism
  • Sensitivity and Specificity
  • Static Electricity


  • Cytokines
  • Receptors, Granulocyte Colony-Stimulating Factor
  • Granulocyte Colony-Stimulating Factor
  • Histidine