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Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching".
- Source :
-
Nature biotechnology [Nat Biotechnol] 2002 Sep; Vol. 20 (9), pp. 908-13. Date of Electronic Publication: 2002 Aug 05. - Publication Year :
- 2002
-
Abstract
- 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.
- Subjects :
- Computer Simulation
Cytokines chemistry
Cytokines genetics
Cytokines metabolism
Escherichia coli genetics
Escherichia coli metabolism
Genes, Switch
Granulocyte Colony-Stimulating Factor metabolism
Histidine metabolism
Hydrogen-Ion Concentration
Mutagenesis, Site-Directed
Protein Binding
Protein Conformation
Receptors, Granulocyte Colony-Stimulating Factor chemistry
Receptors, Granulocyte Colony-Stimulating Factor genetics
Receptors, Granulocyte Colony-Stimulating Factor metabolism
Sensitivity and Specificity
Static Electricity
Granulocyte Colony-Stimulating Factor chemical synthesis
Granulocyte Colony-Stimulating Factor genetics
Histidine chemistry
Histidine genetics
Models, Molecular
Protein Engineering methods
Subjects
Details
- Language :
- English
- ISSN :
- 1087-0156
- Volume :
- 20
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Nature biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 12161759
- Full Text :
- https://doi.org/10.1038/nbt725