title: A lysine-free mutant of epidermal growth factor as targeting moiety of a targeted toxin.
abstract: OBJECTIVE
  Elevated levels of epidermal growth factor (EGF) receptor are observed on several human tumors, e.g. cervical carcinoma and mamma carcinomas. The natural ligand EGF is an alternative to established antibodies and tyrosine kinase inhibitors for targeting EGF receptor-overexpressing tumor cells for therapy. Conjugations of compounds to EGF lack the necessary homogeneity for an intended application, since several amino acids may react with the chemical linker.
  
  
  METHODS
  We designed an EGF variant (EGF(RR)) in which the two lysines were substituted with arginine (K28R and K48R). EGF(RR) was fused to the protein toxin saporin to obtain a model protein for detailed analyses on EGF receptor binding and on both the enzymatic activity of saporin and the cytotoxicity of the fusion protein.
  
  
  RESULTS
  The mutation decreased the enzymatic activity of saporin 2.3-fold and the binding of EGF(RR) retained its specificity for EGF receptor while increasing the Kd 5.5-fold. In spite of these differences the cytotoxicity of the fusion protein was unchanged in comparison to a fusion protein with EGF both when applied alone and in combination with cytotoxicity augmenting saponin.
  
  
  CONCLUSIONS
  We conclude that EGF(RR) retained its ability to bind with high specificity to EGF receptor and is thus suitable for a number of chemical linkage applications such as targeting drugs or dyes to EGF receptor-expressing cells.
authors.affiliation: Charité - Universitätsmedizin Berlin, Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bachran
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schneider
authors.forename: Sebastian B
authors.initials: SB
authors.lastname: Riese
authors.forename: Diana
authors.initials: D
authors.lastname: Bachran
authors.forename: Romy
authors.initials: R
authors.lastname: Urban
authors.forename: Nicole
authors.initials: N
authors.lastname: Schellmann
authors.forename: Claudia
authors.initials: C
authors.lastname: Zahn
authors.forename: Mark
authors.initials: M
authors.lastname: Sutherland
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
mesh_terms: D000818:Animals; D001120:Arginine; D016503:Drug Delivery Systems; D004815:Epidermal Growth Factor / Q000235:genetics*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008239:Lysine; D051379:Mice; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells; D011485:Protein Binding / Q000187:drug effects; D054789:Ribosome Inactivating Proteins, Type 1 / Q000494:pharmacology*; D000076985:Saporins
chemical_list: D000922:Immunotoxins; D054789:Ribosome Inactivating Proteins, Type 1; D004815:Epidermal Growth Factor; D001120:Arginine; D000076985:Saporins; D008239:Lysine