title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "Second Generation" Therapeutic Application.
abstract: Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous "second generation" form of FGF-1 for therapeutic application.
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, Florida 32306.
authors.forename: Xue
authors.initials: X
authors.lastname: Xia
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047.
authors.forename: Ozan S
authors.initials: OS
authors.lastname: Kumru
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, Florida 32306.
authors.forename: Sachiko I
authors.initials: SI
authors.lastname: Blaber
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047.
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.affiliation: Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: David M
authors.initials: DM
authors.lastname: Ornitz
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, Florida 32306.
authors.forename: Mason A
authors.initials: MA
authors.lastname: Sutherland
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, Florida 32306.
authors.forename: Connie A
authors.initials: CA
authors.lastname: Tenorio
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, Florida 32306. Electronic address: michael.blaber@med.fsu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Blaber
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D015202:Protein Engineering; D055550:Protein Stability
chemical_list: D016220:Fibroblast Growth Factor 1; D003545:Cysteine
keywords: FGF-1; X-ray crystallography; cysteine-free mutant; cystine; disulfide; empirical phase diagram; protein engineering; protein stability