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Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "2nd Generation" Therapeutic Application

Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "2nd Generation" Therapeutic Application.
Name(s): Xue, Xia, author
Kumru, Ozan, author
Blaber, Sachiko, author
Middaugh, Russell, author
Li, Ling, author
Ornitz, David, author
Sutherland, Mason, author
Tenorio, Connie, author
Blaber, Michael, author
Type of Resource: text
Genre: text
Date Issued: 2016-02-11
Physical Form: computer
Physical Form: online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and three buried Cys residues (at positions 16, 83 and117) 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 three 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 WT 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 "2nd generation" form of FGF-1 for therapeutic application.
Identifier: FSU_libsubv1_scholarship_submission_1464366396 (IID), 10.1016/j.xphs.2016.02.010 (DOI)
Keywords: FGF-1, disulfide, protein stability, empirical phase diagram, x-ray crystallography, cysteine free mutant, protein engineering, cystine
Publication Note: Publisher's version available at
Preferred Citation: Xia, X., Kumru, O. S., Blaber, S. I., Middaugh, C. R., Li, L., Ornitz, D. M., ... & Blaber, M. (2016). Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application. Journal of pharmaceutical sciences, 105(4), 1444-1453.
Persistent Link to This Record:
Owner Institution: FSU
Is Part Of: Journal of Pharmaceutical Sciences.
Issue: iss. 4, vol. 105