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

Xue Xia, Ozan S. Kumru, Sachiko I. Blaber, C. Russell Middaugh, Ling Li, David M. Ornitz, Mason A. Sutherland, Connie A. Tenorio, Michael Blaber

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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.

Original languageEnglish
Pages (from-to)1444-1453
Number of pages10
JournalJournal of Pharmaceutical Sciences
Volume105
Issue number4
DOIs
StatePublished - Apr 1 2016

Keywords

  • FGF-1
  • X-ray crystallography
  • cysteine-free mutant
  • cystine
  • disulfide
  • empirical phase diagram
  • protein engineering
  • protein stability

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