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Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

Journal Article · · Journal of Pharmaceutical Sciences
 [1];  [2];  [1];  [2];  [3];  [3];  [1];  [1];  [1]
  1. Florida State Univ., Tallahassee, FL (United States)
  2. Univ. of Kansas, Lawrence, KS (United States)
  3. Washington Univ. School of Medicine, St. Louis, MO (United States)
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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. In this work, 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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
Trefoil Therapeutics, LLC; Florida State University; National Institutes of Health (NIH)
Grant/Contract Number:
HL105732
OSTI ID:
1247356
Journal Information:
Journal of Pharmaceutical Sciences, Vol. 105, Issue 4; ISSN 0022-3549
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 12 works
Citation information provided by
Web of Science

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Cited By (2)

Aggrescan3D (A3D) 2.0: prediction and engineering of protein solubility journal May 2019
Unraveling the Connection between Fibroblast Growth Factor and Bone Morphogenetic Protein Signaling journal October 2018

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
FGF-1
disulfide
protein stability
empirical phase diagram
X-ray crystallography
cysteine-free mutant
protein engineering
cystine