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

Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "Second Generation" Therapeutic Application.
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Name(s): Xia, Xue, author
Kumru, Ozan S, author
Blaber, Sachiko I, author
Middaugh, C Russell, author
Li, Ling, author
Ornitz, David M, author
Sutherland, Mason A, author
Tenorio, Connie A, author
Blaber, Michael, author
Type of Resource: text
Genre: Journal Article
Text
Date Issued: 2016-04-01
Physical Form: computer
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 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.
Identifier: FSU_pmch_27019961 (IID), 10.1016/j.xphs.2016.02.010 (DOI), PMC5318998 (PMCID), 27019961 (RID), 27019961 (EID), S0022-3549(16)00366-X (PII)
Keywords: FGF-1, X-ray crystallography, Cysteine-free mutant, Cystine, Disulfide, Empirical phase diagram, Protein engineering, Protein stability
Grant Number: R01 HL076664, R01 HL105732, R01 HL111190, HL105732
Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318998.
Subject(s): Amino Acid Substitution
Crystallography, X-Ray
Cysteine/chemistry
Cysteine/genetics
Fibroblast Growth Factor 1/chemistry
Fibroblast Growth Factor 1/genetics
Humans
Models, Molecular
Protein Engineering
Protein Stability
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_pmch_27019961
Owner Institution: FSU
Is Part Of: Journal of pharmaceutical sciences.
1520-6017
Issue: iss. 4, vol. 105

Choose the citation style.
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. Retrieved from http://purl.flvc.org/fsu/fd/FSU_pmch_27019961