Effect of cysteine substitutions on the mitogenic activity and stability of recombinant human keratinocyte growth factor.
Human Keratinocyte growth factor (hKGF), a member of the FGF family of growth factors, contains five cysteines at amino acid positions 1, 15, 40, 102, and 106. We expressed five cysteine mutants of hKGF in which the cysteines were cumulatively replaced with alanine or serine, starting with cysteine-1. Recombinant hKGF has an inherently higher mitogenic activity and stability to heat and acid than reported for glycosylated hKGF. Mitogenic activity is increased an additional 2.6 fold by substitution of cysteine-1 with alanine. Mutants with the conserved cysteine substituted at position 40 were more susceptible to heat inactivation than rhKGF, but showed no significant difference in acid inactivation. Cysteine-free rhKGF is mitogenic, demonstrating that neither cysteines nor disulfide bonds are required for mitogenic activity. However, cysteine-free rhKGF does not bind Heparin-Sepharose and is unstable to heat and acid compared to rhKGF, suggesting that the cysteines have a role in maintaining KGF's structure. This information will useful in the development of a more stable and more potent wound healing agent from hKGF.
Bare
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Ohmeda, PPD, Murray Hill, NJ 07974.
Brown
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Goyal
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Idler
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Mansson
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000235:genetics*; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006133:Growth Substances / Q000235:genetics / Q000494:pharmacology*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
C496453:FGF7 protein, human; C496454:Fgf7 protein, mouse; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D008934:Mitogens; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D003545:Cysteine