Protease Resistant Variants of FGF1 with Prolonged Biological Activity
Therapeutic potential of human acidic fibroblast growth factor (FGF1) resulting from its undeniable role in angiogenesis
and wound healing processes is questioned due to its low stability and short half-life in vivo. Our previous
studies showed that prolonged biological activity of FGF1 can be achieved by increasing its proteolytic resistance directly
linked to improved global thermostability. In this study, we applied an alternative method of generation of long-lasting
FGF1 variants by rigidification of the growth factor’s segment highly sensitive to proteases action. In order to determine
regions the most prone to enzymatic degradation, we used limited proteolysis by trypsin combined with mass spectrometry
analysis. We found that the initial proteolytic cleavages occurred mainly within the C-terminal region of the wild-type
protein, pointing on its significant role in growth factor degradation. Based on bioinformatic analysis, we introduced two
single mutations (C117P, K118V) within β-strand XI and combined them in a double mutant. We determined resistance to
proteolysis, biophysical properties and biological activities of obtained variants. All of them occurred to be significantly
less susceptible to trypsin (up to 100-fold) and also to chymotrypsin degradation comparing to the wild-type protein. Interestingly,
all variants were not more thermostable than the wild-type FGF1. We attributed this dramatic increase in resistance
to proteolysis to entropic stabilization of C-terminal region.
Keywords: Acidic fibroblast growth factor, biological activity, limited proteolysis, mass spectrometry, proteolytic degradation.
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