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Protein & Peptide Letters
ISSN (Print): 0929-8665
ISSN (Online): 1875-5305
VOLUME: 21
ISSUE: 5
DOI: 10.2174/0929866520666131203102315      Price:  $58









Protease Resistant Variants of FGF1 with Prolonged Biological Activity

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Author(s): Agnieszka Kobielak, Malgorzata Zakrzewska, Michal Kostas, Piotr Jakimowicz, Jacek Otlewski and Daniel Krowarsch
Pages 434-443 (10)
Abstract:
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.
Affiliation:
Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw; ul. Joliot- Curie 14 a, 50-383 Wroclaw, Poland.