@article {Bonadio2023-dv,
	title = {Computational design of matrix metalloprotenaise-9 (MMP-9) resistant to auto-cleavage},
	journal = {Biochem. J.},
	volume = {480},
	number = {14},
	year = {2023},
	month = {jul},
	pages = {1097{\textendash}1107},
	publisher = {Portland Press Ltd.},
	abstract = {Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix. MMP-9 has been implicated in several diseases including neurodegeneration, arthritis, cardiovascular diseases, fibrosis and several types of cancer, resulting in a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9Cat) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9Cat variant that is active but stable to auto-cleavage. For this purpose, we first identified potential auto-cleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the auto-cleavage site by predicting mutations that minimize auto-cleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after 7 days of incubation at 37{\textdegree}C. This MMP-9Cat variant, with an identical with MMP-9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9CAT stabilization could be applied to redesign other proteases to improve their stability for various biotechnological applications.},
	keywords = {auto-cleavage, auto-degradation, enzyme engineering, matrix metalloproteases, MMP-9, protein engineering},
	author = {Bonadio, Alessandro and Oguche, Solomon and Lavy, Tali and Kleifeld, Oded and Shifman, Julia}
}
