Recombinant expression, purification and characterization of human hyaluronidases

摘要: Hyaluronidases and their substrate, hyaluronic acid, are known to play a role in signal transduction events directly correlated with tumor growth cell migration. Yet correlations disease progression controversial due scarce information on enzymatic properties the lack of specific inhibitors as pharmacological tools. Therefore, aim this thesis was establishment recombinant expression systems for human hyaluronidase subtypes, purification enzymes characterization respect biochemical catalytic properties. In first approach subtypes PH-20, Hyal-1, Hyal-2 Hyal-4 were efficiently expressed E. coli. All hyaluronidases formed inclusion bodies cytoplasm coli presence disulfide bridges native proteins. Only Hyal-1 could be re-folded vitro into an enzymatically active protein folding yields strongly depending kind denaturing agent used, shuffling system arginine buffer. However, both yield activity purified by Ni-IMAC under conditions, extremely low. As posed major problems, PH-20 alternatively culture medium Drosophila Schneider-2 cells (DS-2 cells) enabling correct formation bonds mammalian-like glycosylation. Native DS-2 ammonium sulfate precipitation, cation exchange chromatography Ni-IMAC. During use detergents required because extraordinarily high adsorption surfaces. Highly pure, homogeneous (54 kDa 4 comprising glycosidic residues) comparable plasma isolated quantities sufficient biophysical studies. Although characteristic features such pH profile showed similar behavior prokaryotic eukaryotic system, glycosylation shown improve efficiency enzyme solution. A truncation C-terminal domain failed systems. Human (56 kDa) stably chelating-IMAC. determined previously bovine testicular (BTH), commercially available preparation, exhibited typical assay-dependent shift optimum. Furthermore, recognized chondroitin C alternative substrates pH-dependent activities preferred acid. In contrast no detected kDa), cells. Since hydrolytic is discussed controversially literature, variety assays, including electrophoretic viscosimetric techniques, explored. none methods proved any or chondroitinase neutral basic conditions. To gain more detailed insight mechanism comparison BTH capillary zone electrophoresis method established qualitative quantitative detection degradation products and, less significantly, produced different acid at acidic nearly pH, explaining profiles. found degrade molecular weight consecutively di- hexasaccharides final products. The analysis conversion hyaluronan hexa- octasaccharides revealed subtypes: minimum substrate BTH, hexasaccharide, not accepted albeit octasaccharide; transglycosylation, which catalyzed significant extent occurred only great excess substrate. calculation parameters Michaelis-Menten kinetics additional subtypes. model array disaccharide unit binding sites framing site employed explain pH- subtype-specific hyaluronidases. Taken together, production provides basis enzymological studies, crystallization structure-based development inhibitors, can example,