Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
Autor: | Satoshi Wakita, Hirotaka Suzuki, Masayoshi Sakaguchi, Kana Okazaki, Misa Ohno, Fumitaka Oyama, Akinori Kashimura, Yasusato Sugahara, Peter O. Bauer, Masahiro Kimura, Atsushi Ukita, Kazuaki Okawa |
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Jazyk: | angličtina |
Rok vydání: | 2015 |
Předmět: |
Recombinant Fusion Proteins
chitin-binding activity Chitin Biology medicine.disease_cause Catalysis Article catalytic domain chitinolytic activity colloidal and crystalline chitin lcsh:Chemistry Inorganic Chemistry chemistry.chemical_compound Mice medicine food processing Escherichia coli Animals Physical and Theoretical Chemistry Cloning Molecular lcsh:QH301-705.5 Molecular Biology innate immunity Spectroscopy mouse acidic mammalian chitinase chemistry.chemical_classification Organic Chemistry Chitinases Protein primary structure Temperature General Medicine asthma Hydrogen-Ion Concentration allergy Fusion protein Protein tertiary structure Computer Science Applications Enzyme lcsh:Biology (General) lcsh:QD1-999 chemistry Biochemistry Chitinase biology.protein Protein A Protein Binding |
Zdroj: | International Journal of Molecular Sciences Volume 16 Issue 2 Pages 4028-4042 International Journal of Molecular Sciences, Vol 16, Iss 2, Pp 4028-4042 (2015) |
ISSN: | 1422-0067 |
Popis: | Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases. |
Databáze: | OpenAIRE |
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