Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.
Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope a...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3823863?pdf=render |
| _version_ | 1818327438315225088 |
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| author | Akinori Kashimura Kazuaki Okawa Kotarou Ishikawa Yuta Kida Kokoro Iwabuchi Yudai Matsushima Masayoshi Sakaguchi Yasusato Sugahara Fumitaka Oyama |
| author_facet | Akinori Kashimura Kazuaki Okawa Kotarou Ishikawa Yuta Kida Kokoro Iwabuchi Yudai Matsushima Masayoshi Sakaguchi Yasusato Sugahara Fumitaka Oyama |
| author_sort | Akinori Kashimura |
| collection | DOAJ |
| description | Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase. |
| first_indexed | 2024-12-13T12:16:16Z |
| format | Article |
| id | doaj.art-d76125563a9b449f9617b0681749b15e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T12:16:16Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-d76125563a9b449f9617b0681749b15e2022-12-21T23:46:43ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01811e7866910.1371/journal.pone.0078669Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.Akinori KashimuraKazuaki OkawaKotarou IshikawaYuta KidaKokoro IwabuchiYudai MatsushimaMasayoshi SakaguchiYasusato SugaharaFumitaka OyamaAcidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.http://europepmc.org/articles/PMC3823863?pdf=render |
| spellingShingle | Akinori Kashimura Kazuaki Okawa Kotarou Ishikawa Yuta Kida Kokoro Iwabuchi Yudai Matsushima Masayoshi Sakaguchi Yasusato Sugahara Fumitaka Oyama Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. PLoS ONE |
| title | Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. |
| title_full | Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. |
| title_fullStr | Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. |
| title_full_unstemmed | Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. |
| title_short | Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. |
| title_sort | protein a mouse acidic mammalian chitinase v5 his expressed in periplasmic space of escherichia coli possesses chitinase functions comparable to cho expressed protein |
| url | http://europepmc.org/articles/PMC3823863?pdf=render |
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