Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath
<p>Abstract</p> <p>Background</p> <p>2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein c...
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| Format: | Article |
| Language: | English |
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BMC
2010-01-01
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| Series: | BMC Research Notes |
| Online Access: | http://www.biomedcentral.com/1756-0500/3/12 |
| _version_ | 1818644409534644224 |
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| author | Kursula Petri Myllykoski Matti |
| author_facet | Kursula Petri Myllykoski Matti |
| author_sort | Kursula Petri |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.</p> <p>Findings</p> <p>In order to be able to fully characterize CNPase structurally and functionally, we have set up expression systems for different domains of CNPase, using a total of 18 different expression constructs. CNPase was expressed in <it>E. coli </it>with a TEV-cleavable His-tag. Enzymatic activity assays indicated that the purified proteins were active and correctly folded. The folding of both the full-length protein, as well as the N- and C-terminal domains, was also studied by synchrotron CD spectroscopy. A thermal shift assay was used to optimize buffer compositions to be used during purification and storage. The assay also indicated that CNPase was most stable at a pH of 5.5, and could be significantly stabilized by high salt concentrations.</p> <p>Conclusions</p> <p>We have been able to express and purify recombinantly several different domains of CNPase, including the isolated N-terminal domain, which is folded mainly into a β-sheet structure. The expression system can be used as an efficient tool to elucidate the role of CNPase in the myelin sheath.</p> |
| first_indexed | 2024-12-17T00:14:24Z |
| format | Article |
| id | doaj.art-37f484fa3cdf47988c51598e49e1887e |
| institution | Directory Open Access Journal |
| issn | 1756-0500 |
| language | English |
| last_indexed | 2024-12-17T00:14:24Z |
| publishDate | 2010-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Research Notes |
| spelling | doaj.art-37f484fa3cdf47988c51598e49e1887e2022-12-21T22:10:45ZengBMCBMC Research Notes1756-05002010-01-01311210.1186/1756-0500-3-12Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheathKursula PetriMyllykoski Matti<p>Abstract</p> <p>Background</p> <p>2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.</p> <p>Findings</p> <p>In order to be able to fully characterize CNPase structurally and functionally, we have set up expression systems for different domains of CNPase, using a total of 18 different expression constructs. CNPase was expressed in <it>E. coli </it>with a TEV-cleavable His-tag. Enzymatic activity assays indicated that the purified proteins were active and correctly folded. The folding of both the full-length protein, as well as the N- and C-terminal domains, was also studied by synchrotron CD spectroscopy. A thermal shift assay was used to optimize buffer compositions to be used during purification and storage. The assay also indicated that CNPase was most stable at a pH of 5.5, and could be significantly stabilized by high salt concentrations.</p> <p>Conclusions</p> <p>We have been able to express and purify recombinantly several different domains of CNPase, including the isolated N-terminal domain, which is folded mainly into a β-sheet structure. The expression system can be used as an efficient tool to elucidate the role of CNPase in the myelin sheath.</p>http://www.biomedcentral.com/1756-0500/3/12 |
| spellingShingle | Kursula Petri Myllykoski Matti Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath BMC Research Notes |
| title | Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath |
| title_full | Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath |
| title_fullStr | Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath |
| title_full_unstemmed | Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath |
| title_short | Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath |
| title_sort | expression purification and initial characterization of different domains of recombinant mouse 2 3 cyclic nucleotide 3 phosphodiesterase an enigmatic enzyme from the myelin sheath |
| url | http://www.biomedcentral.com/1756-0500/3/12 |
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