Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase
<p>Abstract</p> <p>Background</p> <p>Chitin synthase 3a (CHS3a) from <it>Botrytis cinerea </it>(Bc) catalyses the multiple transfer of <it>N</it>-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homop...
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| Format: | Article |
| Language: | English |
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BMC
2010-11-01
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| Series: | BMC Research Notes |
| Online Access: | http://www.biomedcentral.com/1756-0500/3/299 |
| _version_ | 1818494044142043136 |
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| author | Piffeteau Annie Thellend Annie Drujon Thierry Magellan Hervé Becker Hubert F |
| author_facet | Piffeteau Annie Thellend Annie Drujon Thierry Magellan Hervé Becker Hubert F |
| author_sort | Piffeteau Annie |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Chitin synthase 3a (CHS3a) from <it>Botrytis cinerea </it>(Bc) catalyses the multiple transfer of <it>N</it>-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient.</p> <p>Findings</p> <p>We undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in <it>Escherichia coli</it>. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed.</p> <p>Conclusions</p> <p>Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a.</p> |
| first_indexed | 2024-12-10T18:01:29Z |
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| institution | Directory Open Access Journal |
| issn | 1756-0500 |
| language | English |
| last_indexed | 2024-12-10T18:01:29Z |
| publishDate | 2010-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Research Notes |
| spelling | doaj.art-b358ec8fee414972992cec968616c6d52022-12-22T01:38:46ZengBMCBMC Research Notes1756-05002010-11-013129910.1186/1756-0500-3-299Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthasePiffeteau AnnieThellend AnnieDrujon ThierryMagellan HervéBecker Hubert F<p>Abstract</p> <p>Background</p> <p>Chitin synthase 3a (CHS3a) from <it>Botrytis cinerea </it>(Bc) catalyses the multiple transfer of <it>N</it>-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient.</p> <p>Findings</p> <p>We undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in <it>Escherichia coli</it>. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed.</p> <p>Conclusions</p> <p>Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a.</p>http://www.biomedcentral.com/1756-0500/3/299 |
| spellingShingle | Piffeteau Annie Thellend Annie Drujon Thierry Magellan Hervé Becker Hubert F Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase BMC Research Notes |
| title | Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase |
| title_full | Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase |
| title_fullStr | Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase |
| title_full_unstemmed | Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase |
| title_short | Expression in <it>E. coli </it>and characterization of the catalytic domain of <it>Botrytis cinerea </it>chitin synthase |
| title_sort | expression in it e coli it and characterization of the catalytic domain of it botrytis cinerea it chitin synthase |
| url | http://www.biomedcentral.com/1756-0500/3/299 |
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