Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein
Autor(a) principal: | |
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Data de Publicação: | 2007 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | https://doi.org/10.1016/j.phytochem.2006.11.036 http://www.locus.ufv.br/handle/123456789/19322 |
Resumo: | The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of b-strand structure (42%) over the a-helix (9.9%). The fluorescence emis- sion maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K d ) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K d = 2.79 ± 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6 M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins. |
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Rocha, Carolina S.Luz, Dirce F.Oliveira, Marli L.Baracat-Pereira, Maria C.Medrano, Francisco JavierFontes, Elizabeth P.B.2018-05-03T18:18:24Z2018-05-03T18:18:24Z2007-01-1200319422https://doi.org/10.1016/j.phytochem.2006.11.036http://www.locus.ufv.br/handle/123456789/19322The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of b-strand structure (42%) over the a-helix (9.9%). The fluorescence emis- sion maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K d ) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K d = 2.79 ± 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6 M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.engPhytochemistryv. 68, n. 6, p. 802-810, Março 2007Elsevier Ltdinfo:eu-repo/semantics/openAccessGlycine maxSoybeanLegumesHeterologous expressionProtein renaturationCupin structureSucrose binding proteinExpression of the sucrose binding protein from soybean: renaturation and stability of the recombinant proteininfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALartigo.pdfartigo.pdftexto completoapplication/pdf1074994https://locus.ufv.br//bitstream/123456789/19322/1/artigo.pdf79b2f3f610a38596701877dd12be3c37MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://locus.ufv.br//bitstream/123456789/19322/2/license.txt8a4605be74aa9ea9d79846c1fba20a33MD52THUMBNAILartigo.pdf.jpgartigo.pdf.jpgIM Thumbnailimage/jpeg5159https://locus.ufv.br//bitstream/123456789/19322/3/artigo.pdf.jpg638f484762700b829a175219f3e334a5MD53123456789/193222018-05-03 23:00:44.193oai:locus.ufv.br:123456789/19322Tk9URTogUExBQ0UgWU9VUiBPV04gTElDRU5TRSBIRVJFClRoaXMgc2FtcGxlIGxpY2Vuc2UgaXMgcHJvdmlkZWQgZm9yIGluZm9ybWF0aW9uYWwgcHVycG9zZXMgb25seS4KCk5PTi1FWENMVVNJVkUgRElTVFJJQlVUSU9OIExJQ0VOU0UKCkJ5IHNpZ25pbmcgYW5kIHN1Ym1pdHRpbmcgdGhpcyBsaWNlbnNlLCB5b3UgKHRoZSBhdXRob3Iocykgb3IgY29weXJpZ2h0Cm93bmVyKSBncmFudHMgdG8gRFNwYWNlIFVuaXZlcnNpdHkgKERTVSkgdGhlIG5vbi1leGNsdXNpdmUgcmlnaHQgdG8gcmVwcm9kdWNlLAp0cmFuc2xhdGUgKGFzIGRlZmluZWQgYmVsb3cpLCBhbmQvb3IgZGlzdHJpYnV0ZSB5b3VyIHN1Ym1pc3Npb24gKGluY2x1ZGluZwp0aGUgYWJzdHJhY3QpIHdvcmxkd2lkZSBpbiBwcmludCBhbmQgZWxlY3Ryb25pYyBmb3JtYXQgYW5kIGluIGFueSBtZWRpdW0sCmluY2x1ZGluZyBidXQgbm90IGxpbWl0ZWQgdG8gYXVkaW8gb3IgdmlkZW8uCgpZb3UgYWdyZWUgdGhhdCBEU1UgbWF5LCB3aXRob3V0IGNoYW5naW5nIHRoZSBjb250ZW50LCB0cmFuc2xhdGUgdGhlCnN1Ym1pc3Npb24gdG8gYW55IG1lZGl1bSBvciBmb3JtYXQgZm9yIHRoZSBwdXJwb3NlIG9mIHByZXNlcnZhdGlvbi4KCllvdSBhbHNvIGFncmVlIHRoYXQgRFNVIG1heSBrZWVwIG1vcmUgdGhhbiBvbmUgY29weSBvZiB0aGlzIHN1Ym1pc3Npb24gZm9yCnB1cnBvc2VzIG9mIHNlY3VyaXR5LCBiYWNrLXVwIGFuZCBwcmVzZXJ2YXRpb24uCgpZb3UgcmVwcmVzZW50IHRoYXQgdGhlIHN1Ym1pc3Npb24gaXMgeW91ciBvcmlnaW5hbCB3b3JrLCBhbmQgdGhhdCB5b3UgaGF2ZQp0aGUgcmlnaHQgdG8gZ3JhbnQgdGhlIHJpZ2h0cyBjb250YWluZWQgaW4gdGhpcyBsaWNlbnNlLiBZb3UgYWxzbyByZXByZXNlbnQKdGhhdCB5b3VyIHN1Ym1pc3Npb24gZG9lcyBub3QsIHRvIHRoZSBiZXN0IG9mIHlvdXIga25vd2xlZGdlLCBpbmZyaW5nZSB1cG9uCmFueW9uZSdzIGNvcHlyaWdodC4KCklmIHRoZSBzdWJtaXNzaW9uIGNvbnRhaW5zIG1hdGVyaWFsIGZvciB3aGljaCB5b3UgZG8gbm90IGhvbGQgY29weXJpZ2h0LAp5b3UgcmVwcmVzZW50IHRoYXQgeW91IGhhdmUgb2J0YWluZWQgdGhlIHVucmVzdHJpY3RlZCBwZXJtaXNzaW9uIG9mIHRoZQpjb3B5cmlnaHQgb3duZXIgdG8gZ3JhbnQgRFNVIHRoZSByaWdodHMgcmVxdWlyZWQgYnkgdGhpcyBsaWNlbnNlLCBhbmQgdGhhdApzdWNoIHRoaXJkLXBhcnR5IG93bmVkIG1hdGVyaWFsIGlzIGNsZWFybHkgaWRlbnRpZmllZCBhbmQgYWNrbm93bGVkZ2VkCndpdGhpbiB0aGUgdGV4dCBvciBjb250ZW50IG9mIHRoZSBzdWJtaXNzaW9uLgoKSUYgVEhFIFNVQk1JU1NJT04gSVMgQkFTRUQgVVBPTiBXT1JLIFRIQVQgSEFTIEJFRU4gU1BPTlNPUkVEIE9SIFNVUFBPUlRFRApCWSBBTiBBR0VOQ1kgT1IgT1JHQU5JWkFUSU9OIE9USEVSIFRIQU4gRFNVLCBZT1UgUkVQUkVTRU5UIFRIQVQgWU9VIEhBVkUKRlVMRklMTEVEIEFOWSBSSUdIVCBPRiBSRVZJRVcgT1IgT1RIRVIgT0JMSUdBVElPTlMgUkVRVUlSRUQgQlkgU1VDSApDT05UUkFDVCBPUiBBR1JFRU1FTlQuCgpEU1Ugd2lsbCBjbGVhcmx5IGlkZW50aWZ5IHlvdXIgbmFtZShzKSBhcyB0aGUgYXV0aG9yKHMpIG9yIG93bmVyKHMpIG9mIHRoZQpzdWJtaXNzaW9uLCBhbmQgd2lsbCBub3QgbWFrZSBhbnkgYWx0ZXJhdGlvbiwgb3RoZXIgdGhhbiBhcyBhbGxvd2VkIGJ5IHRoaXMKbGljZW5zZSwgdG8geW91ciBzdWJtaXNzaW9uLgo=Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452018-05-04T02:00:44LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.en.fl_str_mv |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
title |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
spellingShingle |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein Rocha, Carolina S. Glycine max Soybean Legumes Heterologous expression Protein renaturation Cupin structure Sucrose binding protein |
title_short |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
title_full |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
title_fullStr |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
title_full_unstemmed |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
title_sort |
Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein |
author |
Rocha, Carolina S. |
author_facet |
Rocha, Carolina S. Luz, Dirce F. Oliveira, Marli L. Baracat-Pereira, Maria C. Medrano, Francisco Javier Fontes, Elizabeth P.B. |
author_role |
author |
author2 |
Luz, Dirce F. Oliveira, Marli L. Baracat-Pereira, Maria C. Medrano, Francisco Javier Fontes, Elizabeth P.B. |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Rocha, Carolina S. Luz, Dirce F. Oliveira, Marli L. Baracat-Pereira, Maria C. Medrano, Francisco Javier Fontes, Elizabeth P.B. |
dc.subject.pt-BR.fl_str_mv |
Glycine max Soybean Legumes Heterologous expression Protein renaturation Cupin structure Sucrose binding protein |
topic |
Glycine max Soybean Legumes Heterologous expression Protein renaturation Cupin structure Sucrose binding protein |
description |
The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of b-strand structure (42%) over the a-helix (9.9%). The fluorescence emis- sion maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K d ) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K d = 2.79 ± 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6 M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins. |
publishDate |
2007 |
dc.date.issued.fl_str_mv |
2007-01-12 |
dc.date.accessioned.fl_str_mv |
2018-05-03T18:18:24Z |
dc.date.available.fl_str_mv |
2018-05-03T18:18:24Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://doi.org/10.1016/j.phytochem.2006.11.036 http://www.locus.ufv.br/handle/123456789/19322 |
dc.identifier.issn.none.fl_str_mv |
00319422 |
identifier_str_mv |
00319422 |
url |
https://doi.org/10.1016/j.phytochem.2006.11.036 http://www.locus.ufv.br/handle/123456789/19322 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartofseries.pt-BR.fl_str_mv |
v. 68, n. 6, p. 802-810, Março 2007 |
dc.rights.driver.fl_str_mv |
Elsevier Ltd info:eu-repo/semantics/openAccess |
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Elsevier Ltd |
eu_rights_str_mv |
openAccess |
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application/pdf |
dc.publisher.none.fl_str_mv |
Phytochemistry |
publisher.none.fl_str_mv |
Phytochemistry |
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