Optimization of the process of expression in E. coli and purification of the catalytic sites of the ACE1 by the ELP-Intein system

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dc.contributor.authorPRATA, BEATRIZ A.pt_BR
dc.contributor.authorSANTOS, CAROLINA M. dospt_BR
dc.contributor.authorAFFONSO, REGINApt_BR
dc.coverageInternacionalpt_BR
dc.creator.eventoANNUAL MEETING OF THE BRAZILIAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY (SBBq), 51st; CONGRESS OF BRAZILIAN BIOPHYSICAL SOCIETY (SBBf)/LATIN AMERICAN FEDERATION OF BIOPHYSICAL SOCIETIES (Lafebs), 46thpt_BR
dc.date.accessioned2023-03-21T15:15:12Z
dc.date.available2023-03-21T15:15:12Z
dc.date.eventoSeptember 5-8, 2022pt_BR
dc.description.abstractINTRODUCTION: Angiotensin I-converting enzyme (ACE) is a fundamental part of the renin-angiotensin system; this has two domains, N- and C-, each of which has a catalytic site that exhibits 60% sequence identity. Its actions are in the control of blood pressure, protection of the brain by cleavage of beta-amyloid bodies, cell proliferation, formation of hematopoietic stem cells, among others. OBJECTIVES: Obtaining the catalytic sites Ala361 to Gly468 (N domain region, csACEN) and Ala959 to Ser1066 (C domain region, csACEC) in pure form and with their correct structural conformation. MATERIALS AND METHODS: Expression conditions of pE1csACEN and pE1csACEC vectors in E. coli BL21(DE3) strain: cultures grown in Terrific Broth at 37⁰C at 140 rpm for 20–24 h and 0.1 mM IPTG. Purification by Elastin-like Polypeptide (ELP) precipitation: ELP-bound catalytic sites were purified with two ammonium sulfate precipitations (ASp). Remotion of ELP: by autocleavage of the Intein sequence using the buffers: sodium phosphate, sodium cacodylate, MES and Tris-HCl. The ELP/Intein was removed from the sample by ASp. The analyzes of all stages of the process were performed by SDS-PAGE and Dot blotting. DISCUSSION AND RESULTS: The differential for obtaining the pure peptides was the temperature of 37⁰C, with a significant increase in expression concerning the cultivation of 16⁰C. In the ELP purification steps, ammonium sulfate buffer concentrations of 0.57 M and 0.8 M were the most efficient. Intein's self-cleaving was more efficient with MES buffers and Tris-HCl for ELPsACEN and ELPsACEC, respectively. Structural analysis by Circular Dichroism and Fluorescence confirmed the correct structure of the pure peptides. CONCLUSION: In the present work, we defined the most efficient conditions for expression, purification, and obtaining of ACE catalytic sites in pure form. The csACEN and csACEC peptides will allow greater assertiveness in obtaining and characterizing new hypertensive drugs and in the hydrolysis of substrates such as beta-amyloid.pt_BR
dc.format.extent220-220pt_BR
dc.identifier.citationPRATA, BEATRIZ A.; SANTOS, CAROLINA M. dos; AFFONSO, REGINA. Optimization of the process of expression in E. coli and purification of the catalytic sites of the ACE1 by the ELP-Intein system. In: ANNUAL MEETING OF THE BRAZILIAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY (SBBq), 51st; CONGRESS OF BRAZILIAN BIOPHYSICAL SOCIETY (SBBf)/LATIN AMERICAN FEDERATION OF BIOPHYSICAL SOCIETIES (Lafebs), 46th, September 5-8, 2022, Águas de Lindóia, SP. <b>Abstract...</b> São Paulo, SP: Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq, 2022. p. 220-220. Disponível em: http://repositorio.ipen.br/handle/123456789/33911.
dc.identifier.orcidhttps://orcid.org/0000-0002-9264-4262
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/33911
dc.localSão Paulo, SPpt_BR
dc.local.eventoÁguas de Lindóia, SPpt_BR
dc.publisherSociedade Brasileira de Bioquímica e Biologia Molecular - SBBqpt_BR
dc.rightsopenAccesspt_BR
dc.titleOptimization of the process of expression in E. coli and purification of the catalytic sites of the ACE1 by the ELP-Intein systempt_BR
dc.typeResumo de eventos científicospt_BR
dspace.entity.typePublication
ipen.autorCAROLINA MACHADO DOS SANTOS
ipen.autorREGINA AFFONSO
ipen.autorBEATRIZ ANGELO PRATA
ipen.codigoautor14944
ipen.codigoautor1547
ipen.codigoautor15247
ipen.contributor.ipenauthorCAROLINA MACHADO DOS SANTOS
ipen.contributor.ipenauthorREGINA AFFONSO
ipen.contributor.ipenauthorBEATRIZ ANGELO PRATA
ipen.date.recebimento23-03
ipen.event.datapadronizada2022pt_BR
ipen.identifier.ipendoc29545pt_BR
ipen.notas.internasAbstractpt_BR
ipen.type.genreResumo
relation.isAuthorOfPublication82bc9b90-ba55-464b-96cc-8b8b6e6e5290
relation.isAuthorOfPublication97da04b7-6659-49f0-b893-0698c583c338
relation.isAuthorOfPublicationb7396366-6461-4fb8-98c7-477da7c0b2bc
relation.isAuthorOfPublication.latestForDiscoveryb7396366-6461-4fb8-98c7-477da7c0b2bc
sigepi.autor.atividadeAFFONSO, REGINA:1547:810:Npt_BR
sigepi.autor.atividadeSANTOS, CAROLINA M. dos:14944:810:Npt_BR
sigepi.autor.atividadePRATA, BEATRIZ A.:15247:810:Spt_BR
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