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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2022
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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
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INTRODUCTION: 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.
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PRATA, 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. Abstract... São Paulo, SP: Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq, 2022. p. 220-220. Disponível em: http://repositorio.ipen.br/handle/123456789/33911. Acesso em: 19 Apr 2024.
Esta referência é gerada automaticamente de acordo com as normas do estilo IPEN/SP (ABNT NBR 6023) e recomenda-se uma verificação final e ajustes caso necessário.