A new approach for purification of the catalytic site of the Angiotensin Conversion Enzyme, N domain, mediated by the ELP-Inten system

Abstract

Angiotensin-converting enzyme I, ACE, is a key part of the renin-angiotensin system whose main function is to regulate blood pressure and balance of salts in the body. ACE1 has two isoforms, somatic, sACE, and testicular, tACE. sACE possesses two domains, N- C-, with catalytic sites which exhibit 60% sequence identity. These domains differ in terms of chloride-ion activation profiles, rates of peptide hydrolysis and sensitivities to various inhibitors. N-domain has specific action in the hydrolyze of Alzheimer’s diseases beta amyloid bodies and angiotensin 1-7, which active the MAS receptor and triggering anti-thrombotic and anti-inflammatory actions. The objective this work was to obtain catalytic site Ala361 to Gli468 of the N-domain region, csACEN, isolation without chromatographic and denaturant chemical process. For that, a new methodology was used in the expression of the csACEN peptide, in which the peptide was linked to the elastin-like polypeptide, ELP, and Intein, and expressed at 37C. The characterization of catalytic site was made by SDS-PAGE and dot blotting. The culture temperature at 37C significantly increased the expression of the ELP/Intein/csACEN fusion protein. This culture was lysed at a low temperature allowing the fusion protein to become soluble. The precipitation of ELP at high concentrations of ammonium sulfate were obtained in 0.57 M and 0.8 M. Intein autocleavage occurs at acidic pH and it is important to pay attention to: pI 6.65 for csACEN and pI 6.87 for ELPcsACEN, which are very low. The best autocleavage efficiency was with MES and TriHCl buffers, pH 6.3 and 6.8, respectively, in which pure csACEn peptide was obtained. The strategy used to obtain the Ala361 to Gli468 catalytic site in soluble and pure form was obtained with success and the protocol for obtaining similar peptides was established.