| dc.description | INTRODUCTION
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.
OBJECTIVES
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.
MATERIALS AND METHODS
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.
DISCUSSION AND RESULTS
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 withMES and TriHCl buffers, pH 6.3 and 6.8, respectively, in which
pure csACEn peptide was obtained.
CONCLUSION
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. | |
| dc.source | IUPAB International Congress, 20th; SBBf Congress, 45th; Annual Meeting of SBBq, 50th, October 4-8, 2021, S??o Paulo, SP | |
