Resumo de eventos cient??ficos
A new approach to obtain the catalytic site of human Angiotensin Converting Enzyme
Autor
SANTOS, CAROLINA M. dos
FIGUEIREDO, ALINE M. de
LEITE, RODRIGO C.
CAMARGO, NATANE M. de
AFFONSO, REGINA
EUROPEAN SYMPOSIUM ON BIOCHEMICAL ENGINEERING SCIENCES, 12th
Resumen
Angiotensin-converting enzyme I (ACE) is a key part of the renin-angiotensin system whose main function is to
regulate blood pressure. The 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 of
angiotensin I, bradykinin, angiotensina (1-7), beta-amyloid peptide and sensitivities to various inhibitors. A more
detailed analysis shows that these regions are composed of HEMGH and EAIGD sequences, which are the
catalytic sites. Our question is: If the synthesis of catalytic sites with corrects structure and activity could be a
good model per si to study new drugs. In our laboratory the catalytic site of the C-domain was obtained with
correct structural conformation and with enzymatic activity.
The objective this work is to obtain the Ala361 to Gli468 catalytic site, N-domain, in a structural conformation
that resembles its native form.
The 380 pb cDNA to catalytic site was cloned in the pE1 vector (kindly provided by Dr. David Wood), elastinlike-polyptide (ELP) tag sequence was linked with catalytic site, ELP~csACEn recombinant protein, this was
expressed in bacteria with Terrificus broth with 0.1 mM IPTG for 20h. Harvested cells were resuspended in TE
buffer, after sonication and centrifugation the pellet was resuspended the same buffer. The ELP~csACEn was
precipitated with 0.8 M ammonium sulfate and the tag sequence was cleaved by pH change, pH 6.2. All steps
were analyzed by SDS-PAGE, Dot and Western blotting.
The catalytic site was synthesized from bacterial system with ELP sequence tag in soluble form. The purification
process was done by ammonium sulfate precipitation and cleavage of the ELP sequence by changing to acidic
pH. The characterization of catalytic site by SDS-PAGE shows that this is pure and Western blotting
immunological assay confirmed the identity of the protein as csACEn.
The strategy used to obtain the Ala361 to Gli468 catalytic site in soluble and pure form was successful. The next
steps: we will continue with the Maldi-tof and structural conformation analyzes.