Recombinant production of the catalytic sites of the ACE1 enzyme and the ACE2 binding region peptide with SARS-CoV

Abstract

Angiotensin-converting enzymes 1 and 2 (ACE 1 and 2) act decisively in the Renin-Angiotensin System. ACE 1 participates in blood pressure control and brain protection, among others. ACE2 is currently one of the entry points for the SARS-CoV-2 virus, which causes COVID-19. Peptides of ACE1 (catalytic sites) and ACE2 (binding SARS-CoV) can be interesting tools to solve and detect human dysfunctions. In this project, the objectives are: 1) obtain the catalytic sites of pure ACE1 (N and C domains), and 2) obtain the ACE 2 region that binds to the SARS-CoV virus in sufficient quantities for activity studies. The expression of catalytic sites linked to the ELP/Int sequence was at 16ᵒC, and the ELP/Int~capACE2 were at 16ᵒC, 20ᵒC, and 25ᵒC. The purification by precipitation of the three peptides was the only step with 0,8M of ammonium sulfate. The Intein self-cleavage in the three peptides was evaluated in four buffers with acid pH, namely MES pH 6.3; Tris-HCl pH 6.8; Cacodylate pH 6.5; and Bis-Tris pH6.2 in the incubation temperatures at 20ᵒC. Expression and purification by salt precipitation of the three peptides showed good production. The catalytic sites linked to ELP/Int yielded 47% and 70% for N and C in the precipitation step, respectively, and 39% for capACE2 at both temperatures. The expression of these was for N of 0.54 mg/mL.A600 and C of 0.61 mg/mL.A600, while for ELP/Int~capACE2 at 16ᵒC was not observed, and at temperatures of 20ᵒC and 25ᵒC at expression was the same, 0.01 mg/mL.A600. The preliminary results of evaluating the buffers and conditions for self-cleavage of Intein to the removal of ELP showed that the buffers MES and Cacodylate for csACEN, Tris-HCl for csACEC, and Bis-Tris for capACE2, all with incubation at 20ᵒC, had a yield below 10% in the obtaining of the pure peptides. In this present work, the expression of ELP/Int~capACE2 was higher at 25⁰C, and the purification process improved the yield of all ELP/Int peptides and with lower bacterial contaminants. However, the conditions of Intein self-cleavage need more studies, especially regarding the temperature and incubation time of the sample.