Expression,Characterization The N-terminal Domain Of Human Angiotensin Converting Enzyme In Pichia Pastoris And Comparision With C-terminal Domain

Angiotensin converting enzyme(ACE, EC 3.4.15.1) in vertebrates is a kind of zinc metal lopeptidase that plays a key role in regulating blood pressure, body fluid, electrolyte equilibrium, cardiovascular system development and vascular remodeling through rennin-angiotensin system(RAS) and kallikrein-kinin system(KKS). Relative studies showed that somatic angiotensin-converting enzyme involved in the release of angiotensin II (Ang II) and the inactivation of bradykinin (BK), and the C-domain of ACE was the predominant site of angiotensin I cleavage in vivo, In addition, the N-domain of ACE is a main catalytic site of bradykinin cleavage.Currently, the target enzymes used in ACEI screening mainly are ACE which has two domains such as captopril, enalapril, benazepril, fosinopri, et al. Although these drugs were clinically applied as the first-line treatment for hypertension, they all inhibited two highly homologous domains of ACE. These relatively non-selective inhibitors (ACEI) prevented the conversion of Ang I to Ang II and inhibited the generation of bradykinin degradation, resulting in the cough, renal dysfunction and other side effects. In this study we first obtained the human ACE N-domain expression in pichia pastoris yeast. Adding the ACE-C domain protein of high purity and activity, it provided a basis for the in vitro study on the selective inhibition of the two homologous domains.In this paper, we researched human ACE gene in NCBI and selected the N-terminal conserved sequence of 1071 bp as target gene fragment. We obtained the cDNA by RT-PCR from the TE-1 cell line, derived ACE-N gene fragment through PCR, and constructed secretory expression plasmid pPIC9K-ACE-N. The recombinant plasmid was transformed into Pichia pastoris strain GS115, and the positive clones were selected and subjected to electroporation. Antibiotic G418 was used for screening multi-copy inserts. The expressed protein was purified by Ni2+ affinity chromatography. The expression quantity of target protein reached 0.11g/L and its purity reached 99%.Taken HHL(N-Hippuryl-His-Leu) as the substrate, the enzyme activity of ACE, ACE-N, and ACE-C were 21.22 U/mg,1.03 U/mg and 118.64 U/mg respectively, and their Km values were 1.14mM,0.92mM and 1.2mM respectively. Comparing their Vmax, Kcat and Kcat/Km, the value of ACE-C was highest, and the were ACE and ACE-N respectively. It demonstrated that ACE-C had the highest catalytic efficiency for HHL while ACE-N had the lowest catalytic efficiency.In conclusion, we first obtained the human ACE N-domain expression protein in pichia pastoris yeast, and this provided both a basis for the enzyme activity in vitro test and for the establishment of specific inhibitor screening model of angiotensin-converting enzyme C-domain and N-domain.