title: [Expression of human angiotensin converting enzyme-C domain in Pichia pastoris].
abstract: Angiotensin I-converting enzyme (ACE, EC3.4.15.1) plays an important role in regulating blood pressure. The C-domain of ACE has been identified as the main catalytic site of angiotensin I cleavage in vivo. The ACE gene fragment of the C-domain was amplified by PCR and cloned into the pPIC9K secretory expression plasmid. The recombinant plasmid was transformed into Pichia pastoris strain GS115. Positive clones were selected and subject to electroporation. Antibiotic G418 was used for the screening of multicopy inserts. After optimization of the expression system, the protein yield reached 0.5 g/L by flask-shaking culture fermentation, and enzyme activity reached 7.178 U/mL in the fermentation supernatant. The purity of the target protein obtained was 97% after Ni+ affinity chromatography. Enzyme inhibitory activity assay using Captopril showed that it is promising to use ACE-C domain as new generation of target for screening ACE inhibitor antihypertensive drugs.
authors.affiliation: Laboratory of Proteomics & Molecular Enzymology, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Yulan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Jue
authors.initials: J
authors.lastname: Xu
authors.forename: Chuanlian
authors.initials: C
authors.lastname: Xu
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors; D020134:Catalytic Domain / Q000235:genetics*; D018274:Electroporation; D006801:Humans; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D011994:Recombinant Proteins; D007703:Peptidyl-Dipeptidase A