Angiotensin-converting enzyme 2 (ACE2) is a transmembrane protein that is a member of the adrenaline-converting enzyme II (ACE) family. It is a protein containing 805 amino acid residues with structurally multiple domains, including an N-terminal signal peptide, an extracellular region, a transmembrane region, and a C-terminal domain. Catalog No. PA1000-6875
ACE2 binds to peptide molecules associated with angiotensin II primarily through its extracellular domain and metabolizes it to angiotensin 1-7, thereby playing an important role in blood pressure balance and fibrosis and even inflammatory responses.
The preparation and characterization of ACE2 protein is a key step in the study of its biological function and the development of related drugs. The ACE2 protein prepared by recombinant technology can be used for in vitro experiments to simulate the interaction with its ligands or pathogens, and further reveal its molecular mechanism and physiological functions.
2.Experimental preparation.
Step 1: Clone the target sequence.
The ACE2 gene was amplified from human lung tissue using reverse transcription polymerase chain reaction (RT-PCR) technology, and the amplification product was ligated to the expression vector using restriction enzymes to construct a recombinant expression vector of the target ACE2 gene.
Step 2: Transfect host cells.
The recombinant expression vector was transfected into CHO cells or other suitable host cell lines, and after screening and expansion, a cell line stably expressing ACE2 protein was obtained.
Step 3: Expression & Purification of the Protein.
Cells were cultured to the logarithmic growth phase and then an inducer (e.g., isopropyl-D-thiogalactoside, IPTG) was added to stimulate the expression of the ACE2 gene. The cells were harvested, and the cell lysate containing the target protein was obtained after ultrasonic disruption and centrifugation. Purify the target ACE2 protein using techniques such as tracking radioimmunoprecipitation or affinity chromatography.
Step 4: Protein Analysis & Identification.
Techniques such as SDS-PAGE, Western blot, or mass spectrometry were used to analyze the molecular weight, antigenicity, and purity of purified ACE2 protein.
Step 5: Functional qualification.
Methods such as cell binding assays or enzyme activity assays were used to verify that the recombinant ACE2 protein can interact with the coronavirus and perform related functions.