Cloning, Purification, and Characterization of the Catalytic C-Terminal Domain of the Human 3-Hydroxy-3-methyl glutaryl-CoA Reductase: An Effective, Fast, and Easy Method for Testing Hypocholesterolemic Compounds.
Rosita CurcioDonatella AielloAngelo VozzaLuigina MutoEmanuela MartelloAnna Rita CappelloLoredana CapobiancoGiuseppe FiermonteCarlo SicilianoAnna NapoliVincenza DolcePublished in: Molecular biotechnology (2020)
3-hydroxy-3-methyl glutaryl-CoA reductase, also known as HMGR, plays a crucial role in regulating cholesterol biosynthesis and represents the main pharmacological target of statins. In mammals, this enzyme localizes to the endoplasmic reticulum membrane. HMGR includes different regions, an integral N-terminal domain connected by a linker-region to a cytosolic C-terminal domain, the latter being responsible for enzymatic activity. The aim of this work was to design a simple strategy for cloning, expression, and purification of the catalytic C-terminal domain of the human HMGR (cf-HMGR), in order to spectrophotometrically test its enzymatic activity. The recombinant cf-HMGR protein was heterologously expressed in Escherichia coli, purified by Ni+-agarose affinity chromatography and reconstituted in its active form. MALDI mass spectrometry was adopted to monitor purification procedure as a technique orthogonal to the classical Western blot analysis. Protein identity was validated by MS and MS/MS analysis, confirming about 82% of the recombinant sequence. The specific activity of the purified and dialyzed cf-HMGR preparation was enriched about 85-fold with respect to the supernatant obtained from cell lysate. The effective, cheap, and easy method here described could be useful for screening statin-like molecules, so simplifying the search for new drugs with hypocholesterolemic effects.
Keyphrases
- mass spectrometry
- cystic fibrosis
- ms ms
- endothelial cells
- liquid chromatography
- escherichia coli
- endoplasmic reticulum
- cardiovascular disease
- capillary electrophoresis
- cell free
- high performance liquid chromatography
- amino acid
- multiple sclerosis
- hydrogen peroxide
- induced pluripotent stem cells
- binding protein
- fatty acid
- poor prognosis
- type diabetes
- gas chromatography
- stem cells
- recombinant human
- pseudomonas aeruginosa
- bone marrow
- molecularly imprinted
- high resolution mass spectrometry
- klebsiella pneumoniae