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 Curcio
  • Donatella Aiello
  • Angelo Vozza
  • Luigina Muto
  • Emanuela Martello
  • Anna Rita Cappello
  • Loredana Capobianco
  • Giuseppe Fiermonte
  • Carlo Siciliano
  • Anna NapoliEmail author
  • Vincenza DolceEmail author
Original Paper


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.


HMGR Bacterial expression Affinity chromatography MALDI MS and MS/MS Enzymatic activity Screening of statin-like molecules 



This work was supported by Associazione Italiana per la Ricerca sul Cancro (FG Grant No. 15404/2014) and by Ministero Italiano della Ricerca e dell’Università (MIUR–PRIN 2015, Prot. 201545245K_002).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12033_2019_230_MOESM1_ESM.docx (52 kb)
Supplementary material 1 (DOCX 51 kb)


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Authors and Affiliations

  1. 1.Department of Pharmacy, Health, and Nutritional SciencesUniversity of CalabriaArcavacata di RendeItaly
  2. 2.Department of Chemistry and Chemical TechnologiesUniversity of CalabriaArcavacata di RendeItaly
  3. 3.Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, Biotechnologies and BiopharmaceuticsUniversity of BariBariItaly
  4. 4.Department of Biological and Environmental Sciences and TechnologiesUniversity of SalentoLecceItaly

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