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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

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Abstract

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.

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Acknowledgements

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).

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  1. Rosita Curcio, Donatella Aiello and Angelo Vozza have equally contributed to this work.

Authors and Affiliations

  1. Department of Pharmacy, Health, and Nutritional Sciences, University of Calabria, 87036, Arcavacata di Rende, Cosenza, Italy

    Rosita Curcio, Luigina Muto, Emanuela Martello, Anna Rita Cappello, Carlo Siciliano & Vincenza Dolce

  2. Department of Chemistry and Chemical Technologies, University of Calabria, 87036, Arcavacata di Rende, Cosenza, Italy

    Donatella Aiello & Anna Napoli

  3. Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125, Bari, Italy

    Angelo Vozza & Giuseppe Fiermonte

  4. Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100, Lecce, Italy

    Loredana Capobianco

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Curcio, R., Aiello, D., Vozza, A. et al. 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. Mol Biotechnol 62, 119–131 (2020). https://doi.org/10.1007/s12033-019-00230-1

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