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Immunology and Infection

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JoVE Journal Immunology and Infection
Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli
 

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Article DOI: 10.3791/56430-v • 07:35 min • August 3rd, 2017
August 3rd, 2017 •
*1, *1, 2, 2, 1,3, 1,3, 2, 2,4, 2
1Sackler Faculty of Medicine, Tel Aviv University, 2Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 3Department of Ophthalmology, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, 4Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University
* These authors contributed equally

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Resumo
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A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Tags

Overexpression Purification Human Cis-prenyltransferase Escherichia Coli Enzymatic Activity Codon-optimized DHDDS Isoprenoid Synthesis Retinitis Pigmentosa Mutations Cloning Buffer A DNase I Protease Inhibitor Mixture Homogenize Teflon Homogenizer Microfluidizer Centrifuge Cell Lysate Soluble Fraction Cobalt-immobilized Metal Affinity Chromatography Column Imidazole Fast Protein Liquid Chromatography
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