Overexpression and purification of human Cis-prenyltransferase in Escherichia coli

Ilan Edri, Michal Goldenberg, Michal Lisnyansky, Roi Strulovic, Hadas Newman, Anat Loewenstein, Daniel Khananshvili, Moshe Gilad, Yoni Haitin

Research output: Contribution to journalArticlepeer-review

Abstract

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Original languageEnglish
Article numbere56430
JournalJournal of Visualized Experiments
Volume2017
Issue number126
DOIs
StatePublished - 7 Aug 2017

Keywords

  • Codon optimization
  • Dehydrodolichyl diphosphate synthase
  • Heterologous overexpression
  • Immunology
  • Issue 126
  • Polyprenyl
  • Prenyltransferase
  • Protein purification

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