Overexpression of functional human oxidosqualene cyclase in Escherichia coli

Charlotte Kürten, Mathias Uhlén, Per-Olof Syrén

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7mg protein/g cells (hOSC in Pichia pastoris) after 48h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.
Original languageEnglish
JournalProtein Expression and Purification
Volume115
Pages (from-to)46-53
Number of pages8
ISSN1046-5928
DOIs
Publication statusPublished - 2015

Keywords

  • E. coli
  • Expression and purification
  • Membrane protein
  • Oxidosqualene cyclase
  • Triterpene cyclase

Cite this

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title = "Overexpression of functional human oxidosqualene cyclase in Escherichia coli",
abstract = "The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7mg protein/g cells (hOSC in Pichia pastoris) after 48h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.",
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author = "Charlotte K{\"u}rten and Mathias Uhl{\'e}n and Per-Olof Syr{\'e}n",
year = "2015",
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language = "English",
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pages = "46--53",
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Overexpression of functional human oxidosqualene cyclase in Escherichia coli. / Kürten, Charlotte; Uhlén, Mathias; Syrén, Per-Olof.

In: Protein Expression and Purification, Vol. 115, 2015, p. 46-53.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Overexpression of functional human oxidosqualene cyclase in Escherichia coli

AU - Kürten, Charlotte

AU - Uhlén, Mathias

AU - Syrén, Per-Olof

PY - 2015

Y1 - 2015

N2 - The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7mg protein/g cells (hOSC in Pichia pastoris) after 48h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.

AB - The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7mg protein/g cells (hOSC in Pichia pastoris) after 48h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.

KW - E. coli

KW - Expression and purification

KW - Membrane protein

KW - Oxidosqualene cyclase

KW - Triterpene cyclase

U2 - 10.1016/j.pep.2015.04.015

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JO - Protein Expression and Purification

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SN - 1046-5928

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