High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

Kathleen Szeker, Olli Niemitalo, Marco G. Casteleijn, André H. Juffer, Peter Neubauer

Research output: Contribution to journalArticleScientificpeer-review

7 Citations (Scopus)

Abstract

Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression - one example is the formation of stable 5' mRNA secondary structures that can impede an efficient translation initiation.In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5' mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5' mRNA sequence for reduced secondary structures in the translation initiation region.The increase of the cultivation temperature from 30°C to 42°C allowed a more than 10-fold increase of activity per cell and optimizing the 5' mRNA gene sequence further increased the activity per cell 1.7-fold at 42°C. Thus, the combination of high-temperature cultivation and 5' sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5' mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.
Original languageEnglish
Pages (from-to)268-274
Number of pages7
JournalJournal of Biotechnology
Volume156
Issue number4
DOIs
Publication statusPublished - 20 Dec 2010
MoE publication typeA1 Journal article-refereed

Fingerprint

Deinococcus
Purine-Nucleoside Phosphorylase
Escherichia coli
Messenger RNA
Temperature
Genes
Proteins
Recombinant proteins
Biocatalysts
Recombinant Proteins
Nucleosides
Enzymes

Keywords

  • Deinococcus geothermalis
  • MRNA secondary structure
  • Purine nucleoside phosphorylase
  • Recombinant protein expression
  • Thermostable protein

Cite this

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title = "High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli",
abstract = "Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression - one example is the formation of stable 5' mRNA secondary structures that can impede an efficient translation initiation.In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5' mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5' mRNA sequence for reduced secondary structures in the translation initiation region.The increase of the cultivation temperature from 30°C to 42°C allowed a more than 10-fold increase of activity per cell and optimizing the 5' mRNA gene sequence further increased the activity per cell 1.7-fold at 42°C. Thus, the combination of high-temperature cultivation and 5' sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5' mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.",
keywords = "Deinococcus geothermalis, MRNA secondary structure, Purine nucleoside phosphorylase, Recombinant protein expression, Thermostable protein",
author = "Kathleen Szeker and Olli Niemitalo and Casteleijn, {Marco G.} and Juffer, {Andr{\'e} H.} and Peter Neubauer",
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High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli. / Szeker, Kathleen; Niemitalo, Olli; Casteleijn, Marco G.; Juffer, André H.; Neubauer, Peter.

In: Journal of Biotechnology, Vol. 156, No. 4, 20.12.2010, p. 268-274.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

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AU - Juffer, André H.

AU - Neubauer, Peter

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