Synthesis of frataxin genes by direct assembly of serial deoxyoligonucleotide primers and its expression in Escherichia coli

Young Geol Yoon, Sun Hee Park, Jee Suk Lee, Chunlan Yan, Chan Bae Park, Michael D. Koob, Young Hyun Yoo

Research output: Contribution to journalArticlepeer-review

Abstract

Frataxin, a small nuclear-encoded protein targeted to mitochondria, is known to play an important role in both the mitochondrial respiratory chain and iron homeostasis. The protein is highly conserved in most eukaryotic organisms with no major structural changes, suggesting that it serves a crucial function in all organisms. Recently, purified frataxin was used as a therapeutic treatment of Friedreich's ataxia, a common degenerative disorder that results from a frataxin protein deficiency, by directly applying the protein to the diseased cells. In this report, we describe a novel and rapid method of synthesizing genes encoding frataxin proteins for the purpose of efficient protein production. The artificial yeast and human frataxin genes were synthesized by direct assembly of serial deoxyoligonucleotide primers designed based on the optimal nucleotide sequences. When we tested the expression of these synthetic genes in two E. coli host strains, the yeast frataxin gene was expressed 20 folds higher in Rosetta (DE3) cells than in BL21 (DE3) cells, whereas the expression levels of human frataxin were similar in both E. coli strains. Attenuation of the Fenton reactions by the purified yeast and human frataxin proteins was observed under the defined conditions, which suggests that the recombinant frataxin proteins are active and functional. The procedure described here could be applied to many known genes or to generate novel synthetic genes that can be redesigned by arranging functional domains from previously identified genes and to study the structure and function of synthetic recombinant proteins and potential usage.

Original languageEnglish (US)
Pages (from-to)382-389
Number of pages8
JournalBiotechnology and Bioprocess Engineering
Volume18
Issue number2
DOIs
StatePublished - Apr 2013

Bibliographical note

Funding Information:
This work was supported by the National Research Foundation of Korea Grant (2011-0008052 to YGY and 2011-0001262 to YHY) funded by the Korean Government. It was also supported by the ZheJiang Province Natural Science Foundation of China and ZheJiang Province Department of Education Foundation of China (LY12H26006 to CY and Y201120063 to CY).

Keywords

  • expression
  • frataxin
  • friedreich's ataxia
  • gene synthesis
  • rosetta (DE3)

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