Synthesis of frataxin genes by direct assembly of serial deoxyoligonucleotide primers and its expression in Escherichia coli
Tóm tắt
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.
Tài liệu tham khảo
Busi, M. V. and D. F. Gomez-Casati (2012) Exploring frataxin function. IUBMB Life 64: 56–63.
Lill, R., B. Hoffmann, S. Molik, A. J. Pierik, N. Rietzschel, O. Stehling, M. A. Uzarska, H. Webert, C. Wilbrecht, and U. Mühlenhoff (2012) The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism. Biochim. Biophys. Acta 1823: 1491–1508
Gakh, O., P. Cavadini, and G. Isaya (2002) Mitochondrial processing peptidases. Biochim. Biophys. Acta 1592: 63–77.
Schmucker, S. and H. Puccio (2010) Understanding the molecular mechanisms of Friedreich’s ataxia to develop therapeutic approaches. Hum. Mol. Genet. 19: 103–110.
Wilson, R. B. (2012) Therapeutic developments in Friedreich ataxia. J. Child Neurol. 27: 1212–1216.
Babady, N. E., N. Carelle, R. D. Wells, T. A. Rouault, M. Hirano, D. R. Lynch, M. B. Delatycki, R. B. Wilson, G. Isaya, and H. Puccio (2007) Advancements in the pathophysiology of Friedreich’s Ataxia and new prospects for treatments. Mol. Genet. Metab. 92: 23–35.
Whitnall, M. and D. R. Richardson (2006) Iron: A new target for pharmacological intervention in neurodegenerative diseases. Semin. Pediatr. Neurol. 13: 186–197.
Wilson, R. B., D. R. Lynch, and K. H. Fischbeck (1998) Normal serum iron and ferritin concentrations in patients with Friedreich’s ataxia. Ann. Neurol. 44: 132–134.
Lodi, R., B. Rajagopalan, J. L. Bradley, D. J. Taylor, J. G. Crilley, P. E. Hart, A. M. Blamire, D. Manners, P. Styles, A. H. Schapira, and J. M. Cooper (2002) Mitochondrial dysfunction in Friedreich’s ataxia: From pathogenesis to treatment perspectives. Free Radic. Res. 36: 461–466.
Vyas, P. M., W. J. Tomamichel, P. M. Pride, C. M. Babbey, Q. Wang, J. Mercier, E. M. Martin, and R. M. Payne (2012) A TATfrataxin fusion protein increases lifespan and cardiac function in a conditional Friedreich’s ataxia mouse model. Hum. Mol. Genet. 21: 1230–1247.
Kim, W., D. W. Kim, B. N. Shin, D. Y. Yoo, S. M. Nam, M. J. Kim, J. H. Choi, Y. S. Yoon, M. H. Won, S. Y. Choi, and I. K. Hwang (2011) PEP-1-frataxin significantly increases cell proliferation and neuroblast differentiation by reducing lipid peroxidation in the mouse dentate gyrus. Neurochem. Res. 36: 2452–2458.
Sambrook, J., E. Fritsch, and T. Maniatis (1989) Molecular cloning: A Laboratory Manual. 2nd ed. pp. 1.21–1.52. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
Yoon, Y. G., Y. W. Yang, and M. D. Koob (2009) PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli. Biotechnol. Lett. 31: 1671–1676.
Jiang, X., Y. Huo, H. Cheng, X. Zhang, X. Zhu, and M. Wu (2012) Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T. Extremophiles 16: 427–435.
Maliandi, M. V., M. V. Busi, M. Clemente, E. J. Zabaleta, A. Araya, and D. F. Gomez-Casati (2007) Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH). Protein Expr. Purif. 51: 157–161.
Park, S., O. Gakh, S. M. Mooney, and G. Isaya (2002) The ferroxidase activity of yeast frataxin. J. Biol. Chem. 277: 38589–38595.
Osawa, S., T. H. Jukes, K. Watanabe, and A. Muto (1992) Recent evidence for evolution of the genetic code. Microbiol. Rev. 56: 229–264.
Yoon, Y. G. and M. D. Koob (2011) Toward genetic transformation of mitochondria in mammalian cells using a recoded drugresistant selection marker. J. Genet. Genomics 38: 173–179.
Jukes, T. H. and S. Osawa (1993) Evolutionary changes in the genetic code. Comp. Biochem. Physiol. 106: 489–494.
Gustafsson, C., S. Govindarajan, and J. Minshull (2004) Codon bias and heterologous protein expression. Trends Biotechnol. 22: 346–353.
Welch, M., A. Villalobos, C. Gustafsson, and J. Minshull (2011) Designing genes for successful protein expression. Methods Enzymol. 498: 43–66.
Yoon, Y. G. and M. D. Koob (2008) Selection by drug resistance proteins located in the mitochondria of mammalian cells. Mitochondrion 8: 345–351.
Cameron, A. D. S., M. Volar, L. A. Bannister, and R. J. Redfield (2008) RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenza. Nucleic Acids Res. 36: 10–20.
Stenström, C. M., E. Holmgren, and L. A. Isaksson (2001) Cooperative effects by the initiation codon and its flanking regions on translation initiation. Gene 273: 259–265.
Choi, J. W., H. Park, J. W. Yun, C. H. Song, and Y. S. Lee (1999) The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli. Bioproc. Eng. 21: 293–298.
Bencze, K. Z., K. C. Kondapalli, J. D. Cook, S. McMahon, C. Millán-Pacheco, N. Pastor, and T. L. Stemmler (2006) The structure and function of frataxin. Crit. Rev. Biochem. Mol. Biol. 41: 269–291.
Hainfeld, J. F., W. Liu, C. M. Halsey, P. Freimuth, and R. D. Powell (1999) Ni-NTA-gold clusters target His-tagged proteins. J. Struct. Biol. 127: 185–198.