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Prokaryotic Expression and Purification of Soluble Maize Ac Transposase

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Abstract

Transposons are mobile genetic elements that are found in all eukaryotic and prokaryotic species studied to date. The Maize Activator (Ac) transposase recognizes and excises Ac and Dissociation (Ds) elements and mediates insertion elsewhere in the genome. Insertions of Ds can cause disruption in gene sequences and hence are important functional genomics tool for tagging and cloning of unknown gene sequences. The involvement of Ac transposase (AcTPase) in Ds movement is well documented; however, protein structure and function of AcTPase is poorly understood. To express the maize AcTPase in E. coli, Ac cDNA was synthesized with an N-terminal 6xHis tag and cloned in pTrcAc expression vector. The expression cassette was induced in Rosetta2 (DE3) E. coli lines. End-point RT-PCR confirmed the integrity of AcTPase mRNA during cell culture. Autoinducing cultures grown at 37 °C produced prominent partial AcTPase products of ~40 kDa and ~70 kDa. Trypsin digestion and mass spectrometry analyses confirmed AcTPase in both the eluted peptides. When the cultures were grown at 22–25 °C for 24 h the expected ~90 kDa AcTPase soluble product was detected. The successful expression of full length AcTPase in soluble form allows further investigation of its structure and function.

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References

  1. Feschotte, C. (2008). Plant transposable elements: where genetics meets genomics. Nature Reviews Genetics, 9, 397–405.

    Article  CAS  Google Scholar 

  2. Jordan, I. K., Rogozin, I. B., Glazko, G. V., & Koonin, E. V. (2003). Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends in Genetics, 19, 68–72.

    Article  CAS  Google Scholar 

  3. Singh, S., Tan, H.-Q., & Singh, J. (2012). Mutagenesis of barley malting quality QTLs with Ds transposons. Functional and Integrative Genomics, 12, 131–141.

    Article  CAS  Google Scholar 

  4. Singh, J., Zhang, S., Chen, C., Cooper, L., Bregitzer, P., Sturbaum, A., et al. (2006). High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genomes. Plant Molecular Biology, 62, 937–950.

    Article  CAS  Google Scholar 

  5. Qu, S., Desai, A., Wing, R., & Sundaresan, V. (2008). A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants. Plant Physiology, 146, 189–199.

    Article  CAS  Google Scholar 

  6. Qu, S., Jeon, J. S., Ouwerkerk, P. B. F., Bellizzi, M., Leach, J., Ronald, P., et al. (2009). Construction and application of efficient Ac-Ds transposon tagging vectors in rice. Journal of Integrative Plant Biology, 51(11), 982–992.

    Article  CAS  Google Scholar 

  7. Gangadharan, S., Mularononi, L., Fain-Thornton, J., Wheelan, S. J., & Craig, N. L. (2010). DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo. PNAS, 107(51), 21966–21972.

    Article  CAS  Google Scholar 

  8. Emelyanov, A., Gao, Y., Naqvi, N. I., & Parinov, S. (2006). Trans-kingdom transposition of the maize dissociation element. Genetics, 174, 1095–1104.

    Article  CAS  Google Scholar 

  9. Hauser, C., Fusswinkel, H., Li, J., Oellig, C., Kunze, R., Muller-Nuemann, M., et al. (1988). Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector. Molecular and General Genetics, 214, 373–378.

    Article  CAS  Google Scholar 

  10. Weil, C. F., & Kunze, R. (2000). Transposition of maize Ac/Ds transposable elements in yeast Saccharomyces cerevisiae. Nature Genetics, 26, 187–190.

    Article  CAS  Google Scholar 

  11. MacRae, A. F. (2003). Maize activator transposase expressed in Saccharomyces cerevisiae from a genetic clone: detection via ELISA, and proposed use in complementation studies. Journal of Molecular Microbiology and Biotechnology, 5, 78–81.

    Article  CAS  Google Scholar 

  12. Essers, L., Adolphs, R. H., & Kunze, R. (2000). A highly conserved domain of the maize activator transposase is involved in dimerization. Plant Cell, 12, 211–223.

    CAS  Google Scholar 

  13. Yu, J., Marshall, K., Yamaguchi, M., Haber, J. E., & Weil, C. F. (2004). Microhomology-dependent end joining and repair of transposon-induced DNA hairpins by host factors in Saccharomyces cerevisiae. Molecular and Cellular Biology, 24(3), 1351–1364.

    Article  CAS  Google Scholar 

  14. Nesmelova, I. V., & Hackett, P. B. (2010). DDE transposases: structural similarity and diversity. Advanced Drug Delivery Reviews, 62, 1187–1195.

    Article  CAS  Google Scholar 

  15. Rubin, E., Lithwick, G., & Levy, A. (2001). Structure and evolution of the hAT transposon family. Genetics, 158, 949–957.

    CAS  Google Scholar 

  16. Feldmar, S., & Kunze, R. (1991). The ORFa protein, the putative transposase of maize transposable element Ac, has a basic DNA binding domain. EMBO Journal, 10(13), 4003–4010.

    CAS  Google Scholar 

  17. Boehm, U., Heinlein, M., Behrens, U., & Kunze, R. (1995). One of three nuclear localization signals of maize activator transposase overlaps the DNA-binding domain. Plant Journal, 7(3), 441–451.

    Article  CAS  Google Scholar 

  18. Becker, H. A., & Kunze, R. (1997). Maize activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats. Molecular and General Genetics, 254, 219–230.

    Article  CAS  Google Scholar 

  19. Hickman, A. B., Perez, Z. N., Zhou, L., Musingarimi, P., Ghirlando, R., Hinshaw, J. E., et al. (2005). Molecular architecture of a eukaryotic DNA transposase. Nature Structural and Molecular Biology, 12(8), 715–721.

    Article  CAS  Google Scholar 

  20. Arensburger, P., Hice, R. H., Zhou, L., Smith, R. C., Tom, A. C., Wright, J. A., et al. (2011). Phylogenetic and functional characterization of the hAT transposon superfamily. Genetics, 188, 45–57.

    Article  CAS  Google Scholar 

  21. Studier, F. W. (2005). Protein production by auto-induction in high-density shaking cultures. Protein Expression and Purification, 41(1), 207–234.

    Article  CAS  Google Scholar 

  22. Tan, H-Q., & Singh, J. (2011). High-efficiency thermal asymmetric interlaced (HE-TAIL) PCR for amplification of Ds transposon insertion sites in barley. Journal of Plant Molecular Biology and Biotechnology, 2, 9–14.

    Google Scholar 

  23. Singh, M., and Singh, J. (2012) Seed development-related expression of ARGONAUTE4_9 class of genes in barley: possible role in seed dormancy. Euphytica. OnlineFirst, 1–7.

  24. Neuhoff, V., Arold, A., Taube, D., & Ehrhardrt, W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis, 9(6), 255–262.

    Article  CAS  Google Scholar 

  25. Singh, J., Blundell, M., Tanner, G., & Skerritt, J. H. (2001). Albumin and globulin proteins of wheat: immunological and N-terminal sequence characterization. Journal of Cereal Science, 34, 85–103.

    Article  CAS  Google Scholar 

  26. Fusswinkel, H., Schein, S., Courage, U., Starlinger, P., & Kunze, R. (1991). Detection and abundance of mRNA and protein encoded by transposable element activator (Ac) in maize. Molecular and General Genetics, 225, 186–192.

    Article  CAS  Google Scholar 

  27. Dumon-Seignovert, L., Cariot, G., & Vuillard, L. (2004). The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expression and Purification, 37, 203–206.

    Article  CAS  Google Scholar 

  28. Chesshyre, J. A., & Hipkiss, A. R. (1989). Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli. Applied Microbiology and Biotechnology, 31, 158–162.

    Article  CAS  Google Scholar 

  29. Sorensen, H. P., & Mortensen, K. K. (2005). Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factories, 4, 1.

    Article  Google Scholar 

  30. Vasina, J. A., & Baneyx, F. (1997). Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expression and Purification, 9(2), 211–218.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was conducted with the support of the Natural Sciences and Engineering Research Council (NSERC-Discovery) of Canada. We also thank Dr. Eric Bonneil from Research in Immunology and Cancer (IRIC), University of Montreal, for his kind assistance and suggestions in bioinformatics analysis of mass spectra.

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Authors and Affiliations

  1. Plant Science Department, McGill University, 21 111 Rue Lakeshore, Ste-Anne-de-Bellevue, Quebec, H9X 3V9, Canada

    Neil Dylan Lamb-Palmer, Manjit Singh & Jaswinder Singh

  2. Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Quebec, H9X 3V9, Canada

    John P. Dalton

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  1. Neil Dylan Lamb-Palmer
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  2. Manjit Singh
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  4. Jaswinder Singh
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Correspondence to Jaswinder Singh.

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Lamb-Palmer, N.D., Singh, M., Dalton, J.P. et al. Prokaryotic Expression and Purification of Soluble Maize Ac Transposase. Mol Biotechnol 54, 685–691 (2013). https://doi.org/10.1007/s12033-012-9610-z

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  • DOI: https://doi.org/10.1007/s12033-012-9610-z

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