Skip to main content
SpringerLink
Log in

Expression, Purification of Recombinant Human Mitochondrial Transcription Termination Factor 3 (hMTERF3) and Preparation of Polyclonal Antibody Against hMTERF3

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. Mitochondrial transcription termination factor 3 (MTERF3) is the most conserved member of the MTERF family and a negative regulator of mammalian mitochondrial DNA transcription. To create a specific polyclonal antibody against human MTERF3 (hMTERF3), we first cloned hMTERF3 into prokaryotic expression vector pGEX-4T-1, and GST-hMTERF3 was efficiently expressed in Escherichia coli after induction by IPTG. The expressed GST-tagged hMTERF3 fusion protein was purified by passive electro-elution process and then used to immunize BALB/c mice, we obtained anti-GST-hMTERF3 polyclonal antibody purified by protein A column and determined the sensitivity and specificity of the antibody against human MTERF3 by enzyme-linked immunosorbent assay and Western blot assay. Furthermore, the full-length hMTERF3 protein expressed in human embryonic kidney 293T cells was detected by anti-GST-hMTERF3 in western blot analysis and immunofluorescence staining. Taken together, our results demonstrate the functionality of the mouse anti-GST-hMTERF3 polyclonal antibody which will provide a useful tool for further characterization of hMTERF3.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

MTERF:

Mitochondrial transcription termination factor

mtDNA:

Mitochondrial DNA

E. coli :

Escherichia coli

ORF:

Open reading frame

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

OD:

Optical density

IPTG:

Isopropyl-β-d-thiogalactopyranoside

PBS:

Phosphate-buffered saline

LB:

Luria–Bertani

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MALDI-TOF:

Matrix-assisted laser desorption time-of-flight

BSA:

Bovine serum albumin

HRP:

Horseradish peroxodase

ELISA:

Enzyme-linked immunosorbent assay

TMB:

Tetramethylbenzidine

GFP:

Green fluorescent protein

ECL:

Enhanced chemiluminescence

WB:

Western blotting

IF:

Immunofluorescence

References

  1. Fernandez-Silva, P., Martinez-Azorin, F., Micol, V., & Attardi, G. (1997). EMBO Journal, 16, 1066–1079.

    Article  CAS  Google Scholar 

  2. Loguercio-Polosa, P., Roberti, M., Musicco, C., Gadaleta, M. N., Quagliariello, E., & Cantatore, P. (1999). Nucleic Acids Research, 27, 1890–1899.

    Article  CAS  Google Scholar 

  3. Roberti, M., Loguercio-Polosa, P., Bruni, F., Musicco, C., Gadaleta, M. N., & Cantatore, P. (2003). Nucleic Acids Research, 31, 1597–1604.

    Article  CAS  Google Scholar 

  4. Fernandez-Silva, P., Loguercio-Polosa, P., Roberti, M., Di Ponzio, B., Gadaleta, M. N., Montoya, J., et al. (2001). Nucleic Acids Research, 29, 4736–4743.

    Article  CAS  Google Scholar 

  5. Asin-Cayuela, J., Schwend, T., Farge, G., & Gutafsson, C. M. (2005). Journal of Biological Chemistry, 280, 25499–25505.

    Article  CAS  Google Scholar 

  6. Roberti, M., Fernandez-Silva, P., Loguercio-Polosa, P., Fernandez-Vizarra, E., Bruni, B., Deceglie, S., et al. (2005). Biochemical and Biophysical Research Communication, 331, 357–362.

    Article  CAS  Google Scholar 

  7. Roberti, M., Polosa, P.L., Bruni, F., Manzari, C., Deceglie, S., Gadeleta, M. N., et al. (2009). Biochimica et Biophysica Acta, 1787, 303–311.

    Google Scholar 

  8. Linder, T., Park, C. B., Asin-Cayuela, J., Pellegrini, M., Larsson, N. G., Falkenberg, M., et al. (2005). Current Genetics, 48, 265–269.

    Article  CAS  Google Scholar 

  9. Pellegrini, M., Asin-Cayuela, J., Erdjument-Bromace, H., Tempst, P., Larsson, N. G., & Gustafsson, C. M. (2009). Biochimica et Biophysica Acta, 1787, 296–302.

    Article  CAS  Google Scholar 

  10. Wenz, T., Luca, C., Torraco, A., & Moraes, C. T. (2009). Cell Metabolism, 9, 499–511.

    Article  CAS  Google Scholar 

  11. Chen, Y., Zhou, G., Yu, M., He, Y., Tang, W., Lai, J., et al. (2005). Biochemical and Biophysical Research Communication, 337, 1112–1118.

    Article  CAS  Google Scholar 

  12. Camara, Y., Asin-Cayuela, J., Park, C. B., Metodiev, M. D., Shi, Y., Ruzzenente, B., Kukat, C., et al. (2011). Cell Metabolism, 13, 527–539.

    Article  CAS  Google Scholar 

  13. Yu, M., Dai, J., Huang, W., Jiao, Y., Liu, L., & Tan, D. (2011). Acta Biochimica et Biophysica Sinica, 43, 362–371.

    Article  Google Scholar 

  14. Park, C. B., Asin-Cayuela, J., Camara, Y., Shi, Y., Pellegrini, M., Gaspari, M., et al. (2007). Cell, 130, 273–285.

    Article  CAS  Google Scholar 

  15. Plinston, C., Fernie, K., Prior, F. G., & Smith, R. (2007). Journal of Hospital Infection, 66, 52–58.

    Google Scholar 

  16. Xiong, W., Huang, W., Jiao, Y., Ma, J., Yu, M., Ma, M., et al. (2012). Protein Expression and Purification, 89, 11–19.

    Article  Google Scholar 

  17. Asin-Cayuela, J., & Gustafsson, C. M. (2007). Trends in Biochemical Science, 32, 111–117.

    Article  CAS  Google Scholar 

  18. Martin, M., Cho, J., Cesare, A. J., Griffith, J. D., & Attardi, G. (2005). Cell, 123, 1227–1240.

    Article  CAS  Google Scholar 

  19. Spahr, H., Samuelsson, T., Hallberg, B. M., & Gustafsson, C. M. (2010). Biochemica and Biophysica Research Communication, 397, 386–390.

    Article  CAS  Google Scholar 

  20. Hyvarinen, A. K., Pohjoismaki, J. L., Holt, I. J., & Jacobs, H. T. (2011). Molecular Biology Report, 38, 1321–1328.

    Article  Google Scholar 

  21. Zhang, C., & Guy, C. L. (2005). Plant Physiology and Biochemistry, 43, 13–18.

    Article  CAS  Google Scholar 

  22. Filatov, A. V., Krotov, G. I., Zgoda, V. G., & Volkov, Y. (2007). Journal of Immunological Methods, 319, 21–33.

    Article  CAS  Google Scholar 

  23. Larkin, A., Moran, E., Kennedy, S. M., & Clynes, M. (2005). Journal of Immunological Methods, 303, 53–65.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was made possible by generous support from the National Natural Science Foundation of China (Grand numbers 30360040, 30760057, and 30960091) and Science & Technological Key Project of Yunnan Province (Grand nubmers 2008CC003 and 2008CD069). We would like to thank all the members of our laboratory for encouragement and help in this study. We thank Mr. Jian Li (Kunming Zoology Institutes, Academic of Chinese Science, Kunming, People’s Republic of China) for fluorescent microscope analysis.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Dali University, 112 Wanhua Road, Dali, 671000, China

    Wei Xiong, Yonghui Luo, Cuixiang Zhang & Shaoyuan Zuo

  2. Laboratory of Biochemistry and Molecular Biology, School of Life Sciences, Yunnan University, 002 Cuihu Road, Kunming, 650091, China

    Wei Xiong & Deyong Tan

Authors
  1. Wei Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yonghui Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cuixiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Deyong Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shaoyuan Zuo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei Xiong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xiong, W., Luo, Y., Zhang, C. et al. Expression, Purification of Recombinant Human Mitochondrial Transcription Termination Factor 3 (hMTERF3) and Preparation of Polyclonal Antibody Against hMTERF3. Appl Biochem Biotechnol 167, 2318–2329 (2012). https://doi.org/10.1007/s12010-012-9754-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-012-9754-0

Keywords

Search

Navigation