Skip to main content
SpringerLink
Log in

Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×105/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe2+ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS).

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

Similar content being viewed by others

References

  1. You, D. L., X. J. Ren, Y. Xue, G. M. Luo, T. S. Yang, and J. C. Shen (2003) A selenium-containing single-chain abzyme with potent antioxidant activity. Eur. J. Biochem. 270: 4326–4331.

    Article  CAS  Google Scholar 

  2. Castro, L. and B. A. Freeman (2001) Reactive oxygen species in human health and disease. Nutrition 17: 161–165.

    Article  CAS  Google Scholar 

  3. Lin, F., Y. Li, W. K. Yang, B. Liang, Y. Mu, Y. Sun, W. Li, and G. M. Luo (2007) Rapid selection of phage Se-scFv with GPX activity via combination of phage display antibody library with chemical modification. Chem. Res. Chinese U. 23: 58–63.

    Article  CAS  Google Scholar 

  4. Huo, R., J. Y. Wei, J. J. Xu, S. W. Lv, Q. C. Zheng, F. Yan, J. M. Su, J. Fan, J. S. Li, Y. J. Duan, Y. Yu, F. H. Jin, W. G. Sun, Y. Shi, D. L. Cong, W. Li, G. L. Yan, and G. M. Luo (2008) Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity. J. Mol. Recogn. 21: 323–328.

    Article  CAS  Google Scholar 

  5. Xu, J. J., J. Song, J. M. Su, J. Y. Wei, Y. Yu, S. W. Lv, W. Li, and G. J. Nie (2010) A new human catalytic antibody Se-scFv-2D8 and its selenium-containing single domains with high GPX activity. J. Mol. Recogn. 23: 353–359.

    Google Scholar 

  6. Wang, C., P. Wan, P. S. Gong, L. M. Lv, Y. W. Xu, Y. Zhao, B. He, G. Zhao, G. L. Yan, Y. Mu, S. W. Lv, and G. M. Luo (2011) A novel selenium-containing human single chain variable fragment with glutathione peroxidase activity base on computer-aided molecular design. Chem. Res. Chin. U. 27: 813–819.

    CAS  Google Scholar 

  7. Suzuki, M., J. Zhang, M. Liu, N. A. Woychik, and M. Inouye (2005) Single protein production in living cells facilitated by an mRNA interferase. Mol. Cell. 18: 253–261.

    Article  CAS  Google Scholar 

  8. Zhang, Y. L., J. J. Zhang, K. P. Hoeflich, M. Ikura, G. L. Qing, and M. Inouye (2003) MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol. Cell. 12: 913–923.

    Article  CAS  Google Scholar 

  9. Zhang, Y., J. Zhang, H. Hara, I. Kato, and M. Inouye (2005) Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase. J. Biol. Chem. 280: 3143–3150.

    Article  CAS  Google Scholar 

  10. Qing, G., L. C. Ma, A. Khorchid, G. V. Swapna, T. K. Mal, M. M. Takayama, B. Xia, S. Phadtare, H. Ke, T. Acton, G. T. Montelione, M. Ikura, and M. Inouye (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22: 877–882.

    Article  CAS  Google Scholar 

  11. Suzuki, M., L. L. Mao, and M. Inouye (2007) Single protein production (SPP) system in Escherichia coli. Nat. Protocols. 2: 1802–1810.

    Article  CAS  Google Scholar 

  12. Suzuki, M., R. Roy, H. Zheng, N. Woychik, and M. Inouye (2006) Bacterial bioreactors for high yield production of recombinant protein. J. Biol. Chem. 281: 37559–37565.

    Article  CAS  Google Scholar 

  13. Vaiphei, S. T., Y. F. Tang, G. T. Montelione, and M. Inouye (2011) The use of the condensed single protein production system for isotope-labeled outer membrane proteins, OmpA and OmpX in E. coli. Mol. Biotechnol. 47: 205–210.

    Article  CAS  Google Scholar 

  14. Mao, L. L., K. Inoue, Y. S. Tao, G. T. Montelione, A. E. McDermott, and M. Inouye (2011) Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system. J. Biomol. NMR. 49: 131–137.

    Article  CAS  Google Scholar 

  15. Mao, L. L., P. B. Stathopulos, M. Ikura, and M. Inouye (2010) Secretion of human superoxide dismutase in Escherichia coli using the condensed single-protein-production system. Protein Sci. 19: 2330–2335.

    Article  CAS  Google Scholar 

  16. Vaiphei, S. T., L. L. Mao, T. Shimazu, J. H. Park, and M. Inouye (2010) Use of amino acids as inducers for high-level protein expression in the single-protein production system. Appl. Environ. Microb. 76: 6063–6068.

    Article  CAS  Google Scholar 

  17. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

  18. Sun, S. Q., W. J. Mo, Y. P. Ji, and S. Y. Liu (2001) Prepraration and mass spectrometric study of egg yolk antibody (IgY) against rabies virus. Rapid Commun. Mass Spectrom. 15: 708–712.

    Article  CAS  Google Scholar 

  19. Sun, Y., T. Y. Li, H. Chen, K. Zhang, K. Y. Zheng, Y. Mu, G. L. Yan, W. Li, J. C. Shen, and G. M. Luo (2004) Selenium-containing 15-mer peptides with high glutathione peroxidase-like activity. J. Biol. Chem. 279: 37235–37240.

    Article  CAS  Google Scholar 

  20. Lansman, R. A., R. O. Shade, J. F. Shapiro, and J. C. Avise (1981) The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations. III. Techniques and potential applications. J. Mol. Evol. 17: 214–226.

    Article  CAS  Google Scholar 

  21. Shen, N., F. Yan, Y. Guo, S. W. Lv, P. S. Gong, Y. W. Xu, G. L. Yan, Y. Mu, and G. M. Luo (2011) Imprinted human serum albumin with antioxidant activity. Chem. Res. Chinese U. 27: 258–263.

    CAS  Google Scholar 

  22. Gao, N., H. M. Li, Q. S. Li, J. Q. Liu, and G. M. Luo (2011) Synthesis and kinetic evaluation of a trifunctional enzyme mimic with a dimanganese active centre. J. Inorg. Biochem. 105: 283–288.

    Article  CAS  Google Scholar 

  23. Pryor, W. A., J. P. Stanley, and E. Blair (1976) Autoxidation of polyunsaturated fatty acids. II. A suggested mechanism for the formation of TBA-reactive materials from prostaglandin-like endoperoxides. Lipids 11: 370–379.

    CAS  Google Scholar 

  24. Yu, H. J., J. Q. Liu, J. Li, T. Z. Zang, G. M. Luo, and J. C. Shen (2005) Protection of mitochondrial integrity from oxidative stress by selenium-containing glutathione transferase. Appl. Biochem. Biotech. 127: 133–142.

    Article  CAS  Google Scholar 

  25. Shimazu, T., K. Degenhardt, A. Nur-E-Kamal, J. J. Zhang, T. Yoshida, Y. L. Zhang, R. Mathew, E. White, and M. Inouye (2007) NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes Dev. 21: 929–941.

    Article  CAS  Google Scholar 

  26. Hillenkamp, F., M. Karas, R. C. Beavis, and B. T. Chait (1991) Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63: 1193–1203.

    Article  Google Scholar 

  27. Lv, S. W., X. G. Wang, Y. Mu, T. Z. Zang, Y. T. Ji, J. Q. Liu, J. C. Shen, and G. M. Luo (2007) A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity. FEBS J. 274: 3846–3854.

    Article  CAS  Google Scholar 

  28. Kwon, T. and B. Watts (1964) Malonaldehyde in aqueous solution and its role as a measure of lipid oxidation in foods. J. Food Sci. 29: 294–302.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, 310-058, China

    Cheng Wang & Ying Mu

  2. Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, 130-012, China

    Cheng Wang, Shao-Wu Lü, Yang Zhao, Ya-Wei Xu, Gang Zhao, Ping-Sheng Gong, Gui-Min Luo & Ying Mu

  3. Department of Biochemistry, School of Basic Medical Sciences, Jilin Medical College, Jilin, 132-013, China

    Cheng Wang, Gang-Lin Yan & Jun-jie Xu

  4. Department of Chemistry, School of Pharmaceutical Sciences, Jilin Medical College, Jilin, 132-013, China

    Chun-Hong Sui

Authors
  1. Cheng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gang-Lin Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shao-Wu Lü
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chun-Hong Sui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ya-Wei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jun-jie Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ping-Sheng Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gui-Min Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ying Mu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Gang-Lin Yan or Ying Mu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, C., Yan, GL., Lü, SW. et al. Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability. Biotechnol Bioproc E 18, 27–34 (2013). https://doi.org/10.1007/s12257-012-0174-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-012-0174-3

Keywords

Search

Navigation