Skip to main content
SpringerLink
Log in

Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli

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

Abstract

In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

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

References

  1. Kosobokova, E. N., Skrypnik, K. A., & Kosorukov, V. S. (2016). Overview of fusion tags for recombinant proteins. Biochemistry (Mosc), 81, 187–200.

    Article  CAS  Google Scholar 

  2. Raran-Kurussi, S., & Waugh, D. S. (2012). The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated. PloS One, 7, e49589.

    Article  CAS  Google Scholar 

  3. Raran-Kurussi, S., & Waugh, D. S. (2014). Unrelated solubility-enhancing fusion partners MBP and NusA utilize a similar mode of action. Biotechnology and Bioengineering, 111, 2407–2411.

    Article  CAS  Google Scholar 

  4. Kyratsous, C. A., Silverstein, S. J., DeLong, C. R., & Panagiotidis, C. A. (2009). Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli. Gene, 440, 9–15.

    Article  CAS  Google Scholar 

  5. Hansted, J. G., Pietikäinen, L. F., Hög, H., Sperling-Petersen, U., & Mortensen, K. K. (2011). Expressivity tag: a novel tool for increased expression in Escherichia coli. Journal of Biotechnology, 155, 275–283.

    Article  CAS  Google Scholar 

  6. Kavoosi, M., Creagh, A. L., Kilburn, D. G., & Haynes, C. A. (2007). Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli. Biotechnology and Bioengineering, 98, 599–610.

    Article  CAS  Google Scholar 

  7. Kim, S. W., Kim, J. B., Lee, W. S., Jung, W. H., Ryu, J. M., Jang, H. W., Jo, Y. B., Jung, J. K., & Kim, J. H. (2007). Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides. Protein Expression and Purification, 55, 159–165.

    Article  CAS  Google Scholar 

  8. Raran-Kurussi, S., Keefe, K., & Waugh, D. S. (2015). Positional effects of fusion partners on the yield and solubility of MBP fusion proteins. Protein Expression and Purification, 110, 159–164.

    Article  CAS  Google Scholar 

  9. Tsunoda, Y., Sakai, N., Kikuchi, K., Katoh, S., Akagi, K., Miura-Ohnuma, J., Tashiro, Y., Murata, K., Shibuya, N., & Katoh, E. (2005). Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli. Protein Expression and Purification, 42, 268–277.

    Article  CAS  Google Scholar 

  10. Kurz, M., Cowieson, N. P., Robin, G., Hume, D. A., Martin, J. L., Kobe, B., & Listwan, P. (2006). Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins. Protein Expression and Purification, 50, 68–73.

    Article  CAS  Google Scholar 

  11. Waugh, D. S. (2011). An overview of enzymatic reagents for the removal of affinity tags. Protein Expression and Purification, 80, 283–293.

    Article  CAS  Google Scholar 

  12. Kapust, R. B., & Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Science, 8, 1668–1674.

    Article  CAS  Google Scholar 

  13. Wu, X., Wu, D., Lu, Z., Chen, W., Hu, X., & Ding, Y. (2009). A novel method for high-level production of TEV protease by superfolder GFP tag. Journal of Biomedicine & Biotechnology, 2009, 591923.

    Google Scholar 

  14. Miladi, B., Bouallagui, H., Dridi, C., El Marjou, A., Boeuf, G., Di Martino, P., Dufour, F., & Elm’Selmi, A. (2011). A new tagged-TEV protease: construction, optimisation of production, purification and test activity. Protein Expression and Purification, 75, 75–82.

    Article  CAS  Google Scholar 

  15. Costa, S. J., Almeida, A., Castro, A., Domingues, L., & Besir, H. (2013). The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology. Applied Microbiology and Biotechnology, 97, 6779–6791.

    Article  CAS  Google Scholar 

  16. Braud, S., Moutiez, M., Belin, P., Abello, N., Drevet, P., Zinn-Justin, S., Courçon, M., Masson, C., Dassa, J., Charbonnier, J. B., Boulain, J. C., Ménez, A., Genet, R., & Gondry, M. (2005). Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins. Journal of Proteome Research, 4, 2137–2147.

    Article  CAS  Google Scholar 

  17. Fang, J., Zou, L., Zhou, X., Cheng, B., & Fan, J. (2014). Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant. PloS One, 9, e112254.

    Article  Google Scholar 

  18. Miladi, B., Marjou, A. E., Boeuf, G., Bouallagui, H., Dufour, F., Di Martino, P., & Elm’selmi, A. (2012). Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins. Journal of Biotechnology, 158, 97–103.

    Article  CAS  Google Scholar 

  19. Feliciano, J., Liu, Y., & Daunert, S. (2006). Novel reporter gene in a fluorescent-based whole cell sensing system. Biotechnology and Bioengineering, 93, 989–997.

    Article  CAS  Google Scholar 

  20. Buckley, A. M., Petersen, J., Roe, A. J., Douce, G. R., & Christie, J. M. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology, 27, 39–45.

    Article  CAS  Google Scholar 

  21. Kim, J. S., Lim, H. K., Lee, M. H., Park, J. H., Hwang, E. C., Moon, B. J., & Lee, S. W. (2009). Production of porphyrin intermediates in Escherichia coli carrying soil metagenomic genes. FEMS Microbiology Letters, 295, 42–49.

    Article  CAS  Google Scholar 

  22. Do, B. H., Ryu, H. B., Hoang, P., Koo, B. K., & Choe, H. (2014). Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase. PloS One, 9, e89906.

    Article  Google Scholar 

  23. Rosano, G. L., & Ceccarelli, E. A. (2009). Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial Cell Factories, 8, 41.

    Article  Google Scholar 

  24. Fedyunin, I., Lehnhardt, L., Böhmer, N., Kaufmann, P., Zhang, G., & Ignatova, Z. (2012). tRNA concentration fine tunes protein solubility. FEBS Letters, 586, 3336–3340.

    Article  CAS  Google Scholar 

  25. Teerawanichpan, P., Hoffman, T., Ashe, P., Datla, R., & Selvaraj, G. (2007). Investigations of combinations of mutations in the jellyfish green fluorescent protein (GFP) that afford brighter fluorescence, and use of a version (VisGreen) in plant, bacterial, and animal cells. Biochimica et Biophysica Acta, 1770, 1360–1368.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by the Scientific and Technological Project of Anhui Province (1506c085007).

Author information

Authors and Affiliations

  1. Key Laboratory of Crop Biology of Anhui Province, Anhui Agricultural University, Hefei, Anhui, 230036, People’s Republic of China

    Xuelian Yu, Jiaqi Sun, Weiyu Wang, Li Jiang, Beijiu Cheng & Jun Fan

Authors
  1. Xuelian Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiaqi Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Weiyu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Beijiu Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jun Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jun Fan.

Electronic supplementary material

Figure S1

SDS-PAGE analysis of the fusion protein cleaved by the TEVp constructs extracted from the recombinant E. coli BL21(DE3) cells (a) and Rosetta™ (DE3) cells (b). M: protein marker. CK: the protein substrate is incubated with the inactive TEVpC151A variant. The fusion protein substrate and the cleaved products are indicated by arrows. (TIFF 2262 kb)

High Resolution Image (GIF 6 kb)

Figure S2

The pigmented E. coli cells overexpressing the fusions proteins for EcGTR. Recombinant cells carrying pET-28b plasmids are used as the control. (TIFF 4194 kb)

High Resolution Image (GIF 28 kb)

Figure S3

SDS-PAGE analysis of the purified Ex-TEVpH6 and MBP-TEVpH6 proteins by Ni-NTA agarose. M: Protein marker. (TIFF 1971 kb)

High Resolution Image (GIF 4 kb)

Table S1

Primers used in this study (DOC 33 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, X., Sun, J., Wang, W. et al. Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli . Appl Biochem Biotechnol 182, 769–781 (2017). https://doi.org/10.1007/s12010-016-2360-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2360-9

Keywords

Search

Navigation