Biotechnology Letters

, Volume 39, Issue 7, pp 1025–1031 | Cite as

Immobilization of Ulp1 protease on NHS-activated Sepharose: a useful tool for cleavage of the SUMO tag of recombinant proteins

  • Qiujin Liang
  • Zhengzhi Huang
  • Yuan Zhang
  • Hongtao Li
Original Research Paper

Abstract

Objective

To fabricate an active and stable enzyme through covalent immobilization, a Ubl-specific protease (Ulp1) was used to cleave small ubiquitin-like modifier (SUMO) fusion proteins.

Results

We immobilized Ulp1 on N-hydroxysuccinimide (NHS)-activated Sepharose with a coupling efficiency of 1.7 mg/ml. The immobilized Ulp1 maintains 95% substrate-cleavage ability and significantly enhances pH and thermal stability, especially can withstand pH of 10.5. Besides resistance against some small molecules, the immobilized Ulp1 can tolerate 15% (v/v) DMSO and 20% (v/v) ethanol. It can be reused for more than 15 batch reactions with 90% activity retention. This provides a fast purification system to quickly obtain cleaved recombinant proteins with 95% purity from cell lysates with the application of immobilized Ulp1.

Conclusions

Ulp1 used in immobilization form is a potentially useful tool for cleavage of SUMO-tagged proteins and may reduce time and cost of protein purification.

Keywords

Enzymatic cleavage Extreme pHs tolerance Immobilization Thermal stability Ubl-specific protease (Ulp1) 

Notes

Acknowledgments

This work is supported by the Natural Science Foundation of China (31270831 and 30970630), the Outstanding Youth Science Foundation of Chongqing (cstc2011jjjq10003), the Program for New Century Excellent Talents in University (NCET-08-0912), the Fundamental Research Funds for the Central Universities (XDJK2016C158) and the Doctoral Fund of Southwest University (XJKJXM003338).

Supporting information

Supplementary Fig. 1—Different immobilization strategies for Ulp1.

Supplementary Fig. 2—DMSO inhibits the degradation of SUMO.

Supplementary Fig. 3—The influence of small molecules on the activity of Ulp1.

Supplementary Fig. 4—Storage stability of free and immobilized Ulp1 at 30 °C.

Supplementary material

10529_2017_2330_MOESM1_ESM.docx (683 kb)
Supplementary material 1 (DOCX 682 kb)

References

  1. Besselink GAJ, Beugeling T, Bantjes A (1993) N-Hydroxysuccinimide-activated glycine-Sepharose. Appl Biochem Biotechnol 43:227–246CrossRefGoogle Scholar
  2. Ferreira L, Ramos MA, Dordick JS, Gil MH (2003) Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis, protease (Subtilisin Carlsberg). J Mol Catal B Enzym 21:189–199CrossRefGoogle Scholar
  3. Homaei A (2015) Enhanced activity and stability of papain immobilized on CNBr-activated sepharose. Int J Biol Macromol 75:373–377CrossRefPubMedGoogle Scholar
  4. Horchani H, Ouertani S, Gargouri Y, Sayari A (2009) The N-terminal His-tag and the recombination process affect the biochemical properties of Staphylococcus aureus lipase produced in Escherichia coli. J Mol Catal B Enzym 61:194–201CrossRefGoogle Scholar
  5. Hughes MJ, Jost JP, Jiricny J (2001) Purification of sequence-specific DNA-binding proteins by affinity chromatography. Curr Prot Mol Biol 5:221–231Google Scholar
  6. Kokufuta E (1992) Functional immobilized biocatalysts. Prog Polym Sci 17:647–697CrossRefGoogle Scholar
  7. Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398:246–251CrossRefPubMedGoogle Scholar
  8. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genom 5:75–86CrossRefGoogle Scholar
  9. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189CrossRefPubMedPubMedCentralGoogle Scholar
  10. McCarney ER, Armstrong BD, Lingwood MD, Han S (2007) Hyperpolarized water as an authentic magnetic resonance imaging contrast agent. Proc Natl Acad Sci 104:1754–1759CrossRefPubMedPubMedCentralGoogle Scholar
  11. Miladi B, Ei Marjou A, 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. J Biotechnol 158:97–103CrossRefPubMedGoogle Scholar
  12. Rodrigues RC, Ortiz C, Berenguermurcia Á, Torres R, Fernándezlafuente R (2012) Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev 42:6290–6307CrossRefGoogle Scholar
  13. Rutkowska A, Beerbaum M, Rajagopalan N, Fiaux J, Schmieder P, Kramer G, Oschkinat H, Bukau B (2009) Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies. FEBS Lett 583:2407–2413CrossRefPubMedGoogle Scholar
  14. Shainoff JR (1980) Zonal immobilization of proteins. Biochem Biophys Res Commun 95:690–695CrossRefPubMedGoogle Scholar
  15. Vandamme EJ (1989) Peptide antibiotic production through immobilized biocatalyst technology. Enzyme Microb Technol 5:403–416CrossRefGoogle Scholar
  16. Yang M, Wu H, Lian Y, Li X, Lai F, Zhao G (2014) Influence of organic solvents on catalytic behaviors and cell morphology of whole-cell biocatalysts for synthesis of 5′-arabinocytosine laurate. PLoS ONE 9:e104847CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life SciencesSouthwest UniversityChongqingPeople’s Republic of China

Personalised recommendations