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Protein Downstream Processing pp 149–158Cite as

Zbasic: A Purification Tag for Selective Ion-Exchange Recovery

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Part of the Methods in Molecular Biology book series (MIMB,volume 2178)

Abstract

A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived Z-domain, and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchanger resin, and thereby allows solid-phase refolding of the target protein.

Herein, protocols for purification of soluble Zbasic-tagged fusion proteins , as well as for integrated purification and solid-phase refolding of insoluble fusion proteins , are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.

Key words

  • Ion-exchange chromatography
  • Protein A
  • Fusion tag
  • Zbasic
  • Solid-phase refolding
  • Proteolytic cleavage

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  • DOI: 10.1007/978-1-0716-0775-6_12
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References

  1. Asplund M, Ramberg M, Johansson B-L (2000) Development of a cleaning in place protocol and repetitive application of Escherichia coli homogenate on STREAMLINE™ Q XL. Process Biochem 35:1111–1118

    CrossRef  CAS  Google Scholar 

  2. Hale G, Drumm A, Harrison P et al (1994) Repeated cleaning of protein A affinity column with sodium hydroxide. J Immunol Methods 171:15–21

    CrossRef  CAS  Google Scholar 

  3. Anspach FB, Curbelo D, Hartmann R et al (1999) Expanded-bed chromatography in primary protein purification. J Chromatogr A 865:129–144

    CrossRef  CAS  Google Scholar 

  4. Feuser J, Walter J, Kula MR et al (1999) Cell/adsorbent interactions in expanded bed adsorption of proteins. Bioseparation 8:99–109

    CrossRef  CAS  Google Scholar 

  5. Hedhammar M, Gräslund T, Hober S (2005) Protein engineering strategies for selective protein purification. Chem Eng Technol 28:1315–1325

    CrossRef  CAS  Google Scholar 

  6. Nilsson B, Moks T, Jansson B et al (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng 1:107–113

    CrossRef  CAS  Google Scholar 

  7. Graslund T, Lundin G, Uhlen M et al (2000) Charge engineering of a protein domain to allow efficient ion-exchange recovery. Protein Eng 13:703–709

    CrossRef  CAS  PubMed  Google Scholar 

  8. Link AJ, Robison K, Church GM (1997) Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 18:1259–1313

    CrossRef  CAS  PubMed  Google Scholar 

  9. Graslund T, Ehn M, Lundin G et al (2002) Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner. J Chromatogr A 942:157–166

    CrossRef  CAS  PubMed  Google Scholar 

  10. Hedhammar M, Alm T, Graslund T et al (2006) Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag. Biotechnol J 1:187–196

    CrossRef  CAS  PubMed  Google Scholar 

  11. Wiesbauer J, Bolivar JM, Mueller M et al (2011) Oriented immobilization of enzymes made fit for applied biocatalysis: non-covalent attachment to anionic supports using Zbasic2 module. ChemCatChem 3:1299–1303

    CrossRef  CAS  Google Scholar 

  12. Bolivar JM, Nidetzky B (2012) Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass. Biotechnol Bioeng 109:1490–1498

    CrossRef  CAS  PubMed  Google Scholar 

  13. Bolivar JM, Nidetzky B (2012) Positively charged mini-protein Z basic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports. Langmuir 28:10040–10049

    CrossRef  CAS  PubMed  Google Scholar 

  14. Bolivar JM, Luley-Goedl C, Leitner E et al (2017) Production of glucosyl glycerol by immobilized sucrose phosphorylase: options for enzyme fixation on a solid support and application in microscale flow format. J Biotechnol 257:131–138

    CrossRef  CAS  PubMed  Google Scholar 

  15. Hedhammar M, Jung HR, Hober S (2006) Enzymatic cleavage of fusion proteins using immobilised protease 3C. Protein Expr Purif 47:422–426

    CrossRef  CAS  PubMed  Google Scholar 

  16. Shi S, Chen H, Jiang H et al (2017) A novel self-cleavable tag Zbasic–ΔI-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli. Appl Microbiol Biotechnol 101:1133–1142

    CrossRef  CAS  PubMed  Google Scholar 

  17. Yip TT, Nakagawa Y, Porath J (1989) Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography. Anal Biochem 183:159–171

    CrossRef  CAS  PubMed  Google Scholar 

  18. Hedhammar M, Stenvall M, Lonneborg R et al (2005) A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein. J Biotechnol 119:133–146

    CrossRef  CAS  PubMed  Google Scholar 

  19. Alm T, Steen J, Ottosson J et al (2007) High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography. Biotechnol J 2:709–716

    CrossRef  CAS  PubMed  Google Scholar 

  20. Graslund T, Hedhammar M, Uhlen M et al (2002) Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology. J Biotechnol 96:93–102

    CrossRef  CAS  PubMed  Google Scholar 

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Author notes

  1. My Hedhammar and Johan Nilvebrant contributed equally to this work.

Authors and Affiliations

  1. Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), KTH Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden

    My Hedhammar, Johan Nilvebrant & Sophia Hober

Authors
  1. My Hedhammar
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  2. Johan Nilvebrant
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  3. Sophia Hober
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Corresponding author

Correspondence to Sophia Hober .

Editor information

Editors and Affiliations

  1. Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, Athens, Greece

    Dr. Nikolaos E. Labrou

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Hedhammar, M., Nilvebrant, J., Hober, S. (2021). Zbasic: A Purification Tag for Selective Ion-Exchange Recovery. In: Labrou, N.E. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 2178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0775-6_12

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  • DOI: https://doi.org/10.1007/978-1-0716-0775-6_12

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