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Heparin-Binding Affinity Tag: A Novel Affinity Tag for Simple and Efficient Purification of Recombinant Proteins

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Protein Downstream Processing

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

Abstract

Heparin, a polysulfated polyanionic member of the glycosaminoglycan family, is known to specifically bind to a number of functionally important proteins. Based on the available information on structural specificity of heparin–protein interactions, a novel heparin-binding peptide (HB) affinity tag has been designed to achieve simple and cost-effective purification of target recombinant proteins. The HB-fused recombinant target proteins are purified on a heparin-Sepharose column using a stepwise/continuous sodium chloride gradient. A major advantage of the HB tag is that the HB-fused target proteins can be purified under denaturing conditions in the presence of 8 M urea. In addition, polyclonal antibody directed against the HB tag can be used to specifically detect and quantitate the HB-fused recombinant protein(s). Herein, a step-by-step protocol(s) for the purification of different soluble recombinant target proteins is described. In addition, useful tips to troubleshoot potential problems and also suggestions to successfully adopt the HB-tag-based purification to a wide range of target proteins are provided.

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References

  1. Ferrer-Miralles N, Corchero J, Kumar P, Cedano J, Gupta K, Villaverde A, Vazquez E (2011) Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine. Microb Cell Factories 10:1–5

    Article  CAS  Google Scholar 

  2. Andersen DC, Krummen L (2002) Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 13:117–123

    Article  CAS  PubMed  Google Scholar 

  3. De Meyer T, Muyldermans S, Depicker A (2014) Nanobody-based products as research and diagnostic tools. Trends Biotechnol 32:263–270

    Article  CAS  PubMed  Google Scholar 

  4. Lesley SA (2001) High-throughput proteomics: protein expression and purification in the postgenomic world. Protein Expr Purif 22:159–164

    Article  CAS  PubMed  Google Scholar 

  5. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211–222

    Article  CAS  PubMed  Google Scholar 

  6. Arnau J, Lauritzen C, Petersen G, Pedersen J (2006) Current strategies for the use of affinitytags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13

    Article  CAS  PubMed  Google Scholar 

  7. Park N, Ryu J, Jang S, Lee H (2012) Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids. Tetrahedron 68:4649–4654

    Article  CAS  Google Scholar 

  8. Hunt I (2005) From gene to protein: a review of new and enabling technologies for multiparallel protein expression. Protein Expr Purif 40:1–22

    Article  CAS  PubMed  Google Scholar 

  9. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533

    Article  CAS  PubMed  Google Scholar 

  10. Martinez-Ceron M, Targovnik A, Urtasun N, Cascone O, Miranda M, Camperi S (2012) Recombinant protein purification using complementary peptides as affinity tags. New Biotechnol 29:206–210

    Article  CAS  Google Scholar 

  11. Morris J, Jayanthi S, Langston R, Daily A, Kight A, McNabb DS, Kumar TKS (2016) Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins. Protein Expr Purif 126:93–103

    Article  CAS  PubMed  Google Scholar 

  12. Gandhi NS, Mancera RL (2008) The structure of glycosaminoglycans and their interactions with proteins. Chem Biol Drug Des 72:455–482

    Article  CAS  PubMed  Google Scholar 

  13. Xu X, Dai Y (2010) Heparin: an intervenor in cell communication. J Cell Mol Med 14:175–180

    Article  CAS  PubMed  Google Scholar 

  14. Olczyk P, Mencner L, Komosinska-Vassev K (2015) Diverse roles of heparan sulfate and heparin in wound repair. Biomed Res Int 2015:1–7

    Article  CAS  Google Scholar 

  15. Davis JE, Gundampati RK, Jayanthi S, Anderson J, Pickhardt A, Koppolu BP, Zaharoff DA, Kumar TKS (2018) Effect of extension of the heparin binding pocket on the structure, stability, and cell proliferation activity of the human acidic fibroblast growth factor. Biochem Biophys Rep 13:45–57

    PubMed  Google Scholar 

  16. Pomin VH (2014) Heparin-binding proteins (chemokines and Defensins) and their complexes with glycosaminoglycans from the solution NMR perspective. Curr Protein Pept Sci 15:738–744

    Article  CAS  PubMed  Google Scholar 

  17. Tang NH, Chen YL, Wang XQ, Li XJ, Wu Y, Zou QL, Chen YZ (2010) N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells. BMC Cancer 10:552–564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Atha DH, Lormeau JC, Petitou M, Rosenberg RD, Choay J (1985) Contribution of monosaccharide residues in heparin binding to Antithrombin III. Biochemist 24:6723–6729

    Article  CAS  Google Scholar 

  19. Kouzi-Koliakos K, Koliakos GG, Tsilibary EC, FurchtS LT, Charonis AS (1989) Mapping of three major heparin-binding sites on Laminin and identification of a novel heparin-binding site on the B1 chain. J Biol Chem 264:17971–17978

    CAS  PubMed  Google Scholar 

  20. Carter WJ, Cama E, Huntington JA (2005) Crystal structure of thrombin bound to heparin. J Biol Chem 280:2745–2749

    Article  CAS  PubMed  Google Scholar 

  21. Tan K, Duquette M, Liu J, Zhang R, Joachimiak A, Wang J, Lawler J (2006) The structures of the thrombospondin-1 N-terminal domain and its complex with a synthetic pentameric heparin. Structure 14:33–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Margalit H, Fischer N, Ben-Sasson S (1993) Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues. J Biol Chem 268:19228–19231

    CAS  PubMed  Google Scholar 

  23. Weiler J, Linhardt R, Fromm PJ, Hileman R, Caldwell E (1997) Spacing of basic amino acids in heparin binding sites. Arch Biochem Biophys 343:92–100

    Article  PubMed  Google Scholar 

  24. Marty NJ, Rajalingam D, Kight AD, Lewis NE, Fologea D, Kumar TKS, Goforth RL (2009) The membrane-binding motif of the chloroplast signal recognition particle receptor (cpFtsY) regulates GTPase activity. J Biol Chem 284:14891–14903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rajalingam D, Kumar TKS, Yu C (2005) The C2A domain of synaptotagmin exhibits a high binding affinity for copper: implications in the formation of the multiprotein FGF release complex. Biochemist 44:14431–14442

    Article  CAS  Google Scholar 

  26. Sivaraja V, Kumar TKS, Rajalingam D, Graziani I, Prudovsky I, Yu C (2006) Copper binding affinity of S100A13, a key component of the FGF-1 nonclassical copper-dependent release complex. Biophys J 91:1832–1843

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jayanthi S, Gundampati RK, Kumar TKS (2017) Simple and efficient purification of recombinant proteins using the heparin-binding affinity tag. Curr Protoc Protein Sci 90:6–13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cardin AD, Weintraub HJ (1989) Molecular modeling of protein-glycosaminoglycan interactions. Arterioscler Thromb Vasc Biol 9:21–32

    CAS  Google Scholar 

  29. Dempewolf C, Morris J, Chopra M, Jayanthi S, Kumar TKS, Li WN (2013) Identification of consensus glycosaminoglycan binding strings in proteins. Proceedings of the 4th international conference on Information Science & Applications, ISBN#—978-1-4799-0603-1/13/$31, pp 311–314

    Google Scholar 

  30. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172–188

    PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA

    Sanhita Maity, Musaab Al-Ameer, Ravi Kumar Gundampati, Shilpi Agrawal & Thallapuranam Krishnaswamy Suresh Kumar

Authors
  1. Sanhita Maity
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  2. Musaab Al-Ameer
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  3. Ravi Kumar Gundampati
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  4. Shilpi Agrawal
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  5. Thallapuranam Krishnaswamy Suresh Kumar
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Corresponding author

Correspondence to Thallapuranam Krishnaswamy Suresh Kumar .

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

    Nikolaos E. Labrou

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Maity, S., Al-Ameer, M., Gundampati, R.K., Agrawal, S., Kumar, T.K.S. (2021). Heparin-Binding Affinity Tag: A Novel Affinity Tag for Simple and Efficient Purification of Recombinant Proteins. 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_21

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  • DOI: https://doi.org/10.1007/978-1-0716-0775-6_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0774-9

  • Online ISBN: 978-1-0716-0775-6

  • eBook Packages: Springer Protocols

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