Skip to main content
SpringerLink
Log in

Site-specific and effective immobilization of proteins by Npu DnaE split-intein mediated protein trans-splicing reaction

  • Original Article
  • Published:
BioChip Journal Aims and scope Submit manuscript

Abstract

Immobilized proteins on solid supports provide a useful platform for various biological assays including proteomics research, protein-protein interaction studies, and diagnosis. In fabricating protein chips, proteins should be immobilized to maintain activity and specificity towards their targets. Here, we describe an approach to attach a protein of interest onto a solid support through a covalent bond as well as in situ monitoring of protein immobilization and subsequent binding assays. Our system utilized self-assembled monolayers of alkanethiolates on gold as biologically inert solid substrates, and Nostoc punctiforme DnaE splitinteins were used to mediate biospecific interactions and covalently conjugate proteins on the solid support. Use of a gold substrate enabled in situ monitoring of the protein-protein interactions using surface plasmon resonance spectroscopy. This approach provides a flexible method for further protein immobilization applications.

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. Humpel, C. Identifying and validating biomarkers for Alzheimer’s disease. Trends Biotechnol. 29, 26–32 (2011).

    Article  CAS  Google Scholar 

  2. Uttamchandani, M. & Yao, S.Q. Peptide microarrays: next generation biochips for detection, diagnostics and high-throughput screening. Curr. Pharm. Des. 14, 2428–2438 (2008).

    Article  CAS  Google Scholar 

  3. Fabre, S. et al. Protein biochip array technology for cytokine profiling predicts etanercept responsiveness in rheumatoid arthritis. Clin. Exp. Immunol. 153, 188–195 (2008).

    Article  CAS  Google Scholar 

  4. Kwon, Y. et al. Magnetic bead based immunoassay for autonomous detection of toxins. Anal. Chem. 80, 8416–8423 (2008).

    Article  CAS  Google Scholar 

  5. Jung, D.-H., Min, K., Jeon, Y., Jang, W. & Kwon, Y. Optimized magnetic bead-based immunoassay for automated detection of protein toxins. BioChip Journal 6, 293–298 (2012).

    Article  CAS  Google Scholar 

  6. Wang, R., Kim, J.H., Kim, B.S., Park, C.S. & Yoo, S.H. Preparation and characterization of non-covalently immobilized amylosucrase using a pH-dependent autoprecipitating carrier. Bioresour. Technol. 102, 6370–6374 (2011).

    Article  CAS  Google Scholar 

  7. Nakanishi, K., Sakiyama, T., Kumada, Y., Imamura, K. & Imanaka, H. Recent Advances in Controlled Immobilization of Proteins onto the Surface of the Solid Substrate and Its Possible Application to Proteomics. Current Proteomics 5, 161–175 (2008).

    Article  CAS  Google Scholar 

  8. Frederix, F. et al. Reduced nonspecific adsorption on covalently immobilized protein surfaces using poly (ethylene oxide) containing blocking agents. Journal of Biochemical and Biophysical Methods 58, 67–74 (2004).

    Article  CAS  Google Scholar 

  9. Liu, W., Wang, L. & Jiang, R. Specific Enzyme Immobilization Approaches and Their Application with Nanomaterials. Topics in Catalysis 55, 1146–1156 (2012).

    Article  CAS  Google Scholar 

  10. Houseman, B.T., Huh, J.H., Kron, S.J. & Mrksich, M. Peptide chips for the quantitative evaluation of protein kinase activity. Nat. Biotechnol. 20, 270–274 (2002).

    Article  CAS  Google Scholar 

  11. Berrade, L., Garcia, A.E. & Camarero, J.A. Protein microarrays: novel developments and applications. Pharmaceutical Research 28, 1480–1499 (2011).

    Article  CAS  Google Scholar 

  12. Kwon, Y., Coleman, M.A. & Camarero, J.A. Selective immobilization of proteins onto solid supports through split-intein-mediated protein trans-splicing. Angew. Chem. Int. Ed. Engl. 45, 1726–1729 (2006).

    Article  CAS  Google Scholar 

  13. Zettler, J., Schutz, V. & Mootz, H.D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Letters 583, 909–914 (2009).

    Article  CAS  Google Scholar 

  14. Martin, D.D., Xu, M.Q. & Evans, T.C., Jr. Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803. Biochemistry 40, 1393–1402 (2001).

    Article  CAS  Google Scholar 

  15. Nichols, N.M., Benner, J.S., Martin, D.D. & Evans, T.C., Jr. Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression. Biochemistry 42, 5301–5311 (2003).

    Article  CAS  Google Scholar 

  16. Aranko, A.S., Zuger, S., Buchinger, E. & Iwai, H. In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins. PLoS One 4, e5185 (2009).

    Article  Google Scholar 

  17. Volkmann, G. & Iwai, H. Protein trans-splicing and its use in structural biology: opportunities and limitations. Mol. Biosyst. 6, 2110–2121 (2010).

    Article  CAS  Google Scholar 

  18. Oeemig, J.S., Aranko, A.S., Djupsjobacka, J., Heinamaki, K. & Iwai, H. Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Letters 583, 1451–1456 (2009).

    Article  CAS  Google Scholar 

  19. Hodneland, C.D., Lee, Y.S., Min, D.H. & Mrksich, M. Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands. Proc. Natl. Acad. Sci. USA 99, 5048–5052 (2002).

    Article  CAS  Google Scholar 

  20. Kwon, Y., Han, Z., Karatan, E., Mrksich, M. & Kay, B.K. Antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers. Anal. Chem. 76, 5713–5720 (2004).

    Article  CAS  Google Scholar 

  21. Schaeferling, M. et al. Application of self-assembly techniques in the design of biocompatible protein microarray surfaces. Electrophoresis 23, 3097–3105 (2002).

    Article  CAS  Google Scholar 

  22. Hudalla, G.A. & Murphy, W.L. Using “click” chemistry to prepare SAM substrates to study stem cell adhesion. Langmuir 25, 5737–5746, doi:10.1021/la804077t (2009).

    Article  CAS  Google Scholar 

  23. Camarero, J.A. Recent developments in the site-specific immobilization of proteins onto solid supports. Biopolymers 90, 450–458 (2008).

    Article  CAS  Google Scholar 

  24. Lin, P.-C. et al. Site-Specific Protein Modification through CuI-Catalyzed 1,2,3-Triazole Formation and Its Implementation in Protein Microarray Fabrication. Angewandte Chemie 118, 4392–4396 (2006).

    Article  Google Scholar 

  25. Rich, R.L., Day, Y.S., Morton, T.A. & Myszka, D.G. High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE. Anal. Biochem. 296, 197–207 (2001).

    Article  CAS  Google Scholar 

  26. Lockless, S.W. & Muir, T.W. Traceless protein splicing utilizing evolved split inteins. Proc. Natl. Acad. Sci. USA 106, 10999–11004 (2009).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Dongguk University-Seoul, Pildong 3-ga, Seoul, 100-713, Korea

    Kyoungmi Min, Deokho Jung, Yongkwon Jeon & Youngeun Kwon

  2. Department of Chemistry, Gangnung-Wonju National University, Gangneung, Gangwondo, 210-702, Korea

    Eunhee Jeoung

Authors
  1. Kyoungmi Min
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deokho Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yongkwon Jeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eunhee Jeoung
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Youngeun Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Youngeun Kwon.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Min, K., Jung, D., Jeon, Y. et al. Site-specific and effective immobilization of proteins by Npu DnaE split-intein mediated protein trans-splicing reaction. BioChip J 7, 288–294 (2013). https://doi.org/10.1007/s13206-013-7312-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-013-7312-7

Keywords

Search

Navigation