Chemically Induced Self-Assembly of Enzyme Nanorings

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 743)

Abstract

Continued exploration into the field of chemically induced dimerization (CID) has revealed a number of applications for its use in a broader context as a method of structural assembly (1–4). In particular, the use of CID technology to generate self-assembled (and selectively disassembled) protein toroids serves as a key advancement toward developing stable and controllable protein-based platforms. Such structures have broad application to the development of novel therapeutics, lab-on-a-chip technologies, and multi-enzyme assemblies (5, 6). This chapter describes a method of developing an enzymatically active protein nanostructure incorporating both a CID-based assembly region containing dihydrofolate reductase (DHFR) and an enzymatic region consisting of histidine triad nucleotide binding protein 1 (Hint1). Details of both the production and the characterization of this structure are provided.

Key words

Enzyme nanorings DHFR Hint1 nanostructures self-assembly chemically induced dimerization bis-methotrexate gel filtration protein expression protein purification 

References

  1. 1.
    Ballister, E. R., Lai, A. H., Zuckerman, R. N., Cheng, Y., and Mougous, J. D. (2008) In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc. Natl. Acad. Sci. USA 105, 3733.CrossRefGoogle Scholar
  2. 2.
    Carlson, J. C., Jena, S. S., Flenniken, M., Chou, T. F., Siegel, R. A., and Wagner, C. R. (2006) Chemically controlled self-assembly of protein nanorings. J. Am. Chem. Soc. 128, 7630.CrossRefGoogle Scholar
  3. 3.
    Dotan, N., Arad, D., Frolow, F., and Freeman, A. (1999) Self-assembly of a tetrahedral lectin into predesigned diamondlike protein crystals. Angew. Chem. Int. Ed. 38, 2363.CrossRefGoogle Scholar
  4. 4.
    Ringler, P., and Schulz, G. E. (2003) Self-assembly of proteins into designed networks. Science 302, 106.CrossRefGoogle Scholar
  5. 5.
    Chou, T. F., So, C., White, B. R., Carlson, J. C., Sarikaya, M., and Wagner, C. R. (2008) Enzyme nanorings. ACS Nano 2, 2519.CrossRefGoogle Scholar
  6. 6.
    Li, Q., Hapka, D., Chen, H., Vallera, D. A., and Wagner, C. R. (2008) Self-assembling antibodies by chemical induction. Angew. Chem. Int. Ed. 47, 10179.CrossRefGoogle Scholar
  7. 7.
    Quintarelli, C., Vera, J. F., Savoldo, B., Giordano Attanese, G. M. P., Pule, M., Foster, A. E., Heslop, H. E., Rooney, C. M., Brenner, M. K., and Dotti, G. (2007) Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic t lymphocytes. Blood 110, 2793.CrossRefGoogle Scholar
  8. 8.
    Xu, Z. L., Mizuguchi, H., Mayumi, T., and Hayakawa, T. (2003) Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems. Gene 309, 145.CrossRefGoogle Scholar
  9. 9.
    Carlson, J. C. T., Kanter, A., Thuduppathy, G. R., Cody, V., Pineda, P. E., McIvor, R. S., and Wagner, C. R. (2003) Designing protein dimerizers: the importance of ligand conformational equilibria. J. Am. Chem. Soc. 125, 1501.CrossRefGoogle Scholar
  10. 10.
    Chou, T. F., Bieganowski, P., Shilinski, K., Cheng, J., Brenner, C., and Wagner, C. R. (2005) 31P NMR and Genetic analysis establish hinT as the only E. coli purine nucleoside phosphoramidase and essential for growth under high salt conditions. J. Biol. Chem. 280, 15356.CrossRefGoogle Scholar
  11. 11.
    Chou, T. F., Baraniak, J., Kaczmarek, R., Zhou, X., Cheng, J., Ghosh, B., and Wagner, C. R. (2007) Phosphoramidate pronucleotides: a comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins. Mol. Pharm. 4, 208.CrossRefGoogle Scholar
  12. 12.
    Blakley, R. L. (1960) Crystalline dihydropteroylglutamic acid. Nature 188, 231.CrossRefGoogle Scholar
  13. 13.
    Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640.CrossRefGoogle Scholar
  14. 14.
    Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Medicinal ChemistryUniversity of MinnesotaMinneapolisUSA

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