Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Streptavidins with intersubunit crosslinks have enhanced stability

Nature Biotechnology volume 14, pages 1007–1011 (1996)Cite this article

Abstract

Natural tetrameric streptavidin has two subunit interfaces; one is a strong interface between subunits in a tightly associated dimer, and the other is a weak interface between a pair of such dimers (dimer-dimer interface). To test whether strengthening the weak dimer-dimer interface could provide streptavidin with additional structural stability, covalent crosslinks were introduced between adjacent subunits through the dimer-dimer interface. Specific crosslinking sites were designed by site-directed mutations of His-127 residues that are in close proximity in natural streptavidin. The first and second streptavidin constructs have a disulfide bond and an irreversible covalent bond, respectively, between two Cys-127 residues across the dimer-dimer interface. The third variant is a hybrid tetramer consisting of two different streptavidin species, one having lysine and the other aspartic acid at position 127, which are covalently crosslinked. All streptavidin constructs with intersubunit crosslinks showed higher biotin-binding ability than natural core streptavidin after heat treatment. All of these crosslinked Streptavidins retained bound biotin more stably than natural core streptavidin in guanidine hydrochloride at very acidic pH. These results suggest that the introduction of covalent bonds across the dimer-dimer interface enhances the overall stability of streptavidin.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Chalet, L. and Wolf, F.J. 1964. The properties of streptavidin, a biotin-binding protein produced by Streptomycetes . Arch. Biochem. Biophys. 106: 1–5.

    Article  Google Scholar 

  2. Green, N.M. 1975. Avidin Adv. Prot. Chem. 29: 85–133.

    Article  CAS  Google Scholar 

  3. Hendrickson, W.A., Pähler, A., Smith, J.L., Satow, Y., Merrit, E.A. and Phizackerley, R.P. 1989. Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation. Proc. Natl. Acad. Sci. USA 86: 2190–2194.

    Article  CAS  Google Scholar 

  4. Weber, P.C., Ohlendorf, D.H., Wendoloski, J.J. and Salemme, F.R. 1989. Structural origins of high-affinity biotin binding to streptavidin. Science 243: 85–88.

    Article  CAS  Google Scholar 

  5. Chilkoti, A., Tan, P.H. and Stayton, P.S. 1995. Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: Contributions of tryptophan residues 79,108, and 120. Proc. Natl. Acad. Sci. USA 92: 1754–1758.

    Article  CAS  Google Scholar 

  6. Sano, T. and Cantor, C.R. 1995. Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin. Proc. Natl. Acad. Sci. USA 92: 3180–3184.

    Article  CAS  Google Scholar 

  7. Chilkoti, A., Schwartz, B.L., Smith, R.D., Long, C.J. and Stayton, P.S. 1995. Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery. Bio/Technology 11: 1198–1204.

    Google Scholar 

  8. Studier, F.W., Rosenberg, A.M., Dunn, J.J. and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60–89.

    Article  CAS  Google Scholar 

  9. Sano, T. and Cantor, C.R. 1990. Expression of a cloned streptavidin gene in Escherichia coli . Proc. Natl. Acad. Sci. USA 87: 142–146.

    Article  CAS  Google Scholar 

  10. Hofmann, K., Wood, S.W., Brinton, C.C., Montibeller, J.A. and Finn, F.M. 1980. Iminobiotin affinity columns and their application to retrieval of streptavidin. Proc. Natl. Acad. Sci. USA 77: 4666–4668.

    Article  CAS  Google Scholar 

  11. Husain, S.S. and Lowe, G. 1968. Evidence for histidine in the active sites of ficin and stem-bromelain. Biochem. J. 110: 53–57.

    Article  CAS  Google Scholar 

  12. Husain, S.S. and Lowe, G. 1968. Evidence for the presence of histidine-106 in the active site of papain. Chem. Commun. 1968, 310–311.

  13. Sano, T., Pandori, M.W., Smith, C.L. and Cantor, C.R. 1994. Tighter subunit association of streptavidin upon biotin binding, pp. 21–29 in Advances in biomagnetic separation. Uhlén, M., Homes, E., and Olsvik, Ø (eds.). Eaton Publishing, Natick, MA.

    Google Scholar 

  14. Sano, T., Pandori, M.W., Chen, X., Smith, C.L. and Cantor, C.R. 1995. Recombinant core streptavidins. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules. J. Biol. Chem. 270: 28204–28209.

    Article  CAS  Google Scholar 

  15. Sayers, J.R., Schmidt, W. and Eckstein, F. 1988. 5′-3′ exonucleases in phospho-rothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 16: 791–802.

    Article  CAS  Google Scholar 

  16. Sano, T., Smith, C.L. and Cantor, C.R. 1993. A streptavidin mutant containing a cysteine stretch that facilitates production of a variety of specific streptavidin conjugates. Bio/Technology 11: 201–206.

    CAS  PubMed  Google Scholar 

  17. Staros, J.V., Wright, R.W. and Swingle, D.M. 1986. Enhancement by N-hydroxy-sulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156: 220–222.

    Article  CAS  Google Scholar 

  18. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY.

    Google Scholar 

  19. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  Google Scholar 

  20. Wei, R.-D. 1970. Assay of Avidin. Methods Enzymol. 18A: 424–427.

    Article  Google Scholar 

Download references

Author information

Author notes

  1. Charles R. Cantor and Takeshi Sano: Corresponding authors.

Authors and Affiliations

  1. Center for Advanced Biotechnology and Departments of Physics, Biomedical Engineering, and Pharmacology and Experimental Therapeutics, Boston University, Boston, MA, 02215

    Gabriel O. Reznik, Sandor Vajda, Cassandra L. Smith & Charles R. Cantor

Authors
  1. Gabriel O. Reznik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandor Vajda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cassandra L. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles R. Cantor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takeshi Sano
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Reznik, G., Vajda, S., Smith, C. et al. Streptavidins with intersubunit crosslinks have enhanced stability. Nat Biotechnol 14, 1007–1011 (1996). https://doi.org/10.1038/nbt0896-1007

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0896-1007

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing