Hostname: page-component-8448b6f56d-qsmjn Total loading time: 0 Render date: 2024-04-23T22:03:25.353Z Has data issue: false hasContentIssue false
  • English
  • Français

Genetically Engineered Pores as Metal Ion Biosensors

Published online by Cambridge University Press:  15 February 2011

John Kasianowicz ,
Barbara Walker ,
Musti Krishnasastry  and
Hagan Bayley
John Kasianowicz
Affiliation:
NIST, Biotechnology Division, Biosensors Group, Chem-A353, Gaithersburg, MD 20899
Barbara Walker
Affiliation:
Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01545
Musti Krishnasastry
Affiliation:
Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01545
Hagan Bayley
Affiliation:
Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01545
Get access

Abstract

We are adapting proteins that form pores in lipid bilayers for use as components of biosensors. Specifically, we have produced genetically engineered variants of the α hemolysin (αHL) from Staphylococcus aureus with properties that are sensitive to low concentrations of divalent cations. For example, the pore-forming activity of one mutant (αHL-H5: residues 130–134 inclusive replaced with histidine) is inhibited by Zn2+ at concentrations as low as 1 μM, as judged by the reduction in its ability to lyse rabbit red blood cells and to increase the conductance of planar lipid bilayer membranes. When αHL-H5 is added to the aqueous phase bathing one side of a planar membrane, the subsequent addition of 100 μM Zn2+ to either side blocks the pores that form. This result suggests that at least part of the mutated region lines the channel lumen. Ca2+ and Mg2+ do not block the channel and therefore the H5 mutation confers a degree of analyte specificity to the αHL pore. The results suggest that genetically engineered pores have great promise for the rapid and sensitive detection of metal cations and we discuss the merits and potential limitations for their use in this application. Specifically, we examine the issues of selectivity, sensitivity, response time, dynamic range and longevity. Some of these properties are interdependent. For example, the goals of high sensitivity and rapid response time can be in conflict.

Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 330: Symposium S – Biomolecular Materials by Design , 1993 , 217
Copyright
Copyright © Materials Research Society 1994

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

1. Gray, G.S. and Kehoe, M., Infection and Immunity 46, 615 (1984).CrossRefGoogle Scholar
2. Bhakdi, S. and Tranum-Jensen, J., Microbiol. Rev. 55, 733 (1991).CrossRefGoogle Scholar
3. Füssle, R., Bhakdi, S., Sziegoliet, A., Tranum-Jensen, J., Kranz, T. and Wellensiek, H.-J., J. Cell Biol. 91, 83 (1981).CrossRefGoogle Scholar
4. Tobkes, N., Wallace, B.J. and Bayley, H., Biochemistry 24, 1915 (1985).CrossRefGoogle Scholar
5. Menestrina, G., J. Membrane Biol. 90,177 (1986).CrossRefGoogle Scholar
6. Krasilnikov, O., Sabirov, R. Z., Ternovsky, V.I., Merzliak, P.G. and Muratkodjaev, J.N., FEMS Microbiol. ImmunoL. 105, 90 (1992).CrossRefGoogle Scholar
7. Bayley, H., MRS Symp. Proc. 218, 69 (1991).CrossRefGoogle Scholar
8. Hebert, H., Olofsson, A., Thelestam, M. and Skriver, E., FEMS Microbiol. ImmunoL. 105, 5 (1992).Google Scholar
9. Olofsson, A., Kavdus, U., Hacksell, I., Thelestam, M. and Hebert, H., J. Mol. Biol. 214, 299 (1990).CrossRefGoogle Scholar
10. Walker, B., Krishnasastry, M., Zorn, L., Kasianowicz, J. and Bayley, H., J. Biol. Chem. 267, 10902 (1992).CrossRefGoogle Scholar
11. Walker, B., Krishnasastry, M., Zorn, L. and Bayley, H., J. Biol. Chem. 267, 21782 (1992).CrossRefGoogle Scholar
12. Walker, B.J., Krishnasastry, M. and Bayley, H., J. Biol. Chem. 268, 5285 (1993).CrossRefGoogle Scholar
13. Palmer, M., Jursch, R., Weller, U., Valeva, A., Hilger, K., Kehoe, M. and Bhakdi, S., J. Biol. Chem. 268, 11959 (1993).CrossRefGoogle Scholar
14. Palmer, M., Weller, U., Messner, M. and Bhakdi, S., J. Biol. Chem. 268,11963 (1993).CrossRefGoogle Scholar
15. Arnold, F.H. and Haymore, B.L., Science 252, 1796 (1991).CrossRefGoogle Scholar
16. Walker, B., Kasianowicz, J., Krishnasastry, M. and Bayley, H., Submitted for publication.Google Scholar
17. Birnbaum, S. and Mosbach, K., Curr. Opin. Biotechnol. 3, 49 (1992).CrossRefGoogle Scholar
18. Green, L.M. and Berg, J.M., Proc. Natl. Acad. Sci. (USA) 87, 6403 (1990).CrossRefGoogle Scholar
19. Karlin, K.D., Science 261, 701 (1993).CrossRefGoogle Scholar
20. Chen, C.-H.B., Mazumdar, A., Constant, J.-F. and Sigman, D.S., Bioconjugate Chemistry 4, 69 (1993).CrossRefGoogle Scholar
21. Ebright, Y.W., Chen, Y., Pendergrast, P.S. and Ebright, R.H., Biochemistry 31, 10664 (1992).CrossRefGoogle Scholar
22. Stevens, C.F., Nature (London) 270, 391 (1977).CrossRefGoogle Scholar
23. DeFelice, L.J., Introduction to Membrane Noise, (Plenum Press, NY, 1981).CrossRefGoogle Scholar
24. Bezrukov, S.M. and Kasianowicz, J.J., Phys. Rev. Left. 70, 2352 (1993).CrossRefGoogle Scholar
25. Regen, S., J. Am. Chem. Soc. 110, 7463 (1987).Google Scholar
26. Sleytr, U.B., Sara, M., Pum, D., Küpcü, S. and Messner, P., MRS Symp. Proc. 330, this volume (1994).Google Scholar
27. Sleytr, U.B., Sara, M., Pum, D. and Messner, P., J. Cell Biochem. 55, in press (1994).Google Scholar
28. Leitmannova-Ottova, A., Liu, W., Zhou, T.-A. and Tien, H.T., MRS Symp. Proc. 330, this volume (1994).Google Scholar
29. Woghiren, C., Sharma, B. and Stein, S., Bioconjugate Chem. 4:314 (1993).CrossRefGoogle Scholar