Journal of Protein Chemistry

, Volume 14, Issue 3, pp 167–178 | Cite as

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

  • Alexander A. Kortt
  • Robin E. Guthrie
  • Mark G. Hinds
  • Barbara E. Power
  • Neva Ivancic
  • J. Bruce Caldwell
  • L. Clem Gruen
  • Raymond S. Norton
  • Peter J. Hudson
Article

Abstract

The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAGTM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of ∼4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its1H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.

Key words

Antibody VH domain dimerization detergent stabilization of monomer NMR analysis 

Abbreviations

CDR

complementarity-determining region

CHAPS

3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate

2D-TOCSY

two-dimensional total correlation spectroscopy

FLAG

hydrophilic octapeptide tail

DYKDDDDK

Fv, antibody fragment containing variable domains

GuHCL

guanidine hydrochloride

HRP

horseradïsh peroxidase

Mr

relative molecular mass

PBS

phosphatebuffered saline, pH 7.3

scFv

single-chain Fv fragment

VH and VL

variable domains of antibody heavy and light chains, respectively

References

  1. Anglister, J., Grzesiek, S., Ren, H., Klee, C. B., and Bax, A. (1993).J. Biomol. NMR 3, 121–126.CrossRefPubMedGoogle Scholar
  2. Bax, A., and Davis, D. G. (1985).J. Magn. Reson. 65, 355–360.Google Scholar
  3. Bax, A., Griffey, R. H., and Hawkins, B. L. (1983).J. Magn. Reson. 55, 301–315.Google Scholar
  4. Better, M., Chang, C. P., Robinson, R. R., and Horowitz, A. H. (1988).Science 240, 1041–1043.PubMedGoogle Scholar
  5. Bodenhausen, G., and Ruben, D. G. (1980).Chem. Phvs. Lett. 69, 185–189.CrossRefGoogle Scholar
  6. Brahms, S., and Brahms, J. (1980).J. Mol. Biol. 138, 149–178.CrossRefPubMedGoogle Scholar
  7. Braunschweiler, L., and Ernst, R. R. (1983).J. Magn. Reson. 53, 521–528.Google Scholar
  8. Caldwell, J. B., Strike, P. M., and Kortt, A. A. (1990).J. Protein Chem. 9, 493–499.CrossRefPubMedGoogle Scholar
  9. Colman, P. M., Laver, W. G., Varghese, J. N., Baker, A. T., Tulloch, P. A., Air, G. M., and Webster, R. G. (1987).Nature 326, 358–363.CrossRefPubMedGoogle Scholar
  10. Colman, P. M., Tulip, W. R., Varghese, J. N., Tulloch, P. A., Baker, A. T., Laver, W. G., Air, G. M., and Webster, R. G. (1989).Phil. Trans. Roy. Soc. Lond. B 323, 511–518.Google Scholar
  11. Constantine, K. L., Friedrichs, M. S., Metzler, W. J., Wittekind, M., Hensley, P., and Mueller, L. (1994).J. Mol. Biol. 236, 310–327.CrossRefPubMedGoogle Scholar
  12. Crimmins, D. L., McCourt, D. W., and Schwartz, B. D. (1988).Biochem. Biophys. Res. Commun. 156, 910–916.CrossRefPubMedGoogle Scholar
  13. Davies, J., and Riechmann, L. (1994).FEBS Lett. 339, 285–290.CrossRefPubMedGoogle Scholar
  14. Davis, J. (1964).Ann. N. Y. Acad. Sci. 121, 404–427.PubMedGoogle Scholar
  15. Gruen, L. C., McInerney, T. L., Webster, R. G., and Jackson, D. C. (1993).J. Protein Chem. 12, 255–259.CrossRefPubMedGoogle Scholar
  16. Haber, E. (1992).Immun. Rev. 130, 189–212.PubMedGoogle Scholar
  17. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Bajyana Songa, E., Bendahman, N., and Hamers, R. (1993).Nature 363, 446–448.CrossRefPubMedGoogle Scholar
  18. Hjelmeland, L. M., Nebert, D. W., and Osbourne, J. C. (1983).Anal. Biochem. 130, 72–82.CrossRefPubMedGoogle Scholar
  19. Huston, J. S., Mudgett-Hunter, M., Tai, M.-S., McCartney, J., Haber, E., and Opperman, H. (1991).Meth. Enzymol. 203, 46–88.PubMedGoogle Scholar
  20. Kortt, A. A., Malby, R. L., Caldwell, J. B., Gruen, L. C., Ivancic, N., Lawrence, M. C., Howlett, G. J., Webster, R. G., Hudson, P. J., and Colman, P. M. (1994).Eur J. Biochem. 221, 151–157.CrossRefPubMedGoogle Scholar
  21. Laemmli, U. K. (1970).Nature 27, 680–685.Google Scholar
  22. Lilley, G. G., Dolezal, O., Hillyard, C. J., Bernard, C., and Hudson, P. J. (1994).J. Immun. Meth. 171, 211–226.CrossRefGoogle Scholar
  23. Marion, D., and Wüthrich, K. (1983).Biochem. Biophys. Res. Commun. 113, 967–974.PubMedGoogle Scholar
  24. Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J. A. R. G., and Hamers, R. (1994).Protein Eng. 7, 1129–1135.PubMedGoogle Scholar
  25. Neuberger, M. S., Williams, G. T., and Fox, R. O. (1984).Nature 312, 604–608.CrossRefPubMedGoogle Scholar
  26. Orlandi, R., Güssow, D. H., Jones, P. T., and Winter, G. (1989).Proc. Natl. Acad. Sci. USA 86, 3833–3837.PubMedGoogle Scholar
  27. Ornstein, L. (1964).Ann. N. Y. Acad. Sci. 121, 321–341.PubMedGoogle Scholar
  28. Plückthun, A. (1990).Nature 347, 497–498.CrossRefPubMedGoogle Scholar
  29. Power, B. E., Ivancic, N., Harley, V. R., Webster, R. G., Kortt, A. A., Irving, R. A., and Hudson, P. J. (1992).Gene 113, 95–99.CrossRefPubMedGoogle Scholar
  30. Riechmann, L., Foote, J., and Winter, G. (1988).J. Mol. Biol. 203, 825–828.CrossRefPubMedGoogle Scholar
  31. Righetti, P. G., and Drysdale, T. W. (1974).J. Chromatogr. 98, 271–321.CrossRefPubMedGoogle Scholar
  32. Rucker, S. P., and Shaka, A. J. (1989).Mol. Phys. 68, 509–517.Google Scholar
  33. Shaka, A. J., Barker, P. B., and Freeman, R. (1985).J. Magn. Reson. 64, 547–552.Google Scholar
  34. Skerra, A., and Plückthun, A. (1988).Science 240, 1038–1041.PubMedGoogle Scholar
  35. Tulip, W. R., Varghese, J. N., Laver, W. G., Webster, R. G., and Colman, P. M. (1992).J. Mol. Biol. 227, 122–148.CrossRefPubMedGoogle Scholar
  36. Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989).Nature 341, 544–546.CrossRefPubMedGoogle Scholar
  37. Winter, G., and Milstein, C. (1991).Nature 349, 293–299.CrossRefPubMedGoogle Scholar
  38. Wüthrich, K. (1986).NMR of Proteins and Nucleic Acids, Wiley, New York.Google Scholar
  39. Yang, J. T., Wu, C.-S. C., and Martinez, H. M. (1986).Meth. Enzymol. 130, 208–269.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Alexander A. Kortt
    • 1
  • Robin E. Guthrie
    • 1
  • Mark G. Hinds
    • 2
  • Barbara E. Power
    • 1
  • Neva Ivancic
    • 1
  • J. Bruce Caldwell
    • 1
  • L. Clem Gruen
    • 1
  • Raymond S. Norton
    • 2
  • Peter J. Hudson
    • 1
  1. 1.Division of Biomolecular EngineeringCSIROParkvilleAustralia
  2. 2.Biomolecular Research InstituteParkvilleAustralia

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