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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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
Anglister, J., Grzesiek, S., Ren, H., Klee, C. B., and Bax, A. (1993).J. Biomol. NMR 3, 121–126.
Bax, A., and Davis, D. G. (1985).J. Magn. Reson. 65, 355–360.
Bax, A., Griffey, R. H., and Hawkins, B. L. (1983).J. Magn. Reson. 55, 301–315.
Better, M., Chang, C. P., Robinson, R. R., and Horowitz, A. H. (1988).Science 240, 1041–1043.
Bodenhausen, G., and Ruben, D. G. (1980).Chem. Phvs. Lett. 69, 185–189.
Brahms, S., and Brahms, J. (1980).J. Mol. Biol. 138, 149–178.
Braunschweiler, L., and Ernst, R. R. (1983).J. Magn. Reson. 53, 521–528.
Caldwell, J. B., Strike, P. M., and Kortt, A. A. (1990).J. Protein Chem. 9, 493–499.
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.
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.
Constantine, K. L., Friedrichs, M. S., Metzler, W. J., Wittekind, M., Hensley, P., and Mueller, L. (1994).J. Mol. Biol. 236, 310–327.
Crimmins, D. L., McCourt, D. W., and Schwartz, B. D. (1988).Biochem. Biophys. Res. Commun. 156, 910–916.
Davies, J., and Riechmann, L. (1994).FEBS Lett. 339, 285–290.
Davis, J. (1964).Ann. N. Y. Acad. Sci. 121, 404–427.
Gruen, L. C., McInerney, T. L., Webster, R. G., and Jackson, D. C. (1993).J. Protein Chem. 12, 255–259.
Haber, E. (1992).Immun. Rev. 130, 189–212.
Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Bajyana Songa, E., Bendahman, N., and Hamers, R. (1993).Nature 363, 446–448.
Hjelmeland, L. M., Nebert, D. W., and Osbourne, J. C. (1983).Anal. Biochem. 130, 72–82.
Huston, J. S., Mudgett-Hunter, M., Tai, M.-S., McCartney, J., Haber, E., and Opperman, H. (1991).Meth. Enzymol. 203, 46–88.
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.
Laemmli, U. K. (1970).Nature 27, 680–685.
Lilley, G. G., Dolezal, O., Hillyard, C. J., Bernard, C., and Hudson, P. J. (1994).J. Immun. Meth. 171, 211–226.
Marion, D., and Wüthrich, K. (1983).Biochem. Biophys. Res. Commun. 113, 967–974.
Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J. A. R. G., and Hamers, R. (1994).Protein Eng. 7, 1129–1135.
Neuberger, M. S., Williams, G. T., and Fox, R. O. (1984).Nature 312, 604–608.
Orlandi, R., Güssow, D. H., Jones, P. T., and Winter, G. (1989).Proc. Natl. Acad. Sci. USA 86, 3833–3837.
Ornstein, L. (1964).Ann. N. Y. Acad. Sci. 121, 321–341.
Plückthun, A. (1990).Nature 347, 497–498.
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.
Riechmann, L., Foote, J., and Winter, G. (1988).J. Mol. Biol. 203, 825–828.
Righetti, P. G., and Drysdale, T. W. (1974).J. Chromatogr. 98, 271–321.
Rucker, S. P., and Shaka, A. J. (1989).Mol. Phys. 68, 509–517.
Shaka, A. J., Barker, P. B., and Freeman, R. (1985).J. Magn. Reson. 64, 547–552.
Skerra, A., and Plückthun, A. (1988).Science 240, 1038–1041.
Tulip, W. R., Varghese, J. N., Laver, W. G., Webster, R. G., and Colman, P. M. (1992).J. Mol. Biol. 227, 122–148.
Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989).Nature 341, 544–546.
Winter, G., and Milstein, C. (1991).Nature 349, 293–299.
Wüthrich, K. (1986).NMR of Proteins and Nucleic Acids, Wiley, New York.
Yang, J. T., Wu, C.-S. C., and Martinez, H. M. (1986).Meth. Enzymol. 130, 208–269.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kortt, A.A., Guthrie, R.E., Hinds, M.G. et al. Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41. J Protein Chem 14, 167–178 (1995). https://doi.org/10.1007/BF01980329
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01980329