Journal of Protein Chemistry

, Volume 14, Issue 3, pp 167–178

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

DOI: 10.1007/BF01980329

Cite this article as:
Kortt, A.A., Guthrie, R.E., Hinds, M.G. et al. J Protein Chem (1995) 14: 167. doi:10.1007/BF01980329

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

AntibodyVH domaindimerizationdetergent stabilization of monomerNMR 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

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