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


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 



complementarity-determining region




two-dimensional total correlation spectroscopy


hydrophilic octapeptide tail


Fv, antibody fragment containing variable domains


guanidine hydrochloride


horseradïsh peroxidase


relative molecular mass


phosphatebuffered saline, pH 7.3


single-chain Fv fragment

VH and VL

variable domains of antibody heavy and light chains, respectively


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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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