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.
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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
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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
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DOI: https://doi.org/10.1007/BF01980329