| Autor: |
Kortt, Alexander A., Guthrie, Robin E., Hinds, Mark G., Power, Barbara E., Ivancic, Neva, Caldwell, J. Bruce, Gruen, L. Clem, Norton, Raymond S., Hudson, Peter J. |
| Zdroj: |
Journal of Protein Chemistry; April 1995, Vol. 14 Issue: 3 p167-178, 12p |
| Abstrakt: |
The VHdomain 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 VHmolecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VHisoforms 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 VHdomain 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 VHmolecule, prevented dimerization of the VHand permitted good-quality NMR spectra on isotope-labeled protein to be obtained. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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