Solution properties of Escherichia coli-expressed VH domain of anti-neuraminidase antibody NC41.

The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG), has been expressed in high yield (15-27 mg/L) in Escherichia 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 approximately 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 degrees C and concentrations of 5-10 mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its 1H NMR spectrum. Reagents such as CHAPS, n-ocytylglucoside, 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.