Solution properties ofEscherichia coli-expressed V_H domain of anti-neuraminidase antibody NC41

The V-H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG(TM)), 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 2M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V-H-FLAG was composed of three isoforms (pi values of similar to 4.6, 4.9, and 5.3) and the V-H molecule was composed of two isoforms with pi values of 5.1 and 6.7; the difference between the V-H 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 V-H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its H-1 NMR spectrum. Reagents such as CHAPS, n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V-H molecule, prevented dimerization of the V-H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.