Aa. Kortt et al., SOLUTION PROPERTIES OF ESCHERICHIA-COLI - EXPRESSED V-H DOMAIN OF ANTI-NEURAMINIDASE ANTIBODY NC41, Journal of protein chemistry, 14(3), 1995, pp. 167-178
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 w
ere 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-F
LAG 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 val
ues 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 mas
k the exposed hydrophobic interface of the V-H molecule, prevented dim
erization of the V-H and permitted good-quality NMR spectra on isotope
-labeled protein to be obtained.