title: Solution properties of Escherichia coli-expressed VH domain of anti-neuraminidase antibody NC41.
abstract: 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.
authors.affiliation: CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
authors.forename: A A
authors.initials: AA
authors.lastname: Kortt
authors.forename: R E
authors.initials: RE
authors.lastname: Guthrie
authors.forename: M G
authors.initials: MG
authors.lastname: Hinds
authors.forename: B E
authors.initials: BE
authors.lastname: Power
authors.forename: N
authors.initials: N
authors.lastname: Ivancic
authors.forename: J B
authors.initials: JB
authors.lastname: Caldwell
authors.forename: L C
authors.initials: LC
authors.lastname: Gruen
authors.forename: R S
authors.initials: RS
authors.lastname: Norton
authors.forename: P J
authors.initials: PJ
authors.lastname: Hudson
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007526:Isoelectric Point; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D012996:Solutions; D009439:Neuraminidase