Uniform labeling of a recombinant antibody Fv-fragment with 15N and 13C for heteronuclear NMR spectroscopy.
The expression of functional antibody fragments in Escherichia coli enables a detailed analysis by NMR spectroscopy. This is demonstrated with the uniform labeling of an Fv-fragment (25 kDa) comprising the antigen binding site of an antibody against 2-phenyloxazolone with 15N and 13C. The antigen-complexed Fv-fragment was analysed for a potential assignment by heteronuclear multi-dimensional NMR spectroscopy. For almost all backbone amides 15N/1H crosspeaks and for 80% of them TOCSY crosspeaks were observed. In a 13C-edited-HCCH-2D experiment 17 out of 18 threonine spin-systems were identified. Thus detailed assignments are possible, but some amino acid specific labeling in addition to uniform labeling will be required for complete assignments of Fv-fragments.
Riechmann
L
L
MRC Laboratory of Molecular Biology, Cambridge, UK.
Cavanagh
J
J
McManus
S
S
D001483:Base Sequence; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000235:genetics; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010081:Oxazolone / Q000031:analogs & derivatives / Q000276:immunology; D010957:Plasmids; D011994:Recombinant Proteins; D014169:Transformation, Bacterial
D002247:Carbon Isotopes; D007128:Immunoglobulin Fragments; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C018361:2-phenyloxazolone; D010081:Oxazolone