Uniform labeling of a recombinant antibody Fv‐fragment with 15N and 13C for heteronuclear NMR spectroscopy

FEBS Letters

Riechmann, Lutz²  McManus, Siobhan²  Cavanagh, John¹ 

[1] University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, UK;MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

关键词: Antibody;    Fv-fragment;    Protein-engineering;    NMR;    15N;    13C;   

DOI  :  10.1016/0014-5793(91)80047-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 anitbody 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.

【 授权许可】

Unknown