Riechmann L, Cavanagh J, McManus S
MRC Laboratory of Molecular Biology, Cambridge, UK.
FEBS Lett. 1991 Aug 5;287(1-2):185-8. doi: 10.1016/0014-5793(91)80047-7.
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.
在大肠杆菌中表达功能性抗体片段能够通过核磁共振光谱进行详细分析。这通过用15N和13C对包含抗2-苯基恶唑酮抗体抗原结合位点的Fv片段（25 kDa）进行均匀标记得以证明。通过异核多维核磁共振光谱分析了与抗原复合的Fv片段，以进行潜在的归属。对于几乎所有的主链酰胺，观察到了15N/1H交叉峰，其中80%观察到了全相关谱（TOCSY）交叉峰。在一个13C编辑的HCCH-2D实验中，鉴定出了18个苏氨酸自旋系统中的17个。因此可以进行详细的归属，但为了对Fv片段进行完全归属，除了均匀标记外还需要一些氨基酸特异性标记。
