CHARACTERIZATION OF THE LINKER PEPTIDE OF THE SINGLE-CHAIN F(V)-FRAGMENT OF AN ANTIBODY BY NMR-SPECTROSCOPY

A comparison of the single-chain F(v) fragment of the antibody McPC603 (scF(v)) with its corresponding unlinked F(v) fragment has been carried out with N-15-edited NMR spectroscopy. The two F(v) fragments adopt the same structure, indicating that the linker does not perturb the folding of the domains. This also directly demonstrates that folding in vivo (F(v) fragment) and in vitro (scF(v) fragment) leads to the same structure. The main differences in the spectra of the uniformly N-15-labeled scF(v) and F(v) fragments are due to signals of Gly and Ser from the linker peptide of the scF(v) fragment. The linker peptide has been mapped with NMR spectra of N-15-glycine- and N-15-glycine/N-15-serine-labeled scF(v) fragments. The N-15 T2 relaxation data indicate that the linker peptide is more flexible than the rest of the molecule.