Antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells due to the important biological roles of c-Met in the proliferation of malignancies. In particular, fragment antibodies against c-Met such as anti-c-Met single-chain Fv (scFv) antibody can be suitably utilized for the applications such as tumor penetration and imaging due to their small sizes. In this study, we attempted to engineer anti-c-Met scFv in order to introduce unnatural amino acids into the N-terminus of the scFv. Incorporation of non-natural amino acids into proteins is currently of interest since a further dimension can be added to protein structure and function. We generated intracellular chimeric anti-c-Met scFv by a combined rational method of CDR-grafting and structure based framework engineering to produce the scFv as a functional form in the cytoplasm of E. coli. The chimeric anti-c-Met scFv was further engineered to incorporate homopropargylglycine (Hpg), a methionine surrogate containing alkyne group, into the N-terminus of the protein. Finally, the alkynylated chimeric anti-C-Met scFv was labeled with 3-azido-7-hydroxycoumarin by means of a Cu(I)-catalyzed cycloaddition, which yielded a fluorescent anti-C-Met scFv. We expect that the approach used in this study for the site specific alkynylation in the N-terminal region of scFv will have a great impact on the advances in immunology-based diagnosis and therapy.

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