earticle

영어

A single chain Fv (scFv) fragment is a versatile antibody format that is applicable in therapy, diagnostics and so on. In many cases, the scFvs are labeled by fluorescent probes, conjugated by synthetic polymers and immobilized on chip/arrays. These modifications are facilitated
using the site-specific addition of bio-orthogonal reactive groups to the scFv proteins. Especially, the N-terminus of scFv is an attractive target site for the addition of reactive groups in manufacturing scFv-based herapeutics, chips and biosensors. This work describes a strategy for
producing a scFv that bears a bio-orthogonal group that was selectively added onto its N-terminus in the cytoplasm of Escherichia coli. To achieve the production of N-terminal modified scFvs we employed the successfully demonstrated in vivo based methionineresidue
substitution method. The major problem of using the methionine-residue substitution method for N-terminal modification is that all the methionine residues in the target protein are globally
replaced by methionine surrogates, which prevents the N-terminal specific modification. In this study, an approach for engineering a target scFv sequence into a sequence that is methionine-free except for at N-terminus is proposed, and the N-terminal specific incorporation of a methionine analogue into the engineered scFv is demonstrated using the methionine-residue substitution method.