Effect of linkers on immobilization of scFvs with biotin–streptavidin interaction

Abstract Single‐chain variable fragment antibodies (scFvs) are attractive for use in applications that require high specificity and binding to a target, such as biosensors. Previously, we demonstrated that a variety of scFvs can be immobilized onto a streptavidin surface through in vivo biotinylation of the biotin carboxyl carrier protein (BCCP) or smaller AviTag fused to the scFvs. However, the BCCP constructs showed better immobilization than the AviTag constructs. In this work, we investigated whether the discrepancy between the biotinylation tags could be alleviated by incorporating a flexible (G4S)n linker of varying lengths or a rigid (EA3K)3 linker between the biotinylation tags and the scFvs scFv13R4 and scFv5. Fusion of the (G4S)5 linker or the (G4S)3 linker to the AviTag construct of scFv13R4 or scFv5, respectively, and fusion of the (EA3K)3 linkers to the AviTag constructs of both scFvs enhanced immobilization. Meanwhile, the robust immobilization of the BCCP construct of the scFv constructs remained unaffected. The positive to neutral effects of the linkers, with no adverse effects, make them beneficial tools to incorporate into fusion proteins that show poor immobilization without a linker.