Split-GFP labeling of scFvs.

A. GFP complementation vectors. The scFv-D1.3-GFP11 construct was cloned into a pEP based vector (left) or a yeast display vector (right). The GFP11 peptide is cloned at the C-terminus of the scFv in order not to interfere with the scFv/antigen-binding activity. B. Linear representation of the scFv-GFP11 molecule. The sequence of the SV5 tag, followed by the GFP11 sequence and the six histidine tag (6xHis tag) found at the C terminus of the scFv is shown. C. Split GFP fragment complementation. The scFv is fused to the small GFP fragment (strand 11, residues 215-230). The complementary GFP fragment (1-10, residues 1-214) is expressed separately. Neither fragment alone is fluorescent. When mixed, the small and large GFP fragments spontaneously associate, resulting in reconstitution of the fluorophore and fluorescence. D. Split GFP fragment complementation in scFv yeast display. The scFv in fusion with the GFP11 strand is displayed on yeast cells. It is still able to bind the specific antigen and fluorescence is restored when yeast cells are incubated with the GFP1-10 fragment.