Fig. 4. Insertion of a recombinantly expressed peptide into GFP expressed in mammalian cells.

a Schematic presentation of the strategy applied to reconstitute GFP from recombinantly expressed N-/C-terminal fragments and recombinantly expressed peptide X_GFP^REC corresponding to amino acids 65–85 of GFP in HEK293 cells. Inteins A (CfaDnaE) and B (SspDnaB^M86) are indicated by square and round symbols, respectively. b Bright-field (right panels) and fluorescence (left panels) images of HEK293 cells expressing the indicated constructs. Scale bars: 20 µm. GFP fluorescence was only detected when all three constructs (N + X + C) were co-transfected. GFP fluorescence was not detected when one of the three constructs was absent or when +1 extein residues of each split intein is mutated to alanine (C65A for intein A and S86A for intein B) to prevent splicing.