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. 2014 Nov 10;10(2):527–538. doi: 10.1021/cb500689g

Figure 3.

Figure 3

Analysis of surface displayed scFv- and GFP-202–08 fusions. (a) Surface display levels of unfused, wild-type intein fused or 202–08 intein fused scFvs and GFP were analyzed by flow cytometry. The MFI of the FLAG-positive yeast populations was quantified, and all were normalized to the 4–4–20 construct containing the wild-type intein. Reported are the means ± SD of three independent yeast transformants. Statistical analysis was performed by an unpaired Student’s t-test (*p < 0.05; **p < 0.01; NS, not significant p > 0.05). (b) ScFv and GFP per molecule activity was evaluated by detecting binding to the scFv antigens at saturating ligand concentrations or by measuring GFP fluorescence. Activity per molecule was determined by calculating the ratio of the geometric means for activity (binding or fluorescence) to FLAG expression levels and normalizing to the unfused construct lacking intein. Plotted are the means ± SD from three independent yeast transformants, with statistical significance determined by an unpaired Student’s t-test (*p < 0.05; **p < 0.01; NS, not significant p > 0.05) (c) For intein-mediated protein release, MESNA reacts to release the scFv or GFP from the display construct and append a carboxy-terminal thioester. For EPL functionalization, the carboxy-terminal thioester reacts with a biotinylated peptide containing an amino-terminal cysteine to covalently link the scFv or GFP to the biotin by an amide bond. (d) Products of the reaction depicted in panel c resolved and analyzed by Western blotting to detect release of the scFv or GFP (∼30 kDa) from the 202–08 intein construct using an anti-FLAG antibody (F) or biotin functionalization via EPL with an anti-biotin antibody (B). A small amount of uncleaved scFv-intein-Aga2p product can be seen in the anti-FLAG Western blot between ∼80 kDa and 100 kDa due to the glycosylation of Aga2p.