Figure 3
High-throughput de novo screening of EGFR agonists.
(a) A cell wall-anchored form of FLAG-tagged HLH (HLH-FLAG-FLO42) was expressed with the assistance of MFα1-prepro peptide. The HLH consisted of two α-helixes (blue cylinders) linked with a hepta-glycine (G7) loop, in which the second α-helix contained randomized sequences of five amino acids (red dots) on its solvent-accessible surface. (b) Subcellular localization of HLH-FLAG-FLO42 in yeast cells. Intact cells were stained with an anti-FLAG antibody. Bar = 2.5 μm. (c) Flowchart of EGFR agonist screening. Approximately 3.2 × 106 cells co-expressing HLH-FLAG-FLO42 (HLH) and EGFR-V5 (EGFR) were incubated at pH 7 for 1 h, fixed with paraformaldehyde and then treated with zymolyase, followed by incubation with an anti-phospho EGFR antibody. Approximately 2.5 × 105 cells were individually placed into the microchambers of cell array chip by brief centrifugation. The chip was scanned with a CCD camera under UV light by an automated single-cell analysis and isolation system. In the microchamber array chip, positive and negative cells were indicated by red and green circles, respectively. Positive cells were automatically retrieved with a glass capillary equipped on the micromanipulator. Using 20 chips, 83 positive cells were obtained and 65 DNA fragments were amplified by single cell-based PCR. Amino acid sequences were deduced from 13 fragments derived from cells with high fluorescence and then aligned. Finally, eight independent EGFR agonist candidates were obtained, synthesized in E. coli and then evaluated for autophosphorylation of the EGFR in A431 cells.
