Figure 1: Overview of the methodology used for cell-based panning of membrane proteins.

(a) The target membrane gene is cloned into pEGFP-N1 in-frame with GFP. Transfection into CHO or HEK cells results in expression of the membrane protein on the cell surface, with attached intracellular GFP, which gives (b) proportionality between GFP and target expression (shown here by flow cytometry analysis two days post-transfection of CD83-GFP transfected cells binding to anti-CD83 antibody). Expression of the target protein is maximal on Day Two post-transfection (shown here as a time-course flow cytometry assay showing the percentage of cells expressing CD83 and GFP up to 6 days post-transfection). (c) On Day Two post-transfection, the phage library is depleted against non-transfected cells, and then (d) the unbound phage is incubated with the transfected cells. (e) After washing with a low pH buffer, FACS is used to collect high GFP expressing cells, and the phage is eluted from the high-GFP cells and infected into E. coli. (f) The phage is amplified for the next round of panning, where the host transfection cell line in alternated between CHO and HEK cells for 3 to 4 rounds. (g) Individual clones are screened by flow cytometry after 3 to 4 rounds of panning.