Figure 1.

Co-expression of two polypeptide chains with fluorescent markers. (a) Schematic of the expression constructs. Expression is driven by the cytomegalovirus (CMV) promoter. The gene for the heavy chain of the Fab is in blue colors (VH and CH1 D1.3), followed by a hexa-histidine tag (H6) fused to its C-terminus. The light chain, in a separate vector is shown in yellow (VL) and orange (CL D1.3). Expression of the heavy and light chains is coupled to that of green (GFP) and red (RFP) protein, respectively, by an internal ribosome entry site (IRES). (b) Correlation of Fab 2A11 expression levels with fluorescence. Cells were sorted according to their fluorescence profile, and the pools photographed by confocal microscopy in the emission channels corresponding (from top to bottom) to RFP, GFP and the nuclear stain TOTO-3. The resulting Fab is shown under the respective columns, run out, after purification, on a Coomassie blue-stained SDS–PAGE gel. (c) Fluorescence profile of sorted cells. Fluorescence data from 100,000 viable HEK293-T cells expressing 2A11 after antibiotic selection are shown. Fluorescence is plotted in logarithmic scale for RFP (vertical axis) and GFP (horizontal axis). The orange lines are representative of the 4 × 4 grid used to select negative, +, ++ and +++ cells, for RFP fluorescence (horizontal lines from bottom to top, in increasing order) and for GFP fluorescence (vertical lines left to right, in increasing order). (d) Fluorescence profile of a production cell line. A clonal cell line expressing 2A11 Fab was analyzed by FACS as described for panel (c). Reprinted from Protein Expression and Purification, volume 55, Zahra Assur, Ira Schieren, Wayne A. Hendrickson and Filippo Mancia, Two-color selection for amplified co-production of proteins in mammalian cells, pages 319–324, copyright (2007), with permission from Elsevier.