Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS).
Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.
Chen
G
G
Institute for Cell and Molecular Biology, University of Texas, Austin, TX 7812-05, USA.
Hayhurst
A
A
Thomas
J G
JG
Harvey
B R
BR
Iverson
B L
BL
Georgiou
G
G
D001671:Biochemistry / Q000379:methods*; D004076:Digoxigenin / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments / Q000737:chemistry; D008024:Ligands; D009154:Mutation; D019151:Peptide Library; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013997:Time Factors
D007128:Immunoglobulin Fragments; D008024:Ligands; D019151:Peptide Library; D004076:Digoxigenin