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Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

Nature Biotechnology volume 19, pages 537–542 (2001)Cite this article

Abstract

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.

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Figure 1: A schematic diagram showing the principle of periplasmic expression with cytometric screening (PECS).
Figure 2: Optimization of the periplasmic expression screening signal.
Figure 3: Examples of targets visualized by PECS.
Figure 4: Monitoring the phage panning process using FACS via PECS.
Figure 5: Examining clones isolated by phage display and PECS.

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Acknowledgements

We thank the Greg Winter laboratory, Medical Research Council, UK for the Griffin.1 library, Charles H. Patterson Jr. Center for Bio/Molecular Science and Engineering, Naval Research Laboratory (Washington, DC) for the Cy5-TNB probe, and the Jon Beckwith laboratory for strains. We thank Patrick S. Daugherty for critical reading of the manuscript and members of the Georgiou and Iverson laboratories for help and comment. This work was supported by Defense Advanced Research Projects Agency (DARPA). B.R.H. was supported by a US Army Multidisciplinary Research Program of the University Research Initiative (MURI) grant.

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Author notes

  1. George Georgiou

    Present address: Biomedical Engineering Program, University of Texas, Austin, 7812-05, TX

  2. Jeffery G. Thomas

    Present address: EpiGenomics Inc., 1000 Seneca Street, Suite 300, Seattle, WA, 98101

  3. Gang Chen and Andrew Hayhurst: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Cell and Molecular Biology, University of Texas, Austin, 7812-05, TX

    Gang Chen, Andrew Hayhurst, Barrett R. Harvey, Brent L. Iverson & George Georgiou

  2. Chemical Engineering Department, University of Texas, Austin, 7812-05, TX

    Andrew Hayhurst, Jeffery G. Thomas & George Georgiou

  3. Department of Chemistry and Biochemistry, University of Texas, Austin, 7812-05, TX

    Brent L. Iverson

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  1. Gang Chen
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Correspondence to Brent L. Iverson or George Georgiou.

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Chen, G., Hayhurst, A., Thomas, J. et al. Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS). Nat Biotechnol 19, 537–542 (2001). https://doi.org/10.1038/89281

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