Flow Cytometric Sorting of Bacterial Surface‐Displayed Libraries

Sophia Kenrick1, Jeffrey Rice1, Patrick Daugherty1

1 University of California, Santa Barbara, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 4.6
DOI:  10.1002/0471142956.cy0406s42
Online Posting Date:  October, 2007
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Abstract

The protocols herein detail methods for isolating binding peptides from a combinatorial library displayed on the surface of bacterial cells. These methods are appropriate for a variety of display scaffolds and a large range of library sizes, up to ∼5 × 109 or more. Instructions have been provided for isolating peptides that bind to both proteins and non‐protein targets, such as whole cells or inorganic particles. Qualitative analysis by flow cytometry can be exploited for bacterial libraries to characterize a displayed peptide's binding properties with a target of interest, and sorting conditions can be tuned to maximize binding affinity. Curr. Protocol. Cytom. 42:4.6.1‐4.6.27. © 2007 by John Wiley & Sons, Inc.

Keywords: bacterial libraries; surface display; peptide; binding; ligand; E. coli

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Magnetic Cell Selection (MACS) for Large Libraries
  • Basic Protocol 2: Magnetic Cell Selection (MACS) for Libraries Containing ≤108 Members
  • Basic Protocol 3: Growth and Induction of Bacterial Library for FACS
  • Basic Protocol 4: First Round of FACS
  • Basic Protocol 5: Subsequent Cycles of Library Sorting
  • Basic Protocol 6: Kinetic Sorting
  • Basic Protocol 7: Sorting for Binding to Large Particles or Other Cells
  • Alternate Protocol 1: Labeling with Fluorescent Target Protein
  • Alternate Protocol 2: Labeling with Multiple Detection Reagents
  • Alternate Protocol 3: Two‐Color Labeling and Sorting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Magnetic Cell Selection (MACS) for Large Libraries

  Materials
  • LB medium (see recipe)
  • Bacterial display library (e.g., OmpA library constructed by Bessette et al., )
  • 34 mg/ml chloramphenicol (see recipe)
  • 20% (w/v) arabinose, filter sterilized (see recipe)
  • Phosphate buffered saline (PBS), sterile, 4°C
  • Dynabeads MyOne streptavidin C1 1‐µm magnetic beads (see recipe)
  • Biotinylated target protein (See for details)
  • 1 mM biotin in PBS
  • 20% (w/v) glucose, filter sterilized (see recipe)
  • 50% (v/v) glycerol, sterile
  • LB‐agar plates with 34 µg/ml chloramphenicol (see recipe)
  • 125‐ml and 2‐liter baffled flasks, sterile
  • 37°C incubator with shaker
  • Spectrophotometer
  • 15‐ml tubes (Falcon)
  • Inversion shaker, 4°C
  • Magnet, 3 × 1–in. or join three 1 × 1–in. magnets (neodymium, grade N42)
  • 50‐ and 250‐ml centrifuge tubes, sterile
  • 1.5‐ml microcentrifuge tubes, sterile

Basic Protocol 2: Magnetic Cell Selection (MACS) for Libraries Containing ≤108 Members

  Materials
  • Bacterial display library (e.g., enriched library remaining after performing protocol 1)
  • LB medium (see recipe)
  • 34 mg/ml chloramphenicol (see recipe)
  • 2% (w/v) arabinose, filter sterilized (see recipe)
  • Phosphate buffered saline (PBS), sterile and 4°C
  • Dynabeads MyOne streptavidin C1 (1‐µm magnetic beads; see recipe)
  • Biotinylated target protein (see for details)
  • 1 mM biotin in PBS
  • 20% (w/v) glucose, filter sterilized (see recipe)
  • 50% (v/v) glycerol, sterile
  • LB‐agar plates with 34 µg/ml chloramphenicol (see recipe)
  • 34‐ml culture tubes or 125‐ml baffled flasks, sterile
  • Spectrophotometer
  • 37°C shaking incubator
  • 1.5‐ml microcentrifuge tubes, sterile
  • 4°C inversion shaker
  • Magnet, 3 × 1–in. or join three 1 ×1–in. magnets (neodymium, grade N42)

Basic Protocol 3: Growth and Induction of Bacterial Library for FACS

  Materials
  • Bacterial display library (e.g., enriched library remaining after performing protocol 1 or protocol 22)
  • LB medium (see recipe)
  • 34 mg/ml chloramphenicol (see recipe)
  • 2% (w/v) arabinose, filter sterilized (see recipe)
  • 34‐ml culture tubes, sterile
  • Spectrophotometer
  • 37°C incubator with shaker

Basic Protocol 4: First Round of FACS

  Materials
  • Library grown up and induced as in protocol 3
  • Biotinylated target protein
  • Phosphate buffered saline (PBS), sterile and 4°C
  • 10 nM streptavidin‐conjugated R‐phycoerythrin (SAPE) in PBS
  • SOC (see recipe)
  • LB‐agar plates with 34 µg/ml chloramphenicol (see recipe)
  • 34 mg/ml chloramphenicol (see recipe)
  • 50% (v/v) glycerol, sterile
  • 4°C inversion shaker
  • Cytometer (see )
  • 125‐ml flasks, sterile
  • 37°C incubator with shaker
  • 1.5‐ml microcentrifuge tubes, sterile

Basic Protocol 5: Subsequent Cycles of Library Sorting

  Materials
  • Library grown up and induced as in protocol 3
  • Biotinylated target protein
  • Phosphate buffered saline (PBS), sterile and 4°C
  • 10 nM streptavidin‐conjugated R‐phycoerythrin (SAPE) in PBS
  • SOC (see recipe)
  • LB‐agar plates with 34 µg/ml chloramphenicol (see recipe)
  • LB medium (see recipe)
  • 34 mg/ml chloramphenicol (see recipe)
  • 50% (v/v) glycerol, sterile
  • 4°C inversion shaker
  • Cytometer (see )
  • 125‐ml flasks

Basic Protocol 6: Kinetic Sorting

  Materials
  • Library grown up and induced as in protocol 3
  • Fluorescently labeled target protein
  • Phosphate buffered saline (PBS), sterile and 4°C
  • Unlabeled (nonfluorescent) target protein
  • 4°C inversion shaker
  • Cytometer
  • Sterile tubes for cell collection
  • Additional reagents and equipment for plating and saving libraries (see Basic Protocols protocol 44 and protocol 55)

Basic Protocol 7: Sorting for Binding to Large Particles or Other Cells

  Materials
  • Library grown up and induced as in protocol 3
  • Phosphate buffered saline (PBS), sterile and 4°C
  • Target particles of interest
  • 4°C inversion shaker
  • Cytometer
  • Additional reagents and equipment for plating and storing library (see Basic Protocols protocol 44 and protocol 55)

Alternate Protocol 1: Labeling with Fluorescent Target Protein

  • Fluorescently conjugated target protein

Alternate Protocol 2: Labeling with Multiple Detection Reagents

  • Target protein in PBS
  • Biotinylated detection antibody in PBS

Alternate Protocol 3: Two‐Color Labeling and Sorting

  • Fluorescent target protein
  • Second fluorescently conjugated protein
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Figures

Videos

Literature Cited

   Benhar, I. 2001. Biotechnological applications of phage and cell display. Biotechnol. Adv. 19:1‐33.
   Bessette, P.H. Rice, J.J. and Daugherty, P.S. 2004. Rapid isolation of high‐affinity protein binding peptides using bacterial display. Protein Eng. Des. Sel. 17:731‐739.
   Boder, E.T. and Wittrup, K.D. 1998. Optimal screening of surface‐displayed polypeptide libraries. Biotechnol. Prog. 14:55‐62.
   Charbit, A., Boulain, J.C., Ryter, A., and Hofnung, M. 1986. Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; Expression at the cell surface. EMBO J. 5:3029‐3037.
   Christmann, A., Wentzel, A., Meyer, C., Meyers, G., and Kolmar, H. 2001. Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene‐derived random peptide libraries. J. Immunol. Methods 257:163‐173.
   Dane, K.Y., Chan, L.A., Rice, J.J., and Daugherty, P.S. 2006. Isolation of cell specific peptide ligands using fluorescent bacterial display libraries. J. Immunol. Methods 309:120‐129.
   Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single‐cell analysis. Microbiol. Rev. 60:641‐696.
   Francisco, J.A., Earhart, C.F., and Georgiou, G. 1992. Transport and anchoring of beta‐lactamase to the external surface of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 89:2713‐2717.
   Freudl, R., MacIntyre, S., Degen, M., and Henning, U. 1986. Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K‐12. J. Mol. Biol. 188:491‐494.
   Fuchs, P., Breitling, F., Dubel, S., Seehaus, T., and Little, M. 1991. Targeting recombinant antibodies to the surface of Escherichia coli: Fusion to a peptidoglycan associated lipoprotein. Biotechnology 9:1369‐1372.
   Jose, J., Kramer, J., Klauser, T., Pohlner, J., and Meyer, T.F. 1996. Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine‐containing passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway. Gene 178:107‐110.
   Kim, Y.S., Jung, H.C., and Pan, J.G. 2000. Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Appl. Environ. Microbiol. 66:788‐793.
   Lee, S.Y., Choi, J.H., and Xu, Z. 2003. Microbial cell‐surface display. Trends Biotechnol. 21:45‐52.
   Lu, Z., Murray, K.S., Van Cleave, V., LaVallie, E.R., Stahl, M.L., and McCoy, J.M. 1995. Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: A system designed for exploring protein‐protein interactions. Biotechnology 13:366‐372.
   Rice, J.J., Schohn, A., Bessette, P.H., Boulware, K.T., and Daugherty, P.S. 2006. Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci. 15:825‐836.
Internet Resources
  http://www.bdbiosciences.com/pdfs/brochures/SJ‐0003‐00.pdf
  BD FACSAria flow cytometer system's brochure.
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