Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

Keith D. Miller1, Noah B. Pefaur2, Cheryl L. Baird1

1 Pacific Northwest National Laboratory, Richland, Washington, 2 Department of Protein Biochemistry, ZymoGenetics, Seattle, Washington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 4.7
DOI:  10.1002/0471142956.cy0407s45
Online Posting Date:  July, 2008
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Abstract

These protocols describe a yeast surface display–based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single‐chain antibody fragments (scFvs) is created from antigen‐binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic‐activated cell sorting (MACS) and fluorescence‐activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen. Curr. Protocol. Cytom. 45:4.7.1‐4.7.30. © 2008 by John Wiley & Sons, Inc.

Keywords: surface display; yeast; antibody; immune library; scFv

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Antigen‐Specific B Cell Sorting
  • Basic Protocol 2: Construction of Targeted Immune Yeast Display Library
  • Basic Protocol 3: Library Growth and Induction
  • Basic Protocol 4: Library and Clone Archiving
  • Basic Protocol 5: Magnetic‐Activated Cell Sorting (MACS)
  • Basic Protocol 6: FACS Library Sorting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Antigen‐Specific B Cell Sorting

  Materials
  • Pooled splenocytes from the spleens of two immunized mice
  • Splenocytes from one naïve mouse
  • Wash buffer (see recipe)
  • Mouse BD Fc Block (BD Biosciences, cat. no. 553142)
  • FITC‐labeled anti–mouse CD19 (clone 1D3; BD Pharmingen, cat. no. 557398)
  • Streptavidin phycoerythrin (SAPE; Invitrogen, cat. no. S866)
  • FITC‐labeled Rat IgG 2a κ isotype control (BD Pharmingen, cat. no. 553929)
  • Biotinylated antigen
  • RNeasy Mini RNA isolation kit (Qiagen, cat. no. 74106)
  • DEPC dH 2O (Fisher, cat. no. BP561‐1)
  • Centrifuge
  • Fluorescence‐activated cell sorting instrument (e.g., FACSAria, BD Biosciences)

Basic Protocol 2: Construction of Targeted Immune Yeast Display Library

  Materials
  • Mouse scFv assembly primers (see Table 4.7.2)
  • DEPC H 2O (Fisher, cat. no. BP561‐1)
  • RNA (see protocol 1)
  • SuperScript III First Strand cDNA synthesis kit (Invitrogen, cat. no. 18080‐051) containing:
    • 40 U/µl RNaseOUT
    • 25 mM MgCl 2
    • 0.1 M DTT
    • 200 U/µl SuperScript III RT
    • 2 U/µl E. coli RNaseH
    • 10 mM dNTP mix
    • 5× first strand buffer
  • 5× HF buffer (provided with Phusion High‐Fidelity DNA polymerase)Phusion High‐Fidelity DNA Polymerase (NEB, cat. no. F‐530L)
  • TE buffer
  • 1% agarose gel
  • 1× TAE buffer
  • QIAquick gel extraction kit (Qiagen, cat. no. 28706)
  • 10 µg pPNL6 (vector available from PNNL)
  • NheI restriction endonuclease (NEB, cat. no. R0131)
  • 1 M NaCl, sterile
  • BamHI restriction endonuclease (NEB, cat. no. R0136)
  • Sal1 restriction endonuclease (NEB, cat. no. R0138)
  • Saccharomyces cerevisiae strain EBY100 (Invitrogen, cat. no. C83900)
  • 1.5‐ml microcentrifuge tubes
  • Nuclease‐free PCR tube
  • Thermal cycler
  • Ice‐water bath
  • 37°C incubator
  • Spectrophotometer
  • SD‐CAA plates (see recipe)
  • 2‐liter baffled flask
  • Additional reagents and equipment for ethanol precipitation of DNA (Moore and Dowhan, ), agarose gel electrophoresis (Voytas, ), electroporation (Chao et al., ), and transforming yeast using 2× high efficiency yeast lithium acetate (Gietz and Schiestl, )

Basic Protocol 3: Library Growth and Induction

  Materials
  • YSD library (constructed in protocol 2)
  • SD‐CAA medium (see recipe)
  • SGR‐CAA medium (see recipe)
  • Wash buffer (see recipe)
  • Primary anti–c‐Myc antibody (mouse anti–c‐Myc clone 9E10; Novus Biologicals, cat. no. 600‐302; or chicken anti–c‐Myc; Invitrogen, cat. no. A21281)
  • Biotinylated antigen
  • Secondary c‐Myc antibody [Zenon Alexa Fluor 488 mouse IgG1 labeling reagent (Fc‐specific); Invitrogen, cat. no. Z25002; or AlexaFluor 488 goat anti–chicken IgG; Invitrogen, cat. no. A11039]
  • Streptavidin phycoerytherin (SAPE; Invitrogen, cat. no. S866)
  • Baffled flask
  • Spectrophotometer
  • 30°C and 20°C shaking incubator
  • Microcentrifuge

Basic Protocol 4: Library and Clone Archiving

  Materials
  • Late log or early stationary phase yeast culture
  • 30% (w/v) glycerol, autoclaved
  • 2‐ml screw‐cap cryovials, sterile

Basic Protocol 5: Magnetic‐Activated Cell Sorting (MACS)

  Materials
  • YSD library ( protocol 2)
  • Wash buffer (see recipe)
  • Biotinylated antigen
  • Streptavidin magnetic beads (Miltenyi µMACS Streptavidin Kit, cat. no. 130‐074‐101)
  • SD‐CAA medium (see recipe)
  • 37°C shaking incubator
  • Ice bath
  • Miltenyi Midi‐MACS stand (Miltenyi, cat. no. 130‐042‐302) and LS columns (Miltenyi, cat. no. 130‐042‐303) or autoMACS or autoMACS Pro separator (Miltenyi, cat. no. 201‐01)
  • 15‐ml conical tube
  • SD‐CAA plates (see recipe)
  • 30°C incubator
  • 50‐ml Falcon tubes
  • Additional reagents and equipment for checking the level of c‐Myc expression ( protocol 3)

Basic Protocol 6: FACS Library Sorting

  Materials
  • Expanded and induced R1 library output from MACS column (see protocol 5)
  • Wash buffer (see recipe)
  • Primary anti–c‐Myc Antibody: mouse anti–c‐Myc (clone 9E10); Novus Biologicals, cat. no. 600‐302 or chicken anti–c‐Myc; Invitrogen, cat. no. A21281
  • Alexa Fluor 488 labeled Secondary Antibody for c‐Myc: Zenon Alexa Fluor 488 mouse IgG1 labeling reagent (Fc‐specific); Invitrogen, cat. no. Z25002 or AlexaFluor 488 goat anti–chicken IgG; Invitrogen, cat. no. A11039
  • Streptavidin phycoerytherin (SAPE; Invitrogen, cat. no. S866)
  • R‐Phycoerythrin labeled Secondary Antibody for c‐Myc detection (for compensation control): R‐phycoerythrin goat anti–mouse IgG; Invitrogen, cat. no. P852 or comparable anti–chicken reagent
  • Biotinylated antigen
  • YPD medium (see recipe)
  • 2× SD‐CAA medium (see recipe)
  • FACS instrument
  • SD‐CAA plates (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Amersdorfer, P., Wong, C., Chen, S., Smith, T., Deshpande, S., Sheridan, R., Finnern, R., and Marks, J.D. 1997. Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries. Infect. Immun. 65:3743‐3752.
   Bible, J.M., Howard, W., Robbins, H., and Dunn‐Walters, D.K. 2003. IGHV1, IGHV5 and IGHV7 subgroup genes in the rhesus macaque. Immunogenetics 54:867‐873.
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   Bowley, D.R., Labrijn, A.F., Zwick, M.B., and Burton, D.R. 2007. Antigen selection from an HIV‐1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage. Protein Eng. Des. Sel. 20:81‐90.
   Chao, G., Lau, W.L., Hackel, B.J., Sazinsky, S.L., Lippow, S.M., and Wittrup, K.D. 2006. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1:755‐768.
   Colby, D.W., Kellogg, B.A., Graff, C.P., Yeung, Y.A., Swers, J.S., and Wittrup, K.D. 2004. Engineering antibody affinity by yeast surface display. Methods Enzymol. 388:348‐358.
   Feldhaus, M. and Siegel, R. 2004a. Flow cytometric screening of yeast surface display libraries. Methods Mol. Biol. 263:311‐332.
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   Howard, W.A., Bible, J.M., Finlay‐Dijsselbloem, E., Openshaw, S., and Dunn‐Walters, D.K. 2005b. Immunoglobulin light‐chain genes in the rhesus macaque II: Lambda light‐chain germline sequences for subgroups IGLV1, IGLV2, IGLV3, IGLV4 and IGLV5. Immunogenetics 57:655‐664.
   Miller, K.D., Weaver‐Feldhaus, J., Gray, S.A., Siegel, R.W., and Feldhaus, M.J. 2005. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Protein Expr. Purif. 42:255‐267.
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   Seurynck‐Servoss, S., Baird, C., Miller, K., Pefaur, N., Gonzalez, R., Apiyo, D., Engelmann, H., Srivastava, S., Kagan, J., Rodland, K., and Zangar, R. 2008. Immobilization strategies for single chain antibody microarrays. Proteomics In Press.
   Siegel, R.W., Coleman, J.R., Miller, K.D., and Feldhaus, M.J. 2004. High efficiency recovery and epitope‐specific sorting of an scFv yeast display library. J. Immunol. Methods 286:141‐153.
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