Bacteriophage Library Construction and Selection of Recombinant Antibodies

Maria Galanis1, Robert A. Irving1, Peter J. Hudson1

1 Cooperative Research Center for Diagnostic Technologies at CSIRO Molecular Science, Parkville, Victoria, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 17.1
DOI:  10.1002/0471142735.im1701s34
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of E. coli and bacteriophages to display a diverse library of antibody fragments equivalent in complexity to the mammalian immune repertoire, and subsequent screening of the library for antibody fragments with specific binding affinities. The methods are also used for affinity enhancement (maturation), through the display and selection of improved affinity mutants derived from a single parent antibody. This unit discusses the following key components needed in library construction technology: a repertoire of antibody genes, typically amplified by polymerase chain reaction (PCR) technology; construction of scFv genes by PCR assembly; a method for producing a stable library, using bacteriophage that can both display individual antibodies on the viral surface and carry the gene encoding the antibody; a method of growing phage for selection; a method of selecting the highest‐affinity antibody from the phage library; a method for monitoring progress of phage selection; an affinity‐enhancement strategy for improving and manipulating the selected antibody; and expression of affinity‐enhanced antibodies.

     
 
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Table of Contents

  • Strategic Planning
  • PCR Amplification of Antibody Gene Fragments
  • Basic Protocol 1: Amplification of Antibody Gene Regions from cDNA
  • Support Protocol 1: Synthesis of cDNA
  • Support Protocol 2: Purification of DNA Fragments
  • Basic Protocol 2: Construction of scFv Genes by PCR Assembly
  • Library Construction
  • Basic Protocol 3: Cloning scFv Genes into Phage Display Vectors
  • Alternate Protocol 1: Cloning Fab Genes into Phage Display Vectors Using Sequential Cloning
  • Support Protocol 3: Preparation of Electrocompetent E. coli
  • Basic Protocol 4: Determination of Library Complexity
  • Support Protocol 4: PCR‐Based Colony Screening
  • Basic Protocol 5: Growth of Phage for Selection (Phage Rescue) and Phage Titration
  • Selection of High‐Affinity Phage Clones from Library
  • Basic Protocol 6: Phage Selection by Panning on Antigen‐Coated ELISA‐Plate Wells
  • Alternate Protocol 2: Phage Selection on Antigen‐Sepharose Columns
  • Alternate Protocol 3: Phage Affinity Selection in Solution Using Streptavidin‐Coupled Magnetic Beads
  • Alternate Protocol 4: Phage Selection on Whole Cells
  • Basic Protocol 7: Monitoring Progress of Phage Selection by Soluble ELISA
  • Alternate Protocol 5: Monitoring Progress of Selection by Phage ELISA
  • Affinity Enhancement of Recombinant Antibodies
  • Basic Protocol 8: Random Mutagenesis of Antibody Gene Fragments Using Error‐Prone PCR
  • Basic Protocol 9: CDR Shuffling: Window Mutagenesis
  • Basic Protocol 10: In Vivo Mutation by E. coli Mutator Cells
  • Support Protocol 5: Infection of E. coli Mutator Cells
  • Support Protocol 6: Transformation of E. coli Mutator Cells
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Amplification of Antibody Gene Regions from cDNA

  Materials
  • recipe10× PCR buffer (see recipe)
  • recipe10 mM dNTP stock (see recipe)
  • Mixture of oligonucleotide primers (5 pmol/µl each) for PCR amplification of antibody gene regions, synthesized in house or purchased in kit form (e.g., mouse Ig gene–specific kit from Pharmacia Biotech; see )
  • 5 to 20 ng/µl template cDNA (see protocol 2)
  • 5 U/µl Taq DNA polymerase (Perkin‐Elmer)
  • 0.2‐ml thin‐walled polypropylene thermal cycler tubes
  • Thermal cycler

Support Protocol 1: Synthesis of cDNA

  Materials
  • mRNA (unit 10.11), 100 ng per reaction
  • recipe10 mM dNTP stock (see recipe)
  • recipe10× first‐strand buffer (see recipe)
  • 0.1 M dithiothreitol (DTT) in H 2O
  • 200 µg/ml oligo(dT) in H 2O (Promega)
  • 40 U/µl RNasin RNase inhibitor (Promega)
  • AMV reverse transcriptase (Promega)

Support Protocol 2: Purification of DNA Fragments

  Materials
  • PCR products (see protocol 1)
  • recipe6× DNA gel loading buffer (see recipe)
  • HaeIII‐digested φX‐174 DNA molecular weight standards (New England Biolabs) or equivalent
  • TAE buffer (unit 10.4)
  • 1% to 2% agarose gel prepared in 1× TAE buffer containing 3 µg/ml ethidium bromide (unit 10.4)
  • Bresa‐Clean II nucleic acid purification kit (Bresatec; containing Bresa‐Salt solution, Bresa‐Bind silica matrix, and Bresa‐Wash solution) or equivalent (e.g., GeneClean II kit from Bio 101 or Wizard PCR Preps DNA purification system from Promega)
  • TE buffer, pH 8.0 ( appendix 2A)
  • UV light source
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4)

Basic Protocol 2: Construction of scFv Genes by PCR Assembly

  Materials
  • recipe10× PCR buffer (see recipe)
  • recipe10 mM dNTP stock (see recipe)
  • 10 mg/ml BSA in H 2O
  • Oligonucleotide primers for PCR amplification of antibody gene regions (see )
  • V‐domain PCR product as template DNA (see protocol 1)
  • 5 U/µl Taq DNA polymerase (Perkin‐Elmer)
  • 0.2‐ml thin‐walled polypropylene thermal cycler tubes
  • Thermal cycler

Basic Protocol 3: Cloning scFv Genes into Phage Display Vectors

  Materials
  • SfiI and NotI restriction endonucleases (New England Biolabs)
  • 10 µg phagemid display vector
  • ∼2 µg scFv gene construct (see protocol 4)
  • recipe10× T4 DNA ligase buffer (New England Biolabs or see recipe)
  • T4 DNA ligase (New England Biolabs)
  • recipe3 M sodium acetate, pH 5.2 (see recipe)
  • 100% and 70% ice‐cold ethanol
  • 10× TE buffer, pH 8.0 ( appendix 2A)
  • 200 µl E. coli cells (e.g., TGI, Pharmacia Biotech; see Table 17.1.1), electrocompetent (see protocol 7 and appendix 3N)
    Table 7.1.1   MaterialsE. coli Strains Used in Construction of Antibody Phage‐Display Library

    Strain Genotype Source
    TG1 K12 Δ(lacpro), supE, thi, hsdΔ5/F′[traD36, proAB, lacIq, lacZΔM15] Pharmacia Biotech
    HB2151 K12 Δ(lac‐pro), ara, nalr, thi/F′[proAB, lacIq, lacZΔM15] Pharmacia Biotech

  • recipeSOC broth (see recipe)
  • recipe90‐ and 150‐mm YTAG medium plates (see recipe; plates from Nunc)
  • recipe2YTAG broth (see recipe) containing 15% (w/v) glycerol

Alternate Protocol 1: Cloning Fab Genes into Phage Display Vectors Using Sequential Cloning

  • recipe10× NEBuffers 2, 3, and SalI (see recipe)
  • NcoI, NotI, SfiI, and SalI restriction endonucleases (New England Biolabs)
  • Fd gene fragment PCR products (see protocol 1)
  • Phagemid vector pHFA2
  • CsCl and materials for density‐gradient ultracentrifugation (unit 10.3)
  • Wizard DNA Clean‐Up System (Promega) or equivalent
  • recipeYTAG plates (see recipe)
  • recipe2YTAG broth (see recipe)

Support Protocol 3: Preparation of Electrocompetent E. coli

  • E. coli strain TG1 (Pharmacia Biotech; see Table 17.1.1) grown on recipeM9 plates (see recipe)
  • recipeSOB broth (see recipe), prepared without MgCl 2
  • 2 liters 10% (w/v) glycerol in H 2O, ice cold
  • Dry ice
  • Control DNA (e.g., pUC19 DNA; see protocol 9)
  • 250‐ml centrifuge bottles (Beckman)
  • JA‐14 rotor (Beckman) or equivalent, 4°C

Basic Protocol 4: Determination of Library Complexity

  Materials
  • recipe10× Tth reaction buffer (Biotech International or see recipe)
  • 25 mM MgCl 2 (Biotech International)
  • recipe10 mM dNTP stock (see recipe)
  • Forward and reverse primers specific for vector or insert, synthesized in house
  • 5.5 U/µl Tth DNA polymerase (Biotech International)
  • Mineral oil (molecular biology grade, Sigma)
  • Phage display library on agar plates (see protocol 5 and protocol 6)
  • recipe2YTAG plates (see recipe)
  • 0.2‐ml thin‐walled polypropylene thermal cycler tubes
  • Sterile toothpicks (autoclaved)
  • RoboCycler (Stratagene) or other thermal cycler

Support Protocol 4: PCR‐Based Colony Screening

  Materials
  • recipe2YTA broth (see recipe)
  • Phage display library stock in E. coli strain TG1 (see protocol 5)
  • VCS‐M13 helper phage (Stratagene)
  • 50 mg/ml (w/v) kanamycin (see recipe for recipemedium supplement stocks)
  • recipe20% PEG in 2.5 M NaCl (see recipe)
  • PBS, pH 7.4 ( appendix 2A)
  • E. coli strain TG1 (Pharmacia Biotech; see Table 17.1.1)
  • E. coli host strain XL1‐Blue (Stratagene)
  • recipeM9 plates (see recipe)
  • recipe2YT broth (see recipe)
  • recipe2YTAG broth (see recipe)
  • recipeYTAG plates (see recipe)
  • 250‐ml centrifuge bottles (Beckman)
  • JA‐14 rotor (Beckman)

Basic Protocol 5: Growth of Phage for Selection (Phage Rescue) and Phage Titration

  Materials
  • Pure target antigen (at 4 to 400 µg/ml)
  • Coating buffer: 0.1 M NaHCO 3, pH 8.6
  • 10× PBS, pH 7.4 ( appendix 2A)
  • Blocking buffer: 3% (w/v) RIA‐grade BSA (Sigma) in PBS, pH 7.4
  • PBS/0.05% Tween 20
  • Precipitated phage displaying antibody fragments (see protocol 10)
  • 1 M triethylamine stock
  • 1 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • Acid elution buffer: 0.1 M HCl (adjusted to pH 2.2 with glycine)/1 mg/ml BSA
  • 2 M Tris base, pH unadjusted
  • E. coli strain TG1 (in exponential phase of growth; see protocol 10, step )
  • recipe2YT broth (see recipe)
  • recipeYTAG plates (see recipe)
  • 100 mg/ml ampicillin in H 2O
  • 50 mg/ml kanamycin in H 2O
  • Helper phage VCS‐M13 (Stratagene)
  • 20% (w/v) PEG 8000/15% NaCl
  • Costar EIA/RIA 96‐well ELISA plates (Corning Costar)
  • Acetate plate sealer with adhesive back (Linbro ICN)

Basic Protocol 6: Phage Selection by Panning on Antigen‐Coated ELISA‐Plate Wells

  Materials
  • Antigen of interest (∼2 mg)
  • 1× PBS, pH 7.4 ( appendix 2A)
  • 2% (w/v) RIA‐grade BSA (Sigma) in PBS, pH 7.4
  • 1× PBS with 0.05% (w/v) sodium azide, pH 7.4
  • 10‐ml Econo‐column with 1.5‐ml bed volume (Bio‐Rad)
  • Additional reagents and solutions for coupling to CNBr‐activated Sepharose (unit 8.3) and for phage elution (see protocol 11)

Alternate Protocol 2: Phage Selection on Antigen‐Sepharose Columns

  Materials
  • Streptavidin‐coupled Dynabeads (6–7 × 108 beads/ml; Dynal)
  • Precipitated phage display library (typically ∼1011 pfu/ml in BPBS; see protocol 10)
  • BPBS: PBS, pH 7.4, containing 3% (w/v) RIA‐grade BSA and 0.05% (v/v) Tween‐20
  • PBS, pH 7.4 ( appendix 2A)
  • 0.1% (w/v) RIA‐grade BSA (Sigma) in PBS
  • Pure biotinylated target antigen (20 µg per run)
  • Acid elution buffer: 0.1 M HCl, pH 2.2/1 mg/ml BSA
  • 2 M Tris base, pH unadjusted
  • MPC‐E magnetic stand (Dynal)

Alternate Protocol 3: Phage Affinity Selection in Solution Using Streptavidin‐Coupled Magnetic Beads

  Materials
  • scFv phage display library (from protocol 5)
  • Peripheral blood lymphocytes ( appendix 3F) obtained from a melanoma patient, or any cell type displaying the antigen of interest
  • DMEM‐10 ( appendix 2A)
  • PBS, pH 7.4 ( appendix 2A)
  • Elution buffer: 0.1 M glycine, pH 2.2/0.1% (w/v) BSA
  • 1 M Tris⋅Cl, pH 9.1
  • 24‐cm2 tissue culture flask
  • Additional reagents and equipment for preparation of genomic mRNA (unit 10.11), cDNA synthesis (see protocol 2), and preparation of an scFv phage display library (see protocol 2 and Basic Protocols protocol 11 and protocol 42)
NOTE: Perform all tissue culture incubations in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 4: Phage Selection on Whole Cells

  Materials
  • Selected recombinant phage (from protocol 11 or Alternate Protocols protocol 12, protocol 13, or protocol 14)
  • E. coli strain HB2151 (Pharmacia Biotech; see Table 17.1.1)
  • recipe2YT broth (see recipe)
  • recipeYTAG plates (see recipe)
  • 100 mM recipeIPTG (see recipe)
  • recipe2YTAG broth
  • Primary mouse monoclonal antibody specific for the peptide tail, diluted appropriately in 1% (w/v) RIA‐grade BSA in PBS ( appendix 2A)
  • PBS with 0.05% (v/v) Tween 20
  • 1 mg/ml horseradish peroxidase–conjugated anti‐mouse serum in 1% (w/v) RIA‐grade BSA in PBS
  • recipeABTS reagent (see recipe)
  • Sterile toothpicks
  • 96‐well microtiter plates (Nunc or Corning Costar EIA/RIA)
  • Sorvall RT6000 benchtop centrifuge with swing‐out microtiter plate rotor head
  • Acetate plate sealer with adhesive back (Linbro ICN)

Basic Protocol 7: Monitoring Progress of Phage Selection by Soluble ELISA

  Materials
  • Purified target antigen
  • Coating buffer: 0.1 M NaHCO 3, pH 8.6
  • Blocking buffer: 3% BSA in PBS ( appendix 2A)
  • Phage display library (from protocol 11 or Alternate Protocols protocol 122, protocol 133, or protocol 144)
  • 1% (w/v) RIA‐grade BSA (Sigma) in PBS
  • PBS with 0.05% (v/v) Tween‐20
  • 1 mg/ml horseradish peroxidase–conjugated donkey anti‐sheep serum (Pharmacia Biotech)
  • Sheep anti‐M13 antiserum (Pharmacia Biotech)
  • recipeABTS reagent (see recipe)
  • ELISA plates with flat‐bottom wells: e.g., Corning Costar EIA/RIA plates or Nunc Maxisorb
  • Acetate plate sealer with adhesive back (Linbro ICN)

Alternate Protocol 5: Monitoring Progress of Selection by Phage ELISA

  Materials
  • recipe10× PCR buffer (see recipe)
  • recipe10 mM dNTP stock (see recipe)
  • 100 mM MgCl 2
  • 100 mM MnCl 2
  • Oligonucleotide primers for PCR amplification of antibody gene regions (see )
  • Template DNA encoding antibody gene region of interest
  • 5 U/µl Taq thermal DNA polymerase (Perkin‐Elmer)
  • Thermal cycler

Basic Protocol 8: Random Mutagenesis of Antibody Gene Fragments Using Error‐Prone PCR

  Materials
  • recipe10× PCR buffer (see recipe)
  • recipe10 mM dNTP stock (see recipe)
  • 100 mM MgCl 2
  • 100 mM MnCl 2
  • Oligonucleotide primers for PCR amplification of antibody gene regions (see )
  • cDNA isolated from unimmunized donor (see protocol 2)
  • Parent antibody gene (Fab or scFv)
  • Taq thermal DNA polymerase (Perkin‐Elmer)

Basic Protocol 9: CDR Shuffling: Window Mutagenesis

  Materials
  • E. coli mutator strain mutD5‐FIT K12 (see Table 17.1.3; available on request from authors)
    Table 7.1.3   MaterialsE. coli Mutator Strains

    Strain Genotype Source/reference
    mutD5‐FIT K12 araD, (lac‐proAB), thi/F′, [proAB+, lacIq, lacZΔM15] Irving et al. ( )
    XL1‐Red endA1 gyrA96 thi‐1 hsdR17 supE44 relA1 lac mutD5 mutS mutT Tn10 (Tetr) Stratagene

  • Phagemid clone or library ( protocol 20 or protocol 10, respectively)
  • YTAG/Thy/Tet broth: recipeYTAG broth (see recipe) supplemented with 20 µM thymidine and 100 µg/ml tetracycline (see recipe for recipemedium supplement stocks)
  • VCS‐M13 helper phage (Stratagene)

Basic Protocol 10: In Vivo Mutation by E. coli Mutator Cells

  Materials
  • E. colimutD5‐FIT K12 mutator strain (see Table 17.1.3; available on request from the authors)
  • recipe2YT broth (see recipe) supplemented with 10 µg/ml tetracycline and 1% (w/v) glucose (see recipe for recipemedium supplement stocks)
  • Casamino acids (see recipe for recipemedium supplement stocks)
  • Selected phage or phage library (see protocol 10 or Alternate Protocols protocol 122, protocol 133, or protocol 144)
  • 50 mg/ml tetracycline (see recipe for recipemedium supplement stocks)
  • 50% (w/v) glucose (see recipe for recipemedium supplement stocks)
  • 8.5‐mm recipeYTAG plates and broth (see recipe)

Support Protocol 5: Infection of E. coli Mutator Cells

  Materials
  • E. colimutD5‐FIT K12 mutator strain (see Table 17.1.3; available on request from the authors)
  • recipe2YT broth (see recipe) supplemented with 10 µg/ml tetracycline and 1% (w/v) glucose (see recipe for recipemedium supplement stocks)
  • 10% (w/v) casamino acids (see recipe for recipemedium supplement stocks)
  • Phagemid DNA
  • 50 mg/ml tetracycline (see recipe for recipemedium supplement stocks)
  • 50% (w/v) glucose (see recipe for recipemedium supplement stocks)
  • recipeYTAG plates and broth (see recipe)
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Figures

  •   FigureFigure 17.1.1 Flow chart of procedures for bacteriophage library construction and selection of recombinant antibodies, with subsequent screening of the library for antibody fragments with specific binding affinities. Protocols include affinity enhancement through the display and selection of improved affinity mutants derived from a single parent antibody.
  •   FigureFigure 17.1.2 Phage library selection. E. coli host cells are either transformed with the phage display vector or infected with live phage. The phage display library is rescued from the cells as live phage, with the scFv/Fab sequence encoded within the packaged viral genome. Phage encoding the selected antibody are purified by affinity selection on antigen. The process is repeated for several rounds of selection.
  •   FigureFigure 17.1.3 Strategy for scFv construction and vector assembly. Primary PCR amplifies the heavy‐chain (VH) and light‐chain variable regions (VL) of antibody genes from cDNA. In the linker addition step, the VH and VL genes are separately amplified with primers coding for the scFv linker (linker primers) together with the 5′ and 3′ flanking primers (containing SfiI and NotI restriction endonuclease cleavage sites). Linking PCR then joins the VH and VL genes through the overlapping complementary sequences of the linker primers (using only the 5′ and 3′ external primers), forming an scFv gene. The vector assembly process cleaves the scFv with SfiI and NotI restriction endonucleases and ligates it into pHFA at compatible restriction sites.
  •   FigureFigure 17.1.4 Map of bicistronic pHFA2, the phagemid vector for the display of scFv or Fab gene products on the surface of fd phage. p lacZ, promoter; pelB and OmpA, secretion signal sequences; SfiI, SalI, NcoI, and NotI, unique restriction sites for precise insertion of foreign genes; EEF, tripeptide tag (Glu‐Glu‐Phe); FLAG, octapeptide tag (DYKDDDDK); amber, stop codon (TAG); fd‐pIII, fd bacteriophage minor coat protein III.
  •   FigureFigure 17.1.5 Strategy for construction of Fab library in bicistronic phagemid pHFA2. (A) PCR‐amplify Fd chain repertoires (variable heavy‐chain and constant regions) and insert them into pHFA2 between the NcoI and NotI sites. (B) PCR‐amplify light‐chain repertoires and (C) insert them into the double‐stranded library DNA between the SfiI and SalI sites to generate the library.
  •   FigureFigure 17.1.6 Affinity enhancement cycle. After antibody genes (scFv or Fab) are mutated (see Table ) and displayed on the surface of cells in phage libraries, affinity selection (see the section of this unit on ) leads to phage recovery of the highest‐affinity recombinant antibodies. The selected phage are then amplified and taken through further rounds of mutation and selection. After the final affinity enhancement cycle, the scFv or Fab genes are subcloned into vectors designed for high‐level expression.
  •   FigureFigure 17.1.7 CDR shuffling (window mutagenesis). Starting with a VH gene in a Fab or scFv region, the VH fragment encoding just the FR1 through FR3 region is PCR‐amplified. A repertoire of CDR3 through FR4 fragments from cDNA is amplified and then PCR‐linked to the FR1 through FR3 fragment to produce a library of mutated VH genes. Reassembly of the scFv permits phage display and selection.

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Literature Cited

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Key References
   Burton, D.R. and Barbas, C.F. 1994. See above.
  Thorough review of the technology for isolating recombinant human antibody fragments using filamentous phage display libraries, with emphasis on cloning antibodies from preimmune, immune, and memory compartments of the human immune system. Includes discussion on use as laboratory reagents or in human therapy.
   Harrison et al., 1996. See above.
  Methods for constructing and screening antibody phage display libraries.
   Hoogenboom, H.R. 1998. See above.
  Various phage library‐based strategies for isolating high‐affinity antibodies.
   Winter et al., 1994. See above.
  Review of antibody phage display library construction and selection procedures.
   Zhao et al., 1998. See above.
  An important new method that complements the DNA shuffling and homologous recombination strategies reviewed by Patten et al. (1997).
Internet Resources
   http://www.antibodyresource.com/
  The Antibody Resource page, a multidisciplinary resource on antibodies for both novices and experts providing links to other interesting sites.
   http://immuno.bme.nwu.edu
  The Kabat Database of Sequences of Proteins of Immunological Interest.
   http://www.biochem.ucl.ac.uk/∼martin/abs/index.html
  The Kabatman Antibodies—Structure and Sequence page.
   http://www.mrc‐cpe.cam.ac.uk/imt‐doc
  V BASE, a comprehensive directory of all human germline variable region sequences compiled from over 1000 published sequences, including those in the current releases of the Genbank and EMBL data libraries.
   http://www.mgen.uni‐heidelberg.de/SD/SDscFvSite.html
  General recombinant antibody page, with useful links to related background information.
   http://www.mgen.uni‐heidelberg.de/SD/SDscFvSite.html
  Fv structures.
   http://www.mrc‐cpe.cam.ac.uk/imt‐doc
  Vbase; human antibody sequences.
   http://www.antibodyresource.com
  U.S.A. links related to antibodies and antibody fragments.
  http://www.molsci.csiro.au and http://www.crc.sci.qut.edu.au
  Details about the authors of this unit.
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