Phage Display of Single‐Chain Antibody Constructs

Itai Benhar1, Yoram Reiter2

1 The George S. Wise Faculty of Life Sciences, Tel‐Aviv University, Ramat Aviv, Israel, 2 Faculty of Biology Technion, Haifa, Israel
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.19B
DOI:  10.1002/0471142735.im1019bs48
Online Posting Date:  May, 2002
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Abstract

Phage display is based on expressing recombinant proteins fused to a phage coat protein. The genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle. With phage display, tailor‐made proteins (fused peptides, antibodies, enzymes, DNA‐binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity and specificity of binding. This unit provides protocols for the construction of, and selection from antibody phage display libraries, together with useful approaches for the analysis of phage antibodies. This unit focuses on the construction of immune libraries where the sources for antibody genes are spleens of immunized mice and the isolation and initial characterization of single‐chain antibodies (scFvs) from such libraries.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Construction of a scFv Repertoire in a Phagemid Vector
  • Basic Protocol 2: Affinity Selection of scFv Displaying Phage on Plastic‐Immobilized Antigen
  • Alternate Protocol 1: Affinity Selection of scFv‐Displaying Phage on Biotinylated Antigen Using Streptavidin‐Coated Magnetic Beads
  • Basic Protocol 3: Identification of Antigen‐Binding scFv‐Displaying Phage in ELISA
  • Support Protocol 1: Preparation of Helper Phage Stocks
  • Support Protocol 2: Analysis of Clonal Integrity and Diversity by PCR and Fingerprinting
  • Support Protocol 3: Biotinylation of Proteins with NHS‐SS‐Biotin
  • Support Protocol 4: Biotinylation of Proteins with Biotin‐Protein Ligase
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of a scFv Repertoire in a Phagemid Vector

  Materials
  • Immune mouse spleen or 107 hybridoma cells (unit 2.5)
  • Total RNA extraction kit (RNeasy Midi Kit, Qiagen)
  • RT‐PCR kit (Ready‐To‐Go RT‐PCR beads, Amersham Pharmacia Biotech)
  • Oligonucleotide primer set (Table 10.19.1)
  • Appropriate DNA molecular weight marker
  • Agarose‐gel extraction kit (Qiaquick Gel Extraction Kit, Qiagen)
  • 10 mM Tris⋅Cl, pH 8.5 ( appendix 2A)
  • Vent DNA polymerase (e.g., NEB)
  • dNTPs (provided with RT‐PCR kit, or see unit 10.20)
  • PCR buffer (provided with RT‐PCR kit, or see unit 10.20)
  • Phagemid vector (e.g., pCANTAB5E, Amersham Pharmacia Biotech)
  • Restriction enzymes SfiI and NotI and appropriate buffers
  • Bovine serum albumin (BSA)
  • T4 DNA ligase and 10× ligation buffer
  • Electroporation‐competent E. coli TG‐1 cells (TG‐1 cells are included in the RPAS Expression Module, Amersham Pharmacia Biotech) see appendix 3N for preparation of electroporation‐competant cells
  • Electroporation‐competent TG‐1 cells (Stratagene), optional
  • recipeSOC medium (see recipe)
  • YTAG plates: recipe2× YT medium (see recipe) supplemented with 0.4% glucose (agar plates) and 100 µg/ml ampicillin
  • YTAG medium: recipe2× YT medium (see recipe), with and without 15% (v/v) glycerol, and 100 µg/ml ampicillin
  • Thermal cycler
  • Microcon‐PCR centrifugal filter devices (Millipore)
  • 1.5‐ml microcentrifuge tubes
  • Electroporation device for E. coli electroporation (e.g., Gene Pulser II, Bio‐Rad Laboratories)
  • 0.2‐cm cuvette
  • 13‐ml sterile polypropylene tubes
  • Cell scrapers or disposable inoculation loops
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4)

Basic Protocol 2: Affinity Selection of scFv Displaying Phage on Plastic‐Immobilized Antigen

  Materials
  • TG‐1 Bacterial colony grown on a minimal media plate (see protocol 1)
  • recipe2× YT medium (see recipe) with and without 100 µg/ml ampicillin and 1% (v/v) glucose
  • Helper phage (e.g., VCS‐M13, Stratagene; or M13KO7, Bio‐Rad Laboratories; see protocol 6)
  • YTAK medium: recipe2× YT medium (see recipe) supplemented with 100 µg/ml ampicillin and 50 µg/ml kanamycin
  • recipePEG/NaCl (see recipe)
  • recipePBS (see recipe) with and without 15% (v/v) glycerol
  • TG‐1 cells
  • YTAG plates: recipe2× YT medium (see recipe) supplemented 0.4% glucose (agar plates) and 100 µg/ml ampicillin
  • Required antigen in recipePBS
  • recipeCarbonate coating buffer (see recipe)
  • 2% (w/v) MPBS: recipePBS (see recipe) containing 2% skim milk powder, store at 4°C for up to 48 hr
  • PBST: recipePBS (see recipe) containing 0.05% Tween 20, store at room temperature
  • 100 mM triethylamine (Sigma‐Aldrich) in water, prepare fresh
  • 1 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • recipe2× YT medium (see recipe) supplemented with 15% glycerol
  • YTAG medium: recipe2× YT medium (see recipe) supplemented with 1% glucose (liquid medium) and 100 µg/ml ampicillin
  • 4‐ml Maxisorp immunotubes (Nunc), or high capacity protein binding polystyrene tubes from other suppliers
  • 13‐ml polypropylene culture tubes

Alternate Protocol 1: Affinity Selection of scFv‐Displaying Phage on Biotinylated Antigen Using Streptavidin‐Coated Magnetic Beads

  Materials
  • Protein antigen
  • Irrelevant antigen (e.g., BSA)
  • recipePBS (see recipe) or recipecarbonate‐coating buffer (see recipe)
  • MPBS ( recipePBS containing 2% skim milk powder) or 3% BSA‐PBS
  • PBST: recipePBS (see recipe) containing 0.05% Tween 20
  • PEG‐precipitated phage in frozen aliquots (see protocol 2)
  • HRP‐conjugated anti‐M13 antibody (Amersham Pharmacia Biotech) or rabbit anti‐M13 antibody mixed with HRP‐conjugated anti‐rabbit antibodies
  • HRP‐substrate solution (e.g., recipeTMB‐substrate solution, see recipe)
  • 1 M sulfuric acid (H 2SO 4)
  • Output plates (see protocol 2)
  • YTAG medium: recipe2× YT medium (see recipe) supplemented with 1% glucose and 100 µg/ml ampicillin
  • Glycerol
  • YTAG containing 4 × 1010 PFU/ml of helper phage
  • YTAK medium recipe2× YT medium supplemented (see recipe)
  • E. coli HB2151 cells (included in the Amersham Pharmacia Biotech RPAS Expression Module)
  • YTGN medium: recipe2× YT medium (see recipe) supplemented with 1% glucose and 100 µg/ml nalidixic acid
  • YTAGN plates (see recipe for YT medium but prepare plates with YTGN medium prepared as described above)
  • recipe2× YTAGN medium (see recipe)
  • recipe2× YT medium (see recipe) containing 9 mM IPTG
  • Anti‐myc 9E10 monoclonal antibody (when working with phagemids such as pHEN‐1 or pHEN‐2; or anti E‐tag monoclonal antibody (when working with phagemids such as pCANTAB5E)
  • Sterile 96‐well flat‐bottom ELISA assay plates from any supplier
  • Multi‐channel pipettor
  • ELISA plate reader
  • Centrifuge with plate holders

Basic Protocol 3: Identification of Antigen‐Binding scFv‐Displaying Phage in ELISA

  Materials
  • E. coli TG‐1 cells
  • Helper phage
  • YT‐top agar (see recipe for recipeYT medium)
  • YT plates (see recipe for recipeYT medium)
  • recipe2× YT medium (see recipe)
  • Kanamycin
  • TE buffer ( appendix 2A)
  • 2‐liter flasks
  • 0.45‐µm filters (Minisart NML; Sartorius)

Support Protocol 1: Preparation of Helper Phage Stocks

  • PCR primers that are specific to the phagemid used
  • Restriction enzyme BstNI (NEB) or its isoschisomer MvaI (Roche Molecular Biochemicals)

Support Protocol 2: Analysis of Clonal Integrity and Diversity by PCR and Fingerprinting

  • Protein or peptide
  • recipeSodium bicarbonate buffer (see recipe)
  • NHS‐SS‐biotin [sulfosuccinimidyl‐2‐(biotinamido) ethyl‐1,3‐dithiopropionate] (Pierce)
  • 0.1 M sodium phosphate buffer, pH 7.0 ( appendix 2A)
  • 0.1% sodium azide
  • Biotin‐BSA (Sigma‐Aldrich)
  • Peroxidase‐conjugated streptavidin (Dako)
  • DAB (3,3,diamino‐benzidine‐tetrahydrochloride; Sigma‐Aldrich)
  • Centricon 30 or Centricon 10 (Amicon)
  • Dialysis tubing

Support Protocol 3: Biotinylation of Proteins with NHS‐SS‐Biotin

  • Target protein
  • Biomix A (10× concentration: 0.5 M bicine buffer, pH 8.3; Avidity)
  • Biomix B (10× concentration: 100 mM ATP, 100 mM magnesium acetate, 500 µM D‐biotin; Avidity)
  • Biotin ligase (Avidity)
  • Centricon 10 or 30
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Figures

Videos

Literature Cited

Literature Cited
   Barabas, C.F. III, Burton, D., Scott, J.K., and Silverman, G.J. 2001. Phage Display,A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
   Benhar, I. 2001. Biotechnological applications of phage and cell display. Biotechnology Advances. 19:1‐33.
   Berdichevsky, Y., Ben‐Zeev, E., Lamed, R., and Benhar, I. 1999. Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering. J. Immunol. Methods. 228:151‐162.
   Borrebaeck, C.A.K. 1995. Antibody Engineering, Second Edition. Oxford University Press, New York.
   Clackson, T., Hoogenboom, H.R., Griffiths, A.D., and Winter, G. 1991. Making antibody fragments using phage display libraries. Nature 352:624‐628.
   Dübel, S., Breitling, F., Fuchs, P., Braunagel, M., Klewinghaus, I., and Little, M. 1993. A family of vectors for surface display and production of antibodies. Gene. 128:97‐101.
   Griffiths, A.D. and Duncan, A.R. 1998. Strategies for selection of antibodies by phage display. Curr. Opin. Biotechnol. 9:102‐108.
   Hoogenboom, H.R., de Bruine, A.P., Hufton, S.E., Hoet, R.M., Arends, J.W., and Roovers, R.C. 1998. Antibody phage display technology and its applications. Immunotechnology 4:1‐20.
   Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H.R., and Plückthun, A. 1997. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods. 201:35‐55.
   Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D., and Winter, G. 1991. By‐passing immunization. Human antibodies from V‐gene libraries displayed on phage. J. Mol. Biol 222:581‐597.
   Niv, R., Cohen, C., Denkberg, G., Segal, D., and Reiter, Y. 2001. Antibody engineering for targeted therapy of cancer. Current Pharmaceutical Biotechnology 2:19‐46.
   Pini, A., Viti, F., Santucci, A., Carnemolla, B., Zardi, L., Neri, P., and Neri, D. 1998. Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two‐dimensional gel. J. Biol. Chem. 273:21769‐21776.
   Schatz, P.J. 1993. Use of peptide libraries to map the substrate specificity of a peptide‐modifying enzyme: A 13‐residue consensus peptide specifies biotinylation in E. coli. Biotechnology 11:1138‐1143.
   Winter, G., Griffiths, A.D., Hawkins, R.E., and Hoogenboom, H.R. 1994. Making antibodies by phage display technology. Annu. Rev. Immunol. 12:433‐455.
   Yamanaka, H.I., Kirii, Y., and Ohmoto, H. 1995. An improved phage display antibody cloning system using newly designed PCR primers optimized for Pfu DNA polymerase. J. Biochem. 117:1218‐1227.
   Zahn, G., Skerra, A., and Hohne, W. 1999. Investigation of a tetracycline‐regulated phage display system. Protein Eng. 12:1031‐1034.
Key Reference
   Hoogenboom, 1998. See above.
  A thorough review of antibody phage display technology and its applications.
Internet Resources
  http://www.avidity.com
  Avidity's web site.
  http://www.immun.lth.se/TEXTER/MO/MO.html
  A description of useful applications of phage display can be found.
  http://www.mrc-cpe.cam.ac.uk/~phage
  Human synthetic phage display libraries and direction for their use can be found.
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