Use of Monoclonal Antibodies for Expression Cloning

Diane Hollenbaugh1, Alejandro Aruffo1, Bryan Jones1, Peter Linsley1

1 Bristol‐Myers Squibb, Seattle, Washington
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.11
DOI:  10.1002/0471142727.mb0611s62
Online Posting Date:  May, 2003
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Abstract

This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell‐surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell‐surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double‐stranded cDNA.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of cDNA Clones Encoding Cell‐Surface Antigens
  • Support Protocol 1: Preparation of Antibody‐Coated Plates
  • Basic Protocol 2: Isolation of cDNA Clones Encoding Intracellular Antigens
  • Support Protocol 2: Preparation of Polyvinylidene‐Wrapped Plates
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of cDNA Clones Encoding Cell‐Surface Antigens

  Materials
  • Complete Dulbeccos minimum essential medium containing 10% (v/v) NuSerum or 10% (v/v) calf serum (complete DMEM‐10 NS or complete DMEM‐10 CS; appendix 3F)
  • 100‐mm tissue culture plates seeded with COS cells (∼50% confluent)
  • cDNA library plasmid expression vector DNA containing >106 of cDNA clones (unit 5.8; see background information), CsCl‐purified (units 1.7 & 9.1)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • DEAE‐dextran/chloroquine solution: PBS containing 10 mg/ml DEAE‐dextran (Sigma) and 2.5 mM chloroquine (Sigma)
  • 10% (v/v) DMSO in PBS
  • Trypsin/EDTA solution: PBS containing 0.5 mg/ml trypsin + 0.2 mg/ml EDTA
  • 0.5 mM EDTA/0.02% (v/v) azide in PBS
  • 0.5 mM EDTA/0.02% (v/v) azide/5% (v/v) calf serum in PBS
  • 1 µg/ml purified monoclonal antibody (MAb) or 1:100 dilution of ascites fluid (unit 11.1)
  • 0.5 mM EDTA/0.02% (v/v) azide/2% (w/v) Ficoll
  • 60‐mm antibody‐coated plates (first protocol 2support protocol)
  • 5% (v/v) calf serum in PBS
  • 0.6% (w/v) SDS/10 mM EDTA
  • 5 M NaCl ( appendix 22)
  • Phenol (extracted twice with 1 M Tris⋅Cl, pH 7.5)
  • 2 µg/µl linear polyacrylamide
  • TE buffer, pH 7.5 ( appendix 22)
  • Electroporation‐competent E. coli cells (unit 1.8)
  • LB medium (unit 1.1)
  • 100 mg/ml spectinomycin or 35 mg/ml chloramphenicol in ethanol
  • 20% (w/v) sucrose/50 mM Tris⋅Cl, pH 8.0, ice cold
  • 5 mg/ml lysozyme (Sigma #L6876), freshly prepared in 250 mM Tris⋅Cl, pH 8.0
  • 250 mM EDTA, ice cold ( appendix 22)
  • 50 mM Tris⋅Cl, pH 8.0 ( appendix 22)
  • 10% (w/v) sucrose/10 mM MgCl 2 in DMEM (GIBCO/BRL #320‐1960AJ) without serum, filter sterilized
  • 60‐mm tissue culture plates seeded with COS cells (∼50% confluent)
  • 50% (w/w) PEG 1000 or 1450 in DMEM (no serum), adjusted to pH 7 with 7.5% (w/v) sodium bicarbonate (Baker or Kodak)
  • DMEM without serum
  • Complete DMEM‐10 CS ( appendix 3F) containing 15 µg/ml gentamycin sulfate
  • Nylon mesh, 100‐µm pore size (Tetco)
  • Sorvall GSA rotor or equivalent
  • Swinging‐bucket centrifuge (e.g., Sorvall RT‐6000B)
  • Additional reagents and equipment for transformation of E. coli by electroporation (unit 1.8), phenol extraction and ethanol precipitation (unit 2.1), alkaline lysis miniprep (unit 1.7), and immunofluorescence (unit 14.6)

Support Protocol 1: Preparation of Antibody‐Coated Plates

  Additional Materials
  • Anti‐mouse affinity‐purified antibody, sheep or goat (e.g., Cappel)
  • 50 mM Tris⋅Cl, pH 9.5
  • 0.15 M NaCl
  • 1 mg/ml BSA in PBS ( appendix 22)
  • 60‐mm bacteriological plates (e.g., Falcon #1007) or 100‐mm plates (e.g., Fisher #8757‐12)

Basic Protocol 2: Isolation of cDNA Clones Encoding Intracellular Antigens

  Materials
  • 100‐mm tissue culture plates seeded with COS cells
  • cDNA library: plasmid DNA containing >106 cDNA clones (unit 5.8; see background information), CsCl purified (units 1.7 & 9.11)
  • Trypsin/EDTA solution: PBS containing 0.5 mg/ml trypsin + 0.2 mg/ml EDTA
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Methanol
  • 1% (w/v) nonfat dry milk in PBS with and without monoclonal antibody (MAb)
  • 1% (w/v) nonfat dry milk in PBS containing 0.25 µCi/ml of 125I‐labeled protein A
  • 0.6% (w/v) SDS/10 mM EDTA buffer
  • LB medium (unit 1.1)
  • Polyvinylidene‐wrapped plates (second protocol 4support protocol)
  • X‐ray film
  • Polyvinylidene wrap (e.g., Saran Wrap)
  • Rubber cement
  • Luminescent stickers
  • Additional reagents and equipment for alkaline lysis miniprep (unit 1.7) and autoradiography ( appendix 3A)

Support Protocol 2: Preparation of Polyvinylidene‐Wrapped Plates

  Additional Materials
  • Chloroform
  • 70% ethanol
  • 0.1 mg/ml poly‐L‐lysine HCl (Sigma) in 50 mM Tris⋅Cl, pH 8.0, freshly prepared
  • 100‐mm or 60‐mm tissue culture plates
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Figures

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Literature Cited

Literature Cited
   Aruffo, A. and Seed, B. 1987. Molecular cloning of a CD28 cDNA by a high‐efficiency COS cell expression system. Proc. Natl. Acad. Sci. U.S.A.. 84:8753‐8577.
   Dailey, L. and Basilico, C. 1985. Sequence in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression. J. Virol. 54:739‐749.
   Gearing, D.P., King, J.A., Gough, N.M. and Nicola, N.A. 1989. Expression cloning of a receptor for human granulocyte‐macrophage colony‐stimulating factor. EMBO J. 8:3667‐3676.
   Gluzman, Y. 1981. SV40‐Transformed simian cells support the replication of early SV40 mutants. Cell 23:175‐182.
   Goelz, S.E., Hession, C., Goof, D., Griffiths, B., Tizard, R., Newman, B., Chi‐Rosso, G., and Lobb, R. 1990. ELFT: A gene that directs the expression of an ELAM‐1 ligand. Cell 63:1349‐1356.
   Goldfarb, M., Schimizu, K., Perucho, M. and Wigler, M. 1982. Isolation and preliminary characterization of a human transforming gene from T24 bladder carcinoma cells. Nature (Lond.) 296:404‐409.
   Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365‐369.
   Holmes, K. and Fowlkes, B.J. 1991. Preparation of cells and reagents for flow cytometry. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober eds.) pp. 5.3.1‐5.3.11. Greene Publishing and John Wiley & Sons, New York.
   Kaufman, R.J. 1990. Overview of vectors used for expression in mammalian cells. Methods Enzymol. 185:487‐511.
   Kavathas, P. and Herzenberg, L.A. 1983. Stable transformation of mouse L cells for human membrane T‐cell differentiation antigens, HLA and 2‐microglobulin: Selection by fluorescence‐activated cell sorting. Proc. Natl. Acad. Sci. U.S.A. 80:524‐528.
   Kavathas, P., Sukhatme, V.P., Herzenberg, L.A., and Parnes, J.R. 1984. Isolation of the gene encoding the human T‐lymphocyte differentiation antigen Leu‐2(T8) by gene transfer and cDNA subtraction. Proc. Natl. Acad. Sci. U.S.A. 81:7688‐7692.
   Lee, F., Yokota, T., Otsuka, T., Gemmell, L., Larson, N., Luh, J., Arai, K.‐I., and Rennick, D. 1985. Isolation of cDNA for a human granulocyte‐macrophage colony‐stimulating factor by functional expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 82:4360‐4364.
   Littman, D.R., Thomas, Y., Maddon, P.J., Chess, L., and Axel, R. 1985. The isolation and sequence of the gene encoding T8: A molecule defining functional classes of T lymphocytes. Cell 40:237‐246.
   Maddon, P.J. Littman, D.R., Godfrey, M., Maddon, D.E., Chess, L., and Axel, R. 1985. The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: A new member of the immunoglobulin gene family. Cell 42:93‐104.
   McCutchan, J.H. and Pagano, J.S. 1968. Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl‐dextran. J. Natl. Cancer Inst. 40:351‐357.
   McMahan, C.J., Slack, J.L., Mosley, B., Cosman, D., Lupton, S.D., Brunton, L.L., Grubin, C.E., Wignall, J.M., Jenkins, N.A., Brannan, C.I., Copeland, N.G., Huebner, L., Croce, C.M., Cannizzarro, L.A., Benjamin, D., Dower, S.K., Spriggs, M.K., and Sims, J.E. 1991. A novel IL‐1 receptor, cloned from B cell by mammalian expression, is expressed in many cell types. EMBO J. 10:2821‐2832.
   Metzelaar, M.J., Wijngaard, P.L.J., Peters, P.J., Sixma, J.J., Nieuwenhuis, H.K. and Clevers, H.C. 1991. CD63 antigen. J. Biol. Chem. 266:3239‐3245.
   Muller, W.J., Naujokas, M.A., and Hassell, J.A. 1984. Isolation of large T antigen‐producing mouse cell lines capable of supporting replication of polyomavirus‐plasmid recombinants. Mol. Cell. Biol. 4:2406‐2412.
   Munro, S. and Maniatis, T. 1989. Expression cloning of the murine interferon γ receptor cDNA. Proc. Natl. Acad. Sci. U.S.A. 86:9248‐9252.
   Sandri‐Goldin, R.M., Goldin, A.L., Glorioso, J.C., and Levine, M. 1981. High‐frequency transfer of cloned herpes simplex virus type I sequences to mammalian cells by protoplast fusion. Mol. Cell. Biol. 1:743‐752.
   Seed, B. 1987. An LFA‐3 cDNA encodes a phospholipid‐linked membrane protein homologous to its receptor CD2. Nature (Lond.) 329:840‐842.
   Seed, B. and Aruffo, A. 1987. Molecular cloning of the CD2 antigen, the T‐cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:3365‐3369.
   Shih, C. and Weinberg, R.A. 1982. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 29:161‐169.
   Sims, J.E., March, C.J., Cosman, D., Widmer, M.B., MacDonald, H.R., McMahan, C.J., Grubin, C.E., Wignall, J.M., Jackson, J.L., Call, S.M., Friend, D., Alpert, A.R., Gillis, S., Urdal, D.L., and Dower, S.K. 1988. cDNA expression cloning of the IL‐1 receptor, a member of the immunoglobulin superfamily. Science 241:585‐589.
   Tsai, S.F., Martin, D.I., Zon, L.I., D'Andrea, A.D., Wong, G.G., and Orkin, S.H. 1989. Cloning of cDNA for the major DNA‐binding protein of the erythroid lineage through expression cloning. Nature (Lond.) 339:446‐451.
   Wong, G.G., Witek, J.S., Tempel, P.A., Wilkens, K.M., Leary, A.C., Luxenberg, D.P., Jones, S.S., Brown, E.L., Kay, R.M., Orr, E.C., Shoemaker, C., Golde, D.W., Kaufman, R.J., Hewick, R.M., Wang, E.A., and Clark, S.C. 1985. Human GM‐CSF: Molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 228:810‐815.
   Wysocki, L.J. and Sato, V.L. 1978. Panning for lymphocytes: A method for cell selection. Proc. Natl. Acad. Sci. U.S.A. 75:2844‐2848.
   Yamasaki, K., Taga, T., Hirata, Y., Yawata, H., KaWanishi, Y., Seed, B., Taniguchi, T., Hirano, T., and Kishimoto, T. 1988. Cloning and expression of the human interleukin‐6 (BSF‐2/ IFNβ 2) receptor. Science 241:825‐828.
Key References
   Aruffo and Seed 1987.
  Original descriptions of cDNA library construction in CDM8 and isolation of cDNA clones encoding cell‐surface antigens by expression cloning.
   Seed, Aruffo and 1987. see above.
  Contain descriptions of growth of COS cells on wrap and screening for extracellular ligands.
   Metzelaar et al., 1991.
   Munro and Maniatis 1989. See above.
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