Construction of Immunoglobulin Fusion Proteins

Diane Hollenbaugh1, Alejandro Aruffo1

1 Bristol‐Myers Squibb Pharmaceutical Research Institute, Seattle, Washington
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.19A
DOI:  10.1002/0471142735.im1019as48
Online Posting Date:  May, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Recombinant DNA technology has allowed the preparation of chimeric genes encoding proteins with novel properties. This unit describes the construction and subsequent testing of genes encoding immunoglobulin chimeras. The first protocol details fusion of a protein (or protein fragment) of interest onto an immunoglobulin constant region using a modified version of the expression vector pCDM8. The resulting fusion protein generally retains the functional properties of both the protein of interest and the immunoglobulin constant region; this can be demonstrated as described here.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Construction of the immunoglobulin Fusion Gene
  • Basic Protocol 2: Analysis of the Immunoglobulin Fusion Gene
  • Reagents and Solution
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of the immunoglobulin Fusion Gene

  Materials
  • DNA encoding the target gene
  • Electroporation‐competent E. coli cells (Seidman et al., ; unit 10.18, )
  • Oligonucleotide primers with 3′ ends complementary to the sequences flanking the DNA to be amplified and 5′ ends encoding appropriate restriction sites
  • Buffered phenol (unit 10.1)
  • 100% ethanol
  • TE buffer, pH 7.5 ( appendix 2A)
  • 4 M ammonium acetate
  • Expression vector (e.g., modified pCDM8; Fig. )
  • recipe2× T4 DNA ligase buffer
  • T4 DNA ligase
  • LB medium (unit 10.3) and recipeplates (reagents and solutions), both containing an antibiotic selective for the plasmid vector
  • Additional reagents and equipment for PCR (unit 10.20), restriction enzyme digestion (unit 10.8), DNA purification using low gelling/melting temperature agarose gel (unit 10.5), transformation of E. coli (Seidman et al., ), and DNA miniprep by alkaline lysis (unit 10.3)

Basic Protocol 2: Analysis of the Immunoglobulin Fusion Gene

  Materials
  • COS cells (e.g., COS‐7 cells) or other appropriate host cells for the chosen expression vector
  • Complete DMEM‐10 culture medium ( appendix 2A)
  • Complete Dulbeccos minimum essential medium supplemented with 10% NuSerum (complete DMEM‐10 NS; appendix 2A)
  • Purified DNA from expression vector containing the gene fusion of interest, suspended in 20 µl protocol 1TE buffer, pH 7.5 ( protocol 1first basic protocol)
  • Phosphate‐buffered saline (PBS; appendix 2A)DEAE‐dextran/chloroquine solution: PBS containing 10 mg/ml DEAE‐dextran + 2.5 mM chloroquine
  • 10% (v/v) dimethylsulfoxide (DMSO) in PBS
  • 0.2% (w/v) trypsin/ 0.5 mM EDTA in PBS
  • 2% (v/v) formaldehyde in PBS
  • 2% (v/v) formaldehyde/0.1% (v/v) Nonidet P‐40 (NP‐10; appendix 1A) in PBS
  • Antibody directed against the species and isotype of the immunoglobulin constant region conjugated to FITC (unit 1.3) and prepared in complete DMEM‐10 at the concentration recommended by the manufacturer
  • 1 to 2 µg/ml primary antibody (unit 2.6) specific for target protein or 1:100 dilution of ascites fluid (unit 2.6) specific for target protein, both in complete DMEM‐10Secondary antibody directed against the species and isotype of the primary antibody, conjugated to FITC (unit 5.3) and prepared in complete DMEM‐10 at the concentration recommended by the manufacturer.
  • Cysteine‐ and methionine‐free IMDM or DMEM
  • 35S‐labeled cysteine and methionine, in individual or combined preparations (e.g., ICN Biomedicals; Tran35S Label or unit 8.12)
  • Protein A–Sepharose slurry (e.g., Repligen #IPA‐300; unit 8.3)
  • 0.1% (v/v) NP‐40 in PBS
  • Protein gel loading buffer (e.g., 1× SDS sample buffer, unit 8.4)
  • 14C‐labeled protein standards
  • 100‐ and 60‐mm tissue culture plates
  • 25‐cm2 tissue culture flask
  • End‐over‐end mixer or rotary shaker
  • Immunofluorescence microscope
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 8.4), autoradiography ( appendix 3J), and large‐scale plasmid DNA prep (unit 10.3)
NOTE: All incubations are performed in a humidified 37°C, 6% CO 2 incubator unless otherwise stated.CAUTION: Volatile 35S‐containing compounds can be released during the labeling procedure (refer to Safety Precautions for Radioisotopes in appendix 1Q).
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C.B., and Seed, B. 1990. CD44 is the principal cell surface receptor for hyaluronan. Cell 61:1303‐1313.
   Deen, K.C., McDouglas, J.S., Inacker, R., Folena‐Wasserman, G., Arthos, J., Rosenberg, J., Maddon, P.J., Axel, R., and Sweet, R.W. 1988. A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature 331:82‐84.
   Eckert, R.L. 1992. DNA Sequencing by the Chemical Method. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 7.5.1‐7.5.11. Greene Publishing and Wiley‐Interscience, New York.
   Fisher, R.A., Bertonis, J.M., Meier, W., Johnson, V.A., Costopoulos, D.S., Liu, T., Tizard, R., Walker, B.D., Hirsch, M.S., Schooley, R.T., and Flavell, R.A. 1988. HIV infection is blocked by recombinant soluble CD4. Nature 331:76‐78.
   Gascoigne, N.R.J., Goodnow, C., Dudzik, K., Oi, V.T., and Davis, M.M. 1987. Secretion of a chimeric T‐cell receptor‐immunoglobulin protein. Proc. Natl. Acad. Sci. U.S.A. 84:2936‐2940.
   Gascoigne, N.R.J., Goodnow, C., Dudzik, K., Rourke, L., Oi, V.T., and Davis, M.M. 1987. Chimeric proteins produced by T cell receptor‐immunoglobulin gene fusion. UCLA (Univ. Calif. Los Angel.) Symp. Mol. Cell. Biol. New Ser. 41:617‐627.
   Hussey, R.E., Richardson, N.E., Kowalski, M., Brown, N.R., Chang, H.C., Siciliano, R.F., Dorfman, T., Walker, B., Sodroski, J., and Reinherz, E.L. 1988. A soluble CD4 protein selectively inhibits HIV replication and syncytium formation. Nature 331:78‐81.
   Rose, J.K. and Bergmann, J.E. 1983. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell 34:513‐524.
   Rutschmann, R. and Karjalainen, K. 1991. Mouse LFA‐3 studied with chimeric soluble CD2 shows preferential expression on lymphoid cells. Eur. J. Immunol. 21:1379‐1384.
   Seed, B. 1987. An LFA‐3 cDNA encodes a phospholipid‐linked membrane protein homologous to its receptor CD2. Nature 329:840‐842.
   Seidman, C.E., Struhl, K., and Sheen, J. 1989. Introduction of plasmid DNA into cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.8.1‐1.8.8. Greene Publishing and Wiley‐Interscience, New York.
   Slatko, B., Albright, L.M., and Tabor, S. 1991. DNA Sequencing by the Dideoxy Method. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 7.4.1‐7.4.27. Greene Publishing and Wiley‐Interscience, New York.
   Smith, D.H., Byrn, R.A., Marsters, S.A., Gregory, T., Groopman, J.E., and Capon, D.J. 1987. Blocking of HIV‐1 infectivity by a soluble secreted form of the CD4 antigen. Science 238:1704‐1707.
   Traunecker, A., Luke, W., and Karjalainen, K. 1988. Soluble CD4 molecules neutralize human immunodeficiency virus type I. Nature 331:84‐86.
   Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. 1990. Recognition by ELAM‐1 of the Sialyl‐LeX determinant on myeloid and tumor cells. Science 250:1132‐1135.
   Watson, S.R., Imai, Y., Fennie, C., Geoffrey, J., Singer, M., Rossen, S.D., and Lasky, L. 1991. The complement binding‐like domains of the murine homing receptor facilitate lectin activity. J. Exp. Med. 115:235‐243.
   Zettlmeissl, G., Gregersen, J.P., Duport, J.M., Mehdi, S., Reiner, G., and Seed, B. 1990. Expression and characterization of human CD4:immunoglobulin fusion proteins. DNA Cell Biol. 9:347‐353.
Key Reference
   Gascoigne, et al., 1987a. See above.
  The first example of the construction and use of an immunoglobulin fusion protein of the type described herein.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library