Production and Use of Trimeric Isoleucine Zipper Fusion Proteins to Study Surface Receptor Ligand Interactions

Carsten Watzl1

1 University of Heidelberg, Heidelberg
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.11
DOI:  10.1002/0471140864.ps1911s43
Online Posting Date:  March, 2006
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Abstract

Soluble recombinant forms of surface receptors are commonly used to study the interaction between cell surface receptors and their ligands. By fusing the extracellular domain of the receptor to an isoleucine zipper (ILZ) sequence, it is possible to generate recombinant receptor fusion proteins that form trimers in solution. These ILZ fusion proteins demonstrate several advantages over the commonly used Fc fusion proteins. This unit describes the production and purification of recombinant ILZ fusion proteins. A protocol is also provided for the use of ILZ fusion proteins to study cell surface receptor‐ligand interactions on intact cells by flow cytometry.

Keywords: surface receptor; soluble receptor fusion protein; recombinant protein; receptor‐ligand interaction; trimer

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Generation and Purification of ILZ Fusion Proteins
  • Basic Protocol 2: Use of ILZ Fusion Proteins to Detect Receptor‐Ligand Interactions on Living Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation and Purification of ILZ Fusion Proteins

  Materials
  • HEK 293T cells (ATCC no. CRL‐11268)
  • Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1× penicillin/streptomycin (pen/strep)
  • Expression plasmid for the ILZ fusion protein of interest
  • Expression vector for GFP (optional)
  • 2 M CaCl 2 (see recipe), sterilized using 0.22‐µm filter (store up to 2 months at room temperature)
  • 2× HBS solution (see recipe)
  • 1 M imidazole solution (see recipe)
  • 1 M NaOH
  • Ni2+‐NTA agarose beads (e.g., Qiagen)
  • Wash buffer (see recipe)
  • Elution buffer (see recipe)
  • 5× reducing SDS sample buffer (see recipe)
  • SDS polyacrylamide gel (e.g., 10% Nu‐PAGE Bis‐Tris gel, Invitrogen; also see unit 10.1)
  • Coomassie staining solution (e.g., SimplyBlue stain, Invitrogen; also see unit 10.5)
  • 1× phosphate‐buffered saline, pH 7.4 (PBS; see recipe for 10×)
  • BCA protein assay kit (e.g., Pierce; also see unit 3.4)
  • 175‐cm2 tissue culture flasks
  • 15‐ and 50‐ml conical centrifuge tubes
  • End‐over‐end rotator
  • Poly‐Prep disposable chromatography columns (Bio‐Rad)
  • Boiling water bath
  • UV spectrophotometer
  • Dialysis tubing, MWCO 8,000 to 10,000 (also see appendix 3B)
  • Disposable ultrafiltration device, MWCO 10,000 (e.g., Vivaspin 6‐ml concentrator, Vivascience; http://www.vivascience.com/)
  • Additional reagents and equipment for mammalian cell culture ( appendix 3C), SDS‐PAGE (unit 10.1), staining of protein gels (unit 10.5), dialysis ( appendix 3B), and protein assay (unit 3.4)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Use of ILZ Fusion Proteins to Detect Receptor‐Ligand Interactions on Living Cells

  Materials
  • Cells of interest
  • FACS buffer (see recipe)
  • Purified ILZ fusion protein of interest ( protocol 1, step ) or HEK 293T cell supernatant containing the ILZ fusion protein of interest ( protocol 1, step or )
  • FACS buffer (see recipe), ice cold
  • 5 µg/ml anti‐ILZ mouse monoclonal antibody (e.g., anti‐ILZ‐11; Stark et al., ); prepare in ice‐cold FACS buffer just before use
  • Phycoerythrin (R‐PE)–labeled goat anti–mouse IgG antibody (Jackson ImmunoResearch); dilute 1:200 in ice‐cold FACS buffer just before use
  • 2% (v/v) formaldehyde in FACS buffer (optional)
  • 96‐well V‐bottom microtiter plate
  • Refrigerated centrifuge with adapter for 96‐well plates
  • Flow cytometer (e.g., Becton Dickinson FACScan)
NOTE: All solutions should be at 4°C prior to use and all incubations must be done on ice.
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Figures

Videos

Literature Cited

Literature Cited
   Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer‐Williams, M.G., Bell, J.I., McMichael, A.J., and Davis, M.M. 1996. Phenotypic analysis of antigen‐specific T lymphocytes. Science 274:94‐96.
   Brown, M.H., Boles, K., van der Merwe, P.A., Ku mar, V., Mathew, P.A., and Barclay, A.N. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J. Exp. Med. 188:2083‐2090.
   Flaig, R.M., Stark, S., and Watzl, C. 2004. Cutting edge: NTB‐A activates NK cells via homophilic interaction. J. Immunol. 172:6524‐6527.
   Harbury, P.B., Kim, P.S., and Alber, T. 1994. Crystal structure of an isoleucine‐zipper trimer. Nature 371:80‐83.
   Harbury, P.B., Zhang, T., Kim, P.S., and Alber, T. 1993. A switch between two‐, three‐, and four‐stranded coiled coils in GCN4 leucine zipper mutants. Science 262:1401‐1407.
   Landschulz, W.H., Johnson, P.F., and McKnight, S.L. 1988. The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science 240:1759‐1764.
   Stark, S., Flaig, R.M., Sandusky, M., and Watzl, C. 2005. The use of trimeric isoleucine‐zipper fusion proteins to study surface‐receptor‐ligand interactions in natural killer cells. J. Immunol. Methods 296:149‐158.
   Walczak, H., Miller, R.E., Ariail, K., Gliniak, B., Griffith, T.S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R.G., Rauch, C.T., Schuh, J.C., and Lynch, D.H. 1999. Tumoricidal activity of tumor necrosis factor‐related apoptosis‐inducing ligand in vivo. Nat. Med. 5:157‐163.
   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.
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