Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems

Lewis A. Chodosh1, Stephen Buratowski2

1 University of Pennsylvania, Philadelphia, 2 Harvard Medical School, Boston
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.7
DOI:  10.1002/0471140864.ps0907s12
Online Posting Date:  May, 2001
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Abstract

This unit presents purification protocols that exploit the tight and essentially irreversible complex that biotin forms with streptavidin. A DNA fragment containing a high‐affinity binding site for the protein of interest is prepared and a molecule of biotinylated nucleotide is incorporated into one of the ends of the DNA fragment. The protein of interest is allowed to bind to the high‐affinity recognition site present in the biotinylated fragment. The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently removed by adsorption onto a biotin‐containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin‐containing resin. Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA‐protein complex. Finally, the protein of interest is eluted from the resin with a high‐salt buffer. Both batch and column formats are presented, as is a protocol for the use of streptavidin‐agarose. A support protocol describes a mobility shift assay for detecting sequence‐specific DNA‐binding proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Purification using Batch Method
  • Alternate Protocol 1: Purification using a Microcolumn
  • Alternate Protocol 2: Purification using Streptavidin‐Agarose
  • Support Protocol 1: Mobility‐Shift Assay
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Purification using Batch Method

  Materials
  • Plasmid DNA with binding site for the protein of interest
  • Appropriate restriction endonucleases
  • Biotin‐11‐dUTP (Life Technologies)
  • Labeled and unlabeled dNTPs
  • Klenow fragment of E. coli DNA polymerase I
  • TE buffer ( appendix 2E)
  • Biotin‐cellulose (Pierce)
  • recipeBiotin‐cellulose binding buffer (see recipe)
  • BSA
  • Bulk carrier DNA [e.g., poly(dI‐dC)·poly(dI‐dC), salmon sperm DNA, or E. coli DNA]
  • recipeBiotin‐cellulose elution buffer (see recipe)
  • Protein solution
  • Streptavidin (Celltech; may be stored as 5 mg/ml stock for at least 2 months)
  • DEAE membrane (Schleicher & Schuell NA45)
  • 0.025‐µm filter discs (Millipore VS)
  • Additional reagents and equipment for mobility‐shift DNA binding assay (see protocol 4)

Alternate Protocol 1: Purification using a Microcolumn

  • Silanized glass wool
  • 1.0‐ml pipet tip
  • Ring stand

Alternate Protocol 2: Purification using Streptavidin‐Agarose

  • Streptavidin‐agarose

Support Protocol 1: Mobility‐Shift Assay

  Materials
  • 10× electrophoresis buffer: e.g., TAE or TBE electrophoresis buffer ( appendix 2E) or Tris‐glycine electrophoresis buffer (unit 10.3)
  • 30% (w/v) ammonium persulfate, prepared fresh
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED)
  • recipeNondenaturing gel mix (see recipe)
  • Bulk carrier DNA, e.g., poly(dI‐dC)·poly(dI‐dC)
  • BSA
  • Protein preparation containing DNA‐binding protein (crude extract or purified fraction)
  • recipe10× loading buffer (see recipe)
  • Constant‐temperature water bath
  • Two‐head peristaltic pump
  • 10‐µl glass capillary pipet (optional)
  • Clay‐Adams screw‐top loader (optional)
  • Whatman 3MM filter paper (or equivalent)
  • Additional reagents and equipment for digesting DNA with restriction endonucleases, DNA labeling, agarose and nondenaturing polyacrylamide gel electrophoresis, recovery of DNA from gels, oligonucleotide synthesis, PCR, ethanol precipitation, ethidium bromide dot quantitation, and autoradiography (see Ausubel et al., , or Sambrook et al., )
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., (eds.) 1998. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Buratowski, S. and Chodosh, L. 1996. Mobility‐shift DNA‐binding assay using gel electrophoresis. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 12.2.1‐12.2.11. John Wiley & Sons, New York.
   Chodosh, L.A., Carthew, R.W., and Sharp, P.A. 1986. A single polypeptide possesses the binding and transcription activities of the adenovirus major late promoter. Mol. Cell. Biol. 6:4723‐4733.
   Grabowski, P.J. and Sharp, P.A. 1986. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science 233:1294‐1299.
   Haeuptle, M.‐T., Aubert, M.L., Kjiane, J., and Kraehenbuhl, J.‐P. 1983. Binding sites for lactogenic and somatogenic hormones from rabbit mammary gland and liver. J. Biol. Chem. 258:305‐314.
   Kadonaga, J.T. and Tjian, R. 1986. Affinity purification of sequence‐specific DNA binding proteins. Proc. Natl. Acad. Sci. U.S.A. 83:5889‐5893.
   Kasher, M.S., Pintel, D., and Ward, D.C. 1986. Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin‐biotin interactions. Mol. Cell. Biol. 6:3117‐3127.
   Rosenfeld, P.J. and Kelly, T.J. 1986. Purification of nuclear factor I by DNA recognition site affinity chromatography. J. Biol. Chem. 261:1398‐1408.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Key References
   Chodosh et al., 1986. See above.
  Describes the biotin/streptavidin‐agarose microcolumn procedure from which the second alternate protocol of this unit was drawn.
   Kasher et al., 1986. See above.
   Describes a biotin‐cellulose/streptavidin procedure similar to the basic protocol of this unit.
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