Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity Chromatography

Leslie A. Kerrigan1, James T. Kadonaga2

1 Osiris Therapeutics, Baltimore, 2 University of California San Diego, La Jolla
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.6
DOI:  10.1002/0471140864.ps0906s11
Online Posting Date:  May, 2001
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The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. Preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support, is described, and an alternate protocol provides a method to couple DNA to commercially available CNBr‐activated Sepharose. A method for purification of crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin is also provided. A detailed protocol for the actual affinity chromatography procedure is described and a support protocol allows the investigator to determine the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

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Table of Contents

  • Basic Protocol 1: Preparation of DNA Affinity Resin
  • Alternate Protocol 1: Coupling the DNA to Commercially Available CNBr‐Activated Sepharose
  • Support Protocol 1: Purification of Oligonucleotides by Preparative Gel Electrophoresis
  • Basic Protocol 2: DNA Affinity Chromatography
  • Support Protocol 2: Selection and Preparation of Nonspecific Competitor DNA
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Preparation of DNA Affinity Resin

  • 440 µg each of two synthetic oligonucleotides with desired binding site (see protocol 3; or commercial HPLC‐purified)
  • TE buffer, pH 7.8 ( appendix 2E)
  • recipe10× T4 polynucleotide kinase buffer (see recipe)
  • 20 mM ATP (Na+ salt), pH 7.0
  • 150 mCi/ml [γ‐32P]ATP (6000 Ci/mmol)
  • 10 U/µl T4 polynucleotide kinase (New England Biolabs)
  • 10 M ammonium acetate ( appendix 2E)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate ( appendix 2E)
  • 100% and 75% ethanol
  • recipe10× linker/kinase buffer (see recipe)
  • 6000 U/ml T4 DNA ligase (measured in Weiss units; New England Biolabs)
  • Buffered phenol
  • Isopropanol (2‐propanol)
  • Sepharose CL‐2B (Pharmacia Biotech)
  • Cyanogen bromide (CNBr; Aldrich)
  • N,N‐dimethylformamide
  • 5 N NaOH ( appendix 2E)
  • 10 mM and 1 M potassium phosphate buffer, pH 8.0 ( appendix 2E)
  • recipe1 M ethanolamine hydrochloride, pH 8.0 (see recipe)
  • NaOH, solid
  • Glycine
  • 1 M KCl ( appendix 2E)
  • Column storage buffer (see recipe)
  • 15‐ml screw‐cap polypropylene tubes
  • Heating blocks or water baths, 15°C, 37°C, 65°C, and 88°C
  • 60‐ml coarse‐sintered glass funnel
  • Rotating wheel

Alternate Protocol 1: Coupling the DNA to Commercially Available CNBr‐Activated Sepharose

  • 1 mM HCl ( appendix 2E), prepared fresh before use
  • CNBr‐activated Sepharose 4B (Pharmacia Biotech)

Support Protocol 1: Purification of Oligonucleotides by Preparative Gel Electrophoresis

  • recipe16% polyacrylamide‐urea gel (see recipe)
  • Oligonucleotides to be purified
  • recipeFormamide loading buffer (see recipe)
  • sec‐butanol (2‐butanol)
  • Diethyl ether
  • 1 M MgCl 2 ( appendix 2E)
  • Saran wrap or other UV‐transparent plastic wrap
  • Intensifying screen (e.g., Lightning Plus, NEN Life Sciences)
  • Hand‐held short‐wavelength UV light source
  • Silanized glass wool
  • Dry ice/ethanol bath (−78°C)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (unit 10.3)

Basic Protocol 2: DNA Affinity Chromatography

  • Prepared DNA affinity resin (see protocol 1 or protocol 2)
  • recipeBuffer Z or other column buffer (e.g., recipebuffers Ze or recipeTM; see reciperecipes) made with varying KCl concentrations (buffers Z/0.1 M KCl through Z/1 M KCl)
  • Partially purified protein fraction dialyzed against recipebuffer Z/0.1 M KCl
  • Nonspecific competitor DNA (see protocol 5)
  • recipeColumn regeneration buffer (see recipe)
  • recipeColumn storage buffer (see recipe)
  • Disposable chromatography column (Poly‐Prep, Bio‐Rad)
  • Sorvall SS‐34 rotor or equivalent
  • Liquid nitrogen
  • Narrow glass rod, silanized
  • Additional reagents and equipment for DNA‐binding assays (Brenowitz, et al., ; Buratowski and Chodosh, ; Baldwin et al., ), SDS‐PAGE (unit 10.1), and silver staining (unit 10.5)
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Literature Cited

Literature Cited
   Alberts, B. and Herrick, G. 1971. DNA‐cellulose chromatography. Methods Enzymol. 21:198‐217.
   Arndt‐Jovin, D.J., Jovin, T.M., Bähr, W., Frischauf, A.‐M., and Marquardt, M. 1975. Covalent attachment of DNA to agarose: Improved synthesis and use in affinity chromatography. Eur. J. Biochem. 54:411‐418.
   Baldwin, A.S., Oettinger, M., and Struhl, K. 1996. Methylation and uracil interference assays for analysis of protein‐DNA interactions. 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. 12.3.1‐12.3.7. John Wiley & Sons, New York.
   Blanks, R. and McLaughlin, L.W. 1988. An oligodeoxynucleotide affinity column for the isolation of sequence specific DNA binding proteins. Nucl. Acids Res. 16:10283‐10299.
   Brenowitz, M., Senear, D.F., and Kingston, R.E. 1989. DNase I footprint analysis of protein‐DNA binding. 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. 12.4.1‐12.4.16. 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. 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 transcription factor. Mol. Cell. Biol. 6:4723‐4733.
   Duncan, C.H. and Cavalier, S.L. 1988. Affinity chromatography of a sequence‐specific DNA binding protein using Teflon‐linked oligonucleotides. Anal. Biochem. 169:104‐108.
   Galas, D. and Schmitz, A. 1978. DNase footprinting: A simple method for the detection of protein‐DNA binding specificity. Nucl. Acids Res. 5:3157‐3170.
   Gander, I., Foeckler, R., Rogge, L., Meisterernst, M., Schneider, R., Mertz, R., Lottspeich, F., and Winnacker, E.L. 1988. Purification methods for the sequence‐specific DNA‐binding protein nuclear factor I (NFI)—generation of protein sequence information. Biochim. Biophys. Acta 951:411‐418.
   Hager, D.A. and Burgess, R.R. 1980. Elution of proteins from sodium dodecyl sulfate polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: Results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal. Biochem. 109:76‐86.
   Hertzberg, R.P. and Dervan, P.B. 1982. Cleavage of double helical DNA by (methidiumpropyl‐EDTA)iron(II). J. Am. Chem. Soc. 104:313‐315.
   Hoey, T., Welnzierl, R.O., Gill, G., Chen, J.L., Dynlacht, B.D., and Tjian, R. 1993. Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators. Cell 72:247‐260.
   Jackson, S.P. and Tjian, R. 1989. Purification and analysis of RNA polymerase II transcription factors using wheat germ agglutinin affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 86:1781‐1785.
   Janson, J.‐C. and Rydén, L. 1989. Protein Purification. Principles, High Resolution Methods, and Applications. VCH Publishers, New York.
   Kadonaga, J.T. 1991. Purification of sequence‐specific DNA binding proteins by DNA affinity chromatography. Methods Enzymol. 208:10‐23.
   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.
   Kaufman, P.D., Doll, R.F., and Rio, D.S. 1989. Drosophila P element transposase recognizes internal P element DNA sequences. Cell 59:359‐371.
   Leblond‐Francillard, M., Dreyfus, M., and Rougeon, F. 1987. Isolation of DNA‐protein complexes based on streptavidin and biotin interaction. Eur. J. Biochem. 166:351‐355.
   Lichtsteiner, S. and Schibler, U. 1989. A glycosylated liver‐specific transcription factor stimulates transcription of the albumin gene. Cell 57:1179‐1187.
   Mimori, T., Hardin, J.A., and Steitz, J.A. 1986. Characterization of the DNA‐binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J. Biol. Chem. 261:2274‐2278.
   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.
   Slattery, E., Dignam, J.D., Matsui, T., and Roeder, R.G. 1983. Purification and analysis of a factor which suppresses nick‐induced transcription by RNA polymerase II and its identity with poly(ADP‐ribose) polymerase. J. Biol. Chem. 258:5955‐5959.
   Tullius, T.D., Dombroski, B.A., Churchill, M.E.A., and Kam, L. 1987. Hydroxyl radical footprinting: A high resolution method for mapping protein‐DNA contacts. Methods Enzymol. 155:537‐558.
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Key References
   Kadonaga, J.T. 1991. See above.
  Techniques paper, though less descriptive than this unit, containing a table that lists (with references) >50 sequence‐specific proteins that have been purified using the affinity chromatography method described herein.
   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.
  First paper to describe affinity chromatography with multimerized oligonucleotides; details purification of transcription factor Sp1.
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