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

Leslie A. Kerrigan1, James T. Kadonaga1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.10
DOI:  10.1002/0471142727.mb1210s24
Online Posting Date:  May, 2001
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Affinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence‐specific DNA‐binding properties. 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. The first basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An provides a method to couple DNA to commercially available CNBr‐activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second basic protocol outlines the affinity chromatography procedure. A second support protocol describes determination of 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 ( protocol 3support protocol or commercial HPLC‐purified)
  • TE buffer, pH 7.8 ( appendix 22)
  • recipe10× T4 polynucleotide kinase buffer
  • 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; unit 3.10)
  • 10 M ammonium acetate ( appendix 22)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate ( appendix 22)
  • 100% and 75% ethanol
  • recipe10× linker/kinase buffer
  • 6000 U/ml T4 DNA ligase (measured in Weiss units; New England Biolabs; unit 3.14)
  • Buffered phenol (unit 2.1)
  • Isopropanol (2‐propanol)
  • Sepharose CL‐2B (Pharmacia Biotech)
  • Cyanogen bromide (CNBr; Aldrich)
  • N,N‐dimethylformamide
  • 5 N NaOH ( appendix 22)
  • recipe10 mM and 1 M potassium phosphate buffer, pH 8.0
  • 1 M ethanolamine hydrochloride, pH 8.0
  • NaOH, solid
  • Glycine
  • 1 M KCl ( appendix 22)
  • recipeColumn storage buffer
  • 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
  • Additional reagents and equipment for agarose gel electrophoresis of DNA (unit 2.5) and DNA purification (unit 2.1)

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

  Additional Materials
  • 1 mM HCl ( appendix 22), prepared fresh before use
  • CNBr‐activated Sepharose 4B (Pharmacia Biotech)

Support Protocol 1: Purification of Oligonucleotides by Preparative Gel Electrophoresis

  Additional Materials
  • 40% (w/v) 19:1 acrylamide/bisacrylamide
  • 10× TBE buffer ( appendix 22)
  • Urea
  • 10% (w/v) ammonium persulfate (unit 10.3)
  • recipeFormamide loading buffer
  • sec‐butanol (2‐butanol)
  • Diethyl ether
  • 1 M MgCl 2 ( appendix 22)
  • Saran wrap or other UV‐transparent plastic wrap
  • Intensifying screen (e.g., Lightning Plus, DuPont NEN)
  • Hand‐held short‐wavelength UV light source
  • Silanized glass wool ( appendix 3B)
  • Dry ice/ethanol bath (−78°C)
  • Additional reagents and equipment for purification of oligonucleotides by denaturing polyacrylamide gel electrophoresis (unit 2.12), agarose gel electrophoresis of DNA and gel photography (unit 2.5), and butanol concentration and ether extraction of DNA (unit 2.1)

Basic Protocol 2: DNA Affinity Chromatography

  • Prepared DNA affinity resin (first protocol 1basic protocol or protocol 2alternate protocol)
  • recipeBuffer Z or other column buffer (e.g., recipebuffers Ze or recipeTM) 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 ( protocol 5support protocol)
  • recipeColumn regeneration buffer
  • recipeColumn storage buffer
  • Disposable chromatography column (Poly‐Prep, Bio‐Rad)
  • Sorvall SS‐34 rotor or equivalent
  • Liquid nitrogen
  • Narrow glass rod, silanized ( appendix 3A)
  • Additional reagents and equipment for DNA‐binding assays (units 12.2 12.4), SDS‐PAGE (unit 10.2), and silver staining (unit 10.6)
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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.
   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.
   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.
   Ueda, K. and Hayaishi, O. 1985. ADP‐ribosylation. Annu. Rev. Biochem. 54:73‐100.
   Wu, C., Wilson, S., Walker, B., Dawid, I., Paisley, T., Zimarino, V., and Ueda, H. 1987. Purification and properties of Drosophila heat shock activator protein. Science (Wash. DC) 238:1247‐1253.
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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