Molecular Techniques and Methods

Purification of DNA-Binding Proteins
Using Biotin-Cellulose Microcolumn

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The biotin-streptavidin purification system is based on the tight and irreversible complex that biotin forms with streptavidin. The interaction between biotin and avidin is one of the strongest known non-covalent interactions. Avidin, from egg white, and streptavidin, from Streptomyces avidinii, are tetrameric proteins containing four high-affinity binding sites for the biotin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin-containing resin. The strong interaction is extremely useful for purification of DNA-binding proteins, because DNA-affinity columns with streptavidin-biotin bridges can be washed under a wide variety of conditions (i.e., 2 M KCl and 1% SDS) without removing either the streptavidin or the biotinylated DNA fragment from the matrix. Two properties of streptavidin make it more suitable than avidin for use in DNA-affinity purification. The first is that streptavidin, unlike avidin, is not a glycoprotein, and the second is that streptavidin is slightly acidic whereas avidin is basic. Therefore, streptavidin is less likely to bind nonspecifically to cellular glycoproteins and to acidically charged cell components such as nucleic acids.

In this protocol, the following steps are included.

  • A DNA fragment is prepared that contains a high-affinity binding site for the protein of interest.
  • A molecule of biotinylated nucleotide (Biotin-11-dUTP) 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 DNA fragment.
  • The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment.
  • The [Protein-Biotinylated DNA Fragment-Streptavidin] ternary complex is efficiently removed by adsorption onto a biotin-cellulose microcolumn.
  • Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin-cellulose microcolumn.
  • Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA-protein complex.
  • The protein of interest is eluted from the biotin-cellulose microcolumn with a high-salt buffer.




    MATERIALS AND SOLUTIONS

    DEAE Elution Solution (40 ml)
    1 mM Tris-HCl (pH 7.9) ------------------------ 40 ul of 1 M Tris-HCl
    1 mM EDTA (pH 8.0) -------------------------- 80 ul of 0.5 M EDTA
    1 M NaCl --------------------------------------- 8 ml of 5 M NaCl
    Distilled H2O ------------------------------------ 31.88ml
  • Filter through 0.2 um filter.


    Biotin-Cellulose Binding Buffer (100 ml)
    12% Glycerol ------------------------------------- 24 ml of 50% Glycerol
    12 mM HEPES Buffer (pH 7.9) ------------------ 1.2 ml of 1 M HEPES Buffer
    4 mM Tris-HCl (pH 7.9) ------------------------- 0.4 ml of 1 M Tris-HCl
    60 mM KCl -------------------------------------- 6 ml of 1 M KCl
    1 mM EDTA ------------------------------------- 0.2 ml of 0.5 M EDTA
    1 mM DTT --------------------------------------- 0.1 ml of 1 M DTT
    Distilled H2O ------------------------------------- 68.1 ml
  • Store at -20oC.
  • The composition of Binding Buffer-especially with respect to pH, ionic strength, and the presence or absence of MgCl2-should be determined by those conditions which optimize the binding of the protein of interest to its recognition site.


    Biotin-Cellulose Blocking Buffer (1 ml)
    Biotin-Cellulose Binding Buffer 1 ------------------ 1 ml
    0.05% BSA --------------------------------------- 50 ul of 1% BSA
    200 ug of Carrier DNA --------------------------- 10 ul of 20 mg/ml Carrier DNA
  • Poly(dI-dC)-poly(dI-dC), salmon sperm DNA, or E. coli DNA can be served as a carrier DNA.


    Biotin-Cellulose Elution Buffer (100 ml)
    12% Glycerol ------------------------------------ 24 ml of 50% Glycerol
    20 mM Tris-HCl (pH 6.8) ------------------------ 2 ml of 1 M Tris-HCl
    1 M KCl ------------------------------------------7.45 g
    5 mM MgCl2 ------------------------------------- 0.5 ml of 1 M MgCl2
    1 mM EDTA ------------------------------------- 0.2 ml of 0.5 M EDTA
    1 mM DTT --------------------------------------- 0.1 ml of 1 M DTT
    0.02% BSA -------------------------------------- 2 ml of 1% BSA
    Add distilled H2O to make a final volume of ---- 100 ml
  • Store at -20oC.
  • The composition of Elution Buffer-especially with respect to pH, ionic strength, and the presence or absence of MgCl2-should be determined by those conditions that maximize the dissociation rate of the protein from its recognition site.
  • Another carrier protein, such as insulin or hemoglobin, may be substituted for BSA.


    5 x Protein-DNA Binding Buffer (1 ml)
    60% Glycerol ------------------------------------ 600 ul of 100% Glycerol
    60 mM HEPES Buffer (pH 7.9) ----------------- 60 ul of 1 M HEPES Buffer
    20 mM Tris-HCl (pH 7.9) ----------------------- 20 ul of 1 M Tris-HCl
    300 mM KCl ------------------------------------ 300 ul of 1 M KCl
    5 mM EDTA ------------------------------------ 10 ul of 0.5 M EDTA
    5 mM DTT -------------------------------------- 5 ul of 1 M DTT
    Distilled H2O ------------------------------------ 5 ul




    PROCEDURES

    Preparation of Biotinylated-DNA Fragment

    1. Do restriction enzyme digestion of 50 ug plasmid DNA.
  • The plasmid DNA must contain a binding site for the protein of interest and should be digested with one or more restriction endonucleases.

    2. Do Klenow fragment reaction as follow:

    Final Concentration
    10 x Buffer
    1 x
    Restriction Enzyme Digested DNA
    1-50 ug
    Biotin-11-dUTP (BRL)
    20 uM
    [a-32P] dCTP (3,000-6,000 Ci/mmol)
    100 uCi/ 0.2 uM
    Cold dCTP
    20 uM
    Cold dATP
    200 uM
    Cold dGTP
    200 uM
    Klenow Fragment
    5 U
    Final Volume
    10-100 ul

    3. Incubate 20 min at room temperature.

    4. Precipitate the biotinylated DNA by adding 1/10th vol. of 3 M Sodium acetate (pH 7.0) and 2.5 vol. of ethanol.

    5. Centrifuge 15 min at 13,000 rpm and resuspend the DNA pellet in 10-20 ul TE.

    6. Add Gel Loading Buffer to the sample and electrophorese on a agarose minigel (2-4%) containing 5 ug/ml ethidium bromide, or native (nondenaturing) polyacrylamide gel (6-8%).

    7. Purify the DNA band by using any gel purification kit available.
    Alternatively, if gel purification kit is not available, purify the DNA band as follow.

  • Visualize the gel on a UV transilluminator.
  • Using a razor blade, cut a horizontal slit below the band to be recovered.
  • Wet a piece of DEAE membrane in the gel running buffer and slide it into the slit.
  • Squeeze the gel firmly against the paper to close the incision.
  • Insert an extra piece of membrane above the band to keep larger fragments from contaminating it.
  • Alternatively, cut away the region of the gel above the band.
  • Resume electrophoresis until the DNA has run onto the membrane.
  • Check on the UV transilluminator.
  • Electrophoresis longer than necessary is not deleterious because the DNA remains on the paper.
  • Rinse the membrane for a few seconds in the electrophoresis running buffer.
  • Blot momentarily on filter paper to remove excess buffer. Do not allow the membrane to dry.
  • Place the membrane in the bottom of a 1.5-ml, round-bottom screw-cap vial containing 400 ul DEAE Elution Solution. Do not crush the DEAE membrane.
  • Incubate 30 min at 68oC.
  • Remove the elution solution from the paper.
  • Place in a 1.5-ml microcentrifuge tube and spin 15 min in a fixed-angle microcentrifuge at 4oC.
  • Transfer the supernatant in a clean microcentrifuge tube, leaving 10 ul at the bottom of the tube undisturbed.
  • Add 4 ul of 1 M MgCl2 to the supernatant. Precipitate with 1.0 ml of 100% ethanol.

  • Gel purification of the biotinylated fragment removes unreacted Biotin-11-dUTP.

    8. Resuspend in 100 ul TE.

    9. Count 1 ul in a scintillation counter to determine cpm/ ul.

    10. Estimate the DNA concentration by ethidium bromide dot quantitation.
  • Efficient recovery of DNA should yield a DNA concentration of 2-10 ug/ ml.
  • Probe can be used for 4-6 weeks.

    11. Test the biotinylated probe to be certain it will efficiently bind to the protein of interest.
  • Use a standard binding assay with the biotinylated fragment as probe.


    Preparation of Biotin-Cellulose Microcolumn

    12. Place a small plug of siliconized glass wool in the bottom of a 1.0-ml blue pipet tip.
  • Firmly attach the blue pipet tip microcolumn to a ring stand.
  • Prewet the glass wool in Biotin-Cellulose Binding Buffer before insertion into the blue pipet tip to avoid trapping air bubbles which might denature proteins.

    13. Add 500 ul Biotin-Cellulose Binding Buffer to the blue pipet tip microcolumn.
  • Maintain a steady flow through the glass wool plug.
  • If the column does not flow smoothly, a pipettor can be gently inserted into the top of the microcolumn. Slightly depressing the plunger will start the column or increase the flow.

    14. Add 50-100 ul of 1:1 Biotin-Cellulose slurry (Pierce) to the microcolumn.
  • Allow the buffer to run down to the surface of the resin.

    15. Wash with 3 column-volumes of Biotin-Cellulose Binding Buffer.

    16. Wash with 3 column-volumes of Biotin-Cellulose Blocking Buffer.

    17. Wash with 3 column-volumes of Biotin-Cellulose Elution Buffer.

    18. Equilibrate the resin with 3 column-volumes of Biotin-Cellulose Binding Buffer.
  • The biotin-cellulose microcolumn can be washed very rapidly.
  • Each wash takes 2-3 min.
  • The drop size from the pipet tip is 25 ul but this will change with alterations in the ionic strength and protein concentration of the eluate.


    Binding [Protein: DNA: Streptavidin Complex] to [Biotin-Cellulose Microcolumn] and Elution

    19. Determine the molar concentration of the protein to be purified using the gel mobility shift assay.

    20. In a microcentrifuge tube, combine the following:

    5 x Protein-DNA Binding Buffer
    2-3 ul
    Biotinylated DNA fragment
    10-fold molar excess
    relative to the protein to be purified
    poly(dI-dC)-poly(dI-dC)
    0.5-2 ug
    BSA
    3-5 ug
    Protein from a crude cell extract
    5-20 ug
    Final volume
    10-15 ul


    21. Mix gently by tapping the bottom of the tube with finger.

    22. Incubate the binding reaction mix 15 min in a 30oC water bath.

    23. Add a 5-fold molar excess of Streptavidin relative to the biotinylated fragment.

    24. Continue the binding reaction for an additional 5 min in a 30oC water bath.

    25. Load the binding reaction mix onto the microcolumn and collect the flowthrough.
  • The column can be run as fast as 6-10 column-volumes/ hr without affecting the amount of biotinylated fragment bound by the resin.
  • If the flow rate is too slow, use a pipettor to apply pressure to the column.
  • If the flow rate is too fast, plug the lip of the microcolumn with Parafilm in between drops.

    26. Wash with 4 column-volumes of Biotin-Cellulose Binding Buffer.
  • Discard the flowthrough.

    27. Wash with 3 column-volumes of Biotin-Cellulose Elution Buffer.
  • Collect 2-drop fractions and assay.
  • Fractions as small as 5 ul may be rapidly and effectively dialyzed on 0.025 um filter discs with minimal loss of volume and activity.
  • Float a filter (shiny side up) in a petri dish on top of 20 ml dialysis buffer.
  • Allow the filter 10 min to wet.
  • Place the sample (5 to 100 ul) to be dialyzed onto the surface of the filter.
  • Surface tension will keep the sample confined in a drop unless there is a detergent in the sample.
  • After 1 hr remove the sample.




    NOTES

  • Only one end of the DNA fragment should be labeled with biotin. If both ends are labeled, the biotinylated fragment may bind to the resin in such a fashion as to sterically displace the protein bound to the DNA fragment. Thus, one of the restriction enzymes used must be chosen to permit the incorporation of Biotin-11-dUTP in place of TTP.

  • Over time, streptavidin tetramers dissociate into monomers. The integrity of the tetramer may be monitored by performing native (non-denaturing) gel electrophoresis. Since streptavidin monomers will bind to the biotinylated DNA fragment, but will be unable to bind to the biotin-cellulose resin, the monomeric form of streptavidin will inhibit purification.

  • Nonspecific resin binding can be examined by assaying the binding activity in a supernatant containing the protein, the DNA carrier poly(dI-dC)-poly(dI-dC), streptavidin, and biotin-cellulose, but lacking the biotinylated DNA fragment with the protein's binding site. Alternatively, a nonspecific biotinylated DNA fragment can be added. Interestingly, nonspecific protein binding to the resin is minimized with a lower poly(dI-dC)-poly(dI-dC) concentration than is required to generate a discrete band in the gel mobility shift assay.




    KIT INFORMATION




    KIT INFORMATION




    REFERENCES

  • Grabowski, RJ, Sharp, PA (1986) Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science 233: 1294-1299.

  • Kadonaga, IT, Tjian, R (1986) Affinity purification of sequence-specific DNA binding proteins. PNAS 83: 5889- 5893.

  • Kasher, MS, Pintel, D, Ward, DC (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, PJ, Kelly, TJ (1986) Purification of nuclear factor I by DNA recognition site affinity chromatography. J. Biol. Chem. 261: 1398- 1408.



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