1992
Nucleic Acids Research, Vol. 20, No. 7 1815
Oxford University Press
A simple and efficient method for the purification of specific DNA binding proteins Susan E.Rutberg and Zeev Ronai* Molecular Carcinogenesis Program, Naylor Dana Institute for Disease Prevention, I Dana Road, Valhalla, NY 10595, USA Submitted December 26, 1991 In an attempt to purify large amounts of a protein that specifically binds to a newly identified UV-responsive element (URE; 1) we have developed a simple method that enables efficient coupling of specific target DNA sequences to a support matrix. Although several procedures have been reported (2-4), the efficiency of coupling the target DNA sequence to the support matrix is routinely low or the procedures are highly specialized and labor intensive to perform. In the newly developed procedure, the DNA sequence of interest is initially tailed with poly(A) nucleotides and subsequently coupled to oligo-dT cellulose. The affinity columns prepared in this way are quick and simple to construct and the attachment of the DNA to the column occurs at the ends of the oligomer rendering the entire target sequence available for protein binding. In developing this procedure, a double stranded URE sequence (TGACAACA) which has a two bp overhang at the 5' ends (30 ,ug) was tailed using 2 units of terminal transferase (Promega) per jig of DNA in the presence of 0.2 mM dATP at 37°C for 1 hour. The enzyme was deactivated (65°C for 10'), and the reaction mixture was passed through a G-25 (Pharmacia) column prior to precipitation with ethanol. The DNA pellet was resuspended in coupling buffer consisting of 10 mM Tris pH 8.0, 1 mM EDTA, and 1 M NaCl. 100 mg of oligo dT cellulose (Boehringer Mannheim) was hydrated in the same buffer. The resulting mixture was transferred to a Poly Prep Chromatography Column (Bio Rad) and washed with 15 ml coupling buffer. The URE-DNA was applied to the column with 6-8 passes of solution through the matrix. Studies in which we have incorporated 35S-labeled dATP during tailing of the DNA indicate that the majority of coupling occurs during the first 3 passes and coupling efficiency of 98% are routinely achieved. The column is subsequently washed with coupling buffer before proceeding. Nuclear proteins were prepared from UV-treated rat fibroblast cells through a procedure which leaves the proteins in buffer consisting of 20 mM HEPES pH 7.9, 100 mM KCI, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT and 20% glycerol (5). We have found it necessary to modify the composition of this buffer by eliminating the DTT and reducing the glycerol to less than 5 % in order to optimize protein binding and minimize loss of DNA from the affinity column. Subsequent to equilibration with 15 ml of A50 buffer (50 mM Tris pH 8.0, 1 mM EDTA, 50 mM (NH4)2SO4 and 3% glycerol), 1-30 mg of nuclear proteins are passed multiple times through the column for 3 hours at room temperature or alternatively, overnight at 4°C using a peristaltic pump set at low speed. The column is then washed 3 times with A-50 buffer (10 ml each) and the proteins are eluted
with 2 aliquots of 0.5 ml A-600 buffer (50 mM Tris pH 8.0, 1 mM EDTA, 600 mM (NH4)2SO4, 10% glycerol and 0.5 mM DTT). At this point, the column can be re-equilibrated with A-50 buffer and re-used multiple times or the DNA can be eluted from the column with water and re-used for various other purposes. Using this procedure we have obtained a yield of approximately 5 Ag of purified 40 Kd protein from 20 mg of nuclear extract. The profile of proteins before purification, retention of DNA on the oligo dT column and activity of the purified proteins in electrophoretic mobility shift assay (EMSA) are shown in Figure 1.
ACKNOWLEDGEMENTS We wish to thank Yang Ming Yang and David Wivagg for their contributions of nuclear proteins in this study. This research was supported by grants from the A.C.S., N.C.I. and N.I.E.H.S.
REFERENCES 1. 2. 3. 4. 5.
Levens,D. and Howley,P.M. (1985) Mol. Cell. Biol. 5, 2307-2315. Arcangioli,B. et al. (1989) Eur. J. Biochem. 179, 359-363. Kawaguchi,H. et al. (1989) Nucleic Acids Res. 17, 6229-6240. Ronai,Z. and Weinstein,I.B. (1990) Cancer Res. 50, 5374-5381. Dignam,J.D. et al. (1983) Nucleic Acids Res. 11, 1475-1489.
11.~~~~~~~
i
I..
1 2 3 4
e 78
.. .1.1-
.. Z-
X...
Figure 1. (I) Profile of proteins prior to (A) and after (B) purification separated by 10% SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue, (II) retention of DNA on the oligo dT affinity column as determined through monitoring the amount of 35S-labeled tailed DNA that was washed off the column at the indicated stages and (Il) activity of proteins purified through this procedure as determined through EMSA using 32P-labeled URE target DNA reacted with approximately 100, 200, 400 and 800 pg (lanes 1-4) of purified protein yielding approximately 75-fold increase in binding activity over that achieved with 7.2 and 14.4 jg of nuclear extract (shown in lanes 5 and 6, respectively) as determined through quantitation using a radio-imaging blot analyzer (AMBIS). Shown in lane 8 is the migration of probe alone.
Nucleic Acids Research, Vol. 20, No. 7 1815
Oxford University Press
A simple and efficient method for the purification of specific DNA binding proteins Susan E.Rutberg and Zeev Ronai* Molecular Carcinogenesis Program, Naylor Dana Institute for Disease Prevention, I Dana Road, Valhalla, NY 10595, USA Submitted December 26, 1991 In an attempt to purify large amounts of a protein that specifically binds to a newly identified UV-responsive element (URE; 1) we have developed a simple method that enables efficient coupling of specific target DNA sequences to a support matrix. Although several procedures have been reported (2-4), the efficiency of coupling the target DNA sequence to the support matrix is routinely low or the procedures are highly specialized and labor intensive to perform. In the newly developed procedure, the DNA sequence of interest is initially tailed with poly(A) nucleotides and subsequently coupled to oligo-dT cellulose. The affinity columns prepared in this way are quick and simple to construct and the attachment of the DNA to the column occurs at the ends of the oligomer rendering the entire target sequence available for protein binding. In developing this procedure, a double stranded URE sequence (TGACAACA) which has a two bp overhang at the 5' ends (30 ,ug) was tailed using 2 units of terminal transferase (Promega) per jig of DNA in the presence of 0.2 mM dATP at 37°C for 1 hour. The enzyme was deactivated (65°C for 10'), and the reaction mixture was passed through a G-25 (Pharmacia) column prior to precipitation with ethanol. The DNA pellet was resuspended in coupling buffer consisting of 10 mM Tris pH 8.0, 1 mM EDTA, and 1 M NaCl. 100 mg of oligo dT cellulose (Boehringer Mannheim) was hydrated in the same buffer. The resulting mixture was transferred to a Poly Prep Chromatography Column (Bio Rad) and washed with 15 ml coupling buffer. The URE-DNA was applied to the column with 6-8 passes of solution through the matrix. Studies in which we have incorporated 35S-labeled dATP during tailing of the DNA indicate that the majority of coupling occurs during the first 3 passes and coupling efficiency of 98% are routinely achieved. The column is subsequently washed with coupling buffer before proceeding. Nuclear proteins were prepared from UV-treated rat fibroblast cells through a procedure which leaves the proteins in buffer consisting of 20 mM HEPES pH 7.9, 100 mM KCI, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT and 20% glycerol (5). We have found it necessary to modify the composition of this buffer by eliminating the DTT and reducing the glycerol to less than 5 % in order to optimize protein binding and minimize loss of DNA from the affinity column. Subsequent to equilibration with 15 ml of A50 buffer (50 mM Tris pH 8.0, 1 mM EDTA, 50 mM (NH4)2SO4 and 3% glycerol), 1-30 mg of nuclear proteins are passed multiple times through the column for 3 hours at room temperature or alternatively, overnight at 4°C using a peristaltic pump set at low speed. The column is then washed 3 times with A-50 buffer (10 ml each) and the proteins are eluted
with 2 aliquots of 0.5 ml A-600 buffer (50 mM Tris pH 8.0, 1 mM EDTA, 600 mM (NH4)2SO4, 10% glycerol and 0.5 mM DTT). At this point, the column can be re-equilibrated with A-50 buffer and re-used multiple times or the DNA can be eluted from the column with water and re-used for various other purposes. Using this procedure we have obtained a yield of approximately 5 Ag of purified 40 Kd protein from 20 mg of nuclear extract. The profile of proteins before purification, retention of DNA on the oligo dT column and activity of the purified proteins in electrophoretic mobility shift assay (EMSA) are shown in Figure 1.
ACKNOWLEDGEMENTS We wish to thank Yang Ming Yang and David Wivagg for their contributions of nuclear proteins in this study. This research was supported by grants from the A.C.S., N.C.I. and N.I.E.H.S.
REFERENCES 1. 2. 3. 4. 5.
Levens,D. and Howley,P.M. (1985) Mol. Cell. Biol. 5, 2307-2315. Arcangioli,B. et al. (1989) Eur. J. Biochem. 179, 359-363. Kawaguchi,H. et al. (1989) Nucleic Acids Res. 17, 6229-6240. Ronai,Z. and Weinstein,I.B. (1990) Cancer Res. 50, 5374-5381. Dignam,J.D. et al. (1983) Nucleic Acids Res. 11, 1475-1489.
11.~~~~~~~
i
I..
1 2 3 4
e 78
.. .1.1-
.. Z-
X...
Figure 1. (I) Profile of proteins prior to (A) and after (B) purification separated by 10% SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue, (II) retention of DNA on the oligo dT affinity column as determined through monitoring the amount of 35S-labeled tailed DNA that was washed off the column at the indicated stages and (Il) activity of proteins purified through this procedure as determined through EMSA using 32P-labeled URE target DNA reacted with approximately 100, 200, 400 and 800 pg (lanes 1-4) of purified protein yielding approximately 75-fold increase in binding activity over that achieved with 7.2 and 14.4 jg of nuclear extract (shown in lanes 5 and 6, respectively) as determined through quantitation using a radio-imaging blot analyzer (AMBIS). Shown in lane 8 is the migration of probe alone.
