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Short Technical Reports

cleic Acids Res. 16:265-277. constructed several vector plasmids for expression in eukaryotic cells. Hereby,
6.Sambrook, J., E.F. Fritsch and T. Maniatis. overexpression as well as for moderate we report the use of BPV-1 E2-derived
1989. Molecular Cloning: A Laboratory Man-
ual, 2nd ed. CSH Laboratory Press, Cold
expression of single- or double-tagged pro- tags and respective monoclonal antibod-
Spring Harbor, NY. teins in either Escherichia coli or eukaryot- ies for the identification of proteins on
7.Schaefer, B.C. 1995. Revolutions in rapid am- ic cells. The new tags were fused to several Western blots, in the immunofluores-
plification of cDNA ends: new strategies for proteins, and the activity of the tagged pro- cence staining of cells and DNA band-
polymerase chain reaction cloning of full- teins was tested in different assays. The tags shift assay. In addition, we describe an
length cDNA ends. Anal. Biochem. 227:255-
273.
were shown not to interfere with the func- application of the resin-bound tagged
8.Tillett, D. and B.A. Neilan. 1999. Simple n- tion of these proteins in vivo and in vitro. In- protein in DNA binding and DNase I
butanol purification of dye terminator sequenc- teraction of the monoclonal antibodies footprinting assays.
ing reactions. BioTechniques 26:606-610. 3F12 and 1E2 with their respective epitopes
9.Tillett, D. and B.A. Neilan. 1999. Xan-
was specific and had high affinity in a vari-
thogenate nucleic acid isolation from cultured
and environmental cyanobacteria. J. Phycol. ety of conditions. We have demonstrated MATERIALS AND METHODS
(In press.) that the 3F12 antibody-epitope interaction
10.Troutt, A.B., M.G. McHeyzer-Williams, B. tolerates high salt concentrations up to 2 M. Plasmid Constructions
Pulendran and G.J.V. Nossal. 1992. Liga- This permits immunoprecipitation and
tion-anchored PCR: A simple amplification
technique with single-sided specificity. Proc. immunopurification of the tagged proteins For the construction of pBR-3F12
Natl. Acad. Sci. USA 89:9823-9825. in high-salt buffers and reduction of the and pBR-1E2, we inserted the coding
nonspecific binding of the contaminating sequence of XylS with the N-terminal-
This work was supported by grants from proteins. We also provide a protocol for ly fused influenza virus hemagglutinin
the National Health and Medical Research DNA binding and DNase I footprinting as- (HA) epitope (2) from the pET11c-
Council and the Australian Research Coun- says using the tagged, resin-bound DNA- based parent plasmid pETSN117 (4)
cil. We are grateful to M. Cairns for the kind binding proteins. The BPV-1 E2-derived between the NheI and BamHI sites of
gift of the cordecypin modified oligonu- tags can be recommended as useful tools for pBR322. The subcloned fragment con-
cleotide. Address correspondence to Dr. detection and purification of proteins. tained in addition to a ribosome bind-
Brett Neilan, School of Microbiology and ing site, an extra NdeI site and a start
Immunology, University of New South codon preceding the tag sequence. The
Wales, Sydney 2052, Australia. Internet: INTRODUCTION resultant plasmid pBRSN 117 contains
b.neilan@unsw.edu.au XbaI and BamHI sites for cloning of a
Epitope tagging is a recombinant recombinant coding sequence (4).
Received 3 May 1999; accepted 25 DNA technique by which a protein is Then, the coding sequence of the HA
October 1999. made immunoreactive to a preexisting epitope between NdeI and XbaI sites
antibody. This technique simplifies de- was replaced with double-stranded syn-
Daniel Tillett, Brendan P. tection, characterization, purification thetic oligonucleotides encoding pep-
Burns and Brett A. Neilan and in vivo localization of proteins and tides GVSSTSSDFRDR and TTGH-
University of New South Wales has become a standard method of mole- YSVRD, recognized by anti-BPV-1 E2
Sydney, Australia cular genetics (3). However, some tags monoclonal antibodies 3F12 and 1E2,
are not useful in certain applications respectively (7).
due to high background binding. In For the construction of pBR-NC, we
some cases, affinity purification and amplified xylS sequence by PCR using
immunoprecipitation of a tagged pro- the 3-end primer containing a coding
tein is problematic due to co-precipita- sequence for the peptide TSSDFRDR.
tion of contaminating proteins. The peptide recognized by 3F12 Mab
Monitoring and Purifica- Here, we describe the use of two re- was fused in frame to the C-terminus of
cently mapped bovine papillomavirus XylS and flanked by KpnI and BamHI
tion of Proteins Using type 1 (BPV-1) E2 protein epitopes as sites. The resultant PCR fragment was
Bovine Papillomavirus tags. We constructed several vector plas- cloned into pBR-1E2 generating the
mids for over-expression as well as for expression plasmid with cloning sites
E2 Epitope Tags moderate-level expression of either sin- XbaI and KpnI.
gle- or double-tagged proteins in pET-3F12 was generated by cloning
BioTechniques 28:456-462 (March 2000) Escherichia coli and eukaryotic cells. the BamHI/NdeI fragment from pBR-
The new tags were fused to functionally 3F12-xylS into the corresponding sites in
different proteins: a bacterial transcrip- pET-11c. Then, the XylS gene was re-
tional activator, XylS, that agregates and moved by cleavage with XbaI and
ABSTRACT becomes nonfunctional at high levels of BamHI and replaced with coding se-
expression, several mutants of the tumor quences for different mutant p53 proteins
We describe here the use of two newly suppressor protein p53 for overexpres- bearing XbaI and BglII sites at the ends.
mapped bovine papillomavirus type 1 sion in E. coli and rat glyceraldehyde-3- For the construction of pCG-3F12,
(BPV-1) E2 protein epitopes as tags. We phosphate dehydrogenase (GAPDH) for double-stranded synthetic oligonu-

456 BioTechniques Vol. 28, No. 3 (2000)


Table 1. Main Characteristics of Expression Vectors

cleotide encoding 3F12 epitope and in- supplemented with 100 g/mL ampi- Affinity beads were prepared by
corporating multicloning sites was in- cillin at 20C to an A600 of approximate- coupling 3F12 anti-BPV E2 monoclon-
serted between the XbaI and BglII sites ly 1.0. Cells were harvested, washed al antibody to divinylsulfon-activated
of pCG (9). Plasmid pCG-3F12- with TBS and resuspended in 1/10 vol- Toyopearl HW65 TSK-gel (TOSOH,
GAPDH contains rat glyceraldehyde-3- ume of high-salt lysis buffer containing Tokyo, Japan). The 3F12-TSK affinity
phosphate dehydrogenase (GAPDH) 100 mM Tris-HCl, pH7.5, 1.5 M NaCl beads were incubated with crude lysate
inserted between BamHI and BglII sites and 5 mM EDTA, 20% (wt/vol) glyc- at 4C for 1 h with end-over-end agita-
of pCG-3F12. Plasmid pCG-3F12- erol. Cell suspension was frozen in liq- tion. Beads were washed extensively
GAPDH-NLS contains nuclear local- uid nitrogen and stored at -70C. with high-salt lysis buffer on glass fil-
ization signal of human p53 (amino For the preparation of crude extracts, ter. These 3F12-XylS beads were
acids 305327) fused to the C-terminus cells were thawed and dithiothreitol stored at -20C in approximately 10 gel
of the GAPDH protein. (DTT; 10 mM), PMSF (100 g/mL), volumes of storage buffer containing
aprotinin (1 g/mL), and CHAPS 10 mM Tris-HCl, pH 7.5, 100 mM
Immunoaffinity Binding (0.2%) were added. Cells were incubat- KCl, 10 mM MgCl2, 80 M EDTA, 80
of 3F12-XylS ed with lysozyme (0.5 mg/mL) on ice M DTT, 50% (vol/vol) glycerol, 100
for 20 min and disrupted by sonication. g/mL PMSF and 1 g/mL aprotinin.
pBR-3F12-xylS bearing E. coli The lysate was clarified by centrifuga- These beads were used for DNA bind-
DH5 cells were grown in LB medium tion at 40 000 g at 4C for 30 min. ing and footprinting assays.

Vol. 28, No. 3 (2000) BioTechniques 457


Short Technical Reports
DNA Precipitation Assay crude lysate preparations of p53 mu- teins with N-terminally fused peptides
tants. The double-stranded p53 con oli- GVSSTSSDFRDR and TTGHYSVRD,
End-labeled restriction fragments of go (5-GAT CCG GAC ATG CCC recognized by anti-BPV E2 monoclonal
pUPM190 HpaII/HinfI digest and 50 GGG CAT GTC CGG ATC-3) was antibodies 3F12 and 1E2, respectively
L of the suspension of 3F12-XylS used as a probe. Protein-DNA com- (7). Both vectors contain XbaI and
beads were mixed with 0.3 mL of DNA plexes were separated from the un- BamHI sites for cloning of the recombi-
binding buffer containing 50 mM Tris- bound DNA on 5% PAGE (55:1). nant sequence. We also constructed the
HCl, pH 7.5, 100 mM KCl, 2 mM vector pBR-NC for the expression of
EDTA, 10% (wt/vol) sucrose, 0.1% Immunodetection of Proteins proteins with different epitope tags in
CHAPS, 700 g/mL BSA, 1 g/mL both N- and C-termini. The N-terminal
aprotinin and 1 mM meta-toluate. The For Western blot analysis, cells ex- tag was TTGHYSVRD as in pBR-1E2
mixture was incubated for 30 min at pressing the tagged proteins were lyzed and the C-terminal tag was TSSDFR-
room temperature with end-over-end in SDS sample buffer, total protein ex- DR, a shorter version of the epitope rec-
agitation. Beads were washed 3 with 1 tracts were separated on 12% SDS- ognized by 3F12 Mab. The cloning sites
mL of DNA binding buffer and incubat- PAGE and electroblotted onto nitrocel- for a coding sequence in pBR-NC are
ed with Proteinase K (50 g/mL) at lulose membrane filters. Blots were XbaI and KpnI (Table 1).
37C for 30 min in stop solution con- blocked with 1% non-fat dry milk for 1 We transformed E. coli DH5 with
taining 200 mM NaCl, 5 mM EDTA h, incubated with anti-BPV E2 1E2, plasmids expressing the tagged ver-
and 1% SDS. The retained DNA was re- 3F12 or anti-p53 pAb 240 monoclonal sions of XylS and analyzed the expres-
leased by phenol-extraction, ethanol- antibodies, followed by incubation with sion of the tagged XylS proteins by
precipitated, and identified in 5% PAGE anti-mouse IgG alkaline phosphatase- Western blotting. The 3F12 antibody
under non-denaturing conditions. conjugated secondary antibody. recognized both 3F12-XylS and NC-
For immunofluorescence staining, XylS proteins, while the 1E2 antibody
DNase I Footprinting Saos-2 cells were transfected with plas- recognized 1E2-XylS and NC-XylS
mids expressing 3F12-GAPDH-NLS or proteins as single bands on the Western
The Om-containing EcoRI/XhoI 3F12-GAPDH proteins. Cells were blot (Figure 1A, lanes 16). No cross-
fragment of pUPM190 was end-labeled grown on microscopy cover glasses and reaction with cellular proteins was ob-
at XhoI site in the lower strand with fixed 24 h after transfection with served. However, when 3F12 Mab was
Klenow fragment. The labeled probe methanol at -20C. Proteins were detect- used for the detection of the double-
was incubated with 50 L of the sus- ed with 3F12 and anti-mouse Ig anti- tagged NC-XylS protein, a much weak-
pension of 3F12-XylS beads in DNA body conjugated with FITC as a prima- er signal was detected when compared
binding buffer as for the DNA precipi- ry and secondary antibody, respectively. with the 1E2 Mab signal. (Figure 1A,
tation. The beads were washed 3 and lanes 4 and 6). This could be explained
suspended in 100 L of DNase I buffer by the use of the shorter version of the
containing 10 mM Tris-HCl, pH 7.5, RESULTS AND DISCUSSION 3F12-specific epitope in the double-
100 mM KCl, 5 mM MgCl2, 1 mM tagged protein. We studied the effect of
CaCl2, 100 M DTT, 100 M EDTA, Moderate Level Bacterial Expression these tags on the activity of XylS pro-
100 g/mL BSA and 2 g/mL sonicat- of Epitope-Tagged Proteins tein in E. coli strain CC118Pm-lacZ,
ed salmon-sperm DNA. DNase I was which carries a chromosomal copy of
added to concentrations of 0.63.0 The XylS protein, a transcriptional the XylS responsive Pm promoter fused
g/mL, and the reaction mixture was activator from the TOL plasmid pWWO to the lacZ gene (6). The tags had no ef-
incubated for 1 min at 30C. The reac- of the soil bacterium Pseudomonas puti- fect on the transcriptional activation by
tion was stopped by 200 L of stop so- da, like some other AraC/XylS family XylS (data not shown).
lution containing 600 mM NaCl, 120 transcription factors, does not tolerate
mM EDTA, 3% SDS and 150 g/mL high level of over-expression and is Study of Specific DNA Binding
dextran, and the beads were treated prone to aggregation both inside the cell by the Matrix-Attached
with Proteinase K (50 g/mL) at 37C as well as in the solution, in the course Epitope-Tagged Protein
for 30 min. DNA was extracted from of purification (4,5). We expressed XylS
the beads with phenol/chloroform, pre- and several truncated variants of the XylS is a DNA-binding protein,
cipitated with ethanol and analyzed on protein at a near to native level in E. coli which specifically binds to the Om op-
sequencing gel. DNase I cleavage of and tested their physiological activities erator sequence and activates the re-
the unbound template was carried out in vivo. For that, we constructed vectors sponsive Pm promoter. However, suit-
in the same buffer at DNase I concen- for the moderate level of expression of able conditions to study the soluble
trations of 30300 ng/mL. epitope-tagged fusion proteins. The vec- XylS in vitro have not been found, as
tors were based on pBR322, and the tet the protein tends to aggregate. To
Gel-Shift Assay promoter of this plasmid was used for demonstrate the site-specific DNA
expression of recombinant proteins. The binding of epitope-tagged XylS in vit-
Gel-shift assay was performed as vectors pBR-3F12 and pBR-1E2 were ro, we used immunobound 3F12-XylS
previously described (1). We used the constructed for the expression of pro- protein that was attached to the TSK

458 BioTechniques Vol. 28, No. 3 (2000)


beads through the N-terminal tag and single Om-containing fragment of the
3F12 antibody. Western blot analysis pUPM190 digest was retained on the
showed that high salt concentrations, beads, and binding of any other frag-
up to 2 M NaCl, do not hinder the inter- ment could not be detected.
action of 3F12 MAb with the specific Further, we analyzed the interaction
epitope (data not shown). Therefore, to of the immobilized 3F12-XylS protein
avoid aggregation of XylS and coim- with Om by DNase I footprinting. The
munoprecipitation of contaminating specific complexes of the Om-contain-
proteins, the crude lysate was prepared ing DNA fragment and TSK-bound
in a high-salt lysis buffer, containing 3F12-XylS were prepared identically
1.5 M NaCl, and 3F12-XylS was bound as for DNA precipitation and were
to the affinity resin carrying 3F12 Mab treated with DNaseI. After the cleav-
by a single-step, batchwise procedure. age, DNA was extracted from the beads
The DNA binding assay was carried and analyzed on a sequencing gel. As a
out by mixing the 3F12-XylS beads control, the unbound template was
with a mixture of end-labeled restric- cleaved at lower concentrations of
tion fragments of the Om-containing DNaseI to obtain the equal rate of
plasmid pUPM190 (4). After several cleavage. Figure 2B shows that 3F12-
washes, the bound DNA was released XylS protects a 44 bp area on the lower
and identified by gel electrophoresis, strand and four DNase I hypersensitive
using the input mixture of fragments as sites occur within the protected region.
a marker. Figure 2A shows that only a We obtained an identical DNaseI foot-

Figure 1. Detection of the tagged proteins. (A) Western blot analysis of the tagged proteins. Total protein
extracts, separated by 12% SDS-PAGE and electroblotted, were analyzed using 1E2 (lanes 13), 3F12
(lanes 49 and 1315) or anti-p53 pAb 240 (lanes 1012) primary antibodies and alkaline phosphatase-
conjugated secondary antibody. Lanes 16: E. coli DH5 cells producing tagged XylS protein, bearing
pBR-3F12 (lanes 1 and 4), pBR-1E2 (lanes 2 and 5) and pBR-NC (lanes 3 and 6) derived expression plas-
mids. Lanes 712: E. coli BL21 (DE3) cells bearing pET-3F12 derived expression plasmids, producing
3F12-tagged p53 variants N39C362 (lanes 7 and 10), N39C362trp248 (lanes 8 and 11) and
N61C362 (lanes 9 and 12). Lanes 13 and 14: Saos-2 cells transfected with pCG-3F12 derived expression
plasmids producing 3F12-tagged GAPDH (lane 13) and GAPDH-NLS (lane 14). Saos-2 cells expressing
untagged p53 were used for a negative control (lane 15). (B and C) Subcellular localization of 3F12-
GAPDH and 3F12-GAPDH-NLS proteins. Saos-2 cells were transfected with pCG-3F12-GAPDH (B) or
pCG-3F12-GAPDH-NLS (C) and tagged proteins were detected with immmunofluorescence analysis.

Vol. 28, No. 3 (2000) BioTechniques 459


Short Technical Reports
print earlier with HA-epitope tagged binding ability due to the point mutation and produced a shifted band. To show
XylS, using similar technical approach in its DNA-binding domain (8). Plas- that the produced complex really con-
(4). DNA precipitation and DNase I mids, generated for the T7 promoter-di- tains p53, the protein-DNA complexes
footpriting with a resin-bound epitope- rected expression of 3F12-tagged p53 were supershifted with the tag-specific
tagged protein (10) are the methods of fusion proteins were transformed into E. monoclonal antibody 3F12. This way,
choice for proteins that are prone to ag- coli strain BL21 (DE3). epitope tags can be used to verify speci-
gregation and are difficult to purify. Expression of the tagged p53 pro- ficity of the shifted complex in a DNA
teins was monitored by Western blot band-shift assay without purification of
High Level Bacterial Expression analysis. The tag-specific 3F12 anti- the protein.
of Epitope-Tagged Proteins bodies and p53-specific pAb240 anti-
bodies were used for the detection of Expression of Epitope-Tagged
For high-level bacterial expression of the proteins. Using this vector system Proteins in Eukaryotic Cells
N-terminally tagged proteins, we con- resulted in enormous overexpression of
structed the vector pET-3F12, a deriva- the protein detected with 3F12 Mab as For eukaryotic expression, two cod-
tive of pET-11c that contains the tag-en- well as with pAb240 antibodies (Figure ing sequences of rat glyceraldehyde-3-
coding sequence and the cloning sites 1A, lanes 712). phosphate dehydrogenase (GAPDH)
identical to pBR-3F12 (Table 1). The The DNA-binding activity of the gene were cloned into pCG-3F12 plas-
coding sequences for mutant p53 pro- tagged p53 proteins was studied in a mid (Table 1). The first had 3F12 epi-
teins were cloned into the vector. Mutant band-shift assay (Figure 2C). The p53 tope fused in frame to GAPDH amino
proteins N39C362 and N61C362 mutant N39C362trp248, carrying a acids 2333 (pCG-3F12-GAPDH) and
had their transactivation and regulatory point mutation in its DNA-binding do- the second also contained a nuclear lo-
parts deleted, but maintained the ability main, was used as a negative control. calization signal of p53 (amino acids
to bind DNA, while the mutant protein The mutants N39C362 and N61- 305327) fused to the C-terminus of
N39C362trp248 had lost its DNA- C362 are functional in DNA binding the GAPDH protein (pCG-3F12-GAP-
Short Technical Reports
DH-NLS). determinants. J. Virol. 70:6169-6179.
Saos-2 cells were transfected with 1 2.Field, J., J. Nikawa, D. Broek, B. MacDon-
g of expression plasmids and analysed ald, L. Rodgers, I.A. Wilson, R.A. Lerner
and M. Wigler. 1988. Purification of a RAS-
24 h after transfection. Expression and responsive adenylyl cyclase complex from
localization of the proteins were deter- Saccharomyces cerevisiae by use of an epi-
mined by Western blotting and im- tope addition method. Mol. Cell. Biol. 8:2159-
munofluorescence analysis, respective- 2165.
3.Jarvik, J. W. and C. A. Telmer. 1998. Epi-
ly. The 3F12 antibody recognized both tope tagging. Annu. Rev. Genet. 32:601-618.
proteins as single bands on the Western 4.Kaldalu, N., T. Mandel and M. Ustav. 1996.
blot (Figure 1A, lanes 13 and 14) and TOL plasmid transcription factor XylS binds
no cross-reaction with cellular proteins specifically to the Pm operator sequence. Mol.
Microbiol. 20:569-579.
was observed (Figure 1A, lane 15). Im- 5.Kessler, B., M. Herrero, K.N. Timmis and
munofluorescence staining of transfect- V. de Lorenzo. 1994a. Genetic evidence that
ed cells with 3F12 antibody indicated the XylS regulator of the Pseudomonas TOL
that both proteins were localized in the meta operon controls the Pm promoter
appropriate compartment of the cell: through weak DNA-protein interactions. J.
Bacteriol. 176:3171-3176.
The 3F12-GAPDH in the cytoplasm 6.Kessler, B., K.N. Timmis and V. de Lorenzo.
and 3F12-GAPDH-NLS in the nucleus 1994b. The organization of the Pm promoter
(Figure 1, B and C). These results indi- of the TOL plasmid reflects the structure of its
cate that 3F12 epitope-tag can be used cognate activator protein XylS. Mol. Gen.
Genet. 244:596-605.
for the detection and determination of 7.Kurg, R., J. Parik, E. Juronen, T. Sedman,
the localization of proteins expressed in A. Abroi, I. Liiv, U. Langel and M. Ustav.
eukaryotic cells. 1999. Effect of bovine papillomavirus E2 pro-
tein-specific monoclonal antibodies on papil-
lomavirus DNA replication. J. Virol. 73:4670-
Advantages of the BPV E2-Derived 4677.
Epitope Tags 8.Lepik, D., I. Ilves, A. Kristjuhan, T.
Maimets and M. Ustav. 1998. p53 protein is
We analyzed the expression of pro- a suppressor of papillomavirus DNA amplifi-
teins tagged with the BPV E2-derived cational replication. J. Virol. 72:6822-6831.
9.Tanaka, M. and W. Herr. 1990. Differential
Figure 2. Use of the tagged proteins in DNA
epitopes in E. coli and eukaryotic cells. transcriptional activation by Oct-1 and Oct-2:
binding assays. (A) DNA precipitation by 3F12- Detection of the tagged proteins both interdependent activation domains induce
tagged XylS. Radiolabeled HpaII/HinfI digest of on immunoblots and by immunofluo- Oct-2 phosphorylation. Cell 60:375-386.
the Om-containing plasmid pUPM190 was incu- rescence staining of cells indicates low 10.Ustav M., E. Ustav, P. Szymanski and A.
bated with 3F12-beads containing 3F12-XylS. background activity, sensitivity and Stenlund. 1991. Identification of the origin of
Unbound DNA was removed by washing. Bound replication of bovine papillomavirus and char-
DNA was extracted from the beads and analysed good signal-to noise ratio of the used acterization of the viral origin recognition fac-
on non-denaturing TBE/PAGE (5%), using the epitope-antibody combinations. We did tor E1. EMBO J. 10:4321-4329.
fragment mixture as a marker (lane 1). Only a not observe any cross-reaction with cel-
single fragment, which contained the XylS bind- lular proteins. Because of the high
ing site Om, was retained on the beads (lane 2). The study was supported by the Eston-
(B) DNase I footprinting by resin-bound 3F12-
specificity of the epitope-antibody in-
ian Science Foundation Grant Nos. 2496,
tagged XylS. XhoI/EcoRI Om containing frag- teraction, our tagging system is espe-
2497, 2315 and 2316. Address correspon-
ment from pUPM190 was end-labeled in the low- cially useful for the studies of protein
er strand at 3 terminus and incubated with dence to Dr. Mart Ustav, Department of Mi-
localization in the cells. In addition, we
3F12-beads containing 3F12-XylS. Unbound crobiology and Virology, Institute of Molec-
have shown that interaction of 3F12
DNA was removed by washing. Both free and ular and Cell Biology, Tartu University, 23
protein-bound templates were subjected to MAb with the specific epitope is not
Riia Street, Tartu, 51010, Estonia. Internet:
DNase I cleavage. Lane 1, G-specific DNA se- hindered by high salt concentrations.
ustav@ebc.ee
quence marker; lane 2, DNase I digest of the un- That allows us to immunoprecipitate
bound DNA fragment; lane 3, DNase I digest of and immunopurify the tagged proteins
the fragment bound to 3F12-XylS. Brackets indi- Received 14 July 1999; accepted 11
cate the region protected from the DNase I cleav-
in high-salt conditions and to avoid
age by the DNA-bound XylS. (C) Band-shift as- coimmunoprecipitation of contaminat- November 1999.
say of the 3F12-tagged p53 proteins. The ing proteins as well as to avoid aggre-
DNA-binding activity of the p53 proteins was gation of the protein of interest in the Niilo Kaldalu, Dina Lepik,
studied by separating the protein-DNA complex- course of purification.
es from the unbound DNA on 5% PAGE (55:1). Arnold Kristjuhan and
Crude E. coli lysates containing p53 mutants
NC362 and NC362 produce a shifted band Mart Ustav
(lanes 1 and 4). Supershift with tag-specific mon- REFERENCES Tartu University
oclonal antibody 3F12 was used to show that the
protein-DNA complexes contain p53 (lanes 2 and 1.Abroi, A., R. Kurg and M. Ustav. 1996.
Tartu, Estonia
5). The N39C362trp248 p53 protein carrying Transcriptional and replicational activation
a point mutation in its DNA-binding domain was functions in the bovine papillomavirus type 1
used as a negative control (lane 3). E2 protein are encoded by different structural

462 BioTechniques Vol. 28, No. 3 (2000)