Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
PURIFICATION OF POLYOMA D. PAULIN(+),
T
ANTIGEN FROM TRANSFORMED CELLS
P. GAUDRAY(~), and F. CUZIN(~)
D6partement de Biologie mol6culaire, Institut Pasteur, 75015 Paris(+), and Centre de Biochimie, Universit6 de Nice, 06034 Nice, France(%)
Received Julv 9,1975 SUMMARY. The T (tumor) antigen from transformed mouse cells hat been purified 400-fold and prominent polypeptides of molecular weights 85-90,000 and 70-75,000 resolved. Polypeptides exhibiting same molecular weights are adsorbed specifically on insolubilized anti-T antibodies. Study of temperature sensitive early mutants of polyoma and SV 40 viruses has led to the conclusion that the protein product of an early viral gene (ts-a gene of polyoma, A gene of SV 40) plays a critical role in the initiation of autonomous viral DNA replication cess
(3,4).
(1,2), as well as in the transformation pro-
It seems very likely that the protein product of
this gene carries the antigenic site for the virus specific intranuelear T antigen
(5).
As a preliminary step toward the ana-
lysis of the function of polyoma virus ts-a at the biochemical level, we devised a purification procedure for T antigen, using complement fixation
(CF) as an assay. MATERIAL AND METHODS
Polyoma transformed PY6 cells, derived from Swiss mouse 3T3 fibroblasts were obtained from Dr.T.Benjamin. transformed mouse cells
Spontaneously
(3T6) were u s e & as a control. Conditions
for cell growth and the procedure for radioactive labeling were described previously
(6).
T antigen was assayed according to
Sever (7). Immunoadsorbants.
Immunoadsorbants were prepared by the
Cuatrecasas method
(8).
Copyright©19~ ~ Aca~mwPress,~c. AHngh~ ~repro~c~onma~rmrese~ed.
At least 80 to 90% of the proteins were 1418
Vol. 65, No. 4, 1975
found
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
coupled
to the
Sepharose
Gel e l e c t r o p h o r e s i s . Studier
(9), using
The analysis 0.1%
sodium
lyacrylamide
for r u n n i n g
Purification
of T antigen.
and r e s u s p e n d e d
same buffer. homogenized
The
gels
for
PY6 and
a specific trations
homogenizer
radioactivity
not d e t e c t a b l e
and
was
sulfate 40%
was
discarded.
about
40 mg/ml
Mann)
was
discarded.
up to a final
units
contained
with concen-
and were
no d e t e c t a b l e
The p r e c i p i t a t e
was
collected,
sulfate
solution,
pH 7.4,
and d i a l y z e d
DE 52) column
containing
against
the
of T antigen)
gradient
containing
sulfate
fraction
was a p p l i e d
of NaCI
0.05
to the
the a d s o r b e d
concentration
1419
sulfate of
20%
CF a c t i v i t y
to 40% a m m o n i u m
dissolved
(1.2 x 20 em) was
pH 7.4,
ammonium
Ammonium
adjusted
buffer,
buffer,
:
was
50 ml of the same buffer, linear
T antigen
concentration
w~shed
with
a
in 1 ml of
0.05 M NaCI
and 10%
same buffer.
~ = ~ 9 ~ _ ~ 9 ~ 9 ~ g D ~ Y _ ~ D _ ~
The d i a l y z e d
twice
of protein,
supernatant
ammonium
glycerol,
phosphate
times,
sonicated
in PY6 extracts
D 2)
added
The
saturation.
(Whatman
three
Supernatants
Polyoma
from 128 to 256 CF units
0.01 M p h o s p h a t e (w/v)
in the
at 100 W, and c e n t r i f u g e d
of 106 cpm/mg.
The p r e c i p i t a t e
saturated
of 108 cells/ml
in 3T6.
Grade,
saturation.
15% po-
saline
(10 strokes),
A m~_~Di~_~if~_p~i~2~_~ (Enzyme
with
of
in Tris
and thawed
apparatus
contained
(SDS),
were w a s h e d
frozen
The pellet
3T6 cells
ranged
Cells
was
Sonifier
g.
the p r o c e d u r e
(10 cm long).
at a c o n c e n t r a t i o n
in a Potter
30 m at 30,000
from
:
suspension
for 15 s in a Branson
followed
dodecylsulfate
slab
Ce~_ex~ra~s_~s~e~_~ buffer,
beads.
: a DEAE-cellulose equilibrated M NaCI
and
with
0.01 M
10% glycerol.
(4 mg p r o t e i n
and 256 CF
column.
washing
material (0.05
After was
with
eluted w i t h
to 0.6 M)
a
in 100 ml of
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.01 M p h o s p h a t e the T antigen NaCI,
buffer,
activity
pH 7.4,
was
eluted
and II at 0.20 M NaCI.
justed
to 0.15 M NaCI,
of T antigen fractions
were
~
!
selective sheep
was
~
~
extensively
sample
were
~
of contact
!
~
_
~
purified
_
was
~
!
_
pH 7.4,
~
_
prepared
and coupled
I or II, a d j u s t e d
1,
i at 0.12 M
the peaks
~
were
ad-
The ratio
I.
The two
2
to 0.15
T antigen
was r e c o v e r e d
unadsorbed
protein
of the flow through
(6).
4 B.
The
A 2 ml
was
antibodies.
with
from a
protein
M NaCI,
applied
After
1 h
2 ml of 0.01 M phos-
10% glycerol
Polyoma
: a
antibodies
cellular
was w a s h e d
remained
_
using
coupled
0.15 M NaCI,
~
to Sepharose
0.2 ml/m.
protein
under
in Fig.
by CF for T antigen.
with mouse
at 4 ° , the column
of the total
peaks
to 1 in f r a c t i o n
1 x 3 cm of Sepharose
phate buffer,
As shown
separately.
immunized
of fraction
to a c o l u m n
Fractions
and assayed
immunoadsorbant
antibodies
in two main
2 in f r a c t i o n _ l l
pooled _
10% glycerol.
at a flow rate
quantitatively
and wash,
while
of
in the
80 to 90%
on the column.
RESULTS Puri~cation Table
of two d i s t i n c t
1, m a t e r i a l
was p u r i f i e d
with
correspond
to d i f f e r e n t
to one antigen to a viral equilibrium antigen same
antigenic
chromatography
between
seems
were
likely,
of isolated
obtained
separated (Fig.
recognized
complexes
in d i f f e r e n t
the most
conditions
species
antigens
different
As shown
in
as in step 4
with a 25 to 50% recovery.
on D E A E - c e l l u l o s e
in d i f f e r e n t
peptide
species.
T immunospecificity
300 to 400 fold,
Two distinct exchange
antisenic
with
1).
by ionThey might
by the immune cellular
aggregates.
components,
The h y p o t h e s i s
molecular
configurations
because
rechromatography
form
I or II also
1420
serum,
generates
or
of an
of a unique under
the
a bimodal
Vol. 65, No. 4, 1 9 7 . 5
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
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D. O P ~ co ~ J O ~ "-" 0
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Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
m
I
I
z I 0.60.
/
16
s*f /s* 1 , ' /
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o
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O.05J
40 6O FRACTION NUMBER
20
80
I00
Figure 1. C h r o m a t o g r a p h y on DEAE-cellu!ose. Fractions were collected and analyzed for CF activity and 14C r a d i o a c t i v i t y on 25 pl aliquots.
elution profile with peaks c o r r e s p o n d i n g trations
to the same NaCI concen-
(0.12 and 0.20 M).
Characterization
of purified
sition from fractions
polypeptides.
The p o l y p e p t i d e
compo-
I and II was analysed by S D S - p o l y a c r y l a m i d e
gel electrophoresis. As shown in Fig. contained fraction
2, the material
8 main peptides
derived from fraction
with low background,
I was more heterogeneous.
II
while that from
The m o l e c u l a r weights
corres-
ponding to the 4 main bands of I (A to D) and to the 5 bands of II (a to e) are indicated
in Fig.
2.
Similar values
are found for
w
peptides ~ (fraction B~
I) and ~ (fraction
II)
(85-86,000)
and for
b (72-74,000).
Binding of T antigenic labeled protein
material
to i n s o l u b i l i z e d
containing T antigenic
step 2 was adsorbed on i n s o l u b i l i z e d to Del Villano and Defendi
(10).
14
C
activity at p u r i f i c a t i o n
anti-T antibodies
No T antigen
1422
antibodies.
according
could be detected
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
BSA
BSA
OV
BLG
dimer B
I
I
I
t
I
I
I
a
I
0
|
I
2
86 72 68 62 52 I
~
I
4
MIGRATION
I
I
5
i
6
7
IN CM
Figure 2. Gel e l e c t r o p h o r e s i s in SDS of p u r i f i e d fractions (step 4) derived from fractions i (top) and II (bottom) of Fig. 1. 10,000 cpm of labeled p r o t e i n o b t a i n e d after immunoa d s o r p t i o n of host p r o t e i n w e r e a n a l y z e d and the autoradiograms scanned w i t h a densitometer. M a r k e r proteins are d e n o t e d by BSA and BSA dimer : b o v i n e serum a l b u m i n e (MW : 68,000), 0V : o v a l b u m i n e (MW : 43,000), BLG : 6 - 1 a c t o g l o b u l i n e (MW : 18,000).
in the first
supernatant
quantitatively material 0.05%,
was
bound eluted
dissolved
ammonium
to the with
respectively,
or in the wash,
suggesting
immunoadsorbant.
0.2 N H C l - g l y c i n e ,
of the r a d i o a c t i v i t y sulfate
tracts.
Two main bands
phoresis
w i t h MW 7 0 - 7 5 , 0 0 0
precipitates
are r e s o l v e d and
that
The bound pH 2.2
About
(Fig.
0.3 and f r o m re-
3T6 cell
in p o l y a c r y l a m i d e
80-90,000
antibody
were r e c o v e r e d
of PY6 and
it was
gel
exelectro
3).
DISCUSSION Polyoma dure using
virus
T antigen
complement
fixation
was p u r i f i e d (CF)
1423
in a f o u r - s t e p
as an assay,
proce-
and by direct
ad-
VoI. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DIMER
FiGure 3. Gel electrophoresis in SDS of material eluted at pH 2.2 from Sepharose-coupled anti-T immunoglobulin. Marker proteins are the same as in Fig. 2. r
sorption on insolubilized antibodies.
The purified fractions have
in common two polypeptides with molecular weights in the ranges 70-75,000 and 85-90,000. tes
These values agree with previous estima-
(10,12) of the size of the early viral peptide of polyoma and
SV 40 viruses. During a lytic infection, the early viral messenger can eode for 90-100,000 daltons of protein.
RNA complementary to the early
viral region in PY6 cells, however, only corresponds to 70-75,000 daltons of protein.
Additional
sequences are transcribed from the
1424
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E strand in the late region communication). not known.
(antilate)
(13 and R. Kamen, personal
The size of the viral messenger in these cells is
Nor it is known if viral and cellular sequences are
transcribed into a single mRNA molecule. Two hypothes~s can
be advanced
: (i) the 70,000 MW peptide
constitutes the only viral gene product.
The 90,000 MW peptide
could then be a cellular component associated with the viral protein.
(ii) A mRNA corresponding to 70 to 80% of the early viral
region is translated into the 90,000 MW peptide, which is, in turn, partially converted into a smaller antigenic component.
Finger-
prin~ analysis of the peptides is in progress to decide between these hypotheses.
Acknowledgements.
The technical assistance of C. Maczuka, N. Montreau and J. Perreau is gratefully acknowledged. This work was made possible by grants of the Centre National de la Recherche Scientifique, the Commissariat l'Energie Atomique, and the Institut National de la Sant~ et de la Recherche M~dicale (ATP 18 Contracts to F.C. and to G. Meyer). For one of us (P.G.), this work was performed in partial fullfilment of the requirements for the Doctorat degree at the Universities of Paris and Nice.
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P.
(1972)
J.Virol., 10, 591-598.
2. Francke, B., and Eckhart, W. 3. Osborn, M., and Weber, K. 4. Tegtmeyer,
P. (1975)
(1973)
(1975)
7. Sever, J.L.
(1962)
J.Virol., 15,
636-644.
J.Virol., 15, 613-618.
S. Paulin, D., and Cuzin, F. (1975) 6. Pauiin, D., Perreau, 699-705.
Virology, 55, 127-135.
J.Virol., 15,
J., and Cuzin, F. (1974) J.Immunol., 88,
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Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8. Cuatrecasas, P. (1970) 9. Studier, F.W.
(1973)
J.Biol. Chem., 245, 3059-3065. J.Mol. Biol., 79, 237-248.
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1426