Báo cáo khoa học: The viral SV40 T antigen cooperates with dj2 to enhance hsc70 chaperone function docx

The viral SV40 T antigen cooperates with dj2 to enhance
hsc70 chaperone function
Athanasia Salma, Apostolos Tsiapos and Ioannis Lazaridis
Laboratory of General Biology, Medical School, University of Ioannina, Greece
The heat shock protein 70 (hsp70) family of molecu-
lar chaperones consists of stress inducible (e.g. hsp70)
and constitutively expressed (e.g. heat shock cognate
70 kDa; hsc70) members, which actively participate in
a number of vital cellular functions such as protein
folding, translocation, degradation and the acquisition
of cell thermotolerance [1–4]. In order to perform
their functions, the hsp70s cooperate with a wide
range of divergent proteins collectively known as
co-chaperones. Among them, the DnaJ family mem-
bers seem to be the major and critical partners in
regulating at least the folding activity of the hsp70
chaperone machine [5].
DnaJ proteins exert their function by binding to
hsc70 and stimulating its ATPase activity. ATP hydro-
lysis converts hsc70 from an open to a closed state
with low exchange rates, therefore promoting a cycle
of substrate binding and release [6]. In mammals, more
than 20 DnaJ homologues have been reported and
classified into three groups on the basis of their
domain characteristics [7]. All members of the DnaJ
family contain the J domain, which is a 70-amino acid
sequence structured in four helices with a loop between
helices II and III containing the highly conserved tri-
peptide HPD, which is necessary for binding to hsc70
[8]. The type I DnaJs, in addition to the J domain,
contain a Gly ⁄ Phe-rich region followed by a stretch of

cysteine repeats. Type II DnaJs lack the cysteine
repeats and type III DnaJs lack both the G ⁄ F region
and the cysteine repeats. Type I and Type II proteins
seem to have similar functions and they bind non-
native substrates, in contrast to type III proteins which
may not bind denatured polypeptides and thus proba-
bly function as ‘specialized’ molecular chaperones [9].
dj2 is the best characterized type I mammalian DnaJ
and it has been defined as the mammalian homologue of
the bacterial DnaJ and the yeast Ydj1 [10–12]. It is
mainly cytosolic and acts as a cochaperone to hsc70
assisting the folding of denatured proteins and facilitat-
ing the protein import into the mitochondria [13–15]. As
a critical member of the hsc70 chaperone machine, dj2
was found, when overexpressed, to suppress aggregate
Keywords
DnaJ; hsc70; molecular chaperone; protein
folding; T antigen
Correspondence
I. Lazaridis, Department of Biology,
Faculty of Medicine, University of Ioannina,
453 32 Ioannina, Greece
Fax: +302 6510 97863
Tel: +302 6510 97752
E-mail:
(Received 2 April 2007, revised 9 July 2007,
accepted 30 July 2007)
doi:10.1111/j.1742-4658.2007.06019.x
Simian virus 40 large T antigen is a J-domain-containing protein with mul-
tiple functions. Among its numerous activities, T antigen can bind heat

shock cognate 70 (hsc70) but the biological significance of this interaction
has not been fully understood. Here, we show that T antigen can act as an
hsc70 co-chaperone enhancing the protein-folding ability of the hsc70 chap-
erone machine. We also show that T antigen exerts its function in colla-
boration with the mammalian homologue of DnaJ. Moreover, we show
that the participation of T antigen in the hsc70 chaperone machine has
cell-type-specific characteristics.
Abbreviations
dj2, mammalian homologue of DnaJ; GST, glutathione S-transferase; hsc70, heat shock cognate 70 kDa; hsp, heat shock protein;
mutTAg, mutant TAg; SV40, simian virus 40; TAg, SV40 large T antigen; wtTAg, wild-type TAg.
FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5021
formation of androgen receptor and huntingtin caused
by expanded polyglutamine tracts [16,17].
Simian virus 40 (SV40) large T antigen (TAg) is a
multifunctional oncoprotein which is able to induce
transformation in multiple cell types [18]. It has been
shown that T antigen can act as a molecular chaperone
because it contains a functional J domain [19]. It has
also been shown that the J domain is essential for
T antigen to exert its functions related to transforma-
tion, enhancement of cell division, release of Rb ⁄ E2F
complexes and viral DNA replication [20–23]. T antigen
was found to bind hsc70 in an ATP-dependent manner
that requires the ATPase activity to be provided by
hsc70 [24]. Although the biological significance of the
above binding is not yet clear, the prevailing view states
that upregulation of the hsc70 chaperone machine is
required for several viral functions [25].
In this report, we show that T antigen binds to
hsc70 both directly and indirectly via dj2. We also

show that the above binding is ATP independent and
contributes significantly to the folding activity of the
hsc70 chaperone machine. Finally, we show that the
formation of the heterotrimeric complex T antigen–
dj2–hsc70 is cell-type-specific, because it is observed in
cells nonpermissive for SV40 viral lytic infection and
not in cells permissive to lytic infection.
Results
In order to investigate the interactions of T antigen
with the hsc70 chaperone machine, we purified hsc70,
dj2 and T antigen wild-type and mutant proteins as
described in Experimental procedures. Our efforts to
express the full-length forms of wild-type and mutant
T antigen fused to glutathione S-transferase were not
successful, despite the fact that we tested extensively
both the bacterial and the insect cell expression
systems. We believe that the difficulty in purifying full-
length GST-tagged T antigen forms was due to the
large size of the resulting proteins. Therefore, we
decided to purify truncated forms of T antigen which
included the J domain, the Rb-binding domain and the
DNA-binding domain, but lacked the ATPase domain
of the C-terminus. Given that the ATPase domain of
the T antigen has been shown to be dispensable for
hsc70 binding [24], we reasoned that the truncated
T-antigen proteins would mimic the function of their
full-length counterparts.
T antigen binds directly to hsc70
Upon performing GST pull-down experiments, we
observed that the wild-type TAg (wtTAg) bound to

hsc70 in an ATP-dependent manner with a stoichiome-
try of 1 : 1, as reported previously [24]. We also con-
firmed the inability of the mutTAg protein to bind
hsc70, even in the presence of ATP (data not shown).
It is interesting to note that hsp70 exhibited the same
characteristics as hsc70, in that it was found to bind
wtTAg in an ATP-dependent fashion, but with signifi-
cantly reduced affinity (data not shown).
T antigen and dj2 cooperate in binding to hsc70
Having established the assay conditions, we tested
whether the presence of a typical hsc70 co-chaperone,
namely dj2, interfered or altered the TAg ⁄ hsc70-bind-
ing characteristics. The first finding was that dj2 bound
TAg in an ATP-independent manner (Fig. 1A, lanes 3
and 4). Although functional dimerization of DnaJs has
been reported [31–33], our results clearly indicate that
binding between type I and type III DnaJs is possible
in an ATP-independent manner. Moreover, it was
shown that unlike hsc70, dj2 bound mutTAg indicating
AB
Fig. 1. dj2 participation in the TAg ⁄ hsc70
complex. Pull-down assays were performed
using GST-tagged wtTAg and mutTAg as
described in Experimental procedures.
Lanes 1, 2 and 8 represents purified pro-
teins. (A) GST–wtTAg was incubated with
the indicated proteins in the presence or
absence of ATP. Bound proteins were
detected by SDS ⁄ PAGE, stained with Coo-
massie Brilliant Blue (upper), followed by

western blotting (IB). (B) The same experi-
ment as in (A) was performed using GST–
mutTAg instead of GST–wtTAg.
Enhancement of hsc70 chaperone function A. Salma et al.
5022 FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS
once again that dj2 and T antigen, although both
J-domain-containing proteins have clearly distinct
hsc70-binding properties (Fig. 1B, lanes 3 and 4). The
above data also show that dj2 and hsc70 have different
binding properties to wtTAg and mutTAg. Most
importantly, we observed that the presence of dj2 dis-
rupted the stoichiometric balance of hsc70 ⁄ wtTAg
complex reducing the amount of bound hsc70 and
allowing hsc70 to bind wtTAg even in the absence of
ATP (Fig. 1A, lanes 5 and 6). Similar results were
obtained when we used mutTAg instead of wtTAg in
our pull-down assays. More specifically, we observed
that in the presence of dj2, hsc70 bound mutTAg in an
ATP-independent fashion (Fig. 1B, lanes 5 and 6).
These findings led us to hypothesize that dj2 binds
T antigen in an ATP independent manner and the
resulting heterodimer complexes with hsc70 via dj2.
To verify this hypothesis we proceeded in a pull-
down experimental scheme in which wtTAg was
sequentially incubated with hsc70 and dj2. As shown
in Fig. 2, formation of the dj2 ⁄ TAg complex prior to
hsc70 addition resulted in the recruitment of increased
amounts of hsc70 to the glutathione Sepharose-bound
GST–wtTAg (compare lanes 1 and 2). Densitometry
analysis revealed that there is a 2–2.5-fold increase in

the amount of hsc70 collected in the Tag ⁄ dj2 complex.
In contrast, addition of dj2 to the already formed
hsc70 ⁄ TAg complex decreased the amount of bound
hsc70 by 50% (compare lanes 1 and 3). Once again the
absence of ATP in the binding reactions did not influ-
ence hsc70 recruitment to the dj2 ⁄ TAg complex,
confirming our conclusion that the participation of the
T antigen in this complex is mainly mediated by dj2.
The reduced amounts of hsc70 bound to TAg prior to
dj2 addition (lanes 1 and 5) are probably due to the
fact that the binding affinity of dj2 to hsc70 is stron-
ger. The possibility of an antagonistic effect between
dj2 and TAg for hsc70 binding cannot be excluded,
but the finding that formation of the dj2 ⁄ TAg complex
recruits more hsc70 (compare lanes 1, 3 and 5 with
lanes 2, 4 and 6) indicates that the order of inter-
actions is important for the final composition of the
complex.
T antigen enhances the folding activity of the
hsc70 chaperone machine
Because one of the main functions of the dj2 ⁄ hsc70
chaperone complex is the folding of denatured or par-
tially folded proteins [29] we investigated the involve-
ment of T antigen in the above process. Therefore,
we performed luciferase-refolding experiments and
observed that indeed T antigen facilitated hsc70 and
dj2 in recovering of activity for denatured luciferase
(Fig. 3). We also found that T antigen can act as an
hsc70 co-chaperone, albeit with much reduced activity
when compared with dj2. However, we observed that

the presence of wtTAg significantly enhanced the fold-
ing activity of the dj2 ⁄ hsc70 chaperone machine. This
was verified by the finding that the maximal refolding
of luciferase achieved by the hsc70 ⁄ dj2 dimer, was fur-
ther increased with the addition of wtTAg. Impor-
tantly, the mutTAg had no effect on the function of
hsc70 alone or hsc70 with dj2 suggesting that the effect
of wtTAg is dependent on its co-chaperone activity.
Fig. 2. dj2 recruits increased amounts of hsc70 to the heterotrimer-
ic complex. Pull-down experiments were performed using GST–
wtTAg and in the assay mixture hsc70, dj2 and ATP were added
sequentially as indicated. The composition of the complexes
formed was analysed by SDS ⁄ PAGE followed by Coomassie Bril-
liant Blue staining.
Fig. 3. wtTAg enhances the folding activity of the hsc70 ⁄ dj2 chap-
erone pair. Refolding assays were performed as described and
luciferase activity was monitored at several time points ranging
from 0 to 60 min. This diagram depicts values taken after 60 min
incubation with the indicated proteins and expressed as percentage
of the native luciferase activity.
A. Salma et al. Enhancement of hsc70 chaperone function
FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5023
The ATP independence of the heterotrimeric
TAg–dj2–hsc70 complex formation is cell type
specific
The in vitro binding data raised the question of how
T antigen, dj2 and hsc70 may interact in the cellular
context. In order to investigate these interactions we
chose two transformed cell lines, namely Cos7 and
SVTT1, which constitutively express T antigen but dif-

fer in that the Cos7 cells are permissive for SV40 lytic
infection, whereas the SVTT1 are not. Cell extracts
were immunoprecipitated with an anti-TAg coupled to
protein G beads (as described in Experimental proce-
dures) and the immunoprecipitates were analysed for
the presence of TAg, dj2 and hsc70. In accordance with
previous findings [30], we detected the formation of
the hsc70 ⁄ TAg complex only in nonpermissive cells
(Fig. 4B, +ATP). The amount of hsc70 bound to TAg
was quantitated as 50% of the input. We also detected
the presence of dj2 in the immunoprecipitates of both
cell types in the presence of ATP and our quantification
measurements showed that 15–20% of the input was
retained in the complex. However, to our surprise, we
found that in the absence of an ATP regeneration sys-
tem, both dj2 and hsc70 were not able to associate with
T antigen in the extracts of non permissive cells
(Fig. 4B, –ATP). In contrast under the same conditions
the association of T antigen with dj2 persisted in per-
missive cells (Fig. 4A, ±ATP) in accordance with our
in vitro findings that this binding is ATP independent.
Collectively, the above results led us to conclude that
the interactions between T antigen, dj2 and hsc70 are
cell-type dependent.
Discussion
Although the ability of T antigen to bind hsc70 was
extensively studied, the exact mechanism by which this
binding contributes to the T-antigen-mediated cellular
transformation remains elusive [34,35]. However, a
model has been suggested according to which the

J-domain-dependent association of T antigen with
hsc70 stimulates chaperone activity, which in turn
releases E2F from its pRb complex allowing the ‘free’
E2F to transactivate the genes required for cell prolif-
eration [20,22,36]. Our results, without disputing the
above model, indicate that T antigen might not exert
its hsc70-related functions alone but in combination
with dj2, a classical hsc70 co-chaperone. This sugges-
tion is based on our findings that, in vitro, T antigen
associates with dj2 in an ATP-independent manner
and this binding persists even in the presence of hsc70
(Fig. 1A). As a result, T antigen is recruited to the
hsc70 chaperone complex via dj2. Therefore, the pres-
ence of T antigen allows hsc70 to utilize yet another
J-domain-containing protein and enhance its chaperon-
ing function. It is interesting to note that even a
mutated form of T antigen unable to bind hsc70 can
be recruited to the complex via dj2 (Fig. 1B).
Our interpretation of the above data is that
besides the formation of the expected dj2 ⁄ hsc70
(dimer : monomer) complexes, the presence of T anti-
gen leads to the formation of novel heterotrimeric
complex comprising of monomeric forms of TAg ⁄
dj2 ⁄ hsc70. The formation of these complexes does not
exclude the possibility of direct binding between
T antigen and hsc70. On the contrary, given the abun-
dance of hsc70, one can envisage the presence of all
three types of complex in the cellular context. This
interpretation is supported by the finding that associa-
tion of T antigen with dj2 prior to the addition of

hsc70 leads to the recruitment of increased amounts of
hsc70 to the complex. In contrast, addition of dj2 to
the already formed TAg ⁄ hsc70 dimer does not increase
the levels of hsc70 present in the complex, indicating
that probably dj2 associates with hsc70 in a separate
BC
A
Fig. 4. Cell-type-specific interaction of T antigen with hsc70. Immunoprecipitations of Cos7 cell extracts (A), SVTT1 extracts (B), and NIH 3T3
transfected with mutTAg extracts, as a control (C), were performed using an anti-(T antigen) serum column. Bound proteins were detected
by western blotting. The presence or absence of ATP regeneration system in the lysates is indicated. FL, analysis of the flow through mate-
rial; ctrl, the control IgG-protein G beads; Inp, the whole cellular lysate (10% of total).
Enhancement of hsc70 chaperone function A. Salma et al.
5024 FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS
complex which does not include T antigen. Interest-
ingly, the increased amounts of hsc70 required by the
TAg ⁄ dj2 heterodimer do not seem to be affected by
the presence of ATP, indicating once more that the
ATP-independent dj2 binding to hsc70 is the driving
force for the creation of the heterotrimeric complex
(Fig. 2). Therefore, based on our results, we believe
that T antigen can bind hsc70 in two distinctly differ-
ent ways. First, it can bind directly in an ATP-depen-
dent fashion, and second, it can bind indirectly via dj2
in an ATP-independent manner.
The functional significance of our findings was
investigated by monitoring the chaperone-folding
activity of the detected complexes. Indeed, we found
that the presence of T antigen enhanced the ability of
the hsc70 ⁄ dj2 chaperone pair to refold denatured lucif-
erase. We believe that this increased folding activity

was due to the function of the newly formed
heterotrimeric complex (TAg ⁄ dj2 ⁄ hsc70). In contrast,
the presence of the mutated form of T antigen was
not able to change the folding activity of the hsc70 ⁄ dj2
chaperones. In other words, despite the fact that the
trimeric complex mutTAg ⁄ dj2 ⁄ hsc70 can be clearly
formed (Fig. 1B), mutTAg is unable to further
enhance luciferase folding, probably due to its inability
to stimulate the hsc70 ATPase activity. This last
assumption is supported by the finding that although
wtTAg can act as a weak hsc70 cochaperone, mutTAg
seem to be unable to positively contribute to the
enhancement of the hsc70 folding activity. Collectively,
we concluded that T antigen facilitates the hsc70
chaperone machine by enhancing its folding function.
In order to detect the existence of the described
complexes in vivo, we performed immunoprecipitations
with an anti-(T antigen) serum in cellular extracts of
Cos7 and SVTT1 cells. As shown in Fig. 4, our
approach was quite effective in that our antibody col-
umn depleted most of the T antigen from the cell
lysates. As expected, based on our previous data [30],
formation of the TAg ⁄ hsc70 complex was observed
only in nonpermissive cells, reinforcing our suggestion
that this binding has cell-type-specific characteristics.
Moreover, we detected the presence of dj2 in both the
TAg ⁄ hsc70 complex (nonpermissive cells) and in the
T antigen immunoprecipitate (permissive cells). How-
ever, to our surprise, we observed that in the absence
of ATP the dj2 and hsc70 did not associate with TAg

in SVTT1 cells, in contrast to the Cos7 immuno-
precipitates where the binding of T antigen to dj2
persisted (Fig. 4). Given that the dj2 ⁄ TAg binding is
ATP independent, as shown above, we suspect that
the activation of an additional, yet unidentified,
cellular factor is responsible for the cell type specific
dissociation of the heterotrimeric complex in the
absence of ATP or conversely enabling dj2 to bind
TAg in Cos7 cells. Overall, our data using cellular
extracts clearly suggest the existence of a cell type
specific organization of the hsc70 chaperone machine,
which is probably related to the T antigen J-domain-
mediated viral functions.
Experimental procedures
Cell lines
The Cos7 and SVTT1 (NIH 3T3 cells expressing wtTAg)
cell lines established from monkey or mouse embryos,
respectively, were maintained in minimal essential medium
supplemented with 10% fetal bovine serum at 37 °Cina
humidified 5% CO2 atmosphere. Sf9 insect cells were
grown in Sf900II medium (Gibco, Invitrogen, Carlsbad,
CA) containing 10% fetal bovine serum at 27 °C and used
for the production of baculoviruses.
Cloning, expression and purification of
recombinant proteins
The pQE-32-Mydj2 construct [13] was used to overexpress
Mydj2–6·His in Escherichia coli SG13009. Briefly, over-
night cultures of E. coli carrying the pQE-32-Mydj2
plasmid were diluted 10-fold and cultured for 1 h. After
isopropyl thio-b-d-galactoside induction (1 mm) for 2 h at

37 °C, cells were collected by brief centrifugation at 6000 g
using an Avanti J-25 centrifuge with JA-14 rotor (Beckman
Coulter, Fullerton, CA) and cell lysates were prepared by
sonication. The recombinant protein was purified using a
Ni ⁄ nitrilotriacetic acid column and imidazole elution
(50 ± 250 mm) as described by the manufacturers (Qiagen,
Valencia, CA).
The SV40 large T antigen was subcloned from pSG5-T
(a generous gift from J. A. DeCaprio, Harvard Medical
School, MA) into the BamH1 site of pGEX-4T-1 (AMRAD)
to create the pGEX-4T-1–wtTAg construct. The resulting
plasmid was digested with EcoR1 and HindII, treated with
Klenow and religated to create the pGEX-4T-1–wtTAg282
construct which was used to express the first 282 amino
acid residues of the wild-type T antigen. The mutated form
of T antigen was subcloned from pSG5-T-H42Q (a gener-
ous gift from J. A. DeCaprio) to pGEM and subsequently
inserted to the EcoR1 and Sal1 sites of pGEX-4T-1. The
resulting plasmid was digested with Not1 and HindIII,
blunt ended with Klenow and religated to create the
pGEX-4T-1–mutTAg268, which was used to express the
first 268 amino acid residues of T antigen with a single
amino acid substitution (H42Q). The described constructs,
named GST–wtTAg and GST–mutTAg, respectively, were
expressed in BL21(DE3) bacterial cells and the corresponding
A. Salma et al. Enhancement of hsc70 chaperone function
FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5025
proteins were purified from lysates according to standard
procedures [26].
Baculovirus expressed His6·-tagged hsc70 and hsp70

were prepared according to Bac-To-Bac baculovirus expres-
sion system procedure (Invitrogen).
Pull-down assays
GST fusion proteins (3 lg; wtTAg, mutTAg) or GST alone,
were incubated for 30 min at room temperature with 30 lL
of glutathione–agarose beads and ‘blocked’ in 1% fish gela-
tin in assay buffer (150 mm NaCl, 20 mm Tris ⁄ HCl pH 7.5,
2mm MgCl
2
, 250 mm sucrose, 1% Triton X-100, 0.1 mm
EGTA, 1 mm phenylmethanesulfonyl fluoride, 1 mm DTT).
After washing three times with the same buffer, the beads
were combined with equal amounts of purified His–hsc70
and ⁄ or His–dj2 and further incubated for 1 h at room tem-
perature in the presence or absence of ATP regeneration
buffer (1 mm ATP, 30 mm creatine phosphate, 0.25 mg
phosphokinase per mL, 2 mm MgCl
2
,1mm dithiothreitol).
The beads were washed six times with assay buffer, before
eluting the bound proteins with hot Laemmli buffer [27].
Western blots were performed using the following anti-
bodies: T antigen-specific mAb PAb419, which have been
produced by hybridoma cells. Hsc70-specific antibody
(SPA-815, StressGen), Hsp70-specific antibody (SPA-810,
StressGen) and Dj2-specific antibody (HDJ-2 ⁄ DNAJ Ab-1,
NeoMarkers). Quantitation of the pulled down proteins
was performed using the quantity one densitometry
software provided by Bio-Rad Inc (Hercules, CA).
Immunoprecipitation

Whole-cell extracts were prepared from COS and SVTT1
cells in lysis buffer containing 150 mm NaCl, 20 mm
Tris ⁄ HCl pH 7.5, 2 mm MgCl
2
, 250 mm sucrose, 1%NP40,
0.1 mm EGTA, 1 mm phenylmethanesulfonyl fluoride,
1mm dithiothreitol and 1 lgÆmL
)1
each of protease inhibi-
tors aprotinin, leupeptin and pepstatin. The extracts were
centrifuged at 13 000 g for 15 min using a Sigma 1-14
microcentrifuge with 12094 rotor (Sigma-Aldrich, Munich,
Germany), precleared with 20 lL of IgG–protein G (3 lg)
for 30 min at room temperature and then incubated with
20 lL of anti-TAg–protein G beads (40 lg of anti-TAg
serum) or with 20 lL IgG–protein G beads (40 lg of anti-
[mouse IgG] serum) overnight at 4 °C, in the presence or
absence of ATP regeneration buffer. The antibodies were
covalently coupled to the beads using DMP (Pierce, Rock-
ford, IL) as a cross-linker. After incubation, the beads were
washed three times with lysis buffer. Proteins present in the
immune complexes were eluted with lysis buffer supple-
mented with 1 m NaCl. The eluates were trichloroacetic
acid precipitated and the pellets were resuspended in 30 lL
Laemmli buffer for subsequent analysis by SDS ⁄ PAGE and
western blotting.
Luciferase assays
Luciferase activity was measured as described [28] with
minor modifications. Briefly, 1 mg luciferase (Promega,
Madison, WI) was chemically denatured by diluting

3-fold in buffer containing guanidinium-HCl (6 m)25mm
Hepes (pH 7.6), 50 mm KC1, 5 mm MgCl
2
and 1 mm
dithiothreitol for 40 min at room temperature. Refolding
reactions were performed in 125 lL volumes by diluting
denatured luciferase (1 lL) into refolding buffer contain-
ing 25 mm Hepes (pH 7.6), 50 mm KC1, 5 m m MgCl
2
,
purified chaperones, and ATP regeneration buffer. The
samples were incubated at 30 °C and at the indicated
times, 1 lL of each reaction was diluted into 60 lLof
luciferase assay mixture (Promega). Activity was measured
using a luminometer (Junior LB9509 EG & G Berthold,
Bad Wildbad, Germany). Native luciferase activity was
measured after first diluting the stock solution threefold
into refolding buffer and then diluting 125-fold into the
same buffer. All activities were calculated as a percent of
the native luciferase activity and all data points are the
average of at least two replicates.
Acknowledgements
We thank M. Cheetham and H.H. Kampinga for
critically reading the manuscript. We also thank
Dr P. Kouklis for helpful advice. This work was sup-
ported by grants from the EU (QLRT-1999, #30720)
and the Greek Ministry of Education (Hrakleitos).
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