(Received for publication, October 30, 1995; and in revised form, February 2, 1996)
From the
PreS2-S`--galactosidase, a three-domain fusion protein that
aggregates extensively in the cytoplasm of Escherichia coli,
was used to systematically investigate the effects of heat-shock
protein (hsp) overproduction on protein misfolding and inclusion body
formation. While the co-overexpression of the DnaK and DnaJ molecular
chaperones led to a 3-6-fold increase in the recovery of
enzymatically active preS2-S`-
-galactosidase over a wide range of
growth temperatures (30-42 °C), an increase in the
concentration of the GroEL and GroES chaperonins had a significant
effect at 30 °C only. Co-immunoprecipitation experiments confirmed
that preS2-S`-
-galactosidase formed a stable complex with DnaK,
but not with GroEL, at 42 °C. When the intracellular concentration
of chromosomal heat-shock proteins was increased by overproduction of
the heat-shock transcription factor
, or by addition
of 3% ethanol (v/v) to the growth medium, a 2-3-fold higher
recovery of active enzyme was observed at 30 and 42 °C, but not at
37 °C. The overexpression of all heat-shock proteins or specific
chaperone operons did not significantly affect the synthesis rates or
stability of preS2-S`-
-galactosidase and did not lead to the
disaggregation of preformed inclusion bodies. Rather, the improvements
in the recovery of soluble and active fusion protein resulted primarily
from facilitated folding and assembly. Our findings suggest that
titration of the DnaK-DnaJ early folding factors leads to the formation
of preS2-S`-
-galactosidase inclusion bodies.
It is well established that the high level expression of recombinant proteins in Escherichia coli can result in the formation of insoluble aggregates known as inclusion bodies. Since inclusion bodies consist mainly of the protein of interest and are easily isolated by centrifugation, their formation has often been exploited to simplify purification schemes(1) . The recovery of biologically active products from the aggregated state is typically accomplished by unfolding with chaotropic agents or acids, followed by dilution or dialysis into optimized refolding buffers. However, many polypeptides (e.g. structurally complex oligomeric proteins and those containing multiple disulfide bonds) do not easily adopt an active conformation following chemical denaturation(1) . In such cases, maximizing the yields of recombinant proteins in a soluble and active form in vivo becomes an attractive alternative to in vitro refolding.
Molecular chaperones are a ubiquitous class of proteins that play an essential role in protein folding by helping other polypeptides reach a proper conformation or cellular location without becoming part of the final structure(2, 3, 4) . In E. coli, two molecular chaperone ``machines,'' DnaK-DnaJ-GrpE and GroEL-GroES, have been studied extensively. DnaK and its co-factors, DnaJ and GrpE, have been proposed to interact with nascent polypeptides and to either directly facilitate the proper folding of the newly synthesized proteins (2, 3, 4) or to mediate their transfer to the ``downstream'' GroEL-GroES chaperonins (5, 6, 7) . Overproduction of components of either the DnaK-DnaJ-GrpE (8, 9, 10, 11, 12) or GroEL-GroES chaperone machines(9, 10, 11, 12, 13, 14, 15, 16, 17, 18) has been shown to improve the cytoplasmic solubility and/or secretion of a number of aggregation-prone heterologous proteins in E. coli. However, higher intracellular concentrations of molecular chaperones had little effect on the proper folding or localization of other recombinant polypeptides(8, 10, 11, 16) . To date, the mechanisms by which molecular chaperones favor the proper folding of specific proteins remain unclear.
In this work, we used
the preS2-S`--galactosidase fusion protein (19) as a
model to systematically examine the effects of hsp overexpression on
the synthesis and folding/assembly of a highly aggregation-prone
protein in E. coli. One direct approach to increase the
intracellular concentration of molecular chaperones, overproduction of
plasmid-encoded DnaK-DnaJ or GroEL-GroES, and two indirect approaches,
overproduction of plasmid-encoded
or addition of
ethanol to the growth medium, were investigated. Our results indicate
that increased levels of DnaK and DnaJ lead to higher yields of active
preS2-S`-
-galactosidase by facilitating the proper folding and
assembly of the tetrameric fusion protein.
Figure 4:
Effect of co-overexpression (A) or ethanol addition (B)
on the recovery of preS2-S`-
-galactosidase. A,
-galactosidase activity versus post-induction time in
JM105 cells co-transformed with pTBG(H+) and either pTG10 (shaded bars) or p
(open bars).
Transformants were grown at the indicated temperatures and the
-galactosidase activity assayed 1 and 2 h post-induction. B,
-galactosidase activity versus post-induction
time in JM105 cells co-transformed with pTBG(H+) and pTG10 and
grown in the absence (shaded bars) or presence of 3% (v/v)
ethanol (open bars).
Figure 5:
Evolution of -galactosidase activity
following translational arrest. A,
-galactosidase
activity versus post-addition time in JM105 cells
co-transformed with pTBG(H+) and either pDnaK/J (
),
pGroESL (
), p
(
), or pTG10 in the
absence (
) or presence (
) of ethanol. Mid-exponential phase
cells growing at 30 °C were induced with 1 mM IPTG,
shifted to 42 °C for 1 h, and treated with 200 µg/ml neomycin
to stop translation before being returned to 30 °C. Corresponding
whole cell (B), soluble (C), and insoluble (D) fractions from pDnaK/J transformants are shown. The ratios
of preS2-S`-
-galactosidase at the indicated times to the zero time
point were determined by normalized videodensitometric scanning and are
shown below each panel. The average standard deviation in the values
was ±4.6%. The positions of preS2-S`-
-galactosidase (top arrows) and DnaK (bottom arrows) are
indicated.
Figure 6:
PreS2-S`--galactosidase is
progressively released from DnaK after translational arrest. JM105
cells harboring pTBG(H+) and pDnaK/J were grown at 30 °C,
induced with 1 mM IPTG, and shifted to 42 °C for 1 h.
Following addition of 200 µg/ml neomycin to stop translation, the
cells were returned to 30 °C and aliquots were immunoprecipitated
with anti-DnaK antibodies at various time points. Duplicate samples
were separated on 8% SDS gels and transferred to nitrocellulose before
being probed with antibodies to either
-galactosidase (A)
or DnaK (B). The lanes correspond to the marker (M)
and samples taken 0, 20, 60, and 120 min following neomycin
addition.
Figure 3:
PreS2-S`--galactosidase interacts
stably with DnaK. JM105 cells harboring pTBG(H+) and the indicated
plasmids were induced with 1 mM IPTG, shifted to 42 °C for
1 h, and aliquots were immunoprecipitated with anti-DnaK antibodies as
described under ``Experimental Procedures.'' Duplicate
samples were separated on 8% SDS gels and transferred to nitrocellulose
before being probed with antibodies to either
-galactosidase (A) or DnaK (B). The lanes correspond to the marker (M), and samples from cells harboring pTG10 (10),
pDnaK/J (KJ), or pGroESL (ESL). The ratios of
preS2-S`-
-galactosidase to DnaK estimated by videodensitometric
scanning were 1.2, 0.9, and 1.3 for the pTG10, pDnaK/J, and pGroESL
transformants, respectively.
To investigate the influence
of an increase in the cellular concentration of well characterized
molecular chaperones on the expression of preS2-S`--galactosidase, E. coli JM105 was co-transformed with pTBG(H+) and either
pTG10 (control vector), pDnaK/J (which encodes the dnaKJ operon), or pGroESL (which carries the groE operon).
These constructs place the chaperone operons under control of both
their native and the IPTG-inducible lac promoters. Pulse
labeling experiments followed by normalized videodensitometric scanning
of the fluorograms (Fig. 1, Table 1) showed that cells
harboring pDnaK/J synthesized 3-4-fold more DnaK than the control
strain, but lower amounts of chromosomal GroEL, particularly as the
growth temperature was raised. Similarly, the presence of pGroESL led
to a 4-fold increase in the relative synthesis rates of GroEL and some
variation in the expression of chromosomally-encoded DnaK, although no
temperature-dependent trend was detected. However, the overproduction
of plasmid-encoded molecular chaperones did not significantly affect
the synthesis rates of preS2-S`-
-galactosidase relative to the
control cells under the conditions examined (Table 1).
Figure 1:
Effect of DnaK-DnaJ or GroEL-GroES
overexpression on the synthesis of preS2-S`--galactosidase. JM105
cells co-transformed with pTBG(H+) and either pTG10(10) ,
pDnaK/J (KJ), or pGroESL (ESL) were incubated for 30 min post-induction
at the indicated temperatures and labeled for 2 min. The positions of
preS2-S`-
-galactosidase (arrow 1), DnaK (arrow
2), GroEL (arrow 3), normalization band (arrow
4), and DnaJ (arrow 5) are shown. The 10-kDa GroES
subunits are not resolved on these gels.
Figure 2:
Effect of the co-overexpression of
specific molecular chaperones on the recovery of
preS2-S`--galactosidase. Upper panels,
-galactosidase activity versus post-induction time in
JM105 cells co-transformed with pTBG(H+) and either pTG10 (
),
pDnaK/J (
), or pGroESL (
). Mid-exponential phase cultures
induced with 1 mM IPTG were grown at 30 °C (A),
37 °C (B), or 42 °C (C), and the
-galactosidase activity in clarified extracts was measured after 1
and 2 h. Note the differences in the
-galactosidase activity
scale. Lower panels, SDS-PAGE fractionation of protein samples
corresponding to cells harboring pTBG(H+) and the indicated
plasmids. Whole cells (W) and soluble (S) and
insoluble (I) fractions from samples collected 1 h
post-induction growth at 30 °C (D), 37 °C (E), or 42 °C (F) are shown. Markers (M)
correspond to the following molecular masses: 205, 116.5, 89 and 49.5
kDa (Bio-Rad). The positions of preS2-S`-
-galactosidase (upper
arrow), DnaK (middle arrow), and GroEL (lower
arrow) are shown.
The
co-overexpression of plasmid-encoded DnaK and DnaJ significantly
enhanced the yield of active preS2-S`--galactosidase at all
temperatures (Fig. 2, A-C,
). At both 37
and 42 °C, the enzymatic activity in JM105 cells harboring pDnaK/J
was 4-6-fold higher relative to the control strain, while the
increase in activity was approximately 3-fold at 30 °C. The
synergistic effects of reduced growth temperature and overexpression of
the dnaKJ operon led to enzymatic activity of 28,000 Miller
units 2 h post-induction at 30 °C, compared with 10,000 Miller
units in the control strain (Fig. 2A). Fractionation
experiments (Fig. 2, D-F, lanes 5-7) showed
that the increase in activity was accompanied by the partitioning of
larger amounts of preS2-S`-
-galactosidase into the soluble
fraction of the cells. In contrast, the co-overproduction of the GroEL
and GroES chaperonins (Fig. 2, A-C,
) had
little effect at 37 and 42 °C relative to control cultures, but led
to a 1.5-fold increase in activity at 30 °C.
Plasmid p, a pTG10
derivative encoding the rpoH gene under control of the lac promoter, was constructed for this analysis. Co-transformation of
p
and pTBG(H+) in JM105 cells resulted in a
1.5-2-fold increase in the relative synthesis rates of DnaK, GroEL, and
other hsps, but did not significantly affect the expression of the
fusion protein as judged by pulse labeling experiments (Table 1).
At 30 and 42 °C, the presence of p
led to a
2-3-fold increase in the recovery of active
preS2-S`-
-galactosidase relative to control cells (Fig. 4A), a level intermediate between that obtained
upon overexpression of the dnaKJ or groE operons (see Fig. 2, A and C). At 37 °C, however, the
enzymatic activity in cells harboring p
was only
slightly higher than that measured in the control strain or cells
overexpressing the groE operon (Fig. 4A and
2B). Thus,
overproduction facilitates the
proper folding of preS2-S`-
-galactosidase at certain temperatures,
but remains less efficient than the direct co-overexpression of the dnaKJ operon.
In all cases, the levels of -galactosidase activity underwent a
transient increase in the first 20-40 min following neomycin
addition, but remained essentially constant at later time points (Fig. 5A). In particular, an increase of more than
3,600 Miller units of activity was observed in the strain
overexpressing the dnaKJ operon in the first 40 min following
transfer to 30 °C. Normalized videodensitometric analysis of the
corresponding whole cells (Fig. 5B), soluble (Fig. 5C), and insoluble cell fractions (Fig. 5D) showed that no new
preS2-S`-
-galactosidase was synthesized during this time and that
the intensity of the fusion protein did not vary appreciably in either
fraction. Similar results were observed in the control strain (data not
shown). Since no change in the cellular localization of
preS2-S`-
-galactosidase was observed after translational arrest,
the higher level of active enzyme recovered upon overproduction of DnaK
and DnaJ (Fig. 2) cannot be attributed to the disaggregation of
preformed inclusion bodies. This experiment also indicates that the
active fusion protein is stable in each strain at 30 °C.
The
transient increase in activity observed after neomycin addition
suggests that a fraction of non-native preS2-S`--galactosidase
chains undergo a slow progression toward the native, tetrameric state
upon temperature shift to 30 °C. To determine if the evolution of
activity was coupled to the release of the fusion protein from DnaK,
co-immunoprecipitations were performed with a fixed amount of
monoclonal anti-DnaK antibodies at various times after antibiotic
addition. While we observed an initial increase in the intensity of the
preS2-S`-
-galactosidase band following neomycin treatment, the
fraction of preS2-S`-
-galactosidase associated with DnaK
continuously decreased at later time points (Fig. 6A),
indicating that progressive release of the fusion protein from DnaK was
taking place. Taken together, these results indicate that the transient
increase in activity observed following translational arrest most
likely results from the progressive release and folding/assembly of the
fusion protein from its DnaK-bound state. The more pronounced increase
in activity in pDnaK/J transformants correlates well with the higher
numbers of DnaK
preS2-S`-
-galactosidase binary complexes in
these cells (Fig. 3).
The proper folding of overproduced polypeptides is unlikely
to be a straightforward operation within the highly concentrated and
viscous environment of the cell cytoplasm. Indeed, many recombinant
proteins form insoluble aggregates when synthesized at high levels in E. coli(1) . To date, no definitive correlation has
been established between the amino acid sequence of a protein and its
propensity to aggregate in vivo. Nevertheless, it is clear
that even small changes in primary structure can drastically affect
solubility, presumably by altering folding pathways(32) . For
instance, while a fusion protein between the HBsAg preS2 sequence and
-galactosidase (preS2-
-galactosidase) is essentially soluble
in wild type E. coli strains over a wide range of temperature (19) ,
the insertion of a short hydrophobic
sequence (a 30-amino acid segment from the S` region of HBsAg) between
these two domains makes the resulting preS2-S`-
-galactosidase
fusion protein highly susceptible to aggregation ((19) , Fig. 2). Because molecular chaperones are known to interact with
folding intermediates and facilitate their proper folding, we
systematically investigated the effect of various approaches leading to
higher intracellular concentrations of these folding factors to gain
further insight on the process of inclusion body formation in E.
coli.
While preS2-S`--galactosidase aggregates almost
quantitatively in control cells grown at 42 °C, co-overexpression
of plasmid-encoded DnaK and DnaJ led to a 4-6-fold increase in
the levels of enzymatic activity at this temperature (Fig. 2C) and the recovery of active enzyme could be
further increased by cultivating the cells at 30 or 37 °C (Fig. 2, A and B). In all cases, the higher
yields could be explained by an increased partitioning of
preS2-S`-
-galactosidase into the soluble fraction of the cells (Fig. 2, D-F). Since (i) the presence of pDnaK/J
does not significantly affect the synthesis rates of
preS2-S`-
-galactosidase compared with control cells (Fig. 1, Table 1), and (ii) the active enzyme is stable in
the presence or absence of chaperone overexpression ((19) , Fig. 5), the beneficial effects associated with overproduction
of the dnaKJ operon appear to result primarily from
facilitated folding and assembly of the fusion protein. The fact that
preS2-S`-
-galactosidase co-immunoprecipitates efficiently with
DnaK (Fig. 3) further indicates that the DnaK-DnaJ-GrpE folding
machine exerts a direct influence on the folding of the fusion protein.
DnaK has been shown to recognize short extended peptide sequences of at
least seven residues enriched in hydrophobic amino acids but lacking
acidic residues(33) . Since the S` region of the fusion protein (19) meets this criteria, the strong affinity of DnaK for
preS2-S`-
-galactosidase is consistent with the substrate
specificity of this chaperone.
The DnaK-DnaJ-GrpE folding machine
has been proposed to possess a disaggregation activity based on its
ability to renature heat-inactivated RNA polymerase in vitro(34) and the fact that DnaK can dissociate
DnaA-phospholipid aggregates into a form that is active for chromosomal
replication in vitro(35) . However, our results
indicate that even when present at high concentrations in the cell
cytoplasm, DnaK and DnaJ are unable to disaggregate preformed
preS2-S`--galactosidase inclusion bodies (Fig. 5). This
apparent discrepancy may be explained by the fact that RNA polymerase
and DnaA-phospholipid aggregates are not in a ``true''
inclusion body form (i.e. that resulting from the irreversible
association of misfolded folding intermediates(32) ), but
rather consist of improperly oligomerized polypeptides, as suggested
previously(36) . The transient increase in activity following
translational arrest and temperature downshift (Fig. 5A) is best explained by the slow folding and
oligomerization of fusion protein monomers at 30 °C, possibly
through the progressive clearing of
DnaK
DnaJ
preS2-S`-
-galactosidase ternary complexes by
GrpE, as indicated by immunoprecipitation experiments (Fig. 6).
This mechanism is consistent with the ability of DnaK and DnaJ to bind
unfolding luciferase chains generated upon shift to high temperature
and maintain them in a state capable of regaining an active
conformation upon removal of the stress both in vivo and in vitro(37) .
The overexpression of the groE operon in E. coli facilitates the production of a number
of aggregation-prone proteins in a soluble
form(9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
In the case of preS2-S`--galactosidase, however, co-overproduction
of GroEL and GroES had little effect on the recovery of enzymatic
activity at 37 and 42 °C (Fig. 2, B and C,
), although a more pronounced increase in yield was observed at
30 °C (Fig. 2A,
). The marginal effect of
chaperonin overexpression on the folding and assembly of the fusion
protein can be attributed to either a low availability of
preS2-S`-
-galactosidase folding intermediates discharged from
DnaK-DnaJ in a form that can be captured by GroEL, or the lack of
requirement of this particular substrate for the GroEL-GroES system.
Several lines of evidence support the latter possibility: (i) native
-galactosidase does not interact with GroEL in the presence of
GroES and ATP in vitro(38) , (ii) the presence of the groEL140 or groES30 mutations does not reduce the
yield of active preS2-S`-
-galactosidase relative to isogenic wild
type cells, (
)(iii) the fusion protein does not
co-immunoprecipitate with GroEL at 42 °C, and (iv) the largest
improvements in activity are observed in the strain overexpressing the dnaKJ operon, which synthesizes lower levels of chromosomal
GroEL (Fig. 1, Table 1) relative to control cells.
Since the co-overexpression of the dnaKJ operon, but not
that of the groE operon, significantly enhances the solubility
of the fusion protein without disaggregating preformed inclusion
bodies, we conclude that titration of the DnaK-DnaJ-GrpE folding
machine and the concomitant misfolding of unchaperoned fusion protein
monomers leads to the extensive aggregation of
preS2-S`--galactosidase in control cells. This view is further
supported by our observation that strains bearing mutations in the dnaK, dnaJ, and grpE genes produce lower
levels of enzymatically active preS2-S`-
-galactosidase at all
temperatures compared with their isogenic wild type(40) .
Although the effect of GrpE overexpression was not addressed in
this study, the overproduction of this protein may be less essential
due to its catalytic function in the clearing of DnaK
DnaJ
polypeptide ternary complexes(41) .
We also investigated the
effect of two indirect approaches leading to the overproduction of all
cellular molecular chaperones that are hsps. One strategy to induce hsp
synthesis in E. coli is to overexpress the rpoH gene
encoding the heat-shock sigma factor
(21, 27) . At 30 and 42 °C,
co-overproduction of plasmid-encoded
resulted in a
2-3-fold increase in the recovery of active
preS2-S`-
-galactosidase, while only a marginal improvement was
observed at 37 °C (Fig. 4A). At present, we cannot
explain this temperature-dependent behavior, since similar levels of
hsps were synthesized at all three temperatures relative to the control
cells (Table 1). Nevertheless,
overproduction
may be particularly useful for facilitating the correct folding of
other recombinant proteins that require both the DnaK-DnaJ-GrpE and
GroEL-GroES chaperone machines, or possibly other hsps, for proper
folding.
The influence of ethanol on the aggregation of
preS2-S`--galactosidase was also investigated since this solvent
is a strong elicitor of the heat-shock
response(29, 30) . Addition of 3% (v/v) ethanol to the
growth medium of control cells increased the yield of soluble
preS2-S`-
-galactosidase about 2-fold at both 30 and 42 °C (Fig. 4B), and a further synergistic effect was
observed in strains overexpressing molecular chaperones.
In
agreement with these results, Steczko et al.(39) reported that the presence of ethanol increased the yield
of active lipoxygenase L-1 by up to 40% in E. coli cells grown at 15 °C. The positive effect of ethanol can be
explained by either an increase in the synthesis rates of heat-shock
molecular chaperones or a decrease in the concentration of
aggregation-prone preS2-S`-
-galactosidase folding intermediates
brought about by the 10-20% reduction in growth rates observed in
the presence of 3% ethanol. While these mechanisms are not exclusive,
we favor the first possibility since pulse labeling experiments similar
to those shown in Fig. 1indicate that ethanol addition to
control cells increases the synthesis rates of GroEL and DnaK to levels
comparable with those obtained in the strain harboring
p
.
In addition, induction of
preS2-S`-
-galactosidase synthesis with low levels of IPTG (1 to
100 µM), which should reduce the concentration of folding
intermediates, did not increase the enzymatic activity in control cells
grown at 30 °C (data not shown). It is finally interesting to note
that the activity-temperature relationship observed in ethanol treated
cells is very similar to that obtained upon
overproduction (Fig. 4). Since the synthesis of
is solely under control of the lac promoter in p
, these data suggest that ethanol
elicits the heat-shock response in our system by stimulating the
continuous synthesis of
. Alternatively, the organic
solvent may reduce the rapid proteolytic degradation of this
protein(31) .
Overall, our results indicate that (i) the
formation of preS2-S`--galactosidase inclusion bodies, and
probably that of many other recombinant proteins, results largely from
the reduced availability of molecular chaperones, and in particular of
the DnaK-DnaJ-GrpE folding machine, and (ii) that the positive effect
of chaperone co-overexpression on the solubilization of recombinant
proteins results from facilitated partitioning toward proper protein
folding pathways, but not from the disaggregation of inclusion bodies.
The choice of a particular approach for improving the solubility of a
given protein, however, is likely to remain dependent upon the
intrinsic properties and the folding pathway of the target polypeptide.