©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Misfolding and Inclusion Body Formation in Recombinant Escherichia coli Cells Overexpressing Heat-shock Proteins (*)

(Received for publication, October 30, 1995; and in revised form, February 2, 1996)

Jeffrey G. Thomas (§) François Baneyx (¶)

From the Department of Chemical Engineering, Box 351750, University of Washington, Seattle, Washington 98195-1750

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

PreS2-S`-beta-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`-beta-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`-beta-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`-beta-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`-beta-galactosidase inclusion bodies.


INTRODUCTION

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`-beta-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`-beta-galactosidase by facilitating the proper folding and assembly of the tetrameric fusion protein.


EXPERIMENTAL PROCEDURES

Strains, Plasmids, and Media

E. coli strain JM105 (20) was used as a host strain in all experiments since it carries the Delta(lacZ)M15 deletion (no residual beta-galactosidase activity) and lacI^q mutation (increased synthesis of the LacI repressor protein). Plasmid pTBG(H+), which encodes the preS2-S`-beta-galactosidase fusion protein, has been described previously(19) . Plasmid pTG10 is a pACYC184 derived cloning vector(17) , and pGroESL (17) and pDnaK/J (A. A. Gatenby, E. I. DuPont de Nemours & Co.) are pTG10 derivatives encoding the groE and dnaKJ operons, respectively, under control of both their native and lac promoters. Plasmid p, a pTG10 derivative that encodes the rpoH gene under control of the lac promoter only, was constructed by inserting a 1.3-kilobase pair EcoRV fragment from pFN97 (21) into the SmaI site of pTG10. All transformants were obtained by the RbCl(2) method and selected at 30 °C. LB medium was supplemented with 0.2% glucose, 50 µg/ml ampicillin, and 34 µg/ml chloramphenicol. M9 minimal salts were supplemented with 0.1 mM CaCl(2), 2.5 mM MgSO(4), 0.2% glucose, 1% methionine labeling medium (Difco), and the above concentrations of antibiotics.

Protein Labeling

For protein labeling experiments, cells were grown at 30 °C to mid-exponential phase (A approx 0.4) in supplemented M9 labeling medium, induced with 1 mM IPTG, (^1)and transferred to 30, 37, or 42 °C water baths. Thirty minutes post-induction, 0.8 ml of each culture were transferred to prewarmed Eppendorf tubes containing 10 µCi of [S]methionine (Amersham Corp.). After a 2-min labeling period, the proteins were precipitated by addition of 0.8 ml of ice-cold 10% trichloroacetic acid, solubilized in reducing 1 times SDS loading buffer, and approximately 25,000 cpm/lane were loaded on 10% SDS-PAGE mini-gels. The gels were treated with EN^3HANCE (DuPont) and exposed to x-ray film. Lightly developed fluorograms were scanned using a Sharp JX-325 high resolution color scanner with transparency attachment. Densitometry analysis was performed using the NIH Image 1.56 software. The intensities of the preS2-S`-beta-galactosidase, DnaK and GroEL bands were normalized to an unknown protein that remained essentially constant in all experiments. The choice of other normalization bands had little effect on the results.

Shake Flask Cultures and Cell Fractionation

Overnight cultures grown at 30 °C in LB medium were used to inoculate triplicate shake flasks (1 to 50 dilution) containing 25 ml of supplemented LB medium. The cells were grown at 30 °C to mid-exponential phase (A approx 0.4), induced with 1 mM IPTG, and returned to 30 °C or transferred to water baths at 37 or 42 °C. Immediately before addition of IPTG, and 1 and 2 h following induction, 3-ml samples were withdrawn and the absorbance at 600 nm was recorded. For the experiments of Fig. 4B, ethanol was added to the medium prior to inoculation at a 3% (v/v) final concentration. For the experiments of Fig. 5and Fig. 6, the cultures were shifted to 42 °C following IPTG induction. After 1-h incubation at this temperature, neomycin was added to a final concentration of 200 µg/ml, the cultures were returned to 30 °C, and 3-ml aliquots were collected at the indicated times. In all cases, the cells were pelleted at 8,000 times g for 8 min, resuspended in 3 ml of 50 mM potassium phosphate monobasic, pH 6.5, and disrupted using a French press at 10,000 p.s.i. Soluble fractions were immediately clarified by centrifugation at 10,000 times g for 10 min.


Figure 4: Effect of co-overexpression (A) or ethanol addition (B) on the recovery of preS2-S`-beta-galactosidase. A, beta-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 beta-galactosidase activity assayed 1 and 2 h post-induction. B, beta-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 beta-galactosidase activity following translational arrest. A, beta-galactosidase activity versus post-addition time in JM105 cells co-transformed with pTBG(H+) and either pDnaK/J (box), pGroESL (up triangle), p (), or pTG10 in the absence (bullet) or presence (circle) 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`-beta-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`-beta-galactosidase (top arrows) and DnaK (bottom arrows) are indicated.




Figure 6: PreS2-S`-beta-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 beta-galactosidase (A) or DnaK (B). The lanes correspond to the marker (M) and samples taken 0, 20, 60, and 120 min following neomycin addition.



Enzymatic Assays

beta-Galactosidase assays were performed in triplicate on clarified soluble extracts using the chromogenic substrate o-nitrophenyl-beta-D-galactoside as described by Miller(22) . beta-Galactosidase activities are reported in Miller units (1,000 times DeltaA/A of culture/ml of culture/min of reaction)(22) . beta-Galactosidase activity due to alpha-complementation between JM105 and pTG10 was negligible (data not shown).

Electrophoresis Techniques

The samples used for electrophoresis were prepared from 30-ml LB cultures grown and induced as described above. Following IPTG induction, 10-ml aliquots were transferred to fresh flasks and incubated at either 30, 37, or 42 °C. After 1 h, 1-ml aliquots of whole cells were pelleted and resuspended in reducing 1 times SDS loading buffer, and 3-ml samples were collected and fractionated into soluble and insoluble fractions as described above. The soluble cell extracts were concentrated by a highly efficient methanol-chloroform extraction technique (23) before resuspension, while the insoluble fractions were resuspended directly in reducing 1 times SDS loading buffer. Aliquots corresponding to identical amounts of culture from whole cells and soluble and insoluble fractions were resolved on 8% SDS-PAGE mini-gels and visualized by Coomassie Blue staining.

Immunoprecipitations

For the experiments of Fig. 3, 25-ml cultures grown in LB medium were induced as described above, shifted to 42 °C, and two 1.6-ml aliquots were collected into siliconized Eppendorf tubes (Marsh) after 1 h. The cells were lysed as described (24) except that the resuspension buffer was 30 mM Tris-HCl, pH 8.0, 10 mM EDTA, 20% sucrose, 1 mg/ml lysozyme and the cell lysis buffer was 50 mM Tris-HCl, pH 8.0, 40 mM NaCl, 0.1% Tween 20. Clarified soluble extracts were incubated with gentle shaking for one hour at 4 °C with either 5 µg of murine monoclonal anti-DnaK (StressGen) or 1 µg of rabbit polyclonal anti-GroEL (Epicentre) antibodies. The samples were treated with 5 mg of swollen protein A-agarose (Sigma) and mixed at 4 °C for an additional hour. The Immunobeads were washed(24) , resuspended in 50 µl of reducing 2 times SDS loading buffer, and heated at 95 °C for 3 min. Supernatants (10 µl for Fig. 3; 5 µl for Fig. 6) were resolved on duplicate 8% SDS gels and transferred to nitrocellulose. One membrane was probed with either anti-DnaK or anti-GroEL, while the other was probed with murine polyclonal anti-beta-galactosidase (Sigma). The immunoreactive bands were detected by colorimetric reaction following incubation with alkaline-phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) or goat anti-mouse IgG (Sigma).


Figure 3: PreS2-S`-beta-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 beta-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`-beta-galactosidase to DnaK estimated by videodensitometric scanning were 1.2, 0.9, and 1.3 for the pTG10, pDnaK/J, and pGroESL transformants, respectively.




RESULTS

Effect of Chaperone Overexpression on the Synthesis of PreS2-S`-beta-galactosidase

Plasmid pTBG(H+) encodes preS2-S`-beta-galactosidase, a tripartite fusion protein consisting of the 55-residue preS2 domain and the 30-residue hydrophobic S` domain of the hepatitis B surface antigen (HBsAg) followed by the E. coli enzyme beta-galactosidase(19) . In this construct, the preS2-S`-beta-galactosidase gene is under control of the IPTG-inducible tac promoter. The beta-galactosidase activity present in JM109 cells harboring pTBG(H+) has been shown to decrease with increasing growth temperature due to the formation of preS2-S`-beta-galactosidase inclusion bodies(19) . This relationship between enzymatic activity and aggregation makes this fusion protein a useful model for the study of chaperone-assisted folding pathways in E. coli.

To investigate the influence of an increase in the cellular concentration of well characterized molecular chaperones on the expression of preS2-S`-beta-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`-beta-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`-beta-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`-beta-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.





Effect of Chaperone Overexpression on the Recovery of Enzymatically Active PreS2-S`-beta-galactosidase

The effect of molecular chaperone overproduction on the folding and assembly of the tetrameric fusion protein was investigated by enzymatic assays and fractionation experiments as follows. JM105 cells containing various plasmid combinations were grown to mid-exponential phase in LB medium at 30 °C, induced with 1 mM IPTG, and either returned to 30 °C or shifted to 37 or 42 °C. Soluble extracts were prepared 1 and 2 h post-induction and assayed for beta-galactosidase activity. In the pTG10 control strain grown at 42 °C (Fig. 2F, lane 4), almost all of the recombinant protein was found in an inclusion body form, and less than 800 Miller units of enzymatic activity were present in samples collected two hours post-induction (Fig. 2C, bullet). In agreement with previous results(19) , the activity levels increased 2-3-fold when the growth temperature was lowered to 37 °C, and 7-12-fold when the cells were grown at 30 °C (Fig. 2, A and B, bullet). A concomitant increase in the intensity of the soluble preS2-S`-beta-galactosidase band was detected by SDS-PAGE (Fig. 2, D and E, lanes 3), confirming that aggregation was reduced at lower growth temperatures.


Figure 2: Effect of the co-overexpression of specific molecular chaperones on the recovery of preS2-S`-beta-galactosidase. Upper panels, beta-galactosidase activity versus post-induction time in JM105 cells co-transformed with pTBG(H+) and either pTG10 (circle), pDnaK/J (box), or pGroESL (up triangle). Mid-exponential phase cultures induced with 1 mM IPTG were grown at 30 °C (A), 37 °C (B), or 42 °C (C), and the beta-galactosidase activity in clarified extracts was measured after 1 and 2 h. Note the differences in the beta-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`-beta-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`-beta-galactosidase at all temperatures (Fig. 2, A-C, box). 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`-beta-galactosidase into the soluble fraction of the cells. In contrast, the co-overproduction of the GroEL and GroES chaperonins (Fig. 2, A-C, up triangle) 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.

The DnaK Molecular Chaperone Forms a Stable Complex with PreS2-S`-beta-galactosidase

Since the overexpression of DnaK, but not that of GroEL, strongly favored the proper folding of preS2-S`-beta-galactosidase, we investigated the ability of the fusion protein to form a stable complex with these chaperones by co-immunoprecipitation experiments. One hour post-induction samples collected from cultures grown at 42 °C were immunoprecipitated with a fixed amount of either anti-DnaK or anti-GroEL antibodies, resolved on duplicate SDS gels, and transferred to nitrocellulose. One of the membranes was probed with antibodies against beta-galactosidase, while the duplicate was treated with either anti-DnaK or anti-GroEL antibodies. Fig. 3A shows that the fusion protein could be readily detected in all samples immunoprecipitated with anti-DnaK antibodies. The fraction of DnaK-bound preS2-S`-beta-galactosidase was slightly lower in the strain overexpressing the dnaKJ operon compared with the others, as judged by videodensitometric analysis of the blots (see Fig. 3). However, since the concentration of DnaK is approximately 3-4-fold higher in pDnaK/J transformants ( Fig. 1and Fig. 2F), 2.5-3.5-fold more preS2-S`-beta-galactosidase is complexed with DnaK in these cells relative to those harboring pTG10 or pGroESL. We were unable to detect stable complexes between preS2-S`-beta-galactosidase and GroEL, even in the strain overproducing the groE operon (data not shown). Thus, preS2-S`-beta-galactosidase appears to interact strongly with DnaK, but has little affinity for GroEL under our experimental conditions.

Effect of Overproduction on the Yield of Active PreS2-S`-beta-galactosidase

Since the DnaK-DnaJ-GrpE and GroEL-GroES hsps are believed to act sequentially in the folding of many newly synthesized polypeptides(2, 3, 4, 25, 26) , we sought to determine whether the overexpression of both operons would synergistically increase the yields of active preS2-S`-beta-galactosidase. In E. coli, the high level transcription of hsps is performed by E complexes between the RNA polymerase core enzyme and the heat-shock transcription factor, (21, 27, 28) . We therefore overexpressed in order to raise the intracellular concentration of the DnaK-DnaJ-GrpE and GroEL-GroES chaperones, as well as that of other chromosomally encoded heat-shock molecular chaperones. Although certain proteases (e.g. Lon and Clp) are also transcribed by E, preS2-S`-beta-galactosidase is not a target for rapid degradation in the cell (19; see below).

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`-beta-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`-beta-galactosidase at certain temperatures, but remains less efficient than the direct co-overexpression of the dnaKJ operon.

Effect of Ethanol Addition

In addition to high temperatures, a variety of external stimuli can induce the heat-shock response in E. coli. These include bacteriophage infection, UV irradiation, and the presence of amino acid analogs, heavy metals, or organic solvents in the growth medium(29, 30) . To determine if a stress response elicited by growth in the presence of ethanol could improve the recovery of active preS2-S`-beta-galactosidase, JM105 cells harboring pTG10 and pTBG(H+) were grown in LB medium supplemented with various amounts of ethanol and assayed for beta-galactosidase activity. An ethanol concentration of 3% (v/v) was selected, since lower concentrations had little effect on the recovery of enzymatic activity, (^2)and higher concentrations significantly reduce the growth rate of E. coli(30) . Under these conditions, a 2- to 3-fold increase in the recovery of enzymatically active preS2-S`-beta-galactosidase was observed at 30 and 42 °C, but little improvement was detected at 37 °C (Fig. 4B). This pattern is almost identical to that observed upon overproduction of plasmid-encoded (Fig. 4A). When ethanol was added at the time of IPTG induction, the overall yields of enzymatic activity were reproducibly 10-20% lower than when addition was performed prior to inoculation (data not shown). Whether this slight reduction in yields resulted from the delay necessary to reach a new basal level of hsps (30) was not investigated.

Molecular Chaperones Do Not Disaggregate Preformed PreS2-S`-beta-galactosidase Inclusion Bodies

The improved solubilization of preS2-S`-beta-galactosidase associated with the overproduction of molecular chaperones can be explained by two different, but not necessarily exclusive, mechanisms. The first possibility is that the high level expression of preS2-S`-beta-galactosidase in the control strain results in a partial titration of the available pool of chaperones, leading to the misfolding and subsequent aggregation of the unchaperoned fraction of the recombinant protein. Under these conditions, chaperone co-overproduction would restore adequate levels of folding factors and thereby promote proper folding. The second possibility is that at high intracellular concentrations certain molecular chaperones (most notably DnaK and DnaJ) are able to disaggregate preS2-S`-beta-galactosidase inclusion bodies. In an effort to distinguish between the above mechanisms, the following experiment was performed. JM105 cells harboring various plasmid combinations, and control cells cultured in the presence or absence of 3% ethanol were grown to mid-exponential phase at 30 °C, induced with IPTG, and shifted to 42 °C for 1 h to promote the formation of inclusion bodies. A high concentration of neomycin (200 µg/ml) was added to the cultures to stop protein synthesis. The flasks were returned to 30 °C to facilitate the proper folding of preS2-S`-beta-galactosidase chains, and beta-galactosidase activity was measured at the indicated time points. Cell growth was efficiently inhibited for the duration of the experiment as determined by turbidity measurements (data not shown).

In all cases, the levels of beta-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`-beta-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`-beta-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`-beta-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`-beta-galactosidase band following neomycin treatment, the fraction of preS2-S`-beta-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 DnaKbulletpreS2-S`-beta-galactosidase binary complexes in these cells (Fig. 3).


DISCUSSION

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 beta-galactosidase (preS2-beta-galactosidase) is essentially soluble in wild type E. coli strains over a wide range of temperature (19) ,^2 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`-beta-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`-beta-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`-beta-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`-beta-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`-beta-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`-beta-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`-beta-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 DnaKbulletDnaJbulletpreS2-S`-beta-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`-beta-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, up triangle), although a more pronounced increase in yield was observed at 30 °C (Fig. 2A, up triangle). 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`-beta-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 beta-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`-beta-galactosidase relative to isogenic wild type cells, (^3)(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`-beta-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`-beta-galactosidase at all temperatures compared with their isogenic wild type(40) .^3 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 DnaKbulletDnaJbullet 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`-beta-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`-beta-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`-beta-galactosidase about 2-fold at both 30 and 42 °C (Fig. 4B), and a further synergistic effect was observed in strains overexpressing molecular chaperones.^2 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`-beta-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.^2 In addition, induction of preS2-S`-beta-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`-beta-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.


FOOTNOTES

*
This work was supported in part by the Whitaker Foundation and National Science Foundation Award BES-9501212. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported through National Institutes of Health Biotechnology Training Grant GM088437 to the University of Washington.

To whom correspondence should be addressed. Tel.: 206-685-7659; Fax: 206-685-3451; baneyx{at}cheme.washington.edu.

(^1)
The abbreviations used are: IPTG, isopropyl-beta-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. G. Thomas and F. Baneyx, manuscripts in preparation.

(^3)
J. G. Thomas and F. Baneyx, unpublished data.


ACKNOWLEDGEMENTS

We thank Anthony Gatenby, Carol Gross, and Myeong-Hee Yu for their generous gifts of bacterial plasmids. We are grateful to Amanda Ayling and Jess Vasina for reading this manuscript.


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