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Applied and Environmental Microbiology, December 1998, p. 4891-4896, Vol. 64, No. 12
Molecular Biology
Program1 and
Department of Chemical
Engineering,3 University of Texas, Austin, Texas
78712, and
Department of Cell Culture and Fermentation Research
and Development, Genentech, Inc., South San Francisco, California
940802
Received 25 June 1998/Accepted 1 September 1998
The formation of native disulfide bonds in complex eukaryotic
proteins expressed in Escherichia coli is extremely
inefficient. Tissue plasminogen activator (tPA) is a very important
thrombolytic agent with 17 disulfides, and despite numerous attempts,
its expression in an active form in bacteria has not been reported. To
achieve the production of active tPA in E. coli, we have
investigated the effect of cooverexpressing native (DsbA and DsbC) or
heterologous (rat and yeast protein disulfide isomerases) cysteine
oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an
enzyme which catalyzes disulfide bond isomerization in the periplasm,
was found to dramatically increase the formation of active tPA both in
shake flasks and in fermentors. The active protein was purified with an
overall yield of 25% by using three affinity steps with, in sequence,
lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 µg of
tPA with a specific activity nearly identical to that of the authentic
protein can be obtained per liter of culture in a high-cell-density
fermentation. Thus, heterologous proteins as complex as tPA may be
produced in an active form in bacteria in amounts suitable for
structure-function studies. In addition, these results suggest the
feasibility of commercial production of extremely complex proteins in
E. coli without the need for in vitro refolding.
In Escherichia coli and
other gram-negative bacteria, disulfide bonds form in the periplasmic
space, a compartment topologically equivalent to the endoplasmic
reticulum but much more oxidizing (35, 36). The formation of
disulfide bonds in E. coli is catalyzed by a complex
machinery involving at least two soluble, periplasmic cysteine
oxidoreductases (DsbA and DsbC), two membrane-bound enzymes (DsbB and
DsbD), and cytoplasmic proteins (3, 20, 24, 25, 30, 38, 39).
In vitro, DsbA is a potent catalyst of protein cysteine oxidation,
whereas DsbC exhibits disulfide isomerase activity (30, 39).
The membrane proteins DsbB and DsbD appear to be responsible for
maintaining DsbA and DsbC, respectively, in the proper oxidative state
for optimal function.
Extensive studies over the last 15 years have demonstrated that
multidisulfide proteins generally do not fold correctly in bacteria and
accumulate largely in a misfolded form. Examples of commercially
important proteins that cannot be produced in active form when secreted
in the periplasm include enzymes such as tissue plasminogen activator
(tPA) and kallikreins, the protease inhibitors, and various growth
factors (3, 8, 10, 14, 18, 22, 26, 29, 32, 37). The
production of these and other proteins with three or more disulfides is
complicated and has to rely on either expression in higher eukaryotes
that provide a favorable environment for the formation of disulfide
bonds or refolding from inclusion bodies (8, 14).
Human tPA best exemplifies the challenges associated with the
production of complex proteins in E. coli. It is a
527-amino-acid serine protease with 35 cysteine residues that
participate in the formation of 17 disulfide bonds. tPA is comprised of
five distinct structural domains: a finger region, an epidermal growth factor-like subdomain, two kringle domains, and finally, the catalytic domain. The function of tPA is to convert the zymogen plasminogen to
plasmin, a serine protease of broad specificity that degrades the
fibrin network in thrombi (34). The activity of tPA is
markedly enhanced by binding to fibrin, a property of great
physiological importance, as tPA is less likely than other proteases to
cause inadvertent plasmin activation and internal bleeding. Binding to
fibrin and modulation of the proteolytic activity are primarily mediated by the finger domain and the kringle 2 domain, respectively (21).
tPA secreted in the periplasm of E. coli is misfolded and
completely inactive. Attempts to produce active tPA in
Saccharomyces cerevisiae or in insect cells have been
frustrated by problems due to hyperglycosylation, poor export, and
improper folding (7, 27). Here we demonstrate that
engineering the disulfide bond machinery of the cell through the
high-level expression of DsbC allows the production of active
full-length tPA. After purification from a high-cell-density
fermentation, 180 µg of protein per liter with a specific activity
nearly identical to the authentic tPA can be obtained, with a yield
corresponding to 25% of the active material in cell lysates. To the
best of our knowledge this is the first time full-length tPA has been
expressed in active form in bacteria in significant amounts, and it
bodes well for the production of other complex multidisulfide proteins
in bacteria.
Vector construction.
pAP-stII-tPA is a pBR322 derivative
containing the tPA gene fused in frame to the heat-stable enterotoxin
(stII) leader peptide and placed downstream from the phoA
promoter (Genentech plasmid collection). pBAD-stII-tPA was constructed
by amplifying the tPA gene from pAP-stII-tPA with the primers
5'-CGCGCGATATCATGAAAAAGAATATCGCATTTCTTCTT-3' and
5'-TCTACGCAAAGCTTTCACGCTGGTCGCATGTTGTCA-3'. The PCR product was digested with EcoRV and HindIII and
subcloned into pBAD33 (11) (kindly provided by Jon Beckwith,
Harvard Medical School). pTrc-stII-tPA184 is a pACYC184 derivative in
which the tPA gene with the stII leader peptide is placed downstream
from the strong trc promoter from pTrc-99A (Pharmacia,
Uppsala, Sweden).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Expression of Active Human Tissue-Type Plasminogen
Activator in Escherichia coli
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Expression of tPA in shaker flasks.
To evaluate tPA
expression in shaker flasks, E. coli SF110 (F
lacX74 galE galK thi rpsL phoA degP41 ompT)
(2) cells transformed with the appropriate plasmids were
grown in Luria-Bertani medium at 37°C supplemented with ampicillin
(100 µg/ml), chloramphenicol (20 µg/ml), or kanamycin (40 µg/ml)
as necessary. Synthesis of DsbA, DsbC, or DsbA-DsbC in cells bearing
pSE380dsbA, pSE420dsbC, or pSE380dsbAC, respectively, was induced by
the addition of IPTG (isopropyl-
-D-galactopyranoside; 2 mM, final concentration) when the culture optical density at 600 nm
(OD600) reached between 0.8 and 1.0. Synthesis of rPDI in
cells transformed with pLppsOmpArPDI was induced by the addition of
IPTG to 0.5 mM at a culture OD600 of ca. 0.6. Expression of
tPA from the PBAD promoter was induced 30 min
after the addition of IPTG by adding arabinose to a final concentration
of 0.2% (wt/vol).
Expression of tPA in fermentors.
For inoculum preparation,
1.0 ml of frozen SF110(pBAD-stII-tPA/pSE420dsbC) cells was added to 500 ml of Luria-Bertani medium containing 40 µg of ampicillin and 30 µg
of chloramphenicol per ml. The culture was grown in a 2-liter flask for
10 h, reaching an OD550 of ca. 3.0. The inoculum
culture was added to approximately 6.5 liters of mineral salts medium
containing 1.2% digested casein, 1.2% yeast extract, and 1.5 g
of isoleucine and 1 g of glucose per liter in a 15-liter Biolafite
fermentor. The fermentor was operated at 37°C and 1,000 rpm, with 10 standard liters per min of aeration and a 0.3-bar back pressure to
deliver an oxygen transfer rate of approximately 3.0 mmol/liter-min.
When the initial glucose was depleted, a concentrated glucose solution
was added to maintain a growth rate of 0.32 h1 until the
dissolved oxygen concentration (DO2) reached 30% of air
saturation. At that point glucose feeding was adjusted to maintain a
DO2 of 30%. At an OD550 of 25, a feed
consisting of 13.5% digested casein and 6.5% yeast extract was added
at 0.5 ml/min. When the OD550 reached 80, IPTG was added at
a concentration of 0.05 mM, and 30 min later arabinose was added to
0.1% (wt/vol). When respiration poisoning caused the DO2
to rise, the glucose feed rate was lowered to avoid excessive acetate accumulation.
tPA purification. tPA was purified from cell extracts by sequential L-lysine-Sepharose and Erythrina inhibitor-Sepharose affinity chromatography (26) as described below. Cell paste was resuspended in buffer A (50 mM Tris-HCl, pH 7.5; 5mM EDTA; 0.1% Tween 80). The cells were lysed by sonication on ice. The cell lysates were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was loaded onto an L-lysine-Sepharose column (Pharmacia) preequilibrated with buffer A. The column was washed with 8 column volumes of buffer A, followed by 8 volumes of buffer A containing 0.1 M NaCl. tPA was eluted with buffer A containing 0.5 M NaCl and 0.2 M lysine. The eluant from the L-lysine-Sepharose column was loaded onto an Erythrina caffra inhibitor-Sepharose column prepared by coupling E. caffra inhibitor (ETI; American Diagnostica, Greenwich, Conn.) to cyanogen bromide-activated Sepharose 4B (Pharmacia). The column was washed with 4 column volumes of buffer B (0.5 M NH4HCO3, 0.1% Triton X-100), 4 volumes of buffer C (0.05 M NaH2PO4, pH 7.3), and 4 volumes of buffer C with 0.1 M KSCN. The column was eluted with buffer C containing 0.9 M KSCN. Then, 1-ml aliquots of the eluant from the ETI-Sepharose column were incubated with 100 µl of iminodiacetic acid-Sepharose (Pharmacia) that had been preequilibrated with ZnCl2 at 4°C for 30 min. Incubation of the tPA with Zn-Sepharose was carried out at 4°C for 2 h. The Sepharose was precipitated by centrifugation, washed with buffer E (0.05 M NaH2PO4, 0.5 M NaCl, 0.05% Tween 80 [pH 7.3]), and eluted with buffer E containing 0.05 M imidazole.
General methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels and Western blotting were performed according to standard techniques (1). tPA quantitation was performed by the Imubind total tPA stripwell enzyme-linked immunosorbent assay (American Diagnotica). Glycosylated, single-chain tPA (Sigma Chemical Co., St. Louis, Mo.) was used as a standard.
Assays of tPA activity.
Fibrin plates were prepared
essentially as described previously (23) except that 25 µg
of tetracycline per ml was added to prevent bacterial growth. The rate
of plasminogen activation was determined by using the Spectrolyse
tPA/PAI activity assay kit from American Diagnostica and a total assay
volume of 295 µl. Zymography was performed as described by Heussen
and Dowdle (12) with the following modifications: SDS-12%
polyacrylamide gels were copolymerized with 0.1% (wt/vol) 
-casein
and 10 µg of plasminogen per ml (Calbiochem, La Jolla, Calif.). After
electrophoresis at 4°C, the gels were washed with 2.5% Triton X-100
for 1 h to remove the SDS, washed with distilled water
exhaustively to remove the Triton X-100, incubated in 0.1 M glycine
buffer (pH 8.3) for 5 h, and then stained.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Optimization of tPA expression in shaker flasks. A cDNA encoding the complete amino acid sequence (amino acids 1 to 527) of the human tPA was fused in frame to the stII leader peptide, which had been shown earlier to be useful for the periplasmic expression of a variety of proteins (5). The stII-tPA gene was placed downstream from three different promoters: the arabinose-inducible promoter PBAD(11), the phoA promoter under which the tPA gene is transcribed constitutively at a moderate level in phoT mutant cells grown in high-phosphate medium, and, finally, the IPTG-inducible trc promoter. The respective expression vectors were transformed into several different E. coli strains. In every case, the expression level of tPA was low and could not be visualized by SDS-PAGE; a band corresponding to full-length tPA could only be detected by Western blotting, although, as might be expected, the intensity of the tPA band varied depending on the promoter and strain background. However, no fibrinolytic activity could be detected on the fibrin plates. It should be noted that less than 10 pg of purified tPA can be detected with this assay. Cell fractionation demonstrated that the majority (>70%) of the total tPA accumulates in a soluble yet inactive form.
Under certain conditions, the coexpression of the rPDI secreted in the periplasmic space increased the expression of bovine pancreatic trypsin inhibitor (BPTI), a small eukaryotic protein with three disulfide bonds, by up to 15-fold (28). To test the effect of rPDI coexpression on the formation of active tPA, E. coli SF110 (ompT degP) was transformed with the plasmid pLppsOmpArPDI, which contains the rPDI fused to the OmpA leader peptide downstream from the strong lpp-lac promoter. rPDI is secreted efficiently into the periplasmic space, and upon addition of IPTG it becomes the most abundant protein in the soluble fraction (see Fig. 2). A small yet detectable fibrin clearance was observed when stII-tPA was expressed from the PBAD promoter after induction of rPDI expression (Fig. 1A). However, active tPA was barely detectable in a quantitative assay that measures the rate of activation of plasminogen with a chromogenic substrate (Fig. 1B). The low levels of tPA activity were not due to poor expression of the stII-tPA. Even though coexpression of rPDI reduced the accumulation of stII-tPA relative to control cells (pBAD-stII-tPA plasmid alone), a band corresponding to mature tPA was readily visible by Western blotting (Fig. 1D). In other experiments, coexpression of yeast PDI as a secreted protein in E. coli did not facilitate the formation of active tPA either, even though it was shown to be functional in protein oxidation (data not shown).
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tPA expression in high-cell-density fermentations.
SF110
(pBAD-stII-tPA+pSE420dsbC) cells were grown in a 10-liter fermentor in
a synthetic medium supplemented with casein amino acids. The growth
rate was maintained at 0.32 h1 by controlling the
addition of glucose until a DO2 level of 30% was reached.
Preliminary experiments revealed that vigorous induction (with 2 mM
IPTG) of DsbC expression, followed by induction of tPA expression, led
to a dramatic reduction in the oxygen uptake rate after about 1.5 h (Fig. 3). Growth ceased, and a slow
decline in the OD of the culture soon followed. Therefore, a lower
concentration of IPTG (0.05 mM) was used to minimize the detrimental
effects of DsbC overexpression. IPTG was added when the culture reached an OD550 of around 80; this was followed by a bolus of
arabinose 30 min later. Under these conditions, the oxygen uptake rate
remained constant for 3.5 h and then began to decline. The maximum
specific tPA activity was attained 2.5 h after induction (Fig.
3) and then started to decrease, in part
because the inducer, arabinose, is catabolized by strain SF110. The
peak specific activity obtained in the fermentor was essentially
identical to that in shaker flasks. Approximately 25 g of cell
protein was obtained per liter of culture.
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tPA purification. Clarified cell lysates were loaded onto an L-lysine-Sepharose column. L-Lysine binds tightly and specifically to the kringle 2 domain of tPA assuming, of course, that it is correctly folded. About 50% of the total tPA activity that was loaded onto the L-lysine-Sepharose column was retained even after it was washed with 8 bed volumes of NaCl-containing buffer. The bound tPA was eluted with 0.2 M L-lysine. The eluant from the L-lysine-Sepharose affinity chromatography step was loaded onto a second column containing immobilized E. caffra inhibitor, a protein that binds to the tPA protease domain with a very high affinity (13). Active tPA was eluted with 0.9 M KSCN and was shown to have a specific activity of 373 IU/µg, a value nearly identical to that of the authentic glycosylated protein from mammalian cells (400 IU/µg). The bacterial tPA became bound irreversibly to ultrafiltration membranes and therefore could not be concentrated in this manner from the ETI column eluant. For this purpose and also to remove two contaminating E. coli proteins of 35 kDa, the active fraction from the second column was mixed with Zn-Sepharose beads. tPA was bound to Zn-Sepharose quantitatively and could be eluted with buffer containing 50 mM imidazole. The resulting protein was more than 90% pure, as determined by SDS-PAGE and silver staining (Fig. 4).
The yield of purified tPA was 25% of the total activity in the starting material. Interestingly, although the bacterial tPA bound quantitatively to ETI, it could be eluted from the resin with a lower concentration of KSCN relative to the glycosylated, two-chain protein (0.9 and 1.6 M KSCN, respectively). Consequently, it appears that glycosylation affects the equilibrium dissociation constant for ETI. After ETI chromatography, 14 µg of high-specific-activity tPA was obtained from 2 g of cell protein. Thus, approximately 180 µg of purified tPA per liter can be obtained on the basis of the amount of cell protein produced per liter of culture by high-cell-density fermentation (25 g/liter).| |
DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have identified conditions that allow the production of significant amounts of active, full-length tPA in E. coli. Normally, the formation of disulfide bonds takes place in the periplasmic space. In this study tPA was fused to the stII leader peptide, which was efficient in directing the mature protein into the periplasmic space. The stII leader peptide does not appear to be unique, and a similar yield of proteolytically active tPA was obtained when the tPA gene was fused to the OmpA leader peptide (29a). As discussed in Results and as shown in Fig. 1, in the absence of cysteine oxidoreductase overexpression, active tPA is virtually undetectable. However, a >100-fold increase in the specific rate of fibrinolysis in cell extracts is observed in cultures coexpressing DsbC. To the best of our knowledge, this dramatic increase in the production of active tPA represents by far the most striking improvement in the folding of a foreign protein ever obtained via the coexpression of foldases (8).
The rate-limiting step in the oxidative folding of eukaryotic proteins in the periplasmic space appears to be the isomerization of mismatched disulfides (29). Reduced DsbC has been shown to be an efficient catalyst of disulfide bond isomerization in vitro (6). Moreover, recent studies have shown that DsbC is maintained primarily in a reduced state in vivo, suggesting that its primary role in the cell is the catalysis of disulfide isomerization (17, 31, 33). The dramatic increase in the folding of tPA in cells coexpressing DsbC is consistent with this hypothesis. We believe that the high level of DsbC increased the disulfide isomerization capacity of the periplasmic space, thus facilitating the rearrangement of incorrect disulfides in nascent tPA. Indeed, Joly et al. (16) have shown that DsbC accumulated mostly in its partially reduced form when coexpressed in a fermentor together with insulin-like growth factor 1 (IGF-1). In this case, overexpression of DsbC increased the total yield of IGF-1 but not the amount of soluble active protein. However, this result may be a consequence of the very high level of IGF-1 overexpression (7.3 g of IGF-1 per liter of fermentation broth).
An alternative explanation for our results is that DsbC did not participate directly in the folding of tPA. Instead, its overexpression simply resulted in a higher concentration of protein thiols, thereby altering the redox potential of the periplasm to favor the formation of correct disulfide bonds. However, this hypothesis can be ruled out since neither the overexpression of other cysteine oxidoreductases nor the manipulation of the redox potential of the periplasm through the addition of GSH or GSSG had an effect similar to that of DsbC.
A low level of active tPA was detected in cells coexpressing DsbA, but this effect could not be further enhanced by the addition of reduced or oxidized glutathione. Chromosomally expressed DsbA is found almost exclusively in the oxidized form and is a potent catalyst of disulfide bond formation (19). It is possible that when DsbA is overexpressed, a fraction of the protein fails to be oxidized by DsbB and, instead, is present in the reduced form that can catalyze disulfide bond isomerization (16, 18). This activity of the reduced DsbA may in turn be responsible for the small levels of active tPA. Overexpression of rPDI has been shown to increase the yield of small heterologous proteins (15, 28), but it had a very minor effect on tPA. This may be because rPDI functions as a protein thiol oxidase in the bacterial periplasmic space but was shown to be rather ineffective in catalyzing the rate-limiting isomerization in BPTI in the periplasmic space of E. coli (28).
The highest level of active tPA was observed when the synthesis of DsbC
from a trc promoter was induced first, followed by the
induction of tPA expression 30 min later. Interestingly, high-level induction of DsbC, but not DsbA, rPDI, or tPA alone, was found to be
particularly toxic, resulting in cessation of growth within 3 to 4 h after induction. The precursor form of DsbC was found to accumulate
in the cell, raising the possibility that the observed toxicity is
linked to the saturation of the protein translocation machinery. To
obtain the maximum tPA specific activity in a high-cell-density fermentation, it was necessary to use a low concentration of IPTG, which allowed the production phase to be prolonged. Also, maintaining a
low growth rate through the slow feeding of glucose was found to be
essential in order to attain a high cell density in the fermentor.
SF110 was found to be a prolific producer of acetate, which accumulated
to inhibitory levels when glucose feeding was adjusted to control the
growth rate above 0.65 h1.
The above observations suggest a number of ways in which the expression of active, full-length tPA can be increased further. First, in the present study the level of accumulation of tPA was low regardless of the promoter used. A higher level of tPA synthesis may be beneficial and could be obtained by optimizing translation initiation and by substitution of rare codons in the tPA gene (4). Second, since DsbC overexpression has been found to inhibit growth, conditions that inhibit DsbC toxicity will have to be identified to prolong the production phase and help achieve even higher cell densities. Finally, it may be possible to further enhance the effect of DsbC by manipulating its interaction with DsbD (DipZ) or perhaps by isolating DsbC mutants with higher activity towards tPA.
The ability to produce substantial amounts of a heterologous protein as complex as tPA in E. coli bodes well for the expression of other complex eukaryotic proteins both for commercial purposes and for structure-function studies. When protein glycosylation is not essential, expression in bacteria is clearly advantageous in terms of both cost and simplicity.
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ACKNOWLEDGMENTS |
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We are grateful to S. Raina, J. Beckwith, J. Bardwell, and K. De Sutter for gifts of plasmids. We also thank Susan Leung and the Genentech Fermentation Operations Department for assistance with the fermentation experiments and, finally, Paul Bessette, Jose Cotto, and John Joly for reading the manuscript.
Financial support was provided by Genentech and by NSF grant BES-9634036 to J.R.S. and G.G. and by NIH grant GM47520 to G.G.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Chemical Engineering, University of Texas at Austin, College of Engineering, Austin, TX 78712-1062. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg{at}che.utexas.edu.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Applied and Environmental Microbiology, December 1998, p. 4891-4896, Vol. 64, No. 12
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