APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1998, p. 4891-4896 (cid:9)
`0099-2240/98/$04.00+0
`Copyright ' 1998, American Society for Microbiology. All Rights Reserved.
`
`Vol. 64, No. 12
`
`Expression of Active Human Tissue-Type Plasminogen
`Activator in Escherichia coli
`JI QIU, 1 JAMES R. SWARTZ, 2 AND GEORGE GEORGIOU1,a*
`Molecular Biology Program’ 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 94080 2
`
`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 cooverexpres sing 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 E,ythrina cajfra inhibitor, and Zn-Sepharose resins. After purifica-
`tion, approximately 180 Ftg 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, disul-
`fide bonds form in the periplasmic space, a compartment to-
`pologically 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 main-
`taining 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,
`
`* 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@che.utexas.edu.
`
`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 acti-
`vation 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 Sac-
`charomyces cerevisiae or in insect cells have been frustrated by
`problems due to hyperglycosylation, poor export, and im-
`proper 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 jig of protein per liter with a specific activity nearly iden-
`tical to the authentic tPA can be obtained, with a yield corre-
`sponding 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.
`
`MATERIALS AND METHODS
`
`Vector construction, pAP-stII-tPA is a pBR322 derivative containing the tPA
`gene fused in frame to the heat-stable enterotoxin (stil) 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 ’-CGCGCGATATCATGAAAAAGAATATCGCATYI’C’FF
`CYr-3’ and 5 ’-TCTACGCAAAOCJTTCACGCTGGTCGCATGTTGTCA-3’.
`The PCR product was digested with EcoRV and HtndIII and suhcloned into
`pBAD33 (11) (kindly provided by Jon Beckwith, Harvard Medical School).
`pTrc-stII-IPAI84 is a pACYC184 derivative in which the tPA gene with the stil
`leader peptide is placed downstream from the strong trc promoter from pTrc-
`99A (Pharmscia, Uppsala, Sweden).
`pLppsompArPDl expressing the rat protein disulfide isomerase (rPDI) gene
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`APPL. ENVIRON. MICROBIOL.
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`from the /pp-lac promoter was provided by Kristine Dc Sutter, University of
`Gent. pSE380dsbA and pSE420dsbC are derivatives of pSE380 and pSE420
`(Invitrogen, Carlsbad, Calif.) expressing the dsbA and dsbC genes, respectively,
`and were provided by Satish Raina, Centre Medical Universitaire, Geneva,
`Switzerland. The vector pSE380dsbAC was constructed as follows. pSE420dsbC
`was digested with M/ut, blunt ended by using mung bean nuclease, and further
`digested with Hindtll. Subsequently, the fragment containing the dsbC gene
`trc promoter was isolated and ligated with Soil!
`downstream from the
`Hindltl-treated pSE380dsbA to yield pSE380dsbAC.
`Expression of tPA in shaker flasks. To evaluate tPA expression in shaker
`flasks, E. co/i SF110 (F - .S/acX74 galE ga/K 1/it rpsL 2phoA degP41 iornpT) (2)
`cells transformed with the appropriate plasmids were grown in Luria-Bertani
`medium at 37’C supplemented with ampiciltin (100 wg/ml), chloramphenicol (20
`or kanamycin (40 Vg/ml) as necessary. Synthesis of DsbA, DsbC, or
`DshA-DshC in cells bearing pSE380dsbA, pSE420dsbC, or pSE380dsbAC, re-
`spectively, was induced by the addition of IPTG (isopropyl--o-gatactopyrano-
`side; 2 mM, final concentration) when the culture optical density at 600 nm
`(0D 600) reached between 0.8 and tO. Synthesis of rPDt in cells transformed with
`pLppsOmpArPDl was induced by the addition of IPTG to 0.5 mM at a culture
`OD,,,,, of ca. 0.6. Expression of tPA from the BRAD promoter was induced 30 mm
`after the addition of tPTG by adding arahinoae to a final concentration of 0.2%
`(wt/vol).
`After induction with arabinose, cultures were grown for an additional 3 h and
`harvested by centrifugation. The cells were then resuspended in 0.1 M Tris-HCI
`(pH 8.5) and lysed with a French pressure cell operated at 2,000 lb/in 2. Subse-
`quently, the cell lysates were centrifuged at 12,000 X g for 10 twin at 4C to
`separate the soluble and insoluble fractions.
`Expression of IPA in fermentors. For inoculum preparation, 1.0 ml of frozen
`SF1 lO(pBAD-sttt-tPA/pSE42OdsbC) cells was added to 500 ml of Luria-Bertani
`medium containing 40 gg of ampiciltin and 30 gg of chloramphenicol per ml.
`The culture was grown in a 2-liter flask for 10 h, reaching an OD, ) of ca. 3.0.
`The inoculurn culture was added to approximately 6.5 titers of mineral salts
`medium containing 1.2% digested casein, 1.2% yeast extract, and 1.5 g of iso-
`teucine and t g of glucose per titer in a 15-liter Biolafite fermentor. The fer-
`mentor 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 mmoliliter-min. When the initial glucose was depleted, a
`concentrated glucose solution was added to maintain a growth rate of 0.32 h- ,
`until the dissolved oxygen concentration (D0 2) reached 30% of air saturation.
`At that point glucose feeding was adjusted to maintain a DO, of 30%. At an
`OD55 of 25, a feed consisting of 13,5% digested casein and 6.5% yeast extract
`was added at 0.5 ml/min. When the OD 550 reached 80, IPTG was added at a
`concentration of 0.05 mM, and 30 min later arabinose was added to 0.1%
`(wt/vot). When respiration poisoning caused the DO, to rise, the glucose feed
`rate was towered to avoid excessive acetate accumulation.
`tPA purification. tPA was purified from cell extracts by sequential L-lysine-
`Sepharose and E,ythrina inhibitor-Sepharose affinity chromatography (26) as
`described below. Cell paste was resuspended in buffer A (50 mM Tris-HCI, pH
`7.5; 5mM EDTA; 0.1% Tween 80). The cells were lysed by sonication on ice. The
`cell tysates were centrifuged at 12,000 x g for 15 min at 4 C 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 02 M lysine. The eluant from the L-lysine-Sepharose
`column was loaded Onto an Erythrina caffra inhibitor-Sepharose column pre-
`pared by coupling E. caJj5-a 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 NH 4HCO 3, 0.1% Triton
`X-tOO), 4 volumes of buffer C (0.05 M NaH 2PO4, 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
`i.LI of iminodiacetic acid-Sepharose (Pharmacia) that
`were incubated with 100
`had been preequitibrated with ZnCl 2 at 4C for 30 mm. Incubation of the IPA
`with Zn-Sepharose was carried Out at 4C for 2 h. The Sepharose was precipi-
`tated by centrifugation, washed with buffer F (0.05 M NaH,PO 4, 0.5 M NaCl,
`0.05% Tween 80 [pH 7.3}), and eluted with buffer E containing 0.05 M imidazote.
`General methods. Sodium dodecyl sulfate-polyacrytamide get electrophoresis
`(SDS-PAGE) on 12% potyacrylamide gets and Western blotting were performed
`according to standard techniques (1). tPA quantitation was performed by the
`Imubind total tPA stripwetl enzyme-linked immunosorbent assay (American
`Diagnotica). Glycosylated, single-chain tPA (Sigma Chemical Co., St. Louis,
`Mo.) was used as a standard.
`Assays of 1PA activity. Fibrin plates were prepared essentially as described
`previously (23) except that 25 i.g of tetracycline per ml was added to prevent
`bacterial growth. The rate of plasminogen activation was determined by using the
`Spectrotyse tP.AJPAI activity assay kit from American Diagnostica and a total
`l. Zymography was performed as described by Heussen and
`assay volume of 295
`Dowdle (12) with the following modifications: SDS-12% polyacrytamide gets
`were copolymerized with 0.1% (wt/vol) ce-casein and 10 p.g of plasminogen per
`ml (Calbiochem, La Jolla, Calif.). After electrophoresis at 4 C 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.
`
`RESULTS
`
`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 stil leader
`peptide, which had been shown earlier to be useful for the
`periplasmic expression of a variety of proteins (5). The stil-
`tPA gene was placed downstream from three different promot-
`BAD(11) the phoA pro-
`ers: the arabinose -inducible promoter
`moter 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 in-
`tensity 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 se-
`creted in the periplasmic space increased the expression of
`bovine pancreatic tlypsin 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 pLppsOmpArPDl, which contains the rPDI fused to
`lpp-lac
`the OmpA leader peptide downstream from the strong
`promoter. rPDI is secreted efficiently into the periplasmic
`space, and upon addition of IPTG it becomes the most abun-
`dant protein in the soluble fraction (see Fig. 2). A small yet
`detectable fibrin clearance was observed when stII-tPA was
`expressed from the B4fl promoter after induction of rPDI
`expression (Fig. 1A). However, active tPA was barely detect-
`able in a quantitative assay that measures the rate of activation
`of plasminogen with a chromogenic substrate (Fig. 113). 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 experi-
`ments, 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).
`In contrast, the coexpression of high levels of DsbC in cells
`grown in either rich or minimal medium resulted in a dramatic
`increase in plasminogen activation (Fig. 1). Zymography re-
`vealed that this activity arose from a band with a electro-
`phoretic mobility slightly less than that of the single-chain tPA
`standard (Fig. 1C). This is consistent with the fact that the tPA
`standard is glycosylated whereas the bacterially expressed pro-
`tein is not. No other bands were detectable in zymography gels
`(Fig. 1C), indicating that the activity observed on fibrin plates
`and with the Spectrolyse assay arises from the full-length pro-
`tein and not from degradation products. In these experiments,
`DsbC expression was induced with 2 mM IPTG. However,
`because the trc promoter is leaky, DsbC synthesis occurred
`even in uninduced cultures, resulting in low but detectable tPA
`activities. Manipulation of the redox environment through the
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`EXPRESSION OF TISSUE PLASMINOGEN ACTIVATOR IN E. COLI
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`4893
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`AA BC D E
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`B (cid:9)
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`a
`a
`E
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`a. a (cid:9)
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`12
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`11,
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`6
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`4
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`9
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`IM
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`(cid:176)AB C D E F
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`D (cid:9)
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`A
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`B
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`C
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`D
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`E
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`Is ax
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`FIG. 1, Expression of tPA in E. coli, (A) Fibrin plate analysis of IPA activity.
`Equal amounts of soluble protein from each culture were spotted on the fibrin
`plate. Lanes: A, pBAD-sttI-tPA alone; B, pBAD-stIl-IPA + pLppsOmpArPDl;
`C, pBAD-sttI-IPA + pSE380dsbA; D, pBAD-stII-tPA + pSE420dshC; E,
`pBAD-stll IPA + pSE380dshAC. (B) The specific rate of plasminogen activa-
`tion in soluble fractions from cells harvested 3 h after induction was measured by
`the indirect chromogenic assay. tPA activities are reported as milliunits (mU) per
`microgram of total cell protein. Columns: 1, pBAD-stII-tPA alone; 2, pBAD-
`sttt-IPA + pLppsOnipArPDI; 3, pBAD-stIt-tPA + pSE380dsbA; 4, pBAD-stII-
`tPA + pSE420dsbC; 5, pBAD-stII-tPA + pSE380dsbAC. (C) Zymography of E.
`co/i soluble fractions. DsbC is evident as an intense Coomassie blue-stained
`hand. Lanes: A, single-chain tPA standard; B, pBADstII-tPA alone; C, pBAD-
`stII-1PA/pSE380dshA; D, pBAD-stlt-tPAJpSE420dsbC; E, pBAD-stII-tPA/
`pSE380dsbAC; F, pBAD-stlt-tPAJpLppsOmpArPDl. (D) Western blot of tPA
`expression in different strains. Lanes: A, pBAD-stII-tPA alone; B, pBAD-stll-
`IPA + pSE380dsbA; C, pBAD-stII-tPA + pSE420dshC; D, pBAD-stlI-tPA +
`pSE380dsbAC; E, pBAD-sttl-tPA + pLppsOmpArPDl.
`
`addition of GSH (glutathione, reduced form) and/or GSSG
`(glutathione, oxidized form) was not beneficial in cultures co-
`expressing DsbC and stII-tPA. In fact, the addition of as little
`as 0.5 mM GSSG resulted in a more than 40-fold-lower specific
`activity.
`DsbA also stimulated the formation of active tPA, but the
`specific activity obtained was 25-fold lower than that of cul-
`
`tures expressing DsbC. The addition of 2 mM GSH, 2.0 mM
`GSSG, or 2.0 mM GSH-0.5 mM GSSG at the time of induc-
`tion resulted in reduced tPA activities relative to control cul-
`tures that received no additives (data not shown). In vitro,
`DsbA and DsbC have been shown to act synergistically in the
`oxidative folding of BPTI (39). However, concomitant overex-
`pression of DsbA and DsbC gave a lower specific activity of
`tPA than did DsbC alone. This may be because the level of
`DsbC accumulation in cells coexpressing DsbA-DsbC is
`slightly reduced (Fig. 2).
`DsbC exhibits disulfide isomerase activity in vitro, and there
`is strong evidence that it has a similar role in vivo (19, 24, 30).
`DsbC can function as an isomerase only when its active-site
`cysteine thiols are reduced, presumably by the membrane pro-
`tein DsbD (DipZ) (25, 30). In vitro, DsbC is oxidized readily by
`DsbA, the primary catalyst of cysteine oxidation in the pen-
`plasmic space (39). Bardwell and coworkers showed that sub-
`stitution of the residues within the Cys-X-X-Cys active-site
`motif of DsbA modulates the redox potential of the protein
`(9). Several DsbA active-site mutants with a range of redox
`potentials were coexpressed together with tPA in a dsbA null
`mutant strain background. However, no improvement in tPA
`activity was observed relative to cells transformed with a
`control plasmid containing the wild-type dsbA gene. Similarly,
`coexpression of DsbC in a set of strains containing chromo-
`somally integrated dsbA mutants did not result in any further
`increase in active tPA (3a).
`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 h (cid:151)’ by
`controlling the addition of glucose until a DO, 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 OD 550 of around
`80; this was followed by a bolus of arabinose 30 min later.
`Under these conditions, the oxygen uptake rate remained con-
`stant for 3.5 h and then began to decline. The maximum spe-
`cific tPA activity was attained 2.5 h after induction (Fig. 3) and
`then started to decrease, in part because the inducer, arabi-
`nose, is catabolized by strain S1 7 110. The peak specific activity
`
`FIG. 2. SIDS-PAGE of total cell extracts from cultures coexpressing both IPA
`and DsbA, DsbC, or DsbA-DsbC. Cells were harvested 3 h after induction and
`lysed, and equal amounts of total protein from each sample were loaded on a
`12% SDS(cid:151)PAGE gel as described in Materials and Methods. Lanes: A, pBAD-
`stlI-tPA + pLppsOmpArPDl; B, pBAD-stII-IPA + pSE380dsbA; C, pBAD-stlt.
`IPA + pSE420dsbC; D, pBAD-stll-tPA + pSE380dshAC.
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`4894 (cid:9)
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`QIU ET AL. (cid:9)
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`(cid:151)o--O.D.550
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`I
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`1000
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`0
`0
`a)
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`100
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`10
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`---ACTIVITY
`OUR
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`-O
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`a
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`0 <(cid:149)
`C
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`a
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`0
`a
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`Arabinose
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`\4f (cid:9)
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`Bun Timer(hr)
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`FIG. 3. Production of tPA in a high-cell-density fermentation. The cell den-
`sity, reported as 0D 55 , and the oxygen uptake rate (OUR) are shown together
`with the tPA specific activity data from samples taken 0.5 h before and 0.5, 1.5,
`2.5, 3.5, 4.5, and 5.5 h after the addition of arabinose. The tPA activity was
`determined by the indirect chromogenic assay. Comparable levels of activity
`were obtained in different runs (n = 3),
`
`obtained in the fermentor was essentially identical to that in
`shaker flasks. Approximately 25 g of cell protein was obtained
`per liter of culture.
`tPA purification. Clarified cell lysates were loaded onto an
`L-lysine-Sepharose column. L-Lysine binds tightly and specifi-
`cally 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 NaC1-contain-
`ing 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. cajfra 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/ig, a value nearly identical to that of the authentic
`glycosylated protein from mammalian cells (400 IU/j.rg). 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 quantita-
`tively and could be eluted with buffer containing 50 mM imi-
`
`ABCD
`
`-- -94
`
`- -30
`
`FIG. 4. tPA purification. Shown is a silver-stained SDS-PAGE gel on which
`were loaded tPA purified from E. co/i (A), the single-chain tPA standard from
`American Diagnostica (B), the total cell lysate from cells overproducing tPA and
`DshC (C), and molecular size markers (D [in kilodaltons]).
`
`APPL. ENVIRON. MIcR0BI0L.
`
`dazole. 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 glycosy-
`lated, two-chain protein (0.9 and 1.6 M KSCN, respectively).
`Consequently, it appears that glycosylation affects the equilib-
`rium dissociation constant for ETI. After ETI chromatogra-
`phy, 14 pg of high-specific-activity tPA was obtained from 2 g
`of cell protein. Thus, approximately 180 .cg 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
`We have identified conditions that allow the production of
`significant amounts of active, full-length tPA in E. coli. Nor-
`mally, the formation of disulfide bonds takes place in the
`periplasmic space. In this study tPA was fused to the stIl leader
`peptide, which was efficient in directing the mature protein
`into the periplasmic space. The stil 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 coexpres-
`sion of foldases (8).
`The rate-limiting step in the oxidative folding of eukaryotic
`proteins in the periplasmic space appears to be the isomeriza-
`tion 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 isomer-
`ization (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 facilitat-
`ing 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 over-
`expression 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
`
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`VOL. 64, 1998 (cid:9)
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`EXPRESSION OF TISSUE PLASMINOGEN ACTIVATOR IN E. COLI
`
`4895
`
`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 isomeriza-
`tion (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 heter-
`ologous 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 syn-
`thesis 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 pos-
`sibility 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 fer-
`mentor. 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 h’t .
`The above observations suggest a number of ways in which
`the expression of active, full-length tPA can be increased fur -
`ther. 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 overexpres-
`sion has been found to inhibit growth, conditions that inhibit
`DsbC toxicity will have to be identified to prolong the produc-
`tion 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 expres-
`sion of other complex eukaryotic proteins both for commercial
`purposes and for structure-function studies. When protein gly-
`cosylation is not essential, expression in bacteria is clearly
`advantageous in terms of both cost and simplicity.
`
`ACKNOWLEDGMENTS
`
`We are grateful to S. Raina, J. Beckwith, J. Bardwell, and K. Dc
`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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