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Autodisplay of the protease inhibitor aprotinin in Escherichia coli
BBRC Biochemical and Biophysical Research Communications 333 (2005) 1218–1226 www.elsevier.com/locate/ybbrc
Autodisplay of the protease inhibitor aprotinin in Escherichia coli Joachim Jose *, Dirk Zangen 1 Bioanalytics, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universita¨t Du¨sseldorf, Universita¨tsstr. 1, D-40225 Du¨sseldorf, Germany Received 31 May 2005 Available online 17 June 2005
Abstract The Kunitz type protease inhibitor aprotinin, containing three intramolecular disulfide bonds, was expressed on the surface of Escherichia coli by Autodisplay. For this purpose, the aprotinin gene was fused in-frame to the transporter domain encoding DNA region of the AIDA-I autotransporter protein. Culture of cells supplied with the artificial gene at reducing conditions resulted in the translocation of aprotinin to the cell surface. Correct folding of aprotinin was shown by high affinity to its target enzyme HLE. No surface translocation was detectable under non-reducing conditions, indicating the degradation of aprotinin in the periplasm. By the use of periplasmic-protease defective E. coli strains PW147, PW151, and PW152, under non-reducing conditions, significant amounts of aprotinin appeared in the periplasm but not at the surface. Our results indicate that aprotinin molecules, reaching stable conformation before transport across the outer membrane, are degraded in the periplasm due to proteolysis. In case folding can be prevented, i.e., by blocking disulfide bond formation in the periplasm, aprotinin is translocated and can adopt its active conformation at the cell surface. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Autodisplay; Autotransporter; Aprotinin; Disulfide bond; Periplasm; Protease inhibitor; Flow cytometry; DegP; DegQ; Protein transport
During recent years, bacterial surface display of heterologous proteins has become a versatile biotechnological tool, which can be used to gain basic insights in molecular biology issues as well [1]. Among other systems used, the autotransporter pathway [2] is a very elegant way to translocate a recombinant protein of choice to the cell surface of a Gram-negative bacterium. The name ‘‘Autodisplay’’ was initially introduced for the use of the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) [3] for outer membrane translocation of the recombinant protein in combination with the signal peptide of the cholera toxin b-subunit (CTB) and an artificial promoter [4]. Some features of the ‘‘Autodisplay’’-system are unique among other cellular surface display systems used so *
Corresponding author. Fax: +49 211 8113847. E-mail address: [email protected] (J. Jose). 1 Present address: Pfizer Global Manufacturing, Illertissen, P.O. Box 2064, D-89252 Illertissen, Germany. 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.028
far, e.g., more than 105 recombinant molecules have been reported per single cell of E. coli [5] or stable multimeric proteins were detectable even in case subunits were expressed from monomeric genes [6,7]. In addition, it is an easy to handle system. The recombinant passenger protein is transported by simply introducing its coding sequence in-frame between the signal peptide and the translocator domain (Fig. 1), while the native passenger (the actual adhesin) is detached [8]. Introduction of a multiple cloning site instead of the native passenger allows the in-frame insertion of various passenger domains. This ‘‘Autodisplay’’ was successfully used for the functional surface expression of a wide variety of recombinant proteins [4,5,7,9–11]. In addition to ‘‘Autodisplay’’ other surface display tools have been reported more recently based on various natural autotransporter proteins different from AIDA-I [12–14]. The first characterized autotransporter protein was IgA1 protease from Neisseria gonorrhoeae [15]. A concept for its secretion mechanism was proposed
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protease inhibitor, was selected as a passenger, since it should exhibit resistance to degradation in the periplasm caused by several well-known serine proteases, e.g., DegP, DegS or DegQ. This study shows that folded aprotinin was not translocated to the surface but remained in the periplasm. The most probable reason is its three-dimensional structure exceeding the size of the b-barrel lumen. In case that folding of aprotinin during transport was prevented by blocking of disulfide bond formation, it was successfully translocated to the cell surface. Finally it adopted active conformation a posteriori, as indicated by high affinity to one of its known target proteins, human leucocyte elastase.
Materials and methods Fig. 1. Autodisplay of recombinant proteins in E. coli. Proteins to be transported by the autotransporter pathway to the cellsÕ surface are synthesized as a single polyprotein precursor containing structural requirements sufficient for surface translocation. SP, signal peptide; IM, inner membrane; PP, periplasm; and OM, outer membrane.
concurrently with its discovery. The mechanism included the passengers translocation through an outer membrane b-barrel, quite similar to the well-known porins. In contrast to the porins, the b-barrel is formed by the autotransporter itself, namely by its C-terminus [16]. It was quickly concluded that some surface proteins from other Gram-negative bacteria were obviously transported by similar means [17] and finally these surface proteins and/or secreted proteins were comprised to a new protein family called ‘‘autotransporter’’ [2]. However, the last step in transport, in detail the outer membrane translocation, remained subject to controversial discussions. Whereas the initial concept by Meyer and co-workers restricted the passage through the b-barrel only to proteins in an unfolded stages [18–20], other studies reported the translocation of proteins in a folded confirmation [21,22], which appears to be incompatible with translocation across the pore formed by the b-barrel. This resulted in an alternative model of transport involving a kind of a higher-level pore, build up of oligomers of 9–11 single b-barrels [22]. Most recently the first crystal structure of an autotransporter protein, NalP from N. gonorrhoeae, became available [23]. The data from this study were rather consistent with the hypothesis that the passenger-domain is transported across the hydrophilic channel formed by the b-barrel. In the present study, we used aprotinin, a rapidly folding, disulfide bond stabilized serine protease inhibitor [24,25], as a passenger in Autodisplay. The aim was to learn more about the fate of the recombinant passenger protein during transport. Aprotinin, a serine
Bacterial strains, plasmids, and culture conditions. Escherichia coli strains JK321 [20] (DompT proC leu6 trpE38 entA zih12::Tn10 dsbA::kan) and UT5600 (F ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi1 DompT-fepC266) [4] have been used in earlier studies on the autotransporter-mediated surface display of recombinant proteins. Periplasmatic-protease negative strains for the expression of autoransporter fusion proteins used in this study, PW147 (HsdR2, araD139, galE15, galK16, rpsL, degQ1, (DEcoRV)), PW151 (HsdR2 araD139 galE15 galK16 rpsL degP41), and PW152 (HsdR2 araD139 galE15 galK16 rpsL DdegQ2::kan DEcoRV::kan) were kindly provided by H. Kolmar and P. Sauer [26]. E. coli TOP10 (F mcrA D(mrr-hsdRMS-mcrBC) /80lacZDM15 DlacX74 deoR recA1 araD139 D(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG) and the vector pCR2.1-TOPO, which were used for subcloning of PCR products, were obtained from Invitrogen (Groningen, the Netherlands). Plasmid pJM1013 is a derivative of pJM7, which encodes the AIDA-I autotransporter domains and both have been described earlier [8,27]. Plasmid pIU27.1.O encoding aprotinin was kindly provided by H. Apeler, A.G. Bayer (Wupperthal, Germany). Bacteria were routinely grown at 37 °C in Luria–Bertani (LB) broth containing 100 mg ampicillin per litre. Recombinant DNA techniques. For the construction of an aprotinin–autotransporter fusion protein, the aprotinin gene was amplified by PCR from plasmid pIU27.1.O with oligonucleotide primers DZ001 (5 0 -gcg tcg acc gtc ctg act tct gcc tcg agc cg-3 0 ) and DZ002 (5 0 -ggg gta cca gca cca ccg caa gta cg-3 0 ). The PCR product was inserted into vector pCR2.1-TOPO and recleaved with SalI and KpnI. The restriction fragment was ligated into pJM7 resulting in plasmid pAT-AP02-7 (Fig. 2). Plasmid pAT-AP02-7 was used as a template for PCR to construct plasmid pAT-AP02-13 with oligonucleotide primers JJ009 (5 0 -gct cta gac gtc ctg act tct gcc tcg agc cg-3 0 ) and JJ010 (5 0 -gaa gat cta gca cca ccg caa gta cgc-3 0 ). The PCR product was inserted into vector pCR2.1-TOPO and recleaved with XbaI/BglII. The restriction fragment was ligated into pJM1013, restricted with the same enzymes, resulting in plasmid pAT-AP02-13. Both ligations yielded an in-frame fusion of aprotinin with the AIDA-I autotransporter unit under the control of a constitutive promoter (PTK) [4]. They differed in the length of the linker region, that is necessary between the recombinant passenger protein and the b-barrel to obtain full-surface exposure of the passenger (Fig. 2). Outer-membrane preparation. E. coli cells were grown overnight and 1 mL culture was used to inoculate fresh LB medium (20 mL). Cells were cultured at 37 °C with shaking (200 rpm) for about 5 h until OD578 of 0.7 was reached. Cells were harvested and outer membranes were prepared according to a modification of the rapid isolation method of Hantke [28], as described earlier.
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Fig. 2. Construction of the aprotinin–autotransporter fusion proteins. (A) Nucleotide and amino acid sequence of aprotinin used in this study. Restriction sites ResI/ResII were modified by PCR to XhoI/SalI sites for the construction of plasmid pAT-AP02-7 and to XbaI/BglII sites for plasmid pAT-AP02-13. Cysteines which are involved in the formation of intramolecular disulfide bonds are written in bold letters. (B) Structure of the fusion proteins AT-AP7 and AT-AP13E. The environment of the fusion sites between aprotinin and the autotransporter domains are given as sequences. Restriction sites are underlined. The cleavage site of the signal peptidase is indicated by an arrowhead as well as the accessible trypsin cleavage site within the linker region, identified in previous experiments. The eight amino acids of CTB origin are marked by asterisks. The linear epitope for a monoclonal antibody used for labelling fusion protein AT-AP13E, encoded by plasmid pAT-AP02-13 is written in bold letters. Between the epitope and the linker region, a specific IgA1 protease cleavage site (PPSP) was inserted. FP, fusion protein.
SDS–PAGE and Western blot analysis. Outer membrane isolates were diluted (1:2) with sample buffer (100 mM Tris–HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, and 20% glycerol). The samples were then boiled for 20 min and analysed on 12.5% SDS–PAGE. Proteins were visualized with Coomassie brilliant blue. Prestained molecular weight markers (Bio-Rad, Munich, Germany) were used to determine the size of the prepared proteins. For Western blot analysis, gels were electroblotted onto polyvinylidene-difluoride (PVDF) membranes and blotted membranes were blocked in TBS with 3% FCS overnight. For immunodetection, membranes were incubated for 3 h either with aprotinin antibody HDH191 (kindly provided by H. Apeler, A.G. Bayer, Wupperthal, Germany) or with monoclonal anti-PEYFK Du¨142 [4], which were diluted 1:1000 or 1:35 in TBS containing 3% FCS. Prior to the addition of secondary antibodies, immunoblots were rinsed three times with TBS containing 0.1% Tween 20. Antigen–antibody conjugates were visualized by reaction with alkaline phosphatase linked goat anti-mouse IgG secondary antibody (KPL, Gaithersburg, MD, USA), diluted 1:10,000 in TBS with 3% FCS. A colour reaction was obtained by adding 10 mL incubation buffer (100 mM NaCl, 5 mM MgCl2, and 100 mM Tris–HCl, pH 9.5) containing 66 lL nitrobluetetrazoliumchloride (50 mg mL1 in 70% dimethylformamide) and 33 lL 5-
brome-4-chlor-3-indolylphosphate-disodium salt (20 mg mL1 in H2O). Flow cytometer analysis. For flow cytometer analysis cultures of E. coli cells with or without plasmids were grown at 37 °C overnight and subcultured in a dilution of 1:20 at 37 °C until they reached OD578 of 0.7. Cells were harvested, washed three times with PBS, and suspended in PBS including 3% FCS to an OD578 of 1.0. After 30 min, 5 lL of biotin-labelled human leucocyte elastase (HLE) was added to yield a total volume of 1 mL PBS/3% FCS and incubation was extended for another 30 min. HLE was obtained from Lee Scientific (St. Louis, USA) and biotin-labelling was performed with the biotinlabelling kit from Roche (Mannheim, Germany) according to the manufacturerÕs recommendations. After incubation, cells were washed three times with PBS and incubated for 30 min with FITC-coupled streptavidin (Roche, Mannheim, FRG). Then, cells were washed three times with PBS and suspended in PBS to a final OD578 of 0.02 for subsequent FACS analysis. For each experiment at least 10,000 cells were analysed with a FacsCalibur Cytometer (Becton Dickinson, Heidelberg, Germany) using 488 nm as excitation wavelength and FACS-FLOW (Becton–Dickinson, Heidelberg, Germany) as sheath fluid. The threshold trigger was set on side scatter to eliminate background noise and to analyse solely intact cells.
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Results Construction and expression of aprotinin–AIDA-I autotransporter fusion proteins For successful surface display of a recombinant passenger protein by the autotransporter pathway, a gene needs to be constructed that encodes an artificial precursor protein. This precursor must contain a signal peptide at the N-terminus, the passenger (in our case: aprotinin), a linker region and the b-barrel (Fig. 1). For this purpose, the coding region of aprotinin (62 aa) was amplified by PCR and inserted into vector pJM7, which has been used before for the surface display of the choleratoxin b-subunit (CTB) [4]. This resulted in plasmid pAT-AP02-7. Due to the ligation procedure the artificial aprotinin–AIDA-I fusion protein (AT-AP7) encoded by this plasmid, contained 8 amino acids of native CTB in addition (Fig. 2). To analyse expression of AT-AP7, the corresponding plasmid pAT-AP02-7 was transformed into E. coli strains JK321 and UT5600. E. coli JK321 is a dsbA derivative of UT5600. Both strains are ompT and have been used in former experiments for the functional surface display of a broad variety of recombinant passengers by the autotransporter pathway [4,6,7,10]. However, in both strains no protein of the correct size was detectable, neither by Coomassie brilliant blue staining nor by Western blot analysis with the aprotinin-specific antiserum HDH191 (not shown). To exclude that this finding was due to misfolded aprotinin, that could not be detected by antibody HDH191, a peptide tag (PEYFK) was added at the C-terminus of aprotinin (Fig. 2), for which a highly specific monoclonal antibody was available [4]. The aim was to establish a method for detection in Western blot experiments, that is independent of the aprotinin passenger being folded in the correct tertiary structure or not. This was done by constructing plasmid pAT-AP02-13 encoding the fusion protein AT-AP13E, that additionally contained the peptide tag PEYFK. The expression of fusion protein AT-AP13E was analysed again in both strains, UT5600 and JK321, and compared to the expression of FP50 encoded by plasmid pJM1013 as a control. FP50 is identical with AT-AP13E, except that it is lacking the aprotinin domain. As shown in Fig. 3, the control protein was detectable in both strains, whereas no trace of AT-AP13E was seen in Western blot experiments. As the translational and transcriptional signals in both plasmids, pAT-AP02-13 and its control pJM1013, were identical and the correct in-frame fusion of aprotinin to the autotransporter domains was verified by DNA sequence analysis, this clearly pointed on a proteolytic degradation of AT-AP13E during transport. This was surprising, since aprotinin was chosen as a passenger due to the assumption that a protease inhibitor could resist such type of degradation.
Fig. 3. Western blot analysis of outer membrane protein preparations from E. coli strains UT5600 and JK321 [20] harbouring pAT-AP02-13 or the control plasmid pJM1013. Immunoblotting was performed with the peptide tag (PEYFK) specific monoclonal antibody Du¨142. The molecular weight of marker proteins applied is indicated in kilodaltons at the right.
Surface display of aprotinin by blocking of disulfide bond formation Folding of free aprotinin has been subjected to previous investigations and was found to proceed via several one- or two-disulfide bond stabilized intermediates, including a so-called ‘‘kinetic trap,’’ which very slowly rearranges to other folding intermediates [29–31]. In the present concept of surface translocation by the autotransporter pathway, any folding of the passenger would hinder surface translocation and would result in degradation by periplasmic proteases, as long as no stable, protease-resistant conformation is obtained. Therefore, preventing the formation of disulfide bonds appeared to be a solution for keeping aprotinin in an unfolded state and directing the passenger molecule to the cell surface. In other words, blocking of disulfide bonding would enable sufficient aprotinin passenger molecules to escape from the periplasmic-protease machinery across the porin-like b-barrel and these molecules should be detectable at the surface. For this purpose, cells of E. coli UT5600 were transformed with plasmid pAT-AP02-7 and cultured in liquid LB medium supplied with 10 mM 2-mercaptoethanol in order to maintain strong reducing conditions. It has been shown in earlier studies, that a concentration of 10 mM 2-mercaptoethanol was sufficient to block disulfide bond formation during transport [5,19,20] and enabled passenger proteins with naturally occurring disulfide bonds to be transported to the surface. After cells of E. coli UT5600 pAT-AP02-7 in growth medium with 10 mM 2-mercaptoethanol had reached an OD578 = 1, outer membrane proteins were prepared and subjected to Western blot analysis. As shown in Fig. 4, a full-size fusion protein AT-AP7 was detectable in UT5600 under these growth conditions.
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Fig. 4. Western blot analysis of outer membrane protein preparations from E. coli UT5600 harbouring pAT-AP02 and grown in the presence of 10 mM 2-mercaptoethanol. Immunoblotting was performed with aprotinin specific antibody HDH191. M, prestained molecular mass marker. The molecular weight is indicated in kilodaltons. Whole cells were treated with trypsin before outer membranes were prepared (+) or not ().
Theoretically two orientations in the outer membrane are possible for the aprotinin domain in the autotransporter fusion protein, either directed to the periplasm or exposed at the cell surface. In previous experiments with other passengers, the orientation of the protein in the outer membrane was assessed by the treatment of whole bacterial cells with trypsin [4,5,7,8]. Digestion of proteins by trypsin can only take place at the surface of the bacterial cell and not in the periplasm since the trypsin molecule is too big to cross the outer membrane. The previous experiments concordantly showed that incubation of whole cells with trypsin removed the passenger domain from the fusion proteins by a cleavage site within the linker region (K/G), located 54 amino acids upstream of the b-barrel structure (Fig. 2). If cells of UT5600 pAT-AP02-7 were incubated for 30 min with trypsin in the same concentration as in the experiments before, however, no size reduction of the aprotinin– autotransporter fusion protein was detectable (not shown). This could have been an indication that aprotinin at the cell surface gained its active conformation as a protease inhibitor and thereby inhibited trypsin. To overcome this effect, the concentration of trypsin was in-
creased 1000-fold before being added to whole cells of UT5600 pAT-AP02-7. As shown in Fig. 4 (lane 2) the fusion protein was no longer detectable under these conditions. Thus, aprotinin appears to be located at the cell surface and is obviously released from the autotransporter fusion protein by trypsin cleavage within the linker domain (Fig. 2), in case the concentration of the protease was substantially increased. From these results it can be summarized, that maintaining reducing conditions during transport—most probably by preventing disulfide bonding—enables sufficient aprotinin passenger molecules to escape to the surface of the cell before being decomposed in the periplasm. In other words, aprotinin in its active structure containing disulfide bonds cannot be translocated to the surface by the autotransporter pathway. Translocation is only possible if aprotinin is kept in an unfolded state during transport by blocking disulfide bond formation. It can be speculated, that this might also be a question of velocity, and the number of aprotinin molecules present on the surface is only a part of the entire amount of molecules produced. Expression of aprotinin in a periplasmic-protease negative host background Escherichia coli contains several enzymes degrading incorrectly folded proteins in the periplasm [32,33]. This is indispensable, as the periplasm can be assumed as a limited space. In former studies of Sauer and co-workers, the role of periplasmic proteases in E. coli was studied by constructing several mutant strains, either defective in periplasmic protease DegP or DegQ [33]. As our presented results indicate that periplasmic degradation could be the cause of the failed expression of the autotransporter–aprotinin fusion protein AP02-7 under non-reducing growth conditions, we examined the expression of the protein in three of these mutant strains. For this purpose, strains PW147 (degQ), PW151 (degP), and PW152 (degQ) were transformed with plasmid pAT-AP02-7 and analysed under identical non-reducing conditions as described before. Outer membrane proteins were prepared and, as shown in Fig. 5A, a full-size fusion protein AP02-7 was detectable
Fig. 5. Western blot analysis of outer membrane preparations from periplasmic-proteases negative E. coli strains harbouring plasmid pAT-AP02-7. (A) Whole cells were treated with trypsin or not before outer membranes were prepared. (B) Outer membrane preparations were subjected to trypsin treatment subsequently. Immunoblotting was performed with the aprotinin specific antibody HDH191. M, prestained molecular mass marker. The molecular weight is indicated in kilodaltons. + and , trypsin was added or was not.
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by Western blot analysis in all strains used. Obviously the absence of one of the two periplasmic proteases DegP or DegQ prevented the aprotinin–autotransporter fusion protein from periplasmic degradation. To verify, whether the aprotinin domain was indeed directed to the periplasm, trypsin was added to whole cells of each plasmid containing strain, before outer membranes were prepared. As described above, the outer membrane is an invincible barrier for large molecules as trypsin and therefore it would protect the passenger from degradation if the passenger is located in the periplasm [4,5]. Since aprotinin is known to be a potent inhibitor of the serine protease trypsin, again a 1000-fold higher protease concentration was applied as usual in similar experiments with other passenger proteins, known to be no protease inhibitors. As can be seen in Fig. 5A, trypsin addition to whole cells of PW147, PW151, and PW152, all expressing AT-AP7, had no effect, neither in size nor in the amount of protein expressed. This means, that under non-reducing conditions in the DegP or DegQ defective strains, the aprotinin domain appears to be not exposed to the cell surface but rather is directed to the periplasm. The reason for this orientation is probably the fact that under these conditions aprotinin folds into its active structure in the periplasm, leading to a molecule size which is too big for the transport across the b-barrel and which is resistant to proteolytic degradation in the periplasm. For verification, immunofluorescence microscopy studies were performed using the anti-aprotinin antibody HDH191 as a primary antibody and a FITC-labelled anti-mouse IgG as a secondary antibody. Cells of PW147, 151, and 152 expressing AT-AP7 showed no fluorescence at all and were identical to control cells without plasmid. This was an additional hint, that under these conditions, the aprotinin domain is not exposed to the cell surface. To exclude a general resistance of the aprotinin–autotransporter fusion protein to trypsin in these strains,
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outer membrane proteins were first prepared from cells of PW147, PW151, and PW152, all expressing AT-AP7, and then applied to trypsin digestion. This treatment would enable trypsin to have access to the aprotinin– autotransporter fusion protein from both sides of the outer membrane. As Fig. 5B shows, that the fusion protein was no longer detectable in Western blot experiments after this treatment. As mentioned above, this is supposed to be due to a known cleavage site of trypsin within the linker region (Fig. 2), resulting in the release of the aprotinin passenger domain. The detection of the remaining autotransporter core was not expected as it is not recognized by the aprotinin-specific antibody HDH191 used in this experiment. Investigation on the functionality of aprotinin displayed on the cell surface To find out, whether the aprotinin molecules translocated to the cellÕs surface gain an active conformation, cells of UT5600 pAT-AP02-7 grown under reducing conditions (10 mM 2-mercaptoethanol) were incubated with biotin-labelled human leukocyte elastase (HLE) and subsequently, after the addition of streptavidinFITC analysed by flow cytometry. Aprotinin has been reported to be a strong inhibitor of HLE (Ki = 3.5 lM) [34]. As a strong inhibitor is supposed to bind its target enzyme to the same extent, we expected FITC-labelled HLE to bind to the bacterial surface via the aprotinin molecule. This should result in a specific and significant increase in whole cell fluorescence compared to control cells without aprotinin. The controls applied in these experiments were cells of E. coli UT5600 with plasmid pJM7, encoding an autotransporter fusion protein with CTB instead of aprotinin as a passenger. As shown in Fig. 6, surface display of aprotinin resulted in a 10.5fold increase in relative fluorescence (mean value UT5600 pJM7 control: 19.78; UT5600 pAT-AP02-7:
Fig. 6. FACS analysis of E. coli UT5600 harbouring plasmid pAT-AP02-7 (B) or pJM7 (aprotinin-negative control), (A) grown in the presence of 10 mM 2-mercaptoethanol. Whole cells were incubated with biotin-labelled human leucocyte elastase (HLE) and then treated with FITC-coupled streptavidin. For each measurement 10,000 cells were analysed. As controls in each experiment cells were treated similarly but not incubated with biotin-labelled HLE (filled histograms). The mean value of fluorescence for aprotinin displaying cells (208; B, open histogram) was more than 10 times higher than for cells without aprotinin (19; A, open histogram).
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208.12). Although it cannot be excluded in principle that HLE binds by a different part than its active site to aprotinin displayed at the cell surface, this result indicates that aprotinin folds into an active conformation after being translocated to the cell surface. The broad distribution of fluorescent UT5600 pAT-AP02-7 cells could be explained by a linker cleavage activity of elastase. When outer membrane proteins were prepared after the addition of HLE to whole cells and were analysed by Western blot, a significant decrease in the observed amount of full-size AT-AP7 protein was detectable, which was due to cleavage within the linker region (not shown). This cleavage can be possible even after the first HLE molecule has already bound to the aprotinin domain and is inhibited thereby. A second HLE could attack the conglomerate by the linker region. Therefore, the cell population seen in Fig. 6B, might be a mixture of cells, with different numbers of aprotinin molecules on the surface, due to proteolytic activities of HLE molecules in excess.
Discussion When the disulfide bond containing choleratoxin B (CTB) was expressed in E. coli as an autotransporter passenger protein under non-reducing conditions [18], no autotransporter–CTB fusion protein was detectable at all. The folding of the protein including the formation of disulfide bonds during transport in the periplasm obviously resulted in a structure that was too voluminous to be transported through the pore formed by the b-barrel [19,20]. Thereby, CTB was obliged to remain in the periplasm where it was subjected to proteolytic degradation. To get a view on this intermediate step during surface translocation, aprotinin was chosen in this study, because
on one hand it also contains disulfide bonds but on the second hand promised to be resistant to periplasmic protease degradation due to its protease inhibitor activity. Autodisplay of the protease inhibitor aprotinin at the cell surface was achieved by fusing its coding region to the autotransporter domain encoding regions of the AIDA-I [3], and growing the cells transformed by the resulting artificial gene construct under reducing conditions with 10 mM 2-mercaptoethanol. Omitting 2-mercaptoethanol from the growth medium resulted in complete degradation of the autotransporter–aprotinin fusion protein. To verify the hypothesis, that this was due to proteolysis by periplasmic proteases, the identical gene construct was expressed in E. coli strains either defective in periplasmic protease DegQ or DegP [33] under non-reducing conditions. This expression resulted in detectable amounts of the autotransporter–aprotinin fusion protein. However, the aprotinin domain was directed to the periplasmic side of the outer membrane. These results indicate that under reducing growth conditions by application of 2-mercaptoethanol, disulfide bond formation is prevented within the aprotinin passenger domain and it can be translocated to the cell surface (Fig. 7B). When non-reducing growth conditions are applied, aprotinin folds during transport, cannot translocate to the cell surface and remains in the periplasm. There it is degraded as long as all of the periplasmic proteases are active. In case that one of these proteases, either DegQ or DegP are missing, aprotinin can be found in the periplasm in a stable confirmation (Fig. 7A). This may be due to the known serine protease-inhibiting activity of aprotinin [35]. From these results, it can be concluded, that in E. coli strains, defective in one of the periplasmic proteases DegQ or DegP, the aprotinin–autotransporter fusion protein is expressed in detectable amounts. The b-barrel seems to
Fig. 7. Scheme of aprotinin autodisplay in E. coli. (A) In periplasmatic-protease negative strains PW147, PW151, or PW152 grown without 2-ME, aprotinin folds within the periplasm and remains located stable at the inner side of the outer membrane. PP, periplasm; OM, outer membrane. (B) In E. coli UT5600 grown under reducing conditions with 10 mM 2-mercaptoethanol (2-ME), it is translocated to the surface and folds to its active conformation. The structure of aprotinin is in analogy to the resolved crystal structure [36].
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be integrated in the outer membrane, whereas the passenger protein is maintained in the periplasm. This periplasmic location, however, did not result in proteolytic degradation, as seen before for other passengers as, e.g., CTB [18–20]. We suppose, that this is due to the passenger domain used in the present experiments, aprotinin. Aprotinin itself is a known inhibitor of a wide variety of serine proteases [35] and presumably inhibits, in its active form, the periplasmic serine proteases DegP or DeqQ too [3]. Interestingly, it had no effect whether DegP or DegQ was missing. In both cases, aprotinin was able to withstand the proteolytic attack. However, if both proteases, DegP and DegQ are working in concert, as, e.g., in strains UT5600 or JK321 (Fig. 3), the fusion protein AT-AP7 is degraded completely and can not be detected at all. This can be interpreted in two ways. One explanation could be that degradation of aprotinin in the periplasm is a matter of velocity. This means, a competition exists between folding of the aprotinin domain to its active and protease-inhibiting conformation on the one hand and its degradation by the periplasmic proteases on the other hand. If one protease is missing, folding gets the upper hand, otherwise degradation is predominant. A second explanation could be that both proteases must interact in common with the aprotinin domain more or less simultaneously to avoid the formation of an active protease inhibitor. Both cases, however, result in a protease-resistant autotransporter fusion protein, when either DegP or DegQ are missing, most probably due to the protease inhibitory potential of the passenger domain aprotinin. In summary our results indicate that the passenger protein transported by the autotransporter pathway needs to cross a size restricted area during transport, otherwise transport stops in the periplasm and the passenger protein is proteolytically degraded as long as it has no protease-inhibiting activity. These results are in concordance with the concept that the passenger protein is translocated through the porin-like structure formed by the b-barrel of the autotransporter domain, as it has been suggested more recently by the first crystal structure of a member of this family of protein transporters, but was subject of controversial discussions for many years before. Beside resolving this intermediate step in the pathway used by the autotransporter family of proteins, we present a new methodical approach, namely that cells displaying a protease inhibitor at the surface can be labelled by the target enzyme and can be analysed by flow cytometry, in case the target enzyme is labelled with a fluorescent dye.
Acknowledgments We thank H. Apeler (A.G. Bayer, Wupperthal, Germany) for providing the monoclonal antibody HDH191
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and the plasmid pIU27.1.O, encoding the aprotinin DNA sequence, H. Kolmar and P. Sauer for providing the E. coli strains PW147, 151, and 152, and R. Maas for critically reading the manuscript.
References [1] G. Georgiou, C. Stathopoulos, P.S. Daugherty, A.R. Nayak, B.L. Iverson, R. Curtiss III, Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines, Nat. Biotechnol. 15 (1997) 29–34. [2] J. Jose, F. Jahnig, T.F. Meyer, Common structural features of IgA1 protease-like outer membrane protein autotransporters, Mol. Microbiol. 18 (1995) 378–380. [3] I. Benz, M.A. Schmidt, AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule, Mol. Microbiol. 6 (1992) 1539–1546. [4] J. Maurer, J. Jose, T.F. Meyer, Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli, J. Bacteriol. 179 (1997) 794–804. [5] J. Jose, F. Hannemann, R. Bernhardt, Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2Fe–2S] cluster, Chembiochem 2 (2001) 695– 701. [6] J. Jose, R. Bernhardt, F. Hannemann, Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli, J. Biotechnol. 95 (2002) 257–268. [7] J. Jose, S. von Schwichow, Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars, Chembiochem 5 (2004) 100–108. [8] J. Maurer, J. Jose, T.F. Meyer, Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions, J. Bacteriol. 181 (1999) 7014– 7020. [9] C.T. Lattemann, J. Maurer, E. Gerland, T.F. Meyer, Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter, J. Bacteriol. 182 (2000) 3726–3733. [10] E. Schultheiss, C. Paar, H. Schwab, J. Jose, Functional esterase surface display by the autotransporter pathway in Escherichia coli, J. Mol. Catal. B. Enzymat. 18 (2002) 89–97. [11] K. Rizos, C.T. Lattemann, D. Bumann, T.F. Meyer, T. Aebischer, Autodisplay: efficacious surface exposure of antigenic UreA fragments from Helicobacter pylori in Salmonella vaccine strains, Infect. Immun. 71 (2003) 6320–6328. [12] W. Fischer, R. Buhrdorf, E. Gerland, R. Haas, Outer membrane targeting of passenger proteins by the vacuolating cytotoxin autotransporter of Helicobacter pylori, Infect. Immun. 69 (2001) 6769–6775. [13] E. Veiga, V. de Lorenzo, L.A. Fernandez, Autotransporters as scaffolds for novel bacterial adhesins: surface properties of Escherichia coli cells displaying Jun/Fos dimerization domains, J. Bacteriol. 185 (2003) 5585–5590. [14] S. Krause, D. Wurdemann, A. Wentzel, A. Christmann, H. Fehr, H. Kolmar, K. Friedrich, Bacteria displaying interleukin-4 mutants stimulate mammalian cells and reflect the biological activities of variant soluble cytokines, Chembiochem 5 (2004) 804–810. [15] T.F. Meyer, R. Halter, J. Pohlner, Mechanism of extracellular secretion of an IgA protease by gram-negative host cells, Adv. Exp. Med. Biol. 216 (1987) 1271–1281.
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[16] J. Pohlner, R. Halter, K. Beyreuther, T.F. Meyer, Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature 325 (1987) 458–462. [17] T. Klauser, J. Pohlner, T.F. Meyer, The secretion pathway of IgA protease-type proteins in gram-negative bacteria, Bioessays 15 (1993) 799–805. [18] T. Klauser, J. Pohlner, T.F. Meyer, Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation, EMBO J. 9 (1990) 1991–1999. [19] T. Klauser, J. Pohlner, T.F. Meyer, Selective extracellular release of cholera toxin B subunit by Escherichia coli: dissection of Neisseria Iga beta-mediated outer membrane transport, EMBO J. 11 (1992) 2327–2335. [20] J. Jose, J. Kramer, T. Klauser, J. Pohlner, T.F. Meyer, Absence of periplasmic DsbA oxidoreductase facilitates export of cysteinecontaining passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway, Gene 178 (1996) 107–110. [21] E. Veiga, V. de Lorenzo, L.A. Fernandez, Probing secretion and translocation of a beta-autotransporter using a reporter singlechain Fv as a cognate passenger domain, Mol. Microbiol. 33 (1999) 1232–1243. [22] E. Veiga, E. Sugawara, H. Nikaido, V. de Lorenzo, L.A. Fernandez, Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains, EMBO J. 21 (2002) 2122–2131. [23] C.J. Oomen, P. Van Ulsen, P. Van Gelder, M. Feijen, J. Tommassen, P. Gros, Structure of the translocator domain of a bacterial autotransporter, EMBO J. 23 (2004) 1257–1266, Epub 2004 Mar 1211. [24] T.E. Creighton, The disulfide folding pathway of BPTI, Science 256 (1992) 111–114. [25] E. Barbar, G. Barany, C. Woodward, Unfolded BPTI variants with a single disulfide bond have diminished non-native structure distant from the crosslink, Fold. Des. 1 (1996) 65–76. [26] H. Kolmar, P.R. Waller, R.T. Sauer, The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for
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Autodisplay of the protease inhibitor aprotinin in Escherichia coli Joachim Jose *, Dirk Zangen 1 Bioanalytics, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universita¨t Du¨sseldorf, Universita¨tsstr. 1, D-40225 Du¨sseldorf, Germany Received 31 May 2005 Available online 17 June 2005
Abstract The Kunitz type protease inhibitor aprotinin, containing three intramolecular disulfide bonds, was expressed on the surface of Escherichia coli by Autodisplay. For this purpose, the aprotinin gene was fused in-frame to the transporter domain encoding DNA region of the AIDA-I autotransporter protein. Culture of cells supplied with the artificial gene at reducing conditions resulted in the translocation of aprotinin to the cell surface. Correct folding of aprotinin was shown by high affinity to its target enzyme HLE. No surface translocation was detectable under non-reducing conditions, indicating the degradation of aprotinin in the periplasm. By the use of periplasmic-protease defective E. coli strains PW147, PW151, and PW152, under non-reducing conditions, significant amounts of aprotinin appeared in the periplasm but not at the surface. Our results indicate that aprotinin molecules, reaching stable conformation before transport across the outer membrane, are degraded in the periplasm due to proteolysis. In case folding can be prevented, i.e., by blocking disulfide bond formation in the periplasm, aprotinin is translocated and can adopt its active conformation at the cell surface. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Autodisplay; Autotransporter; Aprotinin; Disulfide bond; Periplasm; Protease inhibitor; Flow cytometry; DegP; DegQ; Protein transport
During recent years, bacterial surface display of heterologous proteins has become a versatile biotechnological tool, which can be used to gain basic insights in molecular biology issues as well [1]. Among other systems used, the autotransporter pathway [2] is a very elegant way to translocate a recombinant protein of choice to the cell surface of a Gram-negative bacterium. The name ‘‘Autodisplay’’ was initially introduced for the use of the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) [3] for outer membrane translocation of the recombinant protein in combination with the signal peptide of the cholera toxin b-subunit (CTB) and an artificial promoter [4]. Some features of the ‘‘Autodisplay’’-system are unique among other cellular surface display systems used so *
Corresponding author. Fax: +49 211 8113847. E-mail address: [email protected] (J. Jose). 1 Present address: Pfizer Global Manufacturing, Illertissen, P.O. Box 2064, D-89252 Illertissen, Germany. 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.028
far, e.g., more than 105 recombinant molecules have been reported per single cell of E. coli [5] or stable multimeric proteins were detectable even in case subunits were expressed from monomeric genes [6,7]. In addition, it is an easy to handle system. The recombinant passenger protein is transported by simply introducing its coding sequence in-frame between the signal peptide and the translocator domain (Fig. 1), while the native passenger (the actual adhesin) is detached [8]. Introduction of a multiple cloning site instead of the native passenger allows the in-frame insertion of various passenger domains. This ‘‘Autodisplay’’ was successfully used for the functional surface expression of a wide variety of recombinant proteins [4,5,7,9–11]. In addition to ‘‘Autodisplay’’ other surface display tools have been reported more recently based on various natural autotransporter proteins different from AIDA-I [12–14]. The first characterized autotransporter protein was IgA1 protease from Neisseria gonorrhoeae [15]. A concept for its secretion mechanism was proposed
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protease inhibitor, was selected as a passenger, since it should exhibit resistance to degradation in the periplasm caused by several well-known serine proteases, e.g., DegP, DegS or DegQ. This study shows that folded aprotinin was not translocated to the surface but remained in the periplasm. The most probable reason is its three-dimensional structure exceeding the size of the b-barrel lumen. In case that folding of aprotinin during transport was prevented by blocking of disulfide bond formation, it was successfully translocated to the cell surface. Finally it adopted active conformation a posteriori, as indicated by high affinity to one of its known target proteins, human leucocyte elastase.
Materials and methods Fig. 1. Autodisplay of recombinant proteins in E. coli. Proteins to be transported by the autotransporter pathway to the cellsÕ surface are synthesized as a single polyprotein precursor containing structural requirements sufficient for surface translocation. SP, signal peptide; IM, inner membrane; PP, periplasm; and OM, outer membrane.
concurrently with its discovery. The mechanism included the passengers translocation through an outer membrane b-barrel, quite similar to the well-known porins. In contrast to the porins, the b-barrel is formed by the autotransporter itself, namely by its C-terminus [16]. It was quickly concluded that some surface proteins from other Gram-negative bacteria were obviously transported by similar means [17] and finally these surface proteins and/or secreted proteins were comprised to a new protein family called ‘‘autotransporter’’ [2]. However, the last step in transport, in detail the outer membrane translocation, remained subject to controversial discussions. Whereas the initial concept by Meyer and co-workers restricted the passage through the b-barrel only to proteins in an unfolded stages [18–20], other studies reported the translocation of proteins in a folded confirmation [21,22], which appears to be incompatible with translocation across the pore formed by the b-barrel. This resulted in an alternative model of transport involving a kind of a higher-level pore, build up of oligomers of 9–11 single b-barrels [22]. Most recently the first crystal structure of an autotransporter protein, NalP from N. gonorrhoeae, became available [23]. The data from this study were rather consistent with the hypothesis that the passenger-domain is transported across the hydrophilic channel formed by the b-barrel. In the present study, we used aprotinin, a rapidly folding, disulfide bond stabilized serine protease inhibitor [24,25], as a passenger in Autodisplay. The aim was to learn more about the fate of the recombinant passenger protein during transport. Aprotinin, a serine
Bacterial strains, plasmids, and culture conditions. Escherichia coli strains JK321 [20] (DompT proC leu6 trpE38 entA zih12::Tn10 dsbA::kan) and UT5600 (F ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi1 DompT-fepC266) [4] have been used in earlier studies on the autotransporter-mediated surface display of recombinant proteins. Periplasmatic-protease negative strains for the expression of autoransporter fusion proteins used in this study, PW147 (HsdR2, araD139, galE15, galK16, rpsL, degQ1, (DEcoRV)), PW151 (HsdR2 araD139 galE15 galK16 rpsL degP41), and PW152 (HsdR2 araD139 galE15 galK16 rpsL DdegQ2::kan DEcoRV::kan) were kindly provided by H. Kolmar and P. Sauer [26]. E. coli TOP10 (F mcrA D(mrr-hsdRMS-mcrBC) /80lacZDM15 DlacX74 deoR recA1 araD139 D(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG) and the vector pCR2.1-TOPO, which were used for subcloning of PCR products, were obtained from Invitrogen (Groningen, the Netherlands). Plasmid pJM1013 is a derivative of pJM7, which encodes the AIDA-I autotransporter domains and both have been described earlier [8,27]. Plasmid pIU27.1.O encoding aprotinin was kindly provided by H. Apeler, A.G. Bayer (Wupperthal, Germany). Bacteria were routinely grown at 37 °C in Luria–Bertani (LB) broth containing 100 mg ampicillin per litre. Recombinant DNA techniques. For the construction of an aprotinin–autotransporter fusion protein, the aprotinin gene was amplified by PCR from plasmid pIU27.1.O with oligonucleotide primers DZ001 (5 0 -gcg tcg acc gtc ctg act tct gcc tcg agc cg-3 0 ) and DZ002 (5 0 -ggg gta cca gca cca ccg caa gta cg-3 0 ). The PCR product was inserted into vector pCR2.1-TOPO and recleaved with SalI and KpnI. The restriction fragment was ligated into pJM7 resulting in plasmid pAT-AP02-7 (Fig. 2). Plasmid pAT-AP02-7 was used as a template for PCR to construct plasmid pAT-AP02-13 with oligonucleotide primers JJ009 (5 0 -gct cta gac gtc ctg act tct gcc tcg agc cg-3 0 ) and JJ010 (5 0 -gaa gat cta gca cca ccg caa gta cgc-3 0 ). The PCR product was inserted into vector pCR2.1-TOPO and recleaved with XbaI/BglII. The restriction fragment was ligated into pJM1013, restricted with the same enzymes, resulting in plasmid pAT-AP02-13. Both ligations yielded an in-frame fusion of aprotinin with the AIDA-I autotransporter unit under the control of a constitutive promoter (PTK) [4]. They differed in the length of the linker region, that is necessary between the recombinant passenger protein and the b-barrel to obtain full-surface exposure of the passenger (Fig. 2). Outer-membrane preparation. E. coli cells were grown overnight and 1 mL culture was used to inoculate fresh LB medium (20 mL). Cells were cultured at 37 °C with shaking (200 rpm) for about 5 h until OD578 of 0.7 was reached. Cells were harvested and outer membranes were prepared according to a modification of the rapid isolation method of Hantke [28], as described earlier.
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Fig. 2. Construction of the aprotinin–autotransporter fusion proteins. (A) Nucleotide and amino acid sequence of aprotinin used in this study. Restriction sites ResI/ResII were modified by PCR to XhoI/SalI sites for the construction of plasmid pAT-AP02-7 and to XbaI/BglII sites for plasmid pAT-AP02-13. Cysteines which are involved in the formation of intramolecular disulfide bonds are written in bold letters. (B) Structure of the fusion proteins AT-AP7 and AT-AP13E. The environment of the fusion sites between aprotinin and the autotransporter domains are given as sequences. Restriction sites are underlined. The cleavage site of the signal peptidase is indicated by an arrowhead as well as the accessible trypsin cleavage site within the linker region, identified in previous experiments. The eight amino acids of CTB origin are marked by asterisks. The linear epitope for a monoclonal antibody used for labelling fusion protein AT-AP13E, encoded by plasmid pAT-AP02-13 is written in bold letters. Between the epitope and the linker region, a specific IgA1 protease cleavage site (PPSP) was inserted. FP, fusion protein.
SDS–PAGE and Western blot analysis. Outer membrane isolates were diluted (1:2) with sample buffer (100 mM Tris–HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, and 20% glycerol). The samples were then boiled for 20 min and analysed on 12.5% SDS–PAGE. Proteins were visualized with Coomassie brilliant blue. Prestained molecular weight markers (Bio-Rad, Munich, Germany) were used to determine the size of the prepared proteins. For Western blot analysis, gels were electroblotted onto polyvinylidene-difluoride (PVDF) membranes and blotted membranes were blocked in TBS with 3% FCS overnight. For immunodetection, membranes were incubated for 3 h either with aprotinin antibody HDH191 (kindly provided by H. Apeler, A.G. Bayer, Wupperthal, Germany) or with monoclonal anti-PEYFK Du¨142 [4], which were diluted 1:1000 or 1:35 in TBS containing 3% FCS. Prior to the addition of secondary antibodies, immunoblots were rinsed three times with TBS containing 0.1% Tween 20. Antigen–antibody conjugates were visualized by reaction with alkaline phosphatase linked goat anti-mouse IgG secondary antibody (KPL, Gaithersburg, MD, USA), diluted 1:10,000 in TBS with 3% FCS. A colour reaction was obtained by adding 10 mL incubation buffer (100 mM NaCl, 5 mM MgCl2, and 100 mM Tris–HCl, pH 9.5) containing 66 lL nitrobluetetrazoliumchloride (50 mg mL1 in 70% dimethylformamide) and 33 lL 5-
brome-4-chlor-3-indolylphosphate-disodium salt (20 mg mL1 in H2O). Flow cytometer analysis. For flow cytometer analysis cultures of E. coli cells with or without plasmids were grown at 37 °C overnight and subcultured in a dilution of 1:20 at 37 °C until they reached OD578 of 0.7. Cells were harvested, washed three times with PBS, and suspended in PBS including 3% FCS to an OD578 of 1.0. After 30 min, 5 lL of biotin-labelled human leucocyte elastase (HLE) was added to yield a total volume of 1 mL PBS/3% FCS and incubation was extended for another 30 min. HLE was obtained from Lee Scientific (St. Louis, USA) and biotin-labelling was performed with the biotinlabelling kit from Roche (Mannheim, Germany) according to the manufacturerÕs recommendations. After incubation, cells were washed three times with PBS and incubated for 30 min with FITC-coupled streptavidin (Roche, Mannheim, FRG). Then, cells were washed three times with PBS and suspended in PBS to a final OD578 of 0.02 for subsequent FACS analysis. For each experiment at least 10,000 cells were analysed with a FacsCalibur Cytometer (Becton Dickinson, Heidelberg, Germany) using 488 nm as excitation wavelength and FACS-FLOW (Becton–Dickinson, Heidelberg, Germany) as sheath fluid. The threshold trigger was set on side scatter to eliminate background noise and to analyse solely intact cells.
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Results Construction and expression of aprotinin–AIDA-I autotransporter fusion proteins For successful surface display of a recombinant passenger protein by the autotransporter pathway, a gene needs to be constructed that encodes an artificial precursor protein. This precursor must contain a signal peptide at the N-terminus, the passenger (in our case: aprotinin), a linker region and the b-barrel (Fig. 1). For this purpose, the coding region of aprotinin (62 aa) was amplified by PCR and inserted into vector pJM7, which has been used before for the surface display of the choleratoxin b-subunit (CTB) [4]. This resulted in plasmid pAT-AP02-7. Due to the ligation procedure the artificial aprotinin–AIDA-I fusion protein (AT-AP7) encoded by this plasmid, contained 8 amino acids of native CTB in addition (Fig. 2). To analyse expression of AT-AP7, the corresponding plasmid pAT-AP02-7 was transformed into E. coli strains JK321 and UT5600. E. coli JK321 is a dsbA derivative of UT5600. Both strains are ompT and have been used in former experiments for the functional surface display of a broad variety of recombinant passengers by the autotransporter pathway [4,6,7,10]. However, in both strains no protein of the correct size was detectable, neither by Coomassie brilliant blue staining nor by Western blot analysis with the aprotinin-specific antiserum HDH191 (not shown). To exclude that this finding was due to misfolded aprotinin, that could not be detected by antibody HDH191, a peptide tag (PEYFK) was added at the C-terminus of aprotinin (Fig. 2), for which a highly specific monoclonal antibody was available [4]. The aim was to establish a method for detection in Western blot experiments, that is independent of the aprotinin passenger being folded in the correct tertiary structure or not. This was done by constructing plasmid pAT-AP02-13 encoding the fusion protein AT-AP13E, that additionally contained the peptide tag PEYFK. The expression of fusion protein AT-AP13E was analysed again in both strains, UT5600 and JK321, and compared to the expression of FP50 encoded by plasmid pJM1013 as a control. FP50 is identical with AT-AP13E, except that it is lacking the aprotinin domain. As shown in Fig. 3, the control protein was detectable in both strains, whereas no trace of AT-AP13E was seen in Western blot experiments. As the translational and transcriptional signals in both plasmids, pAT-AP02-13 and its control pJM1013, were identical and the correct in-frame fusion of aprotinin to the autotransporter domains was verified by DNA sequence analysis, this clearly pointed on a proteolytic degradation of AT-AP13E during transport. This was surprising, since aprotinin was chosen as a passenger due to the assumption that a protease inhibitor could resist such type of degradation.
Fig. 3. Western blot analysis of outer membrane protein preparations from E. coli strains UT5600 and JK321 [20] harbouring pAT-AP02-13 or the control plasmid pJM1013. Immunoblotting was performed with the peptide tag (PEYFK) specific monoclonal antibody Du¨142. The molecular weight of marker proteins applied is indicated in kilodaltons at the right.
Surface display of aprotinin by blocking of disulfide bond formation Folding of free aprotinin has been subjected to previous investigations and was found to proceed via several one- or two-disulfide bond stabilized intermediates, including a so-called ‘‘kinetic trap,’’ which very slowly rearranges to other folding intermediates [29–31]. In the present concept of surface translocation by the autotransporter pathway, any folding of the passenger would hinder surface translocation and would result in degradation by periplasmic proteases, as long as no stable, protease-resistant conformation is obtained. Therefore, preventing the formation of disulfide bonds appeared to be a solution for keeping aprotinin in an unfolded state and directing the passenger molecule to the cell surface. In other words, blocking of disulfide bonding would enable sufficient aprotinin passenger molecules to escape from the periplasmic-protease machinery across the porin-like b-barrel and these molecules should be detectable at the surface. For this purpose, cells of E. coli UT5600 were transformed with plasmid pAT-AP02-7 and cultured in liquid LB medium supplied with 10 mM 2-mercaptoethanol in order to maintain strong reducing conditions. It has been shown in earlier studies, that a concentration of 10 mM 2-mercaptoethanol was sufficient to block disulfide bond formation during transport [5,19,20] and enabled passenger proteins with naturally occurring disulfide bonds to be transported to the surface. After cells of E. coli UT5600 pAT-AP02-7 in growth medium with 10 mM 2-mercaptoethanol had reached an OD578 = 1, outer membrane proteins were prepared and subjected to Western blot analysis. As shown in Fig. 4, a full-size fusion protein AT-AP7 was detectable in UT5600 under these growth conditions.
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Fig. 4. Western blot analysis of outer membrane protein preparations from E. coli UT5600 harbouring pAT-AP02 and grown in the presence of 10 mM 2-mercaptoethanol. Immunoblotting was performed with aprotinin specific antibody HDH191. M, prestained molecular mass marker. The molecular weight is indicated in kilodaltons. Whole cells were treated with trypsin before outer membranes were prepared (+) or not ().
Theoretically two orientations in the outer membrane are possible for the aprotinin domain in the autotransporter fusion protein, either directed to the periplasm or exposed at the cell surface. In previous experiments with other passengers, the orientation of the protein in the outer membrane was assessed by the treatment of whole bacterial cells with trypsin [4,5,7,8]. Digestion of proteins by trypsin can only take place at the surface of the bacterial cell and not in the periplasm since the trypsin molecule is too big to cross the outer membrane. The previous experiments concordantly showed that incubation of whole cells with trypsin removed the passenger domain from the fusion proteins by a cleavage site within the linker region (K/G), located 54 amino acids upstream of the b-barrel structure (Fig. 2). If cells of UT5600 pAT-AP02-7 were incubated for 30 min with trypsin in the same concentration as in the experiments before, however, no size reduction of the aprotinin– autotransporter fusion protein was detectable (not shown). This could have been an indication that aprotinin at the cell surface gained its active conformation as a protease inhibitor and thereby inhibited trypsin. To overcome this effect, the concentration of trypsin was in-
creased 1000-fold before being added to whole cells of UT5600 pAT-AP02-7. As shown in Fig. 4 (lane 2) the fusion protein was no longer detectable under these conditions. Thus, aprotinin appears to be located at the cell surface and is obviously released from the autotransporter fusion protein by trypsin cleavage within the linker domain (Fig. 2), in case the concentration of the protease was substantially increased. From these results it can be summarized, that maintaining reducing conditions during transport—most probably by preventing disulfide bonding—enables sufficient aprotinin passenger molecules to escape to the surface of the cell before being decomposed in the periplasm. In other words, aprotinin in its active structure containing disulfide bonds cannot be translocated to the surface by the autotransporter pathway. Translocation is only possible if aprotinin is kept in an unfolded state during transport by blocking disulfide bond formation. It can be speculated, that this might also be a question of velocity, and the number of aprotinin molecules present on the surface is only a part of the entire amount of molecules produced. Expression of aprotinin in a periplasmic-protease negative host background Escherichia coli contains several enzymes degrading incorrectly folded proteins in the periplasm [32,33]. This is indispensable, as the periplasm can be assumed as a limited space. In former studies of Sauer and co-workers, the role of periplasmic proteases in E. coli was studied by constructing several mutant strains, either defective in periplasmic protease DegP or DegQ [33]. As our presented results indicate that periplasmic degradation could be the cause of the failed expression of the autotransporter–aprotinin fusion protein AP02-7 under non-reducing growth conditions, we examined the expression of the protein in three of these mutant strains. For this purpose, strains PW147 (degQ), PW151 (degP), and PW152 (degQ) were transformed with plasmid pAT-AP02-7 and analysed under identical non-reducing conditions as described before. Outer membrane proteins were prepared and, as shown in Fig. 5A, a full-size fusion protein AP02-7 was detectable
Fig. 5. Western blot analysis of outer membrane preparations from periplasmic-proteases negative E. coli strains harbouring plasmid pAT-AP02-7. (A) Whole cells were treated with trypsin or not before outer membranes were prepared. (B) Outer membrane preparations were subjected to trypsin treatment subsequently. Immunoblotting was performed with the aprotinin specific antibody HDH191. M, prestained molecular mass marker. The molecular weight is indicated in kilodaltons. + and , trypsin was added or was not.
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by Western blot analysis in all strains used. Obviously the absence of one of the two periplasmic proteases DegP or DegQ prevented the aprotinin–autotransporter fusion protein from periplasmic degradation. To verify, whether the aprotinin domain was indeed directed to the periplasm, trypsin was added to whole cells of each plasmid containing strain, before outer membranes were prepared. As described above, the outer membrane is an invincible barrier for large molecules as trypsin and therefore it would protect the passenger from degradation if the passenger is located in the periplasm [4,5]. Since aprotinin is known to be a potent inhibitor of the serine protease trypsin, again a 1000-fold higher protease concentration was applied as usual in similar experiments with other passenger proteins, known to be no protease inhibitors. As can be seen in Fig. 5A, trypsin addition to whole cells of PW147, PW151, and PW152, all expressing AT-AP7, had no effect, neither in size nor in the amount of protein expressed. This means, that under non-reducing conditions in the DegP or DegQ defective strains, the aprotinin domain appears to be not exposed to the cell surface but rather is directed to the periplasm. The reason for this orientation is probably the fact that under these conditions aprotinin folds into its active structure in the periplasm, leading to a molecule size which is too big for the transport across the b-barrel and which is resistant to proteolytic degradation in the periplasm. For verification, immunofluorescence microscopy studies were performed using the anti-aprotinin antibody HDH191 as a primary antibody and a FITC-labelled anti-mouse IgG as a secondary antibody. Cells of PW147, 151, and 152 expressing AT-AP7 showed no fluorescence at all and were identical to control cells without plasmid. This was an additional hint, that under these conditions, the aprotinin domain is not exposed to the cell surface. To exclude a general resistance of the aprotinin–autotransporter fusion protein to trypsin in these strains,
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outer membrane proteins were first prepared from cells of PW147, PW151, and PW152, all expressing AT-AP7, and then applied to trypsin digestion. This treatment would enable trypsin to have access to the aprotinin– autotransporter fusion protein from both sides of the outer membrane. As Fig. 5B shows, that the fusion protein was no longer detectable in Western blot experiments after this treatment. As mentioned above, this is supposed to be due to a known cleavage site of trypsin within the linker region (Fig. 2), resulting in the release of the aprotinin passenger domain. The detection of the remaining autotransporter core was not expected as it is not recognized by the aprotinin-specific antibody HDH191 used in this experiment. Investigation on the functionality of aprotinin displayed on the cell surface To find out, whether the aprotinin molecules translocated to the cellÕs surface gain an active conformation, cells of UT5600 pAT-AP02-7 grown under reducing conditions (10 mM 2-mercaptoethanol) were incubated with biotin-labelled human leukocyte elastase (HLE) and subsequently, after the addition of streptavidinFITC analysed by flow cytometry. Aprotinin has been reported to be a strong inhibitor of HLE (Ki = 3.5 lM) [34]. As a strong inhibitor is supposed to bind its target enzyme to the same extent, we expected FITC-labelled HLE to bind to the bacterial surface via the aprotinin molecule. This should result in a specific and significant increase in whole cell fluorescence compared to control cells without aprotinin. The controls applied in these experiments were cells of E. coli UT5600 with plasmid pJM7, encoding an autotransporter fusion protein with CTB instead of aprotinin as a passenger. As shown in Fig. 6, surface display of aprotinin resulted in a 10.5fold increase in relative fluorescence (mean value UT5600 pJM7 control: 19.78; UT5600 pAT-AP02-7:
Fig. 6. FACS analysis of E. coli UT5600 harbouring plasmid pAT-AP02-7 (B) or pJM7 (aprotinin-negative control), (A) grown in the presence of 10 mM 2-mercaptoethanol. Whole cells were incubated with biotin-labelled human leucocyte elastase (HLE) and then treated with FITC-coupled streptavidin. For each measurement 10,000 cells were analysed. As controls in each experiment cells were treated similarly but not incubated with biotin-labelled HLE (filled histograms). The mean value of fluorescence for aprotinin displaying cells (208; B, open histogram) was more than 10 times higher than for cells without aprotinin (19; A, open histogram).
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208.12). Although it cannot be excluded in principle that HLE binds by a different part than its active site to aprotinin displayed at the cell surface, this result indicates that aprotinin folds into an active conformation after being translocated to the cell surface. The broad distribution of fluorescent UT5600 pAT-AP02-7 cells could be explained by a linker cleavage activity of elastase. When outer membrane proteins were prepared after the addition of HLE to whole cells and were analysed by Western blot, a significant decrease in the observed amount of full-size AT-AP7 protein was detectable, which was due to cleavage within the linker region (not shown). This cleavage can be possible even after the first HLE molecule has already bound to the aprotinin domain and is inhibited thereby. A second HLE could attack the conglomerate by the linker region. Therefore, the cell population seen in Fig. 6B, might be a mixture of cells, with different numbers of aprotinin molecules on the surface, due to proteolytic activities of HLE molecules in excess.
Discussion When the disulfide bond containing choleratoxin B (CTB) was expressed in E. coli as an autotransporter passenger protein under non-reducing conditions [18], no autotransporter–CTB fusion protein was detectable at all. The folding of the protein including the formation of disulfide bonds during transport in the periplasm obviously resulted in a structure that was too voluminous to be transported through the pore formed by the b-barrel [19,20]. Thereby, CTB was obliged to remain in the periplasm where it was subjected to proteolytic degradation. To get a view on this intermediate step during surface translocation, aprotinin was chosen in this study, because
on one hand it also contains disulfide bonds but on the second hand promised to be resistant to periplasmic protease degradation due to its protease inhibitor activity. Autodisplay of the protease inhibitor aprotinin at the cell surface was achieved by fusing its coding region to the autotransporter domain encoding regions of the AIDA-I [3], and growing the cells transformed by the resulting artificial gene construct under reducing conditions with 10 mM 2-mercaptoethanol. Omitting 2-mercaptoethanol from the growth medium resulted in complete degradation of the autotransporter–aprotinin fusion protein. To verify the hypothesis, that this was due to proteolysis by periplasmic proteases, the identical gene construct was expressed in E. coli strains either defective in periplasmic protease DegQ or DegP [33] under non-reducing conditions. This expression resulted in detectable amounts of the autotransporter–aprotinin fusion protein. However, the aprotinin domain was directed to the periplasmic side of the outer membrane. These results indicate that under reducing growth conditions by application of 2-mercaptoethanol, disulfide bond formation is prevented within the aprotinin passenger domain and it can be translocated to the cell surface (Fig. 7B). When non-reducing growth conditions are applied, aprotinin folds during transport, cannot translocate to the cell surface and remains in the periplasm. There it is degraded as long as all of the periplasmic proteases are active. In case that one of these proteases, either DegQ or DegP are missing, aprotinin can be found in the periplasm in a stable confirmation (Fig. 7A). This may be due to the known serine protease-inhibiting activity of aprotinin [35]. From these results, it can be concluded, that in E. coli strains, defective in one of the periplasmic proteases DegQ or DegP, the aprotinin–autotransporter fusion protein is expressed in detectable amounts. The b-barrel seems to
Fig. 7. Scheme of aprotinin autodisplay in E. coli. (A) In periplasmatic-protease negative strains PW147, PW151, or PW152 grown without 2-ME, aprotinin folds within the periplasm and remains located stable at the inner side of the outer membrane. PP, periplasm; OM, outer membrane. (B) In E. coli UT5600 grown under reducing conditions with 10 mM 2-mercaptoethanol (2-ME), it is translocated to the surface and folds to its active conformation. The structure of aprotinin is in analogy to the resolved crystal structure [36].
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be integrated in the outer membrane, whereas the passenger protein is maintained in the periplasm. This periplasmic location, however, did not result in proteolytic degradation, as seen before for other passengers as, e.g., CTB [18–20]. We suppose, that this is due to the passenger domain used in the present experiments, aprotinin. Aprotinin itself is a known inhibitor of a wide variety of serine proteases [35] and presumably inhibits, in its active form, the periplasmic serine proteases DegP or DeqQ too [3]. Interestingly, it had no effect whether DegP or DegQ was missing. In both cases, aprotinin was able to withstand the proteolytic attack. However, if both proteases, DegP and DegQ are working in concert, as, e.g., in strains UT5600 or JK321 (Fig. 3), the fusion protein AT-AP7 is degraded completely and can not be detected at all. This can be interpreted in two ways. One explanation could be that degradation of aprotinin in the periplasm is a matter of velocity. This means, a competition exists between folding of the aprotinin domain to its active and protease-inhibiting conformation on the one hand and its degradation by the periplasmic proteases on the other hand. If one protease is missing, folding gets the upper hand, otherwise degradation is predominant. A second explanation could be that both proteases must interact in common with the aprotinin domain more or less simultaneously to avoid the formation of an active protease inhibitor. Both cases, however, result in a protease-resistant autotransporter fusion protein, when either DegP or DegQ are missing, most probably due to the protease inhibitory potential of the passenger domain aprotinin. In summary our results indicate that the passenger protein transported by the autotransporter pathway needs to cross a size restricted area during transport, otherwise transport stops in the periplasm and the passenger protein is proteolytically degraded as long as it has no protease-inhibiting activity. These results are in concordance with the concept that the passenger protein is translocated through the porin-like structure formed by the b-barrel of the autotransporter domain, as it has been suggested more recently by the first crystal structure of a member of this family of protein transporters, but was subject of controversial discussions for many years before. Beside resolving this intermediate step in the pathway used by the autotransporter family of proteins, we present a new methodical approach, namely that cells displaying a protease inhibitor at the surface can be labelled by the target enzyme and can be analysed by flow cytometry, in case the target enzyme is labelled with a fluorescent dye.
Acknowledgments We thank H. Apeler (A.G. Bayer, Wupperthal, Germany) for providing the monoclonal antibody HDH191
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and the plasmid pIU27.1.O, encoding the aprotinin DNA sequence, H. Kolmar and P. Sauer for providing the E. coli strains PW147, 151, and 152, and R. Maas for critically reading the manuscript.
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