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Biochem. J. (2005) 385, 181–187 (Printed in Great Britain)
Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli Benedykt WLADYKA1 , Katarzyna PUZIA and Adam DUBIN Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387, Krakow, Poland
Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was coexpressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme.
Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+ -nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.
INTRODUCTION
host, which results in growth inhibition and lack of expression of the target protein. A significantly improved yield of the papainlike proteases is commonly achieved by their expression together with prodomains, which facilitates correct folding and/or inhibits enzymic activity [15]. Mutagenesis within the active sites may also be a method of choice to abolish the enzymic activity of recombinant proteases, and it is especially applicable to structural studies [11]. It was shown that co-expression of some recombinant proteins with their ligands [16] or chaperones [17] could increase both the amount and solubility of expressed target proteins. Herein, we present a method for efficient co-expression of the recombinant staphopain A together with its specific inhibitor, staphostatin A, in Escherichia coli and an optimized method for refolding of the protease. In the developed system, the yield of the active staphopain A was over 10-fold higher in comparison with isolation from natural sources [2]. This allows for the production of the enzyme in amounts large enough for further detailed studies of staphopain A–staphostatin A interactions.
Staphylococcus aureus produces a large number of extracellular proteins, many of which are important virulence factors toxic to humans and animals [1]. Among the proteolytic enzymes secreted by this bacterium, there are two cysteine proteases, referred to as staphopain A (ScpA) and staphopain B (SspB) [2,3]. Genes coding for the staphopains are located in operons with downstream genes scpB and sspC encoding staphostatins A and B respectively, which were recently described as inhibitors of the staphopains [4,5]. Staphostatins inhibit their target proteases by forming tight, non-covalent complexes in a 1:1 molar ratio. These inhibitors are most probably localized inside the cell, guarding cytoplasmic proteins from non-specific degradation by prematurely folded staphopains [4]. Staphopain A is produced in a zymogen form and must undergo proteolytic cleavage to get activated, but its activation occurs in an as yet unidentified manner [6]. Broadspecificity proteases, such as staphopain A, pose a danger to the cells that produce them, and thus many micro-organisms have developed mechanisms to control the proteolytic activity of the enzymes. The ways to achieve this is to secrete proteases as inactive zymogens [7] or to produce inhibitors of the proteases [8,9]. Although the structures of both staphopains [10,11] and staphostatins [12,13] were solved, there are some details of the enzyme–inhibitor interactions that remain unknown. Therefore the availability of an efficient source of staphopain A is essential for further kinetic studies of staphopain–staphostatin complex formation. It is advisable to use a heterologous expression to obtain cysteine proteases from hazardous and pathogenic organisms [14]. Unfortunately, the expression of proteases in a bacterial system is often problematic, since the recombinant proteins are toxic to the
Key words: co-expression, cysteine protease, inhibitor, refolding, staphopain A, staphostatin A.
EXPERIMENTAL Materials
S. aureus V8-BC10 genomic DNA was kindly provided by Dr A. Sabat (Polish Institute of Public Health, Warsaw, Poland). The recombinant staphostatin A was a gift from G. Dubin (Department of Microbiology, Jagiellonian University). Enzymes for molecular biology were obtained from MBI Fermentas (Vilnius, Lithuania) and New England Biolabs (Beverly, MA, U.S.A.). pETDuet-1 and pACYCDuet-1 vectors were obtained from Novagen (Darmstadt, Germany). All other reagents were
Abbreviations used: DTT, dithiothreitol; GdmHCl, guanidinium chloride; IPTG, isopropyl β-D-thiogalactoside; LB, Luria–Bertani; Ni-NTA, Ni2+ nitrilotriacetate. 1 To whom correspondence should be addressed (email
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from Sigma (Sigma–Aldrich, St. Louis, MO, U.S.A.) or as mentioned in the text. Bacterial strains and growth conditions
E. coli DH5α strain (Invitrogen, Carlsbad, CA, U.S.A.) was used for the propagation of recombinant forms of the plasmids pETDuet-1 and pACYCDuet-1. E. coli strains BL21(DE3) and BL21(DE3)pLysS (Novagen) were used for the expression of recombinant proteins. All the E. coli strains were grown in LB (Luria–Bertani) medium supplemented with appropriate antibiotics. Cloning and mutagenesis of staphopain A and staphostatin A
The coding sequence of the mature form of staphopain A (amino acids 214–388; NCBI accession no. CAD61962) was amplified using PCR with S. aureus genomic DNA as a template. The primers used were ScpA F (5 -GCGAATTCTTACAACGAGCAATATGTA-3 ) and ScpA R (5 -CGCAAGCTTAATAACCATAAATAGATGA-3 ), carrying restriction sites (underlined) for ligation to the expression vector pETDuet-1. The reaction used 1 unit of the proof-reading DNA polymerase (Finnzymes, Espoo, Finland) with the following cycling: 1 cycle at 94 ◦C for 3 min, 30 cycles at 94 ◦C for 30 s, 46 ◦C for 30 s and 72 ◦C for 90 s, and an additional step at 72 ◦C for 5 min. The PCR product was digested with EcoRI and HindIII, gel-purified and cloned into pETDuet-1 previously digested with the same restriction enzymes to yield pETDuet1-scpA. The plasmid encodes the N-terminally Histagged mature form of staphopain A. Similarly, the staphostatin A coding sequence was amplified with the primers ScpB F (5 -CGCCATATGGAACAATTTGAATTATTTATG-3 ) and ScpB R (5 -GCGCTCGAGTTATTATTTATGCTTAATGAA-3 ) and S. aureus V8-BC10 genomic DNA as a template. PCR parameters were similar to those described above, except for the annealing temperature, which was set at 50 ◦C. The product of the reaction was digested with NdeI and XhoI, gel-purified and cloned into pACYCDuet-1 predigested with the same enzymes to give the plasmid pACYCDuet-scpB, encoding staphostatin A. This allowed for expression of the inhibitor in the native sequence. Thus we obtained the separate plasmids encoding staphopain A and staphostatin A, the enzyme and its inhibitor. If necessary, these constructs could exist and be expressed together in one host cell line. Additionally, the staphostatin A coding sequence was cloned into the previously obtained plasmid, pETDuet1-scpA, under the second T7 promoter at NdeI and XhoI restriction sites to give plasmid pETDuet1-scpA&scpB. The plasmid encodes the enzyme and the inhibitor under the control of two independent T7 promoters. All plasmids were sequenced through the regions of the inserts to confirm that no mutations had been introduced during PCR. Mutagenesis studies were performed with a site-directed mutagenesis kit (Stratagene, La Jolla, CA, U.S.A.) according to the manufacturer’s instructions. Briefly, the primer ScpAala F (5 -CTCAAGGTAACAATGGTTGGGCGGCAGGCTATACGATGTCT-3 ) and a reverse complementary primer were used for a PCR with pETDuet1-scpA as a template to exchange the TGT triplet encoding Cys238 in the active site of staphopain A into GCG encoding alanine (indicated in boldface). Then the reaction mixture was digested with DpnI to remove the parental DNA template. The nicked vector, pETDuet1-scpAala, containing the desired mutation was then transformed into DH5α competent cells. Similarly, the primer ScpBala F (5 -GAACACAGAAGCTTTAGCGACTTCTCCTAGAATGAC-3 ) and a reverse complemen c 2005 Biochemical Society
tary primer were used to produce a Gly98 → Ala substitution in the sequence of staphostatin A [12]. The obtained plasmid, pETDuet1-scpA&scpBala, encodes the active, mature form of staphopain A and the inactive form of staphostatin A. All plasmids were sequenced to confirm generation of the desired mutations. In all cloning steps, T7 RNA polymerase-deficient E. coli DH5α cells were used, thus eliminating the possibility of plasmid instability. Expression of recombinant proteins
The desired plasmids were transformed into E. coli BL21(DE3) and bacteria were plated on LB agar plates supplemented with appropriate antibiotics, unless otherwise mentioned. Freshly transformed colonies were inoculated into LB medium and grown at 37 ◦C until an absorbance A600 0.8 was reached; then, the temperature was lowered to 30 ◦C and recombinant expression was induced by the addition of IPTG (isopropyl β-D-thiogalactoside) at a final concentration of 1 mM, and then culturing was continued for 4 h. Subsequently, bacteria were centrifuged (15 000 g, 20 min, 4 ◦C) and the pellet was stored at − 20 ◦C. Purification and refolding of staphopain A
Bacteria were thawed, suspended in water and sonicated on ice to release recombinant proteins and to lower the viscosity. The insoluble fraction, which contained mostly co-precipitated staphopain A and staphostatin A, was removed from the soluble material by centrifugation at 20 000 g for 30 min. The insoluble complex was solubilized overnight at room temperature with vigorous stirring in Buffer A [6 M GdmHCl (guanidinium chloride), 100 mM sodium phosphate, 10 mM Tris/HCl and 5 mM 2-mercaptoethanol, pH 8.0]. After centrifugation at 20 000 g for 30 min, the supernatant was circulated for 2 h through an NiNTA (Ni2+ -nitrilotriacetate; Qiagen, Hilden, Germany) column equilibrated with Buffer A. Then, the column was washed with Buffer A followed by Buffer B (6 M GdmHCl, 100 mM sodium phosphate, 10 mM Tris/HCl, 5 mM 2-mercaptoethanol, pH 6.5) to remove unbound proteins (mostly staphostatin A). Staphopain A was step-eluted with Buffer C (6 M GdmHCl, 100 mM sodium phosphate and 10 mM Tris/HCl, pH 4.5). Fractions containing staphopain A were pooled and concentrated to 10 mg/ml. For large-scale refolding, 50 mg of the Ni-NTA-purified staphopain A was diluted 100-fold in 500 ml of ice-cold optimized refolding buffer [50 mM sodium phosphate, pH 6.0, 150 mM NaCl, 1 mM EDTA, 0.5 M L-arginine, 30 % (v/v) glycerol and 0.1 % (v/v) Tween 20] and incubated at 4 ◦C for at least 72 h. The refolded sample was centrifuged at 20 000 g for 30 min and concentrated to 10 ml using Stirred Cells (models 8400 and 8050; Amicon, Bedford, MA, U.S.A.) with a 10 kDa cut-off membrane. After 5-fold dilution with 50 mM sodium phosphate buffer (pH 6.0) containing 150 mM NaCl and 1 mM EDTA, the sample was stirred with a thiopropyl-Sepharose resin (Amersham Biosciences, Uppsala, Sweden) for 2 h at 4 ◦C. Then, the column was packed and washed with the binding buffer to remove the unbound inactive staphopain A. The active enzyme was eluted with 50 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, 1 mM EDTA and 25 mM 2-mercaptoethanol, concentrated to the desired volume, divided into aliquots and frozen for long-term storage. Protein quantity and quality analysis
Protein concentrations were determined by the method of Bradford [18]. Before induction and every 1 h after induction with IPTG, 1 ml of bacterial culture was taken, pelleted, dissolved in 100 µl of
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SDS sample buffer and boiled in a water bath for 5 min. Fractions (5 µl) were subjected to SDS/PAGE [19] and then either Coomassie Blue-stained or electrotransferred [20] on to a PVDF Western blotting membrane (Applied Biosystems, Foster City, CA, U.S.A.). The membrane was incubated with anti-His-tag (Alexis Biochemicals, Carlsbad, CA, U.S.A.) or anti-staphostatin A rabbit polyclonal IgG [4] to detect staphopain A or staphostatin A respectively. After incubation with the peroxidase-conjugated secondary antibodies (Fisher Scientific, Pittsburgh, PA, U.S.A.), the blot was developed using 4-chloro-1-naphthol as a substrate. Staphopain A proteolytic activity was determined using azocasein as a substrate. Aliquots of 150 µl were incubated at 37 ◦C for 90 min in a final volume of 1 ml of 25 mM phosphate buffer (pH 7.0), containing 2 mM DTT (dithiothreitol), 5 mM EDTA and 0.5 % (w/v) azocasein. The reaction was stopped by the addition of 300 µl of 10 % (w/v) trichloroacetic acid, and the undigested azocasein was removed by centrifugation at 15 000 g for 5 min at room temperature. The absorption of the coloured azopeptides in the supernatant was measured photometrically at 360 nm.
RESULTS Recombinant staphopain A is toxic for E. coli
Target protein expression may be initiated by transferring the plasmid into the expression host, e.g. BL21(DE3) cells, containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control. Expression is induced by the addition of IPTG to the bacterial culture. First, an effort was made to transform the pETDuet1-scpA construct into BL21(DE3) cells. Unfortunately, no colonies were observed after the transformation, even when the medium was supplemented with glucose [21]. Therefore we attempted to transform the construct to the host strain with a tight control of protein expression. BL21(DE3)pLysS cells were chosen, since they contained a compatible plasmid that provided a small amount of T7 lysozyme, a natural inhibitor of T7 RNA polymerase. Indeed, when pETDuet1-scpA was transformed into this host strain, we were able to observe colonies. These colonies were growing at a slower rate in comparison with the BL21(DE3)pLysS cells transformed with the pETDuet-1 vector without any insert. When transformants were grown in the liquid LB media, inhibition of the growth was also observed (results not shown). When the A600 of the culture reached 0.8, IPTG was added and bacteria were allowed to grow for an additional 4 h. No expression of staphopain A was observed after induction, which has been confirmed by SDS/PAGE and Western-blot analysis (Figure 1A) with His-tag-specific antibodies. These results suggest that the active form of staphopain A is toxic for E. coli and could efficiently inhibit the growth of the bacteria even in amounts as low as the basal, non-induced expression level.
Figure 1 Recombinant staphopain A (ScpA) is not expressed in E. coli unless together with its inhibitor, staphostatin A (ScpB) Electrophoretic analysis of (A) total cell proteins of BL21(DE3)pLysS cells transformed with pETDuet1-scpA . Lane M, molecular-mass standards in kDa. Lanes 1 and 2, lysed bacteria before and after induction with IPTG respectively. Lane 3, anti-His-tag probed Western blot of protein from lane 2. (B) Lysate of BL21(DE3) cells transformed with pACYCDuet1-scpB . Lanes 1–3, same as in (A), except that anti-staphostatin A (ScpB) antibodies were used. (C, D) Total cell proteins of BL21(DE3) cells transformed with (C) pETDuet1-scpA and pACYCDuet1-scpB or (D) pETDuet1-scpA&scpB . Lanes M, 1, 2 and 3, same as in (A). The co-expressed proteins are marked with arrows. (E) The enzymically inactive mutant of staphopain A (ScpA*) is expressed well in bacteria. Lane M, molecular-mass standards in kDa. Lanes 1 and 2 represent electrophoretically separated total cell proteins of BL21(DE3) cells transformed with pETDuet1-scpAala before and after induction with IPTG respectively. Lane 3, anti-His-tag probed Western blot of protein from lane 2. (F) Neither staphopain A nor staphostatin A is expressed when the inhibitory property of staphostatin A is abolished. Electrophoretic analysis of total cell proteins of E. coli BL21(DE3)pLysS transformed with pETDuet1-scpA&scpBala . Lanes 1–3, same as in (E).
Staphopain A could be expressed only with its inhibitor
The following expression experiments were performed in BL21(DE3) cells. The pACYCDuet-1 vector, which was the base for the pACYCDuet1-scpB construct, carries the chloramphenicol resistance gene (cat), and therefore it is not compatible with hosts containing pLysS. The cells transformed with the encoding staphostatin A plasmid could grow without any noticeable inhibition, and after induction with IPTG, they expressed staphostatin A very efficiently (Figure 1B). These results indicate that the expression of staphostatin A has no remarkable negative effect on E. coli metabolism.
The first thought when considering the toxicity of staphopain A for E. coli is the proteolytic activity of the enzyme. Staphopain A could degrade the proteins of the host, which might be essential for growth of the bacteria. Secondly, hints of the existence of staphostatin A, the inhibitor of staphopain A, in the cytoplasm of S. aureus, which naturally produces and secretes staphopains, may lead to the conclusion that co-expression of the enzyme and the inhibitor may be the way to obtain the recombinant staphopain A in the heterologous expression system. pETDuet1-scpA and c 2005 Biochemical Society
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pACYCDuet1-scpB encoding staphopain A and staphostatin A respectively were co-transformed to the bacteria. Double transformants grown on the plate were transferred to liquid LB media and induced when the appropriate absorbance was reached. After the induction step, the expression of both the enzyme and the inhibitor was observed (Figure 1C). Similarly, when BL21(DE3) cells were transformed with pETDuet1-scpA&scpB, the transformant colonies were noticed. Moreover, after the addition of IPTG to the media, the expression of both target proteins was observed (Figure 1D). Judging from SDS/PAGE analysis, there was no significant difference between the level of expression of proteins from the plasmids encoding the enzyme and the inhibitor separately and the plasmid encoding both staphopain A and staphostatin A. These findings strongly suggest that, during the expression, staphopain A binds to staphostatin A, generating the enzyme–inhibitor complex, in which the enzymic activity of staphopain A is abolished. The inhibition of the enzyme by staphostatin A results in a decrease in the toxicity of staphopain A for E. coli, which, in turn, made possible the expression of staphopain A. The above results, suggesting toxicity of the enzymically active staphopain A for E. coli, were confirmed by the next set of experiments, in which the inactive mutant of staphopain A was expressed. pETDuet1-scpAala, in which the codon corresponding to cysteine in the active centre was replaced by the alanine one, was transformed efficiently into the expression strain of bacteria. After the inoculation of the liquid LB medium with the obtained colonies, no noticeable growth inhibition was observed. After the induction step, staphopain A was expressed with a good yield (Figure 1E). However, most of the expressed protein was found in the insoluble fraction. Additionally, lowering both the temperature after induction and the concentration of the inducer improved the amount of mutated staphopain A in the soluble form (results not shown). The profragments of the CA clan of cysteine proteases have been suggested to participate in proper folding [22]. Our findings indicate that a proper folding of the mature form of staphopain A, although difficult to obtain, is not impossible. To determine whether staphostatin A is really responsible for the staphopain A inactivation during the co-expression, we mutated pETDuet1-scpA&scpB, generating pETDuet1-scpA&scp Bala, in which the reactive centre Gly98 of the inhibitor was exchanged with an alanine residue. Such a mutation abolishes the inhibitory properties of staphostatin A, and thus the mutated staphostatin A acts as a substrate for staphopain A [12]. Indeed, after the mutation, we were unable to transform pETDuet1scpA&scpBala into BL21(DE3) cells. These results provide the additional evidence that the non-inhibited staphopain A is toxic to the cells. Herein, similar to the attempted transformation of pETDuet1-scpA into BL21(DE3) cells, the basal expression of the active staphopain A was sufficiently toxic for E. coli to prevent the growth of the transformants. To support this finding, when pETDuet1-scpA&scpBala has been transformed into BL21(DE3)pLysS cells, after the induction step, the expression of neither the enzyme nor the inhibitor was observed (Figure 1F). Purification of recombinant staphopain A
After the co-expression of staphopain A and staphostatin A from pETDuet1-scpA&scpB, cells were collected by centrifugation and lysed by sonication. Both soluble and insoluble fractions of the cell lysates were analysed by SDS/PAGE. Coomassie Blue staining as well as Western-blot analysis showed that staphopain A was located exclusively in the insoluble fraction (Figure 2, lanes 3 and 4). Since conditions for the mild dissociation of the enzyme–inhibitor complex are still not known, we did not make c 2005 Biochemical Society
Figure 2 Staphopain A purification by Ni-NTA-affinity chromatography under denaturing conditions SDS/PAGE (A) and Western-blot analysis (B) of the fractions obtained during purification of the recombinant staphopain A. Lane M, molecular-mass standards in kDa. Lanes 1 and 2, total cell proteins of BL21(DE3) cells transformed with pETDuet1-scpA&scpB before and after induction with IPTG respectively. Lane 3, a soluble protein fraction of cells from lane 2. Lane 4, solubilized inclusion bodies containing staphopain A and staphostatin A. Lane 5, Ni-NTA–Sepharose column flow-through with unbounded staphostatin A and other impurities. Lane 6, Ni-NTA column eluate containing purified staphopain A.
efforts to optimize the conditions for the expression of the proteins of interest in the soluble form. The recombinant staphopain A was expressed as an N-terminal His-tagged fusion protein. Therefore we applied Ni-NTA metalaffinity chromatography under denaturing conditions for the purification of staphopain A. In the presence of 6 M GdmHCl, the enzyme–inhibitor complex dissociates, which allows one to separate the His-tagged staphopain A, which binds to the NiNTA resin, from the non-fused staphostatin A. A Coomassie Blue-stained gel is presented in Figure 2(A) and a Western-blot analysis of the fractions obtained during purification is presented in Figure 2(B). Lane 4 represents solubilized inclusion bodies that contained the enzyme and the inhibitor. This finding supports the thesis that, during the co-expression, the staphopain–staphostatin complex is produced. Lane 5 represents the column flow-through, indicating that staphostatin A and the endogenous E. coli proteins did not bind to the Ni-NTA resin, whereas staphopain A did. After the washing step, the protein could be eluted from the column (lane 6) by lowering the pH of the buffer to 4.5. After the elution, almost completely pure staphopain A was obtained, but unfortunately in an unfolded conformation and therefore inactive. From 1 litre of bacterial culture after the Ni-NTA purification step, approx. 50 mg of staphopain A was obtained. To recover the enzymic activity of staphopain A, attempts were made to refold the protein. Of the methods tested, dilution was the most effective. To achieve the high refolding efficiency, different buffer compositions were tested. The optimization steps
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interesting that the restoration of the native conformation of the enzyme took at least 48 h (Figure 3C). Such a long time of renaturation could be explained by the lack of the prodomain, which is known to support the proper folding of papain-like proteases [22]. In the optimized refolding buffer, little, if any, protein aggregation was observed. However, when the activity of the refolded enzyme was measured by titration with staphostatin A, we noticed that only a minor part of the initial pool of the protein was active. Therefore separation of the misfolded form of staphopain A from the active one was necessary. Thiopropyl-Sepharose is uniquely suitable for binding to activated cysteine residues, as present in the ion pair of active sites in cysteine proteases. At acidic pH, only these proteases bind to this resin and can be easily eluted from the column at neutral pH with an excess of a reducing agent. Taking advantage of this feature, we were able to obtain approx. 3.5 mg of the active fraction of the enzyme from 50 mg of the protein subjected to refolding.
DISCUSSION
Figure 3 Refolding of staphopain A proceeds under conditions that are rather unusual for cysteine proteases (A) Dependence of refolding yield on the pH of the refolding buffer (50 mM buffering agent, 150 mM NaCl, 1 mM EDTA and 20 % glycerol). Protease activity was determined by measuring the A 360 of azopeptides liberated by 15 µg of staphopain A exposed to refolding after 90 min incubation with azocasein. (B) SDS/PAGE of staphopain A after refolding. Lane M represents the molecular-mass standards in kDa. Lanes 1 and 2, staphopain A refolded in buffers without and with reducing agents respectively. (C) Time progress curves for the refolding yield. The following buffers were used: 50 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA and 20 % glycerol (pH 6.0) (䊏) and the optimized buffer (䉱).
included the evaluation of factors such as pH, salt concentration and addition of stabilizing agents and detergents. The optimal pH was found to be 6.0. Both the higher and lower pH values of the refolding buffer caused a significant decrease in the yield of the active protein (Figure 3A). The addition of glycerol up to 40 % (v/v) improved the recovery of the active staphopain A, but led to a slight increase in viscosity of the refolding buffer, which interfered with the following purification step. Additionally, supplementation of the buffer with L-arginine and Tween 20 increased the refolding efficiency. Staphopain A possesses one cysteine residue in the sequence; therefore, the influence of reducing agents, such as cysteine and DTT, on proper protein folding was also tested. We found that these agents did not improve the refolding yield; however, they activated the refolded staphopain A (the native enzyme needs reducing agents for its activity), which in turn led to the degradation of the protein (Figure 3B). It seems
Co-expression of proteins is a commonly applied procedure [16,23]. In the present study, we showed for the first time, to our knowledge, the expression of proteolytic enzyme together with its specific reversible inhibitor. Such a system allowed us to overexpress successfully the mature form of staphopain A in E. coli and routinely obtain approx. 3.5 mg of the highly pure, active enzyme from 1 litre of bacterial culture using the method developed. Heterologous expression of cysteine proteases has been described by many authors [24–27]. In a bacterial system, recombinant proteins are often produced in an inactive form, and a workable refolding method should be at hand to permit the generation of active protein [28–31]. Papain-like proteases are naturally produced as inactive zymogens, and their proregions play a dual role: they facilitate the proper folding of the catalytic domain [32,33] and act like competitive inhibitors of proteolytic activity [34,35]. These features of the prodomain have been applied to express cysteine proteases in heterologous expression systems. Recombinant cathepsins are routinely expressed as proenzymes and then activated by acidic pH and reducing agents [36]. Similarly, the recombinant staphopain B was produced in a zymogen form and subsequently processed to the active form by digestion with the staphylococcal glutamyl endopeptidase, but with a low yield [11]. The mechanism of activation of staphopain A remains unknown. Therefore we were not able to take advantage of producing staphopain A in a proenzyme form. It is noteworthy that trials to express recombinant zymogen of staphopain A were unsuccessful (G. Dubin, personal communication). Our initial attempts to express the mature form of staphopain A failed. Staphostatins, which are probably localized within the S. aureus cell, are very specific inhibitors of staphopains [13]. Staphopains and staphostatins are co-expressed in S. aureus [4]. The above data led us to the conclusion that co-expression of these proteins in E. coli might also be a means of producing recombinant staphopain A. Indeed, staphopain A could be expressed only together with its inhibitor. Co-expression of multiple proteins in E. coli can be achieved by using a single vector that carries two or more genes. Another approach utilizes multiple plasmids containing compatible replication origins and drug resistance markers that allow for stable maintenance in the same cell. Herein, we used Duet vectors (Novagen) that can be used independently for the co-expression of two genes or used together for the coexpression of up to four proteins. c 2005 Biochemical Society
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B. Wladyka, K. Puzia and A. Dubin
It has been shown that staphopain B and staphostatin B, homologues of staphopain A and staphostatin A respectively, form a non-covalent complex that dissociates under denaturing conditions [4]. Mild conditions for dissociation of the staphopain– staphostatin complex remain unknown. Therefore we applied NiNTA-affinity chromatography under denaturing conditions for the separation of the His-tagged staphopain A from the non-fused inhibitor and other impurities. To recover the enzymic activity of staphopain A, we attempted to refold the enzyme. However, the accuracy of the refolding of recombinant proteins is unpredictable and very much dependent on experimental conditions [37]. From a multitude of parameters affecting refolding efficiency, pH of the refolding buffer has been shown to be crucial. Staphopain A refolded most efficiently at pH 6.0. This is, somehow, in contrast with other data suggesting rather alkaline to neutral pH values for renaturation of cysteine proteases [25,26,38]. Supplementation of the refolding buffer with a mixture of oxidized and reduced thiols was shown to facilitate the correct folding of cysteine proteases in vitro, especially the proper disulphide bond formation [39]. For staphopain A, where no disulphides are present, the addition of cysteine or DTT to refolding buffer did not improve the refolding yield. On the contrary, it resulted in enzyme activation and autodegradation of the refolded active protein. It is noteworthy that staphopain A was expressed and then refolded in the mature form, without its prodomain. With the exception of falcipain-2 [40], it is the first successful expression and refolding of a cysteine protease without the proregion. In summary, we applied the co-expression method for efficient production of the staphylococcal cysteine protease together with its specific inhibitor in E. coli. The recombinant staphopain A was expressed and refolded without its prodomain. The developed method allows for production of the enzyme with the yield over 10-fold higher as compared with isolation from natural sources, facilitating further detailed studies of staphopain A–staphostatin A interactions. We thank G. Dubin (Department of Microbiology, Faculty of Biotechnology) and T. Dylag (Mass Spectrometry Laboratory, Faculty of Chemistry, Jagiellonian University) for their critical reading of this paper. A. D. was supported by grant no. 2P04A 00127 from the State Commission for Scientific Research (Poland).
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Received 7 June 2004/17 August 2004; accepted 23 August 2004 Published as BJ Immediate Publication 23 August 2004, DOI 10.1042/BJ20040958
c 2005 Biochemical Society
