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Applied and Environmental Microbiology, June 2008, p. 3601-3604, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02576-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High-Yield Production of Secreted Active Proteins by the Pseudomonas aeruginosa Type III Secretion System ^,

M. Derouazi, B. Toussaint, L. Quénée, O. Epaulard, M. Guillaume, R. Marlu, and B. Polack^*

Laboratoire TIMC-IMAG GREPI-TheREx, Université Joseph Fourier-Grenoble 1 UMR CNRS 5525, IFR INSERM 130, Grenoble, France

Received 15 November 2007/ Accepted 22 March 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.

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Recombinant proteins are becoming increasingly valuable in different areas of research, including structural analysis studies, diagnostic investigation, and the treatment and prevention of various human diseases. Among the different expression systems commonly used for recombinant protein production in research and industry, the Escherichia coli system is often preferred if glycosylation and other posttranslational modifications are not required for the biological activity of the protein. The advantages of bacterial expression methods result from the ability of bacteria to reproduce rapidly to high densities on inexpensive substrates, their ease of manipulation, and the high protein yields that can be obtained in bacterial expression systems (11). Due to intracytosolic conditions not adapted for proper folding of polypeptide chains, however, some difficult-to-express proteins are often produced in E. coli in an undesirable insoluble form in inclusion bodies. To avoid the problem of insolubility and to facilitate purification, Mergulhão et al. have proposed use of a bacterial expression system with the intrinsic ability to secrete recombinant protein (11). Furthermore, protein secretion during bacterial growth could permit the development of a continuous process for recombinant protein production. However, the process of secretion in bacteria is complex, and the yields of secreted recombinant proteins are often low.

Gram-negative bacteria use different types of secretion systems for their own purposes. For example, the type III secretion system (TTSS) is involved in the cytotoxicity of several pathogenic bacteria (Salmonella spp., Yersinia spp., Shigella spp., Pseudomonas spp.) (1). The flagellar TTSS of E. coli has been previously shown to be a powerful tool for the production of recombinant proteins (10). The mechanisms of substrate recognition and secretion by TTSSs are complex for two reasons. First, it has been proposed that during the synthesis of the naturally secreted type III proteins, their correct folding is maintained by a specific interaction with a small acidic chaperone (2, 19). Second, once the proteins are secreted, they could be more easily folded with appropriate disulfide bonding, since the extracellular space is an oxidative environment (17). Although secretion signals seem to be localized in the N-terminal parts of proteins, these termini are not conserved between proteins secreted by the same TTSS (7). The Pseudomonas aeruginosa TTSS naturally delivers four types of large effector proteins (exotoxins S, T, Y, and U, with molecular masses of 49, 53, 42, and 74 kDa, respectively) into the cytoplasm of target cells in vivo. It has also been previously shown that direct TTSS secretion in the surrounding media occurs in vitro when P. aeruginosa is grown in calcium-depleted conditions (6). Taking advantage of these properties, we engineered an attenuated P. aeruginosa strain and a dedicated expression plasmid to secrete recombinant proteins into bacterial growth medium.

We previously showed that there were high levels of secretion when each of the following reporter proteins was fused with the 54 N-terminal amino acids of ExoS: P. aeruginosa inhibitor of vertebrate lysozyme (IVY), Pseudomonas putida catechol 2,3-dioxygenase, and green fluorescent protein (4). In the present study, we checked whether shorter tags could also allow the secretion of fusion proteins. As shown in Fig. 1A, the 17, 30, or 42 N-terminal amino acids of ExoS did not allow secretion of MRP8, a human soluble small protein with a molecular mass of 11 kDa belonging to the S100 family (Table 1). Considering the high homology of ExoT with ExoS, MRP8 was also fused to the 129, 96, 54, or 17 N-terminal amino acids of ExoT. The largest three constructs were secreted, while the small ExoT17-MRP8 construct was not (Fig. 1B), and the secretion levels were comparable to those seen for the ExoS42 fusion protein. Therefore, we considered the first 54 residues of ExoS the optimized N-terminal domain required for the secretion of fusion protein. We then designed and constructed the expression plasmid pEAI-S54 as follows. First, exsA, the gene coding for the common TTSS regulator of P. aeruginosa, was amplified by PCR using PfuUltra (Stratagene) with P. aeruginosa chromosomal DNA as the template and primers EXSAS and EXSAR (see Table S1 in the supplemental material). The PCR product was cloned as an EcoRI/HindIII fragment into EcoRI/HindIII-digested pTTQ18 (16) to produce pTTQ18exsA. The expression of the exsA gene was under the control of the inducible promoter Ptac. The whole inducible system, including the genes coding for the Plac repressor lacI^q, exsA, and the Ptac promoter, was amplified from pTTQ18exsA with the Xbaptac and KpnlacIq primers (see Table S1 in the supplemental material) and cloned as an XbaI/KpnI fragment into pUCP20 (18) to produce pExaInd. Then the orf1 gene and the fragment of the exoS gene coding for the 54 N-terminal amino acids of ExoS was amplified from P. aeruginosa chromosomal DNA with primers Orf1-Exos54S and Orf1-Exos54R and cloned as an XbaI/SphI fragment into XbaI/SphI-digested pExaInd. The resulting plasmid was designated pEAI-S54-BS and contains unique BamHI and SphI restriction sites. The final vector was obtained by insertion of the hybridized oligonucleotides LinkS and LinkR into BamHI/SphI-digested pEAI-S54-BS. The final cloning plasmid possessed a polylinker containing unique sites for BamHI, AgeI, NheI, BsrGI, BglI, and SphI and allowed easy cloning of genes coding for proteins of interest; this plasmid was designated pEAI-S54 (Fig. 1C). Plasmid pEAI-S54 is easily introduced into P. aeruginosa either by conjugation with E. coli strain S17.1 (see Table S1 in the supplemental material) or by electroporation of plasmid DNA (3). This plasmid allows, with stimulation by isopropyl-β-D-thiogalactopyranoside (IPTG), activation of the whole TTSS. The synthesized protein of interest can then be secreted under calcium deprivation conditions, which are induced by addition of EGTA to the culture medium.

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FIG. 1. (A) Secretion of MRP8 fused to the 54, 42, 30, and 17 N-terminal amino acids of ExoS. The P. aeruginosa CHA strain was transformed with the designed constructs by electroporation. A single colony from Pseudomonas isolation agar plates was subsequently inoculated into 1.5 ml Luria-Bertani medium supplemented with 300 µg·ml–1 carbenicillin and grown overnight at 37°C. After harvesting by centrifugation and a wash step, bacteria were resuspended in fresh Luria-Bertani medium to an optical density of 0.2. Protein expression was induced by addition of 0.5 mM IPTG, and protein secretion was induced by addition of 5 mM EGTA and 20 mM MgCl2, followed by 3 h of incubation at 37°C. Cells were pelleted from 2 ml of a bacterial culture by centrifugation (15 min, 13,000 x g at room temperature). The clarified supernatant was precipitated with 12.5% trichloroacetic acid for 30 min on ice and centrifuged at 18,000 x g at 4°C, and the protein pellets were washed two times at –20°C with cold acetone and resuspended in electrophoresis buffer. Proteins were separated on a 12% sodium dodecyl sulfate-Tricine gel and visualized by Coomassie blue staining. ExoT (53 kDa) and ExoS (49 kDa) are TTSS toxins, and PopB (40 kDa) and PopD (31 kDa) are TTSS structural proteins secreted by the CHA strain. The position of the fusion protein is indicated by an arrow. (B) Secretion of MRP8 fused to the 54, 42, 30, and 17 N-terminal amino acids of ExoT. The experiment was performed as described above for the ExoS-MRP8 fusion. The position of the fusion protein is indicated by arrows. (C) Plasmid map of the pEAI-S54-EC-BS plasmid. pEAI-S54-EC is derived from the pUCP20 P. aeruginosa shuttle vector (GenBank accession no. U07165). Antibiotic resistance is encoded by bla (ampicillin). ori is the ColE1 origin of replication. The lac promoter (ptaq), rnnbT1T2 terminator fragment, and lacIq repressor protein gene (lacI) are from pTTQ18 (GenBank accession no. CS410149). repB is the replication protein gene (Entrez protein no. AAB40009) from pUCP20. ExsA is the common TTSS regulator of P. aeruginosa (6); orf1 encodes the specific ExoS chaperone for TTSS secretion (15); and exoS54 is the sequence coding for the 54 first amino acids of the ExoS toxin that allow the secretion of the protein of interest by the TTSS of P. aeruginosa.

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TABLE 1. Peptides and proteins secreted with the TTSS of P. aeruginosaa

As shown in Fig. 1A and B, ExoS and ExoT, the secreted TTSS toxins of P. aeruginosa, represent the major protein contaminants. Therefore, to increase the purity and yield of recombinant proteins in the supernatant, we used the CHA-OST strain, which has deletions in the genes coding for these toxins, as previously described by Quénée et al. (14). Using this strain, different proteins were secreted using the approach described above. The results are summarized in Table 1. Among these proteins, all four proteins examined were shown to be truly active, suggesting that they were properly folded and disulfide bonded. To further increase the protein yield before purification, we developed a simple process to concentrate the recombinant protein in the supernatant. To avoid any contamination and facilitate downstream processing, the recombinant proteins were produced in chemically defined classical RPMI mammalian cell cultivation media. In the example given here, ExoS54 fused to the human gp100 protein was very efficiently secreted (Fig. 2).

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FIG. 2. Protein secretion with the CHA-OST strain in RPMI medium. Precultures (10 or 20 ml) were started in Luria-Bertani medium supplemented with IPTG. At an optical density at 600 nm of 1.8, bacteria were recovered, washed with RPMI medium, and concentrated in 1 ml of RPMI culture medium. Protein secretion was induced by EGTA. After 1, 2 or 3 h, 20 µl of culture medium was analyzed for protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. The control was 2 ml of culture supernatant precipitated and analyzed as described in the legend to Fig. 1.

In conclusion, we developed a completely novel expression system for recombinant protein production in P. aeruginosa, including a plasmid for inducible expression (pEAI-S54) and a bacterial strain that allows production and secretion of a wide variety of proteins in the active form. This system offers a new alternative solution to one of the main drawbacks of protein production with bacteria: the accumulation of protein as inclusion bodies in the bacterial cytosol. Although it has been demonstrated that coexpression of a chaperone (9) or fusion of the protein of interest with thioredoxin (8, 20) allows the production of recombinant protein in a soluble, active form in E. coli, the proteins are produced in the bacterial cytosol. Therefore, we propose that our system should be exploited to reduce insolubility problems, since it permits the targeted secretion of recombinant proteins through the TTSS of P. aeruginosa. We hypothesize that, due to the presence of dedicated intracellular chaperones acting on the ExoS54 sequence, the formation of inclusion bodies is avoided. In addition, the secretion of proteins into the oxidative extracellular medium could favor natural folding and disulfide bridge formation. Furthermore, the expression yield of the different recombinant proteins tested is sufficient to allow the development of straightforward purification schemes. We suggest that use of the bacterial TTSS constitutes an alternative to the eukaryotic cellular system for laboratory or industrial production of fully soluble recombinant proteins and their easy purification from inexpensive and well-defined culture media.

 
We thank C. Abergel and V. Monchois for the generous gift of the P. aeruginosa IVY gene.

This work was funded by grants from the Agence Nationale de la Recherche and Vaincre la Mucoviscidose.

 
* Corresponding author. Mailing address: Laboratoire TIMC-IMAG GREPI-TheREx, Batiment Jean Roget, Domaine de la Merci, 38706 La Tronche, France. Phone: 33(0) 476 765 967. Fax: 33(0) 476 765 935. E-mail: BPolack{at}chu-grenoble.fr

Published ahead of print on 4 April 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

M.D. and L.Q. contributed equally to this work.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Applied and Environmental Microbiology, June 2008, p. 3601-3604, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02576-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Derouazi, M. Articles by Polack, B. Search for Related Content PubMed PubMed Citation Articles by Derouazi, M. Articles by Polack, B. Agricola Articles by Derouazi, M. Articles by Polack, B.