General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae

Abstract

General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.

Albumin binding protein, Intracellular expression, Outer membrane protein A, Secretion, Klebsiella pneumoniae, Staphylococcus carnosus

Abbreviation

Abbreviation
• ABP

  albumin binding protein from streptococcal protein G

• BB

  albumin binding region from streptococcal protein G

• Gua

  guanidine hydrochloride

• HSA

  human serum albumin

• LPS

  lipopolysaccharide

• MCS

  multiple cloning site

• OmpA

  outer membrane protein A

• OmpA_Kpn

  36-kDa OmpA of Klebsiella pneumoniae

• PP

  propeptide from the Staphylococcus hyicus lipase gene

• TST

  Tris-buffered saline with Tween

1 Introduction

Escherichia coli is the dominating bacterial host for recombinant protein production, with a wide variety of vector systems available, offering different promoters, signal peptides, affinity tag systems etc. [1,2]. A number of other bacteria have also been investigated for recombinant protein production[3], including the Gram-negative Vibrio cholerae[4] and Salmonella typhimurium[5], and certain Gram-positive bacteria including Lactococcus lactis[6], Lactobacillus plantarum, Corynebacterium glutamicum, Streptococcus gordonii[7], Staphylococcus aureus, Bacillus subtilis and Bacillus brevis[3]. One reason to evaluate other bacteria than E. coli for recombinant production is that proteolytic degradation of a particular protein might be avoided[8]. Furthermore, the development of live bacterial vaccine delivery systems represents an area in which various bacteria are investigated [9,10]. In the context of vaccine development but also for recombinant production, food-grade bacteria have attracted much attention as they are generally regarded as safe (GRAS) microorganisms[3].

One particular area in which E. coli has a drawback as being a Gram-negative bacterium, is for the recombinant production of proteins to be investigated in immunological studies [11,12]. The endotoxic effects of residual amounts of contaminating lipopolysaccharides (LPS) can significantly influence immune responses and thus make the results of immunization results uninterpretable [11,12]. For recombinant production of LPS-free protein immunogens, there exists a significant need for versatile expression systems in host bacteria not expressing LPS.

Staphylococcus carnosus is traditionally widely used in food biotechnology, e.g. as starter culture for the fermentation of meat and fish products[13]. Götz and coworkers have developed a host-vector system for recombinant protein production in S. carnosus[14], and this expression system has for example been used for the production of enzymes [15,16] and antibody fragments [17,18]. S. carnosus has a very low extracellular proteolytic activity[14], and is therefore an attractive host organism for recombinant protein production. Furthermore, a system for surface display of heterologous proteins on S. carnosus has been developed[19], and this system has been extensively investigated as a system for live mucosal delivery of subunit vaccines [20,,,,24].

On the basis of the previously presented expression system for recombinant production in S. carnosus[14], we have constructed general expression vectors with a multiple cloning site (MCS) preceded by an albumin binding protein (ABP) affinity fusion tag derived from streptococcal protein G (SpG) [25,26]. The ABP tag has been extensively evaluated as affinity fusion partner in both bacterial and mammalian cell expression systems [27,28]. The MCS is preceded by cleavage sites for both site-specific proteases H₆₄A subtilisin [29,30] and the Coxsackie virus protease 3C[31]. Downstream from the MCS, a hexahistidyl encoding sequence is introduced enabling an in frame C-terminal His₆ fusion when desired. The expression vectors exist in one variant enabling secretion of the gene fusion product, and a second variant designed for intracellular production of target proteins that are found to be inefficiently secreted.

Here, the two different expression vectors have been evaluated for the expression of the outer membrane protein A of Klebsiella pneumoniae (OmpA_Kpn)[32]. The OmpA_Kpn, previously expressed in E. coli[32], has been found to exhibit interesting immunopotentiating properties [33,34]. Since surface proteins such as OmpAs, due to their natural affinity for LPS[35], have been described to be very difficult to recover free from LPS contamination in Gram-negative expression systems[35], it was of interest to investigate the production of the OmpA_Kpn in a LPS-free expression system, to allow further characterization. Although the OmpA_Kpn contains eight transmembrane regions[32], we had reason to also evaluate the secretion strategy, since similar proteins have earlier been reported to be successfully secreted from S. carnosus[35]. The expression levels, solubility and secretion efficiency of the gene products expressed from the general expression vectors and the vectors encoding the OmpA_Kpn gene fusion products have been evaluated. The use of the presented expression systems for production and recovery of recombinant proteins of immunological interest is discussed.

2 Materials and methods

2.1 Bacterial strains and vectors

E. coli strain RRIΔM15[36] was used as bacterial host during vector constructions and E. coli strain RV308[37] was used for expression of an E. coli-produced BB-OmpA_Kpn fusion protein[32]. The S. carnosus strain TM300[14] was used for both intracellular and secreted expression of heterologous proteins. Plasmids used were pSPPmABPXM[19], pAff2c[38], pRIT28OmpA_Kpn[32] and pVABBOmpA_Kpn[32]. The preparation and transformation of protoplasts from S. carnosus cells were performed as described earlier[39].

2.2 Construction of expression vectors

The shuttle vector pSPPmABPXM[19] was cleaved with Bsm I and Bgl II and the deleted fragment was replaced by an oligonucleotide linker (5′-ACA AAA CAT CAA CAC GCT AGC GGG GGG TCC GGA-3′ with complementary sequence 5′-G ATC TCC GGA CCC CCC GCT AGC GTG TTG ATG TTT TGT TG-3′) which restored the sequence encoding the first eight amino acids from the Staphylococcus hyicus lipase signal peptide (Lip′) and introduced two unique restriction sites, Nhe I and Bsp EI, into the resulting vector pSLip′. The vector pAff2c[38] was digested with Nhe I and Bsp EI to release a gene fragment encoding an ABP from SpG [25,26], the recognition sites for H₆₄A subtilisin[29] and Coxsackie virus protease 3C[31], an MCS and a hexahistidyl tag (His₆). The fragment was inserted into the pSLip′ vector, precleaved with Nhe I and Bsp EI, to yield the intracellular expression vector pABPm. For construction of an expression vector adapted for secretion of the encoded protein, an oligonucleotide linker (5′-GAT CCT GCT AGC CCC GGG TCC GGA GGC CTC CC-3′ with complementary sequence 5′-AG GCC TCC GGA CCC GGG GCT AGC AG-3′) was inserted into the pSPPmABPXM vector[19], precleaved with Bam HI and Sfi I. The resulting vector pSPPL was cleaved with Nhe I and Bsp EI, unique restriction sites introduced by the oligonucleotide linker, and the same Nhe I-Bsp EI fragment from the pAff2c vector as described above was introduced into the pSPPL vector, yielding the vector pSPPABPm designed for secretion of expressed target proteins.

The gene encoding the 36-kDa K. pneumoniae OmpA protein [32,33] was isolated by Eco RI-Sal I restriction from plasmid pRIT28OmpA_Kpn[32] and introduced into the expression vectors pABPm and pSPPABPm, respectively, pre-digested with the same enzymes. The resulting expression vectors, pABPOmpA_Kpn and pSPPABPOmpA_Kpn, encode the fusion proteins ABP-OmpA_Kpn and PPABP-OmpA_Kpn, respectively.

2.3 Protein expression and purification

S. carnosus cells harbouring the expressions vectors pABPm, pSPPABPm, pABPOmpA_Kpn and pSPPABPOmpA_Kpn, respectively, were grown at 37°C for 20–40 h in shake flasks containing 125 ml tryptic soy broth (30 g l⁻¹) (Difco, Detroit, MI, USA) supplemented with yeast extract (5 g l⁻¹) (Difco) and chloramphenicol (10 mg l⁻¹). Intracellularly expressed proteins were recovered by centrifugation of the cells at 3000×g for 15 min followed by resuspension of the cells in 1×TST buffer (25 mM Tris, 200 mM NaCl, 1.25 mM EDTA and 0.05% Tween, pH 8.0) supplemented with 150 units Lysostaphine (Sigma-Aldrich, St. Louis, MO, USA). The resuspended cells were incubated at 37°C for 30 min, sonicated and pelleted by centrifugation at 5000×g for 15 min. Soluble proteins were recovered from the supernatant and insoluble proteins were solubilized by resuspending the cell pellet in 3 ml 7 M guanidine hydrochloride (Gua) pH 8.0 and incubated at 4°C overnight. The Gua treated samples were diluted to a final concentration of 0.5 M Gua and clarified by centrifugation at 10 000×g for 15 min. Secreted proteins were recovered from the culture medium after centrifugation at 10 000×g for 15 min. The four different proteins were affinity purified on human serum albumin (HSA)–Sepharose[25]. Prior to sample loading, all samples were filtered (0.45 μm) and TST buffer was added to a final concentration of 1×TST. In addition, Zwittergent 3–14 detergent (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) was added to all samples containing the OmpA_Kpn protein (final concentration 0.1%). Affinity captured proteins were eluted with 0.5 M HAc, pH 2.8 and relevant fractions were collected and lyophilized. For production of the reference protein BB-OmpA_Kpn, E. coli RV308 cells harbouring the plasmid pVABBOmpA_Kpn were grown overnight at 37°C in a shake flask containing 100 ml tryptic soy broth (30 g l⁻¹) supplemented with yeast extract (5 g l⁻¹), ampicillin (100 mg l⁻¹) and tetracycline (8 mg l⁻¹). The cells were harvested at 3000×g for 15 min, resuspended in 1×TST buffer, sonicated and pelleted by centrifugation at 5000×g for 15 min. For solubilization of the insoluble BB-OmpA_Kpn protein, the cells were resuspended in 3 ml 7 M Gua, pH 8.0 and incubated at 4°C overnight. The solubilized protein solution was diluted to a final Gua concentration of 0.5 M, clarified by centrifugation at 10 000×g for 15 min, and subjected to HSA affinity chromatography as described above.

2.4 SDS–PAGE and Western blotting analyses

The affinity purified proteins were analysed by SDS–PAGE and Western blotting using a XCell II™ Mini-Cell (Novex, San Diego, CA, USA) and a Panther™ Semidry Electroblotter (Owl, Portsmouth, NH, USA). The proteins were separated under reducing conditions on precasted 10–20% Tris–glycine gels (Novex) according to the supplier's recommendations. The gels were either stained with Coomassie brilliant blue (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the supplier's recommendations or subjected to electroblotting for transfer of proteins onto nitrocellulose membranes (Novex) according to the supplier's recommendations. The membranes were blocked with 1% milk powder (Semper, Stockholm, Sweden) in 1×TST buffer for 30 min at room temperature (RT) and thereafter incubated for 30 min at RT with either (i) biotinylated HSA (biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin from Pierce, Rockford, IL, USA according to the supplier's recommendations) (4 mg l⁻¹ in 1×TST) or (ii) OmpA_Kpn-reactive rabbit antisera (kindly provided by Centre d'Immunologie Pierre Fabre) diluted 1/10 000 in 1×TST. The membranes were washed three times in 1×TST, followed by incubation with (i) streptavidin–alkaline phosphatase conjugate (0.5 U ml⁻¹ in 1×TST) (Roche Diagnostics, Basel, Switzerland) or (ii) goat anti-rabbit whole IgG–alkaline phosphatase conjugate (Sigma-Aldrich) diluted 1/10 000 in 1×TST, for 30 min at RT. After washing three times with 1×TST, the membranes were developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma-Aldrich) according to the supplier's recommendations.

3 Results

3.1 General expression vectors for recombinant protein production in S. carnosus

Two novel general expression vectors, pABPm and pSPPABPm (Fig. 1A, B), designed for recombinant protein production in the food-grade Gram-positive bacterium S. carnosus, have been constructed. The expression vector pABPm (Fig. 1A) is designed for intracellular expression, while the vector pSPPABPm (Fig. 1B) is designed for secretion of the expressed gene products, since it encodes the signal peptide (S) and propeptide (PP) of a S. hyicus lipase gene[14], which has been shown to enable efficient secretion in S. carnosus[14,19]. The propeptide was included since it has previously been demonstrated, in other staphylococcal expression systems, to mediate efficient translocation of gene products that are normally poorly secreted [40,41]. The pABPm plasmid was constructed to encode only the first eight amino acids (Lip′) of the signal sequence from the S. hyicus lipase gene construct, since we wanted to avoid modification in the transcription and translation initiation sequence of the S. hyicus lipase promoter (p_Lip) that previously has shown the give high expression levels in S. carnosus[14,15,19]. The two E. coli–staphylococcal shuttle vectors (Fig. 1A, B) share the following parts: (i) origin of replication for E. coli and the β-lactamase gene (bla) giving ampicillin resistance in transformed E. coli cells; (ii) the origin of replication from S. aureus and the chloramphenicol acetyltransferase gene (cat) for staphylococcal expression; (iii) the origin of replication for the E. coli phage f1; (iv) a gene fragment encoding a serum ABP from SpG [25,26]; (v) cleavage sites for the site-specific proteases His64Ala subtilisin (H₆₄A)[29] and Coxsackie virus protease 3C[31]; (vi) an MCS containing five unique recognition sites for restriction endonucleases; and (vii) a hexahistidyl (His₆) tag for immobilized metal ion affinity chromatography. Two expression vectors pABPm (Fig. 1A) and pSPPABPm (Fig. 1B) encode, prior to any gene insertion, the intracellularly expressed ABP affinity tag (Fig. 1C) and the PP-ABP (Fig. 1D) fusion protein, designed for secretion.

1

The general expression vectors and encoded fusion proteins. The general expression vectors pABPm (A) and pSPPABPm (B) designed for intracellular protein production and secretion of the encoded gene products, respectively, in S. carnosus. The five unique restriction sites in the MCS are indicated. Schematic presentation of the intracellularly produced gene products ABP and ABP-OmpA_Kpn (C), and the secreted fusion proteins PPABP and PPABP-OmpA_Kpn (D) with their respective molecular masses indicated.

3.2 Expression vectors for recombinant production of the outer membrane protein A of K. pneumoniae in S. carnosus

The gene encoding the 36-kDa OmpA_Kpn[32,33] was introduced into the general expression vectors pABPm and pSPPABPm, respectively. The OmpA_Kpn gene was introduced with its natural stop codon meaning that the optional His₆ tag was not utilized in this particular case. The resulting vectors pABPOmpA_Kpn and pSPPABPOmpA_Kpn encode the fusion proteins ABP-OmpA_Kpn (Fig. 1C) and PPABP-OmpA_Kpn (Fig. 1D), respectively. The fusion protein ABP-OmpA_Kpn should be produced intracellularly, while the fusion protein PPABP-OmpA_Kpn is designed for secretion to the culture medium when produced in S. carnosus.

3.3 Protein expression and affinity purification

S. carnosus cells were transformed with the expression vectors pABPm, pABPOmpA_Kpn, pSPPABPm and pSPPABPOmpA_Kpn, respectively, and subsequently cultivated for various times (20–40 h) in shake flasks. Soluble proteins, produced intracellularly or secreted, were directly recovered by ABP-mediated affinity chromatography on HSA–Sepharose [25,27], while proteins that had precipitated intracellularly as inclusion bodies were solubilized in Gua, prior to affinity purification. The protein solutions were in these cases diluted to 0.5 M Gua, which has earlier been shown to be compatible with HSA affinity chromatography[42]. In order to further minimize the risk of co-purification of hydrophobic proteins interacting with the highly hydrophobic OmpA_Kpn protein, a zwitterionic detergent was included in the protein solutions, the washing buffer and the elution buffer when affinity purifying the OmpA_Kpn fusion proteins. Protein concentrations after affinity recovery were estimated by OD_280nm measurements and verified by correlation to Coomassie staining of SDS–PAGE gels. The ABP protein was produced intracellularly as a soluble protein at a production level of 0.4–1.0 mg l⁻¹ (Table 1). As expected for this highly soluble protein[27], no detectable amounts of insoluble ABP could be recovered (data not shown). The ABP-OmpA_Kpn fusion protein was produced intracellularly at a production level of 0.7–2.5 mg l⁻¹ and the majority of the protein was found to be insoluble (Table 1). A higher percentage of insoluble ABP-OmpA_Kpn protein and an overall higher production level was detected when the cells were grown for a longer period of time (40 h).

1

Protein characteristics and production data

Fusion protein	Molecular mass (kDa)	Expression levela (mg l−1)	Solubility
ABP	18.4	0.4–1.0	>95%
ABP-OmpAKpn	52.5	0.7–2.5	5–50%
PPABP	41.3	2.5–4.5b	Secreted
PPABP-OmpAKpn	75.4	0.3–0.5b	Secreted

^aExpression levels defined as the amount of protein recovered by affinity chromatography after various cultivation times (20–40 h).

^bAmount of protein recovered from the culture medium.

The PPABP protein was, as expected, found to be efficiently secreted to the culture medium upon expression in S. carnosus cells to a level of 2.5–4.5 mg l⁻¹. Interestingly, also the PPABP-OmpA_Kpn fusion protein was found to be secreted to a level of 0.3–0.5 mg l⁻¹. This indicates a highly efficient secretion system since the OmpA_Kpn contains eight transmembrane regions[32], and such sequences are generally inefficiently translocated through cellular membranes [27,43]. The significant translocation efficiency in our secretion system is most probably due to the S. hyicus lipase propeptide (PP)[14], which has earlier been demonstrated to be important for translocation and surface exposure of inefficiently secreted proteins in S. carnosus[40,41]. In order to investigate whether the PPABP-OmpA_Kpn protein was present also in other compartments of the S. carnosus cells, the cell wall fraction was separated from the cytoplasmic and membrane fraction according to Samuelsson and co-workers[19] and the different fractions were analysed by SDS–PAGE. Interestingly, no detectable amounts of PPABP-OmpA_Kpn could be recovered from these fractions (data not shown), demonstrating that the fusion protein was efficiently directed to the secretion machinery, despite the significant hydrophobicity of the OmpA_Kpn. In general, higher production levels were achieved when the S. carnosus cells were grown for a longer period of time (40 h).

3.4 SDS–PAGE analysis and immunoblotting

The recombinant proteins recovered by single-step affinity chromatography were analysed by SDS–PAGE (Fig. 2), with E. coli-produced BB-OmpA_Kpn (63 kDa)[32] fusion protein included as a control (Fig. 2, lane 5). BB, also derived from SpG[25], represents a larger (25 kDa) serum albumin binding affinity tag[27] than ABP. The SDS–PAGE analysis demonstrated that the S. carnosus-produced proteins could be efficiently recovered to a high degree of purity by single-step affinity chromatography. The proteins were furthermore found to be recovered as predominantly full-length products of expected sizes (see Fig. 1). A protein band of approximately 15 kDa could be seen as a potential degradation product of PPABP-OmpA_Kpn (Fig. 2, lane 4). In accordance with earlier observations for proteins containing the lipase propeptide PP [14,19], the recovered chimeric proteins PPABP (Fig. 2, lane 2) and PPABP-OmpA_Kpn (Fig. 2, lane 4) migrated as slightly larger proteins.

2

SDS–PAGE analysis of affinity purified recombinant proteins on a 10–20% polyacrylamide gradient gel under reducing conditions. S. carnosus produced and HSA affinity purified gene products; lanes: 1, intracellularly expressed ABP protein; 2, intracellularly expressed ABP-OmpA_Kpn fusion protein; 3, secreted PPABP protein; 4, secreted PPABP-OmpA_Kpn protein; 5, E. coli expressed BB-OmpA_Kpn. Lanes M, marker proteins with molecular masses in kDa.

In order to further investigate the purity and to verify the identity of the isolated proteins, blotting analyses, using biotinylated HSA (Fig. 3A) and OmpA_Kpn-specific polyclonal antibodies (Fig. 3B), were performed. The affinity blotting strategy using biotinylated HSA for detection of the albumin binding reporter proteins (ABP or BB) [26,,28] is a highly specific and sensitive system and also convenient since it does not require the generation of target protein-specific antibodies. As expected, all the recovered proteins were efficiently stained (Fig. 3A) and the absence of additional staining further supported that indeed a high degree of purity was obtained. In the immunoblotting using OmpA_Kpn-specific polyclonal antibodies (Fig. 3B), as expected, only the fusion proteins ABP-OmpA_Kpn (Fig. 3B, lane 2), PPABP-OmpA_Kpn (Fig. 3B, lane 4) and BB-OmpA_Kpn (Fig. 3B, lane 5) were stained. When comparing the ABP (Fig. 3A) and anti-OmpA_Kpn (Fig. 3B) blotting experiments, it can be concluded that the 15-kDa impurity in the PPABP-P40 sample (Fig. 2, lane 4) was indeed an ABP-derived degradation product, since it clearly binds HSA (Fig. 3A, lane 4).

3

Electroblotting analysis of the affinity purified recombinant proteins using (A) an affinity blotting strategy using biotinylated HSA followed by incubation with a streptavidin–alkaline phosphatase conjugate for detection of proteins containing serum albumin binding (ABP/BB) affinity tags or (B) an immunoblotting procedure with OmpA_Kpn-reactive rabbit antibodies and goat anti-rabbit–alkaline phosphatase conjugate for specific detection of OmpA_Kpn. Lanes: 1, S. carnosus produced ABP; 2, S. carnosus produced ABP-OmpA_Kpn; 3, S. carnosus produced PPABP; 4, S. carnosus produced PPABP-OmpA_Kpn; 5, E. coli produced BB-OmpA_Kpn.

4 Discussion

Two general expression vectors for recombinant protein production in the Gram-positive food-grade bacterium S. carnosus are described. One vector is designed for intracellular production, while the second is adapted for secretion of the gene products. Both vector systems encode two different affinity tags, an upstream ABP and a downstream hexahistidyl peptide. To evaluate the novel vectors, the gene encoding the outer membrane protein A of K. pneumoniae (OmpA_Kpn) was introduced. Efficient production was demonstrated in both systems, and full-length fusion proteins could be recovered by ABP-mediated single-step affinity chromatography. Interestingly, the PPABP-OmpA_Kpn fusion protein was found to be secreted to significant levels despite that the OmpA_Kpn contains eight hydrophobic transmembrane regions, thus indicating that the presented secretion vector indeed can enable secretion of normally poorly translocated proteins. In the presented example we did not utilize the possibility to also include a downstream His₆ tag. This option might, however, be a suitable strategy for the production of target proteins that are proteolytically sensitive. It has earlier been demonstrated in a number of systems, that by employing a dual affinity approach (having two different affinity tags fused at each end of the target protein), a significant stabilizing effect can be obtained on proteolytically sensitive proteins [44,,46]. Furthermore, this strategy allows two successive affinity purification steps to obtain proteins that per definition contain both tags, and therefore also the central target protein[2]. The two vector systems are in addition furnished with cleavage sites for two site-specific proteases, His64Ala subtilisin (H₆₄A)[29] and Coxsackie virus protease 3C (3C^pro)[31], for affinity tag removal. These were not evaluated here but earlier studies have shown that H₆₄A subtilisin can be efficiently used for a variety of target fusions[30]. For cleavage using the 3C^pro, an affinity tagged protease ABP-3C^pro is available[31] that thus would allow removal of the protease simultaneously to the removal of the cleaved off ABP tag[2]. Taken together, two versatile expression vector systems for recombinant production in S. carnosus have been presented. The host-vector systems should be particularly suited for production and recovery of proteins of immunological interest, due to the LPS-free host, and their general applicability in this context will be evaluated in the near future.

Acknowledgements

This work has been supported by grants from the Cell Factory for Functional Genomics within the Swedish Foundation for Strategic Research. The Centre d'Immunologie Pierre Fabre is acknowledged for providing the OmpA_Kpn-reactive rabbit antiserum. All recombinant work was performed in accordance with the national guidelines.