A generalised module for the selective extracellular accumulation of recombinant proteins
© Sevastsyanovich et al.; licensee BioMed Central Ltd 2012
Received: 8 April 2012
Accepted: 11 May 2012
Published: 28 May 2012
It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu.
Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems.
The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.
KeywordsAutotransporter Escherhichia coli Recombinant protein production Secretion
Escherichia coli is the preferred host for recombinant protein production (RPP) in both a research and industrial setting. The popularity of E. coli stems from attributes that include high growth rates in inexpensive media, high product yields, simple process scale-up and safety . The choice of alternative hosts for RPP is predicated on the inability of E. coli to achieve adequate production of a target protein. A predominant reason for the selection of an alternative host is the apparent inability of laboratory strains of E. coli to secrete proteins to the extracellular milieu. Targeting recombinant proteins to the culture medium has several advantages over intracellular accumulation of the desired protein including overcoming problems with product toxicity, degradation, aggregation and incorrect folding [1, 2]. In principle, it will reduce the number of downstream processing steps due to the ease of product recovery, the reduction in the number and quantity of process impurities and absence of laborious refolding experiments to isolate an active molecule .
Several non-specific strategies for extracellular accumulation of recombinant proteins have been developed for E. coli including genetically or chemically altering strains to promote protein leakage from the periplasmic space to the culture medium [3, 4]. Unfortunately, this results in large numbers of process impurities in the form of lipids, polysaccharides and proteins derived from the periplasm space and outer membrane (OM). Conversely, if bacterial secretion systems could be manipulated to selectively secrete a desired target protein into the culture medium, in a controlled and predictable manner, it would drastically reduce costs and increase efficiency in bioprocessing . The bacterial type 1, 2, 3 and chaperone-usher systems have been manipulated to secrete foreign proteins from E. coli and other Gram-negative bacteria [6–9]. However, their use for RPP is hampered by the debatable nature of the secretion signals, their molecular complexity (which results in species and/or substrate specificity) and the limited accumulation of the target protein . Extensive genetic manipulation is required to make these systems tractable.
In contrast, the Type 5, or Autotransporter (AT), system has been utilised widely to successfully secrete a variety of heterologous target molecules to the bacterial cell surface in a process called Autodisplay [10–14]. ATs are widely distributed among Gram-negative bacteria [15–17]. The precursor protein contains an N-terminal signal sequence, which mediates Sec-dependent protein export into the periplasm, a passenger domain encoding the effector function and a C-terminal domain mediating translocation of the passenger domain across the OM [16, 18, 19]. The effector portion of the molecule displays functional and structural heterogeneity and can be substituted with heterologous proteins [14, 16]. Whilst successful in delivering a diverse variety of molecules to the cell surface, the AT system has not been successfully adapted for accumulation of heterologous proteins in the culture medium. The system can be engineered to release the heterologous passenger protein into the culture medium with the use of a protease , but the use of such proteases is undesirable for production technologies. Here we demonstrate that an AT module can be utilised not only for cell surface display but also for the accumulation of heterologous proteins in the culture medium without the addition of exogenous protease.
Extracellular accumulation of heterologous proteins
Other groups have demonstrated the utility of ATs for Autodisplay of heterologous proteins on the bacterial cell surface . In this case the passenger domain remains covalently attached to the β-barrel translocating subunit. Unlike the ATs used for Autodisplay, cleavage of the passenger domain of the serine protease ATs of the Enterobacteriaceae (SPATEs) from their cognate β-barrel is effected by nucleophilic attack of β-barrel residues on a single residue in the α-helix . As such, no exogenous protease is required for liberation of the passenger domain from the β-barrel and in theory the passenger domain can be completely replaced with a target protein. Thus, we hypothesised that the SPATEs could be used to target heterologous proteins to the extracellular milieu rather than the cell surface. To test this hypothesis, initial experiments focused on the E. coli SPATE protein, Pet . When compared to other members of the SPATE family Pet possesses high amino acid sequence identity and structural similarity: the passenger domain consists of a central β-helical stem decorated with several discursive subdomains and is connected to the characteristic β-barrel by a short α-helical peptide (Additional file 1: Figure S1). The gene encoding Pet was synthesised de novo (Additional file 2: Figure S2) and cloned into the pASK-IBA33plus or pET22b expression vectors to create plasmid templates onto which the genes encoding heterolgous proteins could be grafted for further experiments.
Heterologous proteins are secreted
Secreted proteins are soluble, folded and can be modified
To be useful as a method of RPP, the AT system must be able to secrete soluble, folded and functional proteins into the culture medium. To test if the chimeric proteins were natively folded after secretion, we harvested the proteins from the culture supernatant fractions and subjected them to analysis by circular dichroism (CD) (Figure 1C). The structure of YapA is unknown, however bioinformatic analyses predicted YapA to possess a mixed α-helical/β-strand conformation whereas structural data reveal mCherry adopts a β-barrel conformation . CD spectra of YapA showed minima at 222 nm and 208 nm and maxima at 195 nm indicative of a folded protein with mixed α-helical/β-strand content. Consistent with their natively folded β-strand conformations, CD spectra for Pet and mCherry show minima at 218 nm and maxima at 195 nm. Additionally, mCherry purified from the culture supernatant fraction shows fluorescence indicating a folded protein with functional activity (Figure 1C).
Determination of the minimal construct mediating secretion
Next we investigated whether other SPATE proteins could support secretion of heterologous proteins in a manner analogous to Pet. Pic  belongs to a clade of the SPATEs that is evolutionarily distinct from that harbouring Pet. Alignment of Pet and Pic protein sequences from the beginning of the predicted AC domains shows 68% identity and 80% similarity (Additional file 8: Figure S7). Based on the alignment we constructed ESAT6-Pic fusions containing C-terminal Pic fragments equivalent to those for ESAT6-Pet variants. All ESAT6-Pic fusions were efficiently secreted (Figure 4C) allowing us to conclude that β-barrels from SPATE proteins other than Pet can be used to secrete proteins to the culture medium.
E. coli was the first organism to be used for industrial scale production of recombinant proteins. Since then, a large number of proteins produced in E. coli have successfully reached the market including human interferons, interleukins, and granulocyte stimulating factor. However, the dominance of E. coli in industrial bioprocessing is waning with only ca. 40% of new recombinant protein pharmaceuticals being produced using prokaryotic cells. The diminution of E. coli is strongly related to that fact that strains of E. coli used for industrial scale protein production do not effectively secrete proteins into the extracellular environment. Solutions to this problem would effectively reposition E. coli as the host of choice for industrial recombinant protein production. To achieve selective accumulation of recombinant proteins in the extracellular milieu, a Gram-negative protein secretion system must be harnessed. While secretion of heterologous proteins through the Type 1–3 systems has been achieved, the complexity of these systems limits the nature and size of the proteins that can be targeted for secretion. The work described here demonstrates that an AT module, based on SPATE proteins, can be used for the targeted secretion of chimeric proteins into the culture media of Gram-negative bacteria. Importantly, we have demonstrated that this platform may be used for the specific accumulation of folded heterologous proteins with functional, structural and size heterogeneity and for multicomponent complexes (Figure 1). The ability to selectively accumulate target proteins in the culture medium, away from the majority of the process impurities associated with expression in other compartments, makes this system attractive for adoption in industrial RPP applications since extracellular expression of proteins in a folded active form enables simplification of the purification process and significantly reduces downstream processing. However, effective utilisation of the AT module for generalised RPP necessitates achieving yields of target proteins at industrial scale and at concentrations competitive with alternative technologies. Importantly, the yields achieved here are consistent with levels achieved for other E. coli protein secretion systems [1, 36]. However, in the experiments conducted here, only ~ 50% of the expressed protein is targeted to the extracellular milieu, the remainder accumulating at the outer membrane. Furthermore, these yields were obtained in small-scale non-optimised conditions. Thus, an optimised secretion platform, in a controlled optimised fermentation process, would be expected to generate higher protein yields.
Live attenuated bacterial vectors offer the opportunity to deliver vaccine candidates inside human cells thereby eliciting a protective immune response against both infectious and non-infectious disease e.g. tumours. While the induction of antibody has been demonstrated in many animal models, the anticipated induction of cell-mediated immunity has been disappointing . There are several reasons for this including (1) inefficient production of the recombinant heterologous antigen and (2) after invading professional phagocytic cells the live vector remains in the phagosome such that its antigens do not reach the cytosolic processing pathway. Surface display of AT-fusions in live-attenuated vaccine strains offers potential to overcome these problems . Here we have demonstrated that the AT-module can be used in conjunction with live-attenuated Salmonella strains and demonstrated the successful surface display of folded functional heterologous proteins (Figure 2 and 3). Unfortunately, previous experiments using surface display have also failed to induce substantial protective immunity . However, the ability of live attenuated vectors to secrete antigens intracellularly may enhance presentation of the antigens to the immune system and provide the desired protective response . Thus, our demonstrated expression of secreted heterologous protein constructs in live attenuated Salmonella strains offers the possibility of developing a platform for the delivery of multivalent vaccines based on the continuous secretion of proteins in vivo in which significant cell mediated immunity is generated.
Finally, this work reveals novel insight into the biology of the AT secretion system. Several articles have described the importance of specific amino acid residues for the secretion of passenger domain proteins notably residues present in the AC domain [33, 34]. Indeed, recent investigations of BrkA suggested that during secretion portions of the AC domain are sequestered by a specific domain within the β-barrel which initiates translocation of the passenger domain to the external environment . Work provided here clearly demonstrates that the AC domain is not essential for secretion since the smallest secretion-competent constructs completely lack this domain and mutations within the domain do not affect secretion levels (Figure 4 and Additional file 8: Figure S7). These investigations reinforce the concept that the AC domain is required specifically for folding of the β-helical passenger domain, although folding of the passenger domain may enhance the rate at which secretion occurs .
In conclusion, we have developed a versatile platform, based on an AT module, which can be used for secretion of heterologous fusion proteins to the culture medium in a soluble folded form. Additionally, this system can be used for surface display of heterologous fusions on the bacterial cell surface.
Bacterial strains and growth conditions
E. coli strains TOP10 (Invitrogen), NEB 5αF’Iq and JM110 (NEB) were used for cloning. E. coli TOP10, TOP10 dsbA and BL21* (Novagen) and S. enterica SL1344 and SL3261 strains  were used for protein expression and secretion. Bacteria were grown at 37°C in Luria-Bertani liquid or solid media supplemented with carbenicillin (100 and 80 μg/ml, respectively) or kanamycin (50 μg/ml) when appropriate. Protein expression was induced by adding anhydrotetracycline (200 μg/l) or IPTG (0.5 mM) as appropriate and the cultures were incubated for a further 1.5–2 h.
General molecular biology techniques and plasmid construction
Recombinant DNA manipulations were described elsewhere . Phusion High-Fidelity DNA polymerase (Finnzymes), DNA modifying enzymes, plasmid mini-prep and PCR/gel extraction kits (Fermentas, Qiagen and NEB) were used according to the manufacturer’s instructions. Oligonucleotides were synthesized by Alta Bioscience and Eurogentec. Sequencing, mass spectrometry, flow cytometry and gel imaging/densitometry were done using the University of Birmingham Functional genomics facility. Codon optimization for the most commonly used codons and de novo synthesis of DNA was done by GenScript, GenArt or Epoch Life Science. Alignments were generated with ClustalX  and phylogenetic trees generated with Geneious software (http://www.geneious.com/).
Plasmids used in this study are listed in Additional file 9: Table S2. Primers used for PCR are listed in Additional file 10: Table S3. To construct pASK-Pet, the pet gene was PCR-amplified from pBAD-Pet with BsaI-pet-F and HindIII-pet-R primers and cloned between Bsa I/Hind III sites in pASK-IBA33plus (IBA BioTAGnology). pET-Pet was constructed by cloning the pet gene into the Nde I-Hind III sites of pET22b. pASK-His6-Pet and pASK-His6-Pet-ΔD1 were constructed by replacing the Sac I-Bgl II or Sac I-BstB I pet fragment in pASK-Pet with an amplicon generated by PCR with SacI-pet-F and PetSS-BglII-AflII-BstBI-R primers, the latter encoding a His6-tag sequence. To construct pet chimeras the heterologous genes were amplified by PCR using appropriate primer pairs and relevant DNA templates listed in Additional file 2: Figure S2. The PCR-amplified heterologous genes were cloned between Bgl II/BstB I and Bgl II/Pst I sites in the pet gene in pASK-Pet, pASK-Pet* or pET-Pet. pASK-Ag85B-ESAT6-Pet was constructed by inserting PCR-amplified esxA gene (ESAT-6) between BstB I-Pst I sites in pASK-Ag85B-Pet-BB. Constructs pASK-ESAT6-Pet Δ*1 to Δ*20 were made by replacing the Pst I-Hind III fragment in pASK-ESAT6-Pet-BP with the shorter pet gene fragments generated by PCR with one of the forward primers (PstI-TSYQ-del1-F to PstI-YKAF-del20-F) and HindIII-pet-R as a reverse primer. Equivalent constructs encoding ESAT6-Pic chimeras were generated by replacing the Pst I-Hind III pet fragment in pASK-ESAT6-Pet-BP with the pic fragment amplified from pPic1  using one of the forward primers SbfI-FKAG-Pic-del6-F to SbfI-YKNF-Pic-del20-F) and HindIII-Pic-end-R as a reverse primer. Codons encoding I974, W985, L987 and G989 were mutated by site directed mutagenesis using primers BglII-ESAT6-F and HindIII-pet-R as previously described . All constructs generated in this study were sequenced to confirm the veracity of the nucleotide modifications.
Preparation and analysis of proteins
Proteins were visualised by Coomassie staining after SDS-PAGE on standard  or precast (Precise 4-20% Tris/HEPES, ThermoFisher; NuPAGE 4-12% Bis-Tris/MES, Invitrogen) polyacrylamide protein gels or by western immunoblotting as previously described . Rabbit polyclonal antibodies against Pet passenger domain (1:5000 dilution) , ESAT-6 (Abcam; 1:2000 dilution) and mCherry (anti-RFP, Abcam; 1:2000) were used for western immunoblotting. Secondary alkaline phosphatase-conjugated goat anti-rabbit antibodies and NBT/BCIP (Nitro blue tetrazolium chloride/5-Bromo-4-chloro-3-indolylphosphate) substrate were purchased from Sigma. Protein concentrations were determined spectrophotometrically and by SDS-PAGE comparisons with known quantities of purified protein: Bovine serum albumin, Ovalbumin and Lysozyme (Sigma).
Cellular fractions were prepared essentially as described previously . His6-tagged proteins were purified by affinity chromatography on Ni-agarose following manufacturer’s instructions (WebScientific). Briefly, 400 ml cultures were grown and protein expression was induced as described above. Culture supernatants were harvested and sterilised as above, supplemented with 1 mM PMSF and then concentrated through Vivaspin centrifugation device (Sartorius). The concentrated supernatant fractions were passed over a Ni-agarose affinity chromatography column under native conditions using 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole (pH7.5) as binding buffer and 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole (pH7.5) as elution buffer.
To test viability, bacterial cells (~105–106 cells/ml) were diluted in 1 ml filter-sterilised Dulbecco’s PBS supplemented with 10 μl of working solutions of PI and BOX (5 and 10 μg/ml respectively; Sigma) and analysed immediately on FACSAria II (BD Biosciences) using 488 nm laser . Side and forward scatter data and fluorescence data from 104 particles were collected. Optical filters used to measure green and red fluorescence were 502LP, 530/30BP (FITC) and 610LP, 616/23BP (PE-Texas Red), respectively. To analyse surface localisation of proteins by indirect flow cytometry, cells were washed in PBS and incubated at RT with 1% BSA in PBS. Cells were then incubated for 1 h at RT with primary antibody diluted in PBS (anti-Pet, 1:500; anti-ESAT6, 1:500; anti-mCherry, 1:800) followed by 3 PBS washes and final incubation with Alexa Fluor® 488 goat anti-rabbit IgG (1:500; Invitrogen). Cells were washed as before and analysed on a FACSAria II as above.
Proteins in live or fixed bacterial cells were detected by indirect Immunofluorescence as previously described . Cells were visualized using either phase contrast or fluorescence using a Leica DMRE fluorescence microscope-DC200 digital camera system. Exposure time was 118 ms. The Garen and Levinthal  assay of Alkaline Phosphatase activity was used based on conversion of p-nitrophenylphosphate (pNPP) substrate into yellow product with absorbance at 410 nm. Far-UV CD measurements from 190 to 260 nm were collected on a JASCO J-715 spectropolarimeter at room temperature, as described previously . Protein structures were modelled in Swiss-Model  or Phyre  and were visualised using PyMol (http:\\http://www.pymol.org). Secondary structures were predicted with PsiPred .
IRH, JAC and AFC designed the project. DLL, YRS and KT designed and constructed the expression vectors. YRS, DLL and TJK purified proteins and performed CD analyses. YRS, TJW, FCM and CAW prepared and analysed bacterial fractions. TJW performed the immunofluorescence studies. YRS performed the alkaline phosphatase experiments and flow cytometry analyses. All authors contributed to the preparation of the manuscript. All authors read and approved the final manuscript.
recombinant protein production
Serine Protease Autotransporter of the Enterobacteriaceae
Bis-(1,3-dibutylbarbituric acid) trimethine oxonol
This work was supported by grants from BBSRC to IRH and MRC to IRH, AFC and JAC. We thank Dr Lewis E. H. Bingle (University of Sunderland) for critical reading of the manuscript and Dr Raul Pacheco-Gomez for advice on CD.
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