Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis
© Park et al.; licensee BioMed Central Ltd. 2013
Received: 3 January 2013
Accepted: 17 September 2013
Published: 22 September 2013
The anchoring motif is one of the most important aspects of cell surface display as well as efficient and stable display of target proteins. Thus, there is currently a need for the identification and isolation of novel anchoring motifs.
A system for the display of recombinant proteins on the surface of Escherichia coli was developed using the Bacillus anthracis exosporal protein (BclA) as a new anchoring motif. For the surface display of recombinant proteins, the BAN display platform was constructed in which a target protein is linked to the C-terminus of N-terminal domain (21 amino acids) of BclA. The potential application of BAN platform for cell surface display was demonstrated with two model proteins of different size, the Bacillus sp. endoxylanase (XynA) and monooxygenase (P450 BM3m2). Through experimental analysis including outer membrane fractionation, confocal microscopy and activity assay, it was clearly confirmed that both model proteins were successfully displayed with high activities on the E. coli cell surface.
These results of this study suggest that the strategy employing the B. anthracis BclA as an anchoring motif is suitable for the display of heterologous proteins on the surface of E. coli and consequently for various biocatalytic applications as well as protein engineering.
Cell surface display allows expression of proteins or peptides on the surface of cells in a stable manner using the surface proteins of bacteria, yeast, or even mammalian cells as anchoring motifs [1–4]. This powerful tool has been used in a wide range of biotechnological and industrial applications, such as live vaccine development , peptide libraries screening [6, 7], whole-cell catalysis , biosensor development [9, 10] and environmental bio adsorption [11, 12]. For the efficient display of recombinant proteins on a surface of host cells, various anchoring motifs have been developed, including OprF, OmpC, OmpX, and many others [12–14]. Although many successful results have been achieved, the use of current anchoring motifs did not always allow efficient display of all target proteins . In cell surface display systems, successful protein display is highly dependent on the choice of the anchoring motif. Therefore, in this study, we decided to explore and develop an alternative cell surface display system for the expression and display of recombinant proteins.
Development of a surface display system with BclA
The native BclA contain 19-residue amino terminal peptide, but this peptide is proteolytically removed during sporulation and the remaining mature BclA is attached to the surface of the developing forespore [15, 16]. The mature BclA protein consists of three parts: NTD, CTD and the central domain, which contains 1 ~ 8 repeating regions of ‘(GPT)XGDTGTT triplet sequence’ (Figure 1a and Additional file 1: Figure S1) [15, 16]. The central domain looks like a mammalian collagen protein (it is also called ‘collagen-like region’, CLR) and, according to the repeating number of CLR, the BclA can be of different sizes (253 ~ 445 amino acids). Each domain has its own unique independent function. In addition, the truncated form of each domain shows different levels of expression and localization on the membrane [8, 16, 17]. Thus, in order to develop the most efficient display system based on the B. anthracis BclA, we examined three different display systems using the three different motifs (BAN, BANC, and BAF) of BclA as anchoring motifs as shown in Figure 1b. The BAN system contains only the NTD (21 amino acids) without the 19-residue amino-terminal peptide of BclA, and the BANC system contains both NTD and CTD without the central CLR (total 178 amino acids). Finally, the BAF system contains a mature form of full-length BclA from B. anthracis RA3 strain (NCBI accession no. CAD56878.1, 233 amino acids). In each system, the target protein (Bacillus sp. TG43 lipase) was fused to the C-terminus of each anchoring motif, and gene expression was controlled under the IPTG-inducible tac promoter (P tac ). Comparison of gene expression in three systems suggested that the BAN expression platform (pTJ1-BAN) allowed significantly higher gene expression, while other expression systems (BANC and BAF) showed rather poor expression levels (Additional file 2: Figure S2). Even though much higher production level could be achieved with the BAN-fused system, the localization of BAN-fused lipase on cell surface gave multiple varying results during the repeated experiments. Thus, the display of other recombinant proteins instead of lipase was studied using the BAN platform as a display system in further experiments.
Display of endoxylanase on the E. coli cell surface
The localization of excess proteins on outer membrane might cause problems in cell wall integrity, and consequently result in cell lysis and possible release of anchored proteins into the culture medium. Thus, the contamination of endoxylanase in the culture supernatant was analyzed during the display of endoxylanase on the surface. After cultivation of E. coli harboring pTJ1-BAN-XynA, the presence of endoxylanase on the surface and culture medium were analyzed by SDS-PAGE followed by western blotting analysis. The BAN-fused endoxylanase was clearly detected in the total cell lysate and membrane fractions, but not in the supernatant fraction (Additional file 3: Figure S3). This result indicates that BAN-fused endoxylanases were displayed on the surface and were not released into the culture medium. The specific endoxylanase activities in whole cells and culture supernatant were determined as described above. A low level of activity from the culture supernatant of E. coli (pTJ1-BAN-XynA) was obtained, but it was much lower than that of the whole-cell suspension (Additional file 4: Figure S4). This result also indicates that the BAN-fused endoxylanase was successfully anchored on the cell surface and was stably maintained without cell lysis.
Display of monooxygenase on the E. coli cell surface
Microbial cell-surface display systems can be used in a wide range of applications as described earlier. The display of active enzymes has been intensively pursued due to its potential use as a whole-cell biocatalyst in the fields of pharmaceuticals, fine chemicals and agrochemicals production. To date, several cell-surface display systems have been developed for the expression of polypeptides or proteins on the surface of E. coli and, as an anchoring motif, outer membrane proteins such as OmpC, OmpX, maltoprotein LamB, the outer membrane protein S, lipoprotein TraT and many others, have been employed, which were fused to the protein of interest . In most of the surface display systems that use outer membrane proteins as anchoring motifs, target proteins are fused to the anchoring motif via a sandwich fusion format. This results in minimal destabilization of the anchoring motif on the outer membrane, and improved efficiency of surface display. In an alternative, which includes the OmpX case and has been called the circularly permutation strategy, both the N and C termini are presented on the external cell-surface . However, in most cases, the passengers displayed have been limited primarily to short peptides or polypeptides. In the present study, the B. anthracis exosporal protein BclA was examined as a new anchoring motif. The C-terminal fusion strategy was employed to display foreign proteins. In this display format, both proteins (endoxylanase and monooxygenase) were successfully displayed with high activities. These results suggest that the C-terminal deletion-fusion strategy employing the B. anthracis BclA is suitable for the display of heterologous polypeptides and proteins, which should prove useful in a wide range of applications. Particularly, the successful display of monooxygenase (P450 BM3m2), which has much bigger size than the average size of passenger proteins , was very promising. In general, most cell display systems have size limitations in regards to the protein to be displayed, and most systems are only suitable for peptides or relative small polypeptides (below 50 kDa). So far, there is only one successful case for the display of P450 on a bacterial cell surface, where an ice-nucleation protein (INP) was used as an anchoring motif . When other well-known anchoring motifs including OmpC and OmpX were tested, such a big-size enzyme (P450 BM3m2) could not be displayed (data not shown). The successful display of P450 using the BAN anchoring motif clearly indicates the potential of the BAN anchoring motif to accommodate many different passenger proteins.
Although two model proteins of different sizes could be successfully displayed on the cell surface using the BclA anchoring motif, the mechanisms for the secretion and anchoring of BclA and BclA-fused proteins are unknown at this time. In general, the protein secretion into periplasm and cell surface including cell surface display requires a signal peptide. The native BclA protein synthesized in the mother cell is attached to the surface of the forespore; the 19-residue amino-terminal peptide is proteolytically removed and the next 20–40 amino acids (NTD) are used for anchoring. Also, it is known that the anchoring of the mature BclA protein on the surface of B. anthracis requires a few specific exosporium receptor proteins (i.e., BxpB or its homologue ExsFB) [20–22]. In the display system reported here, only the NTD (21 amino acids) after the first 19 amino acids of BclA was employed. To our knowledge, the receptor proteins reported for B. anthracis are not produced in E. coli. As demonstrated with two model proteins (endoxylanase and P450 BM3m2), their fusions with the BAN motif resulted in their successful secretion and localization onto the cell surface, which was clearly confirmed by several experiments including membrane protein fractionation, confocal microscopy, and activity assay. The use of the well-known PelB signal peptide for the secretion of BAN-fused proteins also allowed the localization of proteins on the cell surface of E. coli, but the efficiencies were relatively lower than that obtained with the signal peptide-free system (Figures 2, 3 and 4).
Thus, it will be an interesting future study to decipher the detailed mechanisms for such signal peptide-free secretion using the BAN system. However, this is not the only case reported. The ice nucleation protein (INP) of Pseudomonas syringae has been used as a great anchoring motif for the cell surface display of various proteins in E. coli and other gram negative bacteria. The INP and its fusion proteins do not require any signal peptide for their secretion and anchoring on the cell surface [8, 17, 23]. Interestingly, the BclA protein has structural similarity to INP which also comprises three domains (NTD, CTD and CLR). Thus, although there is no proof that INP and BclA could mediate membrane transport and cell attachment in a similar way, the mechanism of BAN-based signal peptide-free secretion might be similar to that of INP. Further studies are needed to understand the mechanism.
We developed a new cell surface display platform based on the BclA of B. anthracis as an anchoring motif. The C-terminal fusion of two model proteins to BclA (especially BAN) allowed their successful secretion and surface display with high efficiency and stability. Although only two proteins (21 and 120 kDa) were examined, the results seem to indicate the versatility of this display system capable of secreting and displaying proteins of different sizes; this is an important advantage of the BAN anchoring motif. Furthermore, both enzymes displayed using the BAN system, endoxylanase (XynA) and monooxygenase (P450 BM3m2), showed high activities and stabilities (as examined for xylanase). By displaying different enzymes of interest using our system, it will be possible to develop cost-effective bio catalytic systems in the fields of pharmaceuticals, fine chemicals, agrochemicals and other demanding industries.
Materials and methods
Bacterial strains and growth conditions
Bacterial strains and plasmids used in this study
Strain or plasmid
Reference or source
E. coli JM109
F’traD36 proA + B + lacIq Δ(lacZ)M15/ Δ(lac-proAB) glnV44 e14- gyrA96 recA1 relA1 endA1 thi hsdR17
New England Biolabsa
E. coli XL1-Blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacI qZ ΔM15 Tn10 (Tetr)].
4.17 kb, bla, trc promoter
5.68 kb, pTrc99A derivative; tac promoter
5.76 kb, pTac99A derivative; N-terminal of bclA
6.23 kb, pTac99A derivative; N- & C-terminal of bclA
6.45 kb, pTac99A derivative; bclA
pUC19 containing endoxylanase gene (xynA)
6.8 kb, pTJ1-BAN derivative; BAN-fused xynA with His6 tag (N-terminus) and FLAG tag (C-terminus)
6.36 kb, pTJ1-BAN derivative; pelB signal peptide, BAN-fused xynA with FLAG tag (C-terminus)
6.28 kb, pTJ1 derivative; pelB signal peptide, xynA with FLAG tag (C-terminus)
9.0 kb, pTJ1-BAN derivative; BAN-fused B. megaterium monooxygenase (P450-BM3m2) gene with His6 tag (N-terminus) and FLAG tag (C-terminus)
8.9 kb, pTJ1-BAN derivative; B. megaterium monooxygenase (P450-BM3m2) with FLAG tag (C-terminus)
Plasmids and DNA manipulation
Oligonucleotides used for PCR amplification in this study
Primer sequences (5′ → 3′)a
Gene to be amplified
Source or references
GGAATTC ATGCACCACCACCACCACCAC GCATTTGACCCTAATCTT
Full bclA gene
B. anthracis RA3
Truncated bclA gene (NTD)
B. anthracis RA3
Truncated bclA gene (NTD + CTD)
B. anthracis RA3
CGTCTAGACTCGAGGC TAGCCCCGG GAGCAACTTTTTCAATAA
Bacillus sp. Endoxylanase (XynA)
Bacillus megaterium Monooxygenase (P450 BM3m2)
Fractionation of outer membrane proteins
Outer membrane proteins were prepared and analyzed as previously described [27, 28]. Briefly, after washing cells with 0.5 mL of 10 mM Na2HPO4 buffer (pH 7.2) twice, cells were disrupted by three cycles of sonication (each for 20 s at 15% of maximum output; High-Intensity Ultrasonic Liquid Processors, Sonics & Material Inc., Newtown, CT). After quick centrifugation (12,000 × g for 2 min) to remove partially disrupted cells, membrane proteins and the lipid layer were isolated by centrifugation at 12,000 × g for 30 min at 4°C and then, pellets were resuspended in 0.5 mL of 10 mM Na2HPO4 buffer (pH 7.2) and 0.5% (w/v) sarcosyl solution. After incubation at 37°C for 30 min, an insoluble pellet containing outer membrane proteins was obtained by centrifugation at 12,000 × g for 30 min at 4°C. After washing the insoluble pellet with 10 mM Na2HPO4 buffer (pH 7.2), the outer membrane proteins were resuspended in 50 μL of TE buffer (pH 8.0).
SDS-PAGE and western blotting analysis
Protein samples were analyzed by electrophoresis on a 10% (w/v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. For immunodetection of the FLAG-tag fused protein, a monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich) and goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate (Invitrogen, Carlsbad, CA) were used. An ECL kit (Amersham ECL Prime Western Blotting Detection Reagent, GE Healthcare) was used for signal detection.
Measurement of endoxylanase activity
Endoxylanase activity was measured using the 3′,5′-dinitrosalicylic acid (DNS) method . After cultivation, cells were washed twice with 1× phosphate-buffered saline (PBS), and then mixed with 2% (w/v) beech wood xylan solution. The reaction mixture was incubated at 37°C, and the supernatant of the mixture was sampled at time intervals by centrifugation for 10 min at 12,000 ×g and room temperature. The supernatant samples were mixed with 3 times the volume of a DNS solution containing 7.5 g 3,5-dinitrosalicylic acid (DNS), 14 g NaOH, 216.1 g Rochelle salt, 5.4 mL phenol and 5.9 g Na2S2O5 per liter. After boiling for 10 min, samples were cooled to room temperature for 5 min, and then the absorbance of the reactant was detected by a spectrophotometer at 550 nm. One unit (U) of endoxylanase activity was defined as the amount of enzymes capable of producing 1 μmol of reducing sugar per min. The specific activity was defined as the endoxylanase activity for the amount of cells with an OD600 of 2.0.
Measurement of monooxygenase (P450 BM3m2) activity
After cultivation, cells were harvested by centrifugation at 6,000 rpm for 10 min. Cell pellets were washed twice with 100 mM potassium phosphate buffer (pH 7.4) and the cells were concentrated in the same buffer to a calculated final OD600 of 100. The activity of the displayed P450 was determined by analyzing the dealkylation of 7-epoxycoumarin to 7-hydroxycomuarin as described previously . The prepared cells were mixed with 7-ethoxycoumarin (1 mM) and NADPH (0.5 mM) in 100 mM potassium phosphate buffer (pH 7.4). The reaction was performed at room temperature for 30 min, the product (7-hydroxycomuarin) was quantified using a multi-well plate fluorometer (VICTOR ×3, Perkin-Elmer, Waltham, MA) with excitation wavelength at 405 nm and emission wavelength at 460 nm.
After cultivation, cells (1 mL) were washed with 1× PBS and resuspended in 1× PBS supplemented with 3% (w/v) bovine serum albumin (BSA; Sigma-Aldrich). The cells were first incubated with the rabbit anti-FLAG probe antibody conjugated with FITC (Invitrogen) at a dilution of 1:250 for 1 h. Cells were washed 2 times with a PBS solution to remove the unbound probes. Finally, cells were mounted on a poly-l-lysine coated microscopic slide and examined by confocal microscopy (Carl Zeiss LSM510 META, Jena, Germany). Samples were excited by a 488 nm argon laser, and images were filtered by a long pass 505 nm filter. Images were acquired with Carl Zeiss LSM 510 software (version 4.2.rk).
This work was supported by the Technology Development Program to Solve Climate Changes on systems metabolic engineering for bio refineries from the Ministry of Science, ICT & Future Planning through the National Research Foundation of Korea (NRF-2012-C1AAA001-2012M1A2A2026556).
- Cornelis P: Expressing gene in different Escherichia coli compartments. Curr Opin Biotechnol. 2000, 11: 450-454. 10.1016/S0958-1669(00)00131-2View Article
- Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss R: Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol. 1997, 15: 29-34. 10.1038/nbt0197-29View Article
- Lee SY, Choi JH, Xu Z: Microbial cell surface display. Trends Biotechnol. 2003, 21: 45-52. 10.1016/S0167-7799(02)00006-9View Article
- Stahl S, Uhlen M: Bacterial surface display: trends and progress. Trends Biotechnol. 1997, 15: 185-192. 10.1016/S0167-7799(97)01034-2View Article
- Lee JS, Shin KS, Pan JG, Kim CJ: Surface-displayed viral antigens on Salmonella carrier vaccine. Nat Biotechnol. 2000, 18: 645-648. 10.1038/76494View Article
- Martineau P, Charbit A, Leclerc C, Werts C, O’Callaghan D, Hofnung M: A genetic system to elicit and monitor anti-peptide antibodies without peptide synthesis. Biotechnology. 1991, 9: 170-172. 10.1038/nbt0291-170View Article
- Boder ET, Wittrup KD: Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997, 15: 553-557. 10.1038/nbt0697-553View Article
- Jung HC, Lebeault JM, Pan JG: Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nat Biotechnol. 1998, 16: 576-580. 10.1038/nbt0698-576View Article
- Dhillon JK, Drew PD, Porter AJ: Bacterial surface display of an anti-pollutant antibody fragment. Lett Appl Microbiol. 1999, 28: 350-354. 10.1046/j.1365-2672.1999.00552.xView Article
- Shibasaki S, Ueda M, Ye K, Shimizu K, Kamasawa N, Osumi M, Tanaka A: Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration. Appl Microbiol Biotechnol. 2001, 57: 528-533. 10.1007/s002530100767View Article
- Sousa C, Kotrba P, Ruml T, Cebolla A, de Lorenzo V: Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB. J Bacteriol. 1998, 180: 2280-2284.
- Xu Z, Lee SY: Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Appl Environ Microbiol. 1999, 65: 5142-5147.
- Lee SH, Lee SY, Park BC: Cell surface display of lipase in Pseudomonas putida KT2442 using OprF as an anchoring motif and its bio catalytic applications. Appl Environ Microbiol. 2005, 71: 8581-8586. 10.1128/AEM.71.12.8581-8586.2005View Article
- Rice JJ, Schohn A, Bessette PH, Boulware KT, Daugherty PS: Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci. 2006, 15: 825-836. 10.1110/ps.051897806View Article
- Synvestre P, Couture-Tosi E, Mock M: A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium. Mol Microbiol. 2002, 45: 169-178. 10.1046/j.1365-2958.2000.03000.xView Article
- Synvestre P, Couture-Tosi E, Mock M: Polymorphism in the collagen-like region of the Bacillus anthracis BclA protein leads to variation in exosporium filament length. J Bacteriol. 2003, 185: 1555-1563. 10.1128/JB.185.5.1555-1563.2003View Article
- Jung HC, Park JH, Park SH, Lebeault JM, Pan JG: Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein. Enzyme Microb Technol. 1998, 22: 348-354. 10.1016/S0141-0229(97)00224-XView Article
- Jeong KJ, Park IY, Kim MS, Kim SC: High-level expression of an endoxylanase gene from Bacillus sp. In Bacillus subtilis DB104 for the production of xylobiose from xylan. Appl Microbiol Biotechnol. 1998, 50: 113-118. 10.1007/s002530051264View Article
- Yim SK, Kim DH, Jung HC, Pan JG, Kang HS, Ahn T, Yun CH: Surface display of heme- and diflavin-containing cytochrome P450-BM3 in Escherichia coli: a whole cell biocatalyst for oxidation. J Microbiol Biotechnol. 2010, 20: 712-717. 10.4014/jmb.0910.10043View Article
- Tan L, Turnbough CL: Sequence motifs and proteolytic cleavage of the collagen-like glycoprotein BclA required for its attachment to the exosporium of Bacillus anthracis. J Bacteriol. 2010, 192: 1259-1268. 10.1128/JB.01003-09View Article
- Steichen CT, Kearney JF, Turnbough CL: Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis. J Bacteriol. 2005, 187: 5868-5876. 10.1128/JB.187.17.5868-5876.2005View Article
- Sylvestre P, Couture-Tosi E, Mock M: Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the Bacillus anthracis exosporium. J Bacteriol. 2005, 187: 5122-5128. 10.1128/JB.187.15.5122-5128.2005View Article
- Li Q, Yan Q, Chen J, He Y, Wang J, Zhang H, Yu Z, Li L: Molecular characterization of an ice nucleation protein variant (inaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli. Int J Biol Sci. 2012, 8: 1097-1108.View Article
- Park SJ, Lee SY: Efficient recovery of secretory recombinant protein from protease negative mutant Escherichia coli strains. Biotechnol Techn. 1998, 12: 815-818. 10.1023/A:1008844013548.View Article
- Lee SH, Kwon YC, Kim DM, Park CB: Cytochrome P450-catalyzed O-dealkylation coupled with photochemical NADPH regeneration. Biotechnol Bioeng. 2013, 110: 383-390. 10.1002/bit.24729View Article
- Sambrook J, Russell D: Molecular cloning: a laboratory manual. 2001, New York: Cold Spring Harbor Laboratory Press, 3,
- Filip C, Fletcher G, Wulff JL, Earhart CF: Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973, 115: 717-722.
- Lee SH, Choi JI, Park SJ, Lee SY, Park BC: Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis. Appl Environ Microbiol. 2004, 70: 5074-5080. 10.1128/AEM.70.9.5074-5080.2004View Article
- Chang CC, Ryu DDY, Park CS, Kim JY: Enhancement of rice R-amylase production in recombinant Yarrowia lipolytica. J Ferment Bioeng. 1997, 84: 421-427. 10.1016/S0922-338X(97)82002-8.View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.