Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein
Cecropin A (CeA), a natural cationic antimicrobial peptide, exerts potent antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, making it an attractive candidate substitute for antimicrobials. However, the low production rate and cumbersome, expensive processes required for both its recombinant and chemical synthesis have seriously hindered the exploitation and application of CeA. Here, we utilized a short β-structured self-aggregating protein, ELK16, as a fusion partner of CeA, which allowed the efficient production of high-purity CeA antibacterial peptide with a simple inexpensive process.
In this study, three different approaches to the production of CeA peptide were investigated: an affinity tag (His-tag)-fused protein expression system (AT-HIS system), a cell-free protein expression system (CF system), and a self-assembling peptide (ELK16)-fused protein expression system (SA-ELK16 system). In the AT-HIS and CF systems, the CeA peptide was obtained with purities of 92.1% and 90.4%, respectively, using one or more affinity-chromatographic purification steps. The procedures were tedious and costly, with CeA yields of only 0.41 and 0.93 μg/mg wet cell weight, respectively. Surprisingly, in the SA-ELK16 system, about 6.2 μg/mg wet cell weight of high-purity (approximately 99.8%) CeA peptide was obtained with a simple low-cost process including steps such as centrifugation and acetic acid treatment. An antimicrobial test showed that the high-purity CeA produced in this study had the same antimicrobial activity as synthetic CeA peptide.
In this study, we designed a suitable expression system (SA-ELK16 system) for the production of the antibacterial peptide CeA and compared it with two other protein expression systems. A high yield of high-purity CeA peptide was obtained with the SA-ELK16 system, which greatly reduced the cost and time required for downstream processing. This system may provide a platform for the laboratory scale production of the CeA antibacterial peptide.
KeywordsSelf-aggregating protein Cecropin A Antimicrobial peptide ELK16 Escherichia coli
Mxe GyrA intein
- RF cloning
Restriction Free cloning
Antimicrobial peptides (AMPs) are small peptides of 10–50 amino acids that are effective in the innate immune systems of a wide variety of organisms, including bacteria, plants, insects, and mammals [1, 2, 3]. In general, they are cationic, amphipathic, β-folded or α-helix peptides that display rapid, strong, and broad-spectrum activities against a wide range of pathogens, including bacteria, fungi, plants, and even insects [4, 5, 6].
In recent decades, the widespread use of antimicrobials has resulted in a rapid increase in antimicrobial resistance, which has increased the worldwide morbidity  and economic losses [8, 9]. This has led to enormous efforts to explore and develop new antimicrobial agents, such as AMPs. Moreover, because of their broad spectrum of antibacterial properties but no hemolytic or cytotoxic activities, AMPs are considered attractive targets for the development of new antimicrobial compounds [10, 11]. Cecropin A (CeA) is a natural linear cationic α-helical AMP isolated from insects [12, 13, 14]. It contains a strongly cationic region at its N-terminus and a large hydrophobic tail at its C-terminus, which allow its interaction with the microbial membrane, and then induces cell lysis by pore formation . It has a wide spectrum of antimicrobial activities against Gram-negative bacteria, Gram-positive bacteria, and fungal phytopathogens . More importantly, this peptide does not induce the lysis of erythrocytes or lymphocytes, even at high concentrations . Therefore, the CeA peptide has become a potentially useful antimicrobial substance in biochemical and pharmaceutical research .
Because the chemical synthesis of the CeA peptide or extraction from natural sources are extremely expensive, with low yields [6, 18, 19, 20], the development of a simple way to efficiently produce high-purity CeA peptide is urgently required. In recent years, there has been much research into the production of CeA and its derivatives [21, 22, 23, 24]. To reduce the production costs, Pichia pastoris and Bacillus subtilis have been used as the expression hosts for the secretion of the CeA peptide [23, 25, 26]. To avoid the formation of inclusion bodies, some researchers have fused the peptide to glutathione S-transferase (GST) or thioredoxin (TRX), which significantly increased the soluble fraction of the peptide [16, 21, 27]. To obtain high-purity CeA peptide, affinity tags such as the hexahistidine (His6) tag, cysteine protease domain tag, and strep tag, have been used, and high-purity peptide produced [24, 28, 29]. However, all these methods still require expensive purification with affinity chromatography or high-performance liquid chromatography and the yields are relatively low. More importantly, the peptide toxicity for host cells and the proteolysis of the product are still two obstacles to the high production of the CeA peptide.
Recently, a small β-strand self-assembling peptide, ELK16, which induces the formation of “active inclusion bodies” in vivo, has attracted the attention of many researchers [30, 31]. Based on the self-assembling property of ELK16, a CeA fusion protein containing Mxe GyrA intein and ELK16 was constructed and expressed in E. coli BL21(DE3) cells (SA-ELK16 system). Besides, two other approaches were also investigated: an affinity tag (His tag) fusion protein expression system (AT-HIS system) and a cell-free protein expression system (CF system). We compared the costs and yields of each process, and thus established a simple, inexpensive, single-step purification method for the efficient production of high-purity CeA peptide. This method provides a novel platform for the laboratory large-scale production of CeA peptide.
Design and construction of three different CeA fusions
Expression, optimization and purification of CeA peptide with the AT-HIS system
Summary of the expression and purification schemes in the three different systems
AT-HIS expression system
CF expression system
SA-ELK16 expression system
Step 1: Expression of fusion protein CeA-Mxe-PT-His6
Step 1: Expression of antibacterial peptide His6-CeA
Step 1: Expression of fusion protein CeA-Mxe-ELK16
Step 2: Cell disruption and supernatant collection
Step 2: Ni2+ affinity chromatography
Step 2: Cell disruption and acquisition of protein aggregates
Step 3: Ni2+ affinity chromatography
Step 3: Intein-mediated cleavage
Step 4: Intein-mediated cleavage
Step 4: acetic acid treatment and collection of CeA peptide.
Step 5: The second Ni2+ affinity chromatography
Final yield of peptide production (μg/mg wet cell pellet) a
Purity of bio-produced Cecropin A peptide (%)
Expression and purification of CeA peptide with the CF system
The CeA peptide was purified with nickel ion affinity chromatography, as reported previously . As shown in Fig. 3b, the amount of CeA peptide successfully purified was about 0.93 μg/ml of reaction mixture, with a purity of approximately 90.4% (Table 1).
High-purity CeA peptide released in the SA-ELK16 system
Quantification of cecropin A peptides cleaved from the fusions in the three different schemes
Fusions yielda (μg/mg wet cell pellet)
Peptide yieldb (μg/mg wet cell pellet)
Cleavage effciencyc (%)
Percent recoveryd (%)
Peptide puritye (%)
Antibacterial properties of the bioproduced CeA peptide
In this study, we investigated three different protein expression systems for the production of the CeA peptide: the AT-HIS system, CF system, and SA-ELK16 system. By comparing the results, we developed a cost-effective and efficient protein expression system for high-purity CeA peptide.
In the AT-HIS system, two rounds of nickel ion affinity chromatography were required which was both expensive and time-consuming (approximately 5 days, Table 1). More importantly, a large amount of CeA peptide was lost in the second round of Ni2+ affinity chromatography, which significantly limited the large-scale production of CeA peptide. Finally, CeA peptide with 92.1% purity of was obtained at a yield of 0.41 μg/mg wet cell pellet, indicating that AT-HIS system is not an ideal system for the production of the CeA peptide. Due to its open nature and membrane-free, cell free protein expression system was considered the most suitable platform for expression of toxic proteins in recent years . We tested the efficacy of the CF expression system for producing the CeA peptide. Table 1 shows that about 0.93 μg/ml reaction mixture of the CeA peptide was eventually collected, with 90.4% purity. When the CF and AT-HIS systems were compared, the CF system performed better than the AT-HIS system in that the final yield of CeA peptide was 2-fold higher than with the AT-HIS system. However, this yield is relatively low compared with the production of other AMPs, and falls far short of the yields required for large scale production [47, 48]. Moreover, the purity of the CeA peptide produced by these two expression systems was less than 95% and is inappropriate for further research. Most importantly, several cumbersome and expensive procedures like nickel affinity chromatography are required, which are not practicable for the large-scale production of the CeA peptide. Therefore, there is an urgent need to investigate and explore a high and efficient system for the production of CeA peptide.
To achieve a highly efficient low-cost production process for high-purity (> 98%) CeA peptide, the small self-assembling protein ELK16 was attempted in this study. In SA-ELK16 system, the recovery of CeA peptide was estimated to be 78.38%, higher than that in AT-HIS system (52.14%, Table 2). Finally, highly pure (~ 99.8%) CeA peptide was obtained after the cleavage of Mxe GyrA intein (Table 1), with a yield of 6.20 μg/mg wet cell pellet, which was 15-fold higher than the yield of the AT-HIS system and 6.7-fold higher than that of the CF protein expression system (Table 2). These results indicated that SA-ELK16 system was more efficient than AT-HIS system and CF system for production of CeA peptide. Furthermore, the CeA peptide was produced rapidly (2 days) with simple centrifugation and acetic acid treatment, eliminating expensive and tedious procedures like nickel affinity chromatography or high-performance liquid chromatography, dramatically reducing the production cost (Table 1). These factors make the SA-ELK16 system an ideal candidate for the laboratory scale production of the CeA peptide.
However, in SA-ELK16 system, much effort is still needed for the industrial production of CeA peptide. Such as the cleavage of CeA–Mxe–ELK16 fusion was induced by DTT in this study which was expensive at industry scale. Some other self-cleavage inteins like Ssp dnaB and sortase, which cleavage reactions were activated by pH and calcium ion, can be used to reduce the costs at industry production of CeA peptide [49, 50, 51]. Besides, the downstream process like dialysis also need to be further optimized to meet the needs of industrial production of CeA peptide.
Cecropin A is a natural linear cationic AMP, with rapid and potent activity against a broad spectrum of pathogens, including bacteria, fungi, viruses, and neoplastic cells. Therefore, it has great potential utility in various biotechnological applications [2, 19, 22]. However, the exploitation of the CeA peptide is seriously limited by the low yields and expensive purification costs of its production. Here, we report a fast, economical and efficient expression system, the SA-ELK16 system, for the laboratory scale production of high-purity CeA peptide, which circumvents the difficulties encountered by ordinary recombinant methods or expensive chemical syntheses. It completely avoids the use of affinity resins and greatly reduces the cost and time required, providing a novel platform for laboratory scale production of the CeA peptide. This study lays a solid foundation for further research into the CeA peptide.
Strains and materials
Competent E. coli DH5α and BL21(DE3) cells were purchased from Tiangen Biotech (Beijing, China). Oligonucleotides for gene manipulation were synthesized by Invitrogen (Shanghai, China) or Tianyi Huiyuan Gene Technology (Guangzhou, China). The restriction endonuclease DpnI used for restriction-free cloning (RF cloning) was obtained from Fermentas Thermo Scientific (Glen Burnie, MD, USA). The Hi Trap 5 mL pre-packed column used for protein purification was purchased from Qiagen (Dusseldorf, Germany). All chemical reagents used in this study were of analytical grade.
Construction of recombinant plasmids
All three recombinant expression vectors used in the study were constructed with RF cloning. Like fusion PCR cloning, RF cloning uses the appropriate DNA fragment as a megaprimer for the linear amplification of the vector to introduce a foreign DNA into the plasmid at a predetermined position . CeA peptide, Mxe GyrA intein, and ELK16 were synthesized with the appropriate DNA sequences by codon-optimized expression in E. coli BL21(DE3) by Sangon Biotech (Shanghai, China). The DNA fragments encoding the CeA peptide, Mxe GyrA intein, and ELK16 were cloned into the vector pET30a with RF cloning to construct pET30a–AM–ELK16. To construct pET21a–AM–His, primers AMH-F and AMH-R (see Additional file 2) were used to amplify the AM sequence encoding both the CeA peptide and the Mxe GyrA intein from plasmid pET30a-AM–ELK16. The amplified DNA fragment was then integrated into the pET21a vector with RF cloning. Plasmid pET21–His6–AMP was obtained by amplifying the CeA gene from pET30a–AM–ELK16 and inserting it into pET21a. Plasmid pET21–AMP–His6 was constructed in the same way as pET21–His6–AMP. All the primers used in this work are listed in Additional file 2.
Expression of fusion proteins in three different systems
In the SA-ELK16 and AT-HIS systems, Luria–Bertani (LB) medium containing 50 μg/ml kanamycin or 100 μg/ml ampicillin was inoculated with E. coli BL21(DE3) cells carrying pET30a–AM–ELK16 or pET21a–AM–His, respectively, and incubated at 37 °C. Different concentrations of IPTG (0.1–1 mM) were added to initiate protein expression when the cell density (OD600) reached 0.6–08. The cultures were then incubated under different conditions (5 h/37 °C, 12 h/25 °C, or 24 h/16 °C) to optimize protein expression. The cells were harvested by centrifugation at 7500×g for 15 min and the pellets were stored at − 80 °C for further analysis. In the CF system, pET21–His6–AMP and its derivative were expressed with a previously described method . The reaction solution, containing 60 μl of reaction mixture (RM) and 900 μl of feeding mixture (FM), was added to the wells of standard 24-well microplates. To optimize the Mg2+ concentration, regenerated cellulose membranes with a molecular-weight cut-off of 14 kDa were used. The reactions were performed in the continuous-exchange cell-free configuration and incubated for 12–16 h at 30 °C with shaking (180 rpm).
Intein-mediated cleavage and peptide purification
In the CF system, the soluble fractions were harvested by centrifuging the reaction mixture at 12,000 g for 10 min. Ten-fold volumes of cold acetone were added to the supernatants to precipitate the protein . The pellets were air dried, dissolved in 1 × loading buffer (20 mM Tris–HCl, 3% [w/v] glycerol, 1.5% [w/v] SDS, 5% [w/v] β-mercaptoethanol, 0.02% [w/v] bromophenol blue, pH 8.5), and denatured by heating at 95 °C for 10 min. The samples were separated and analyzed with 16% tricine SDS-PAGE. The CeA fusion produced with the CF system was purified with nickel ion affinity chromatography, as previously reported .
The harvested cell pellets containing the CeA–Mxe–ELK16 or CeA–Mxe–His fusions were resuspended to 10 OD600 units/ml of culture with lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, pH 8.5) and the suspended cells were lysed with a high-pressure homogenizer (Constant Systems Limited, United Kingdom). The CeA–Mxe–ELK16 aggregates were separated by centrifugation at 12,000 g for 30 min at 4 °C. The soluble fractions containing the CeA–Mxe–His fusions were isolated from the aggregates by centrifugation at 12,000 g for 30 min at 4 °C, and then purified with nickel ion affinity chromatography, as described above. For Mxe GyrA intein mediate cleavage, the CeA–Mxe–ELK16 aggregates or purified CeA–Mxe–His fusions were resuspended in cleavage buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, pH 8.5) containing different concentrations of DTT (40–80 mM). The cleavage reaction was assessed by incubating the samples under different conditions (4 °C or 25 °C for 0–14 h) to optimize the efficiency of cleavage. The quantity of protein was determined with a BCA Protein Assay Kit (Sangon) or the Gel-Pro Analyzer software (Media Cybernetics, Houston, USA), using aprotinin as the standard and adjusting for the loading volume.
Acetic acid treatment for high-purity CeA peptide in the SA-ELK16 system
To obtain high-purity (≥ 98%) CeA peptide, different proportions of acetic acid (0.5–3%) were added to the suspensions of the cleaved CeA–Mxe–ELK16 fusion and incubated at room temperature for 10 min. The supernatant containing high-purity CeA peptide was separated by centrifugation at 12,000 g for 30 min at 4 °C. The soluble and insoluble fractions were analyzed with tricine SDS-PAGE.
Fluorescence confocal microscopy
Escherichia coli BL21(DE3) cells carrying the pET30–GFP–Mxe–ELK16 or pET21–GFP–Mxe–His plasmid were cultured and the expression of the fusion was induced by the addition of 0.2 mM IPTG. After expression, 200 μl of cells were harvested by centrifugation at 7000 g at 4 °C for 5 min, resuspended in sterile PBS (1% w/v), spotted onto a slide, air dried, and visualized with a 63× phase-contrast objective on a Zeiss LSM 710 microscope (Germany).
The MICs of the CeA peptides produced in the three different expression systems against E. coli ATCC 25922 were assessed with the broth microdilution method, as previously reported , and compared with that of a synthetic CeA peptide. For the antimicrobial assays, overnight cultures of E. coli ATCC 25922 cells were diluted with fresh Müller–Hinton broth (MHB) to an OD625 of 0.08–0.13 (0.5 McFarland standards), and further diluted with fresh MHB (1:100) to a final concentration of approximately 107 colony-forming units (CFU) per ml. The bio-produced CeA peptides were serially diluted in MHB medium and 50 μl aliquots of the diluted CeA peptides were added to 96-well plates. The standardized bacterial suspension (50 μl) was then added to each well. Growth controls contained no peptide antimicrobial, and sterility controls contained only broth. The plates were incubated at 37 °C for 16–18 h. The OD600 values were monitored with a microplate reader and the lowest concentration of peptide at which the growth of E. coli ATCC 25922 was inhibited was determined as the MIC.
We thank Janine Miller, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
This work was supported by the financial support of Natural Science Foundation of Guangdong Province, China (grant number: 2015A030310322); the Innovative Program of Department of Education of Guangdong Province, China (grant number: 2013KJCX0013). We are also grateful the important role of the funding in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
All data for this study are included in this published article and its Additional files.
JW and MW designed the experiments. MW and KZ performed the experiments. JL, MH and XL carried out the data analysis. MW wrote the manuscript. SL, YM and JW supervised the whole work attended the discussions and revised the manuscript. All authors have read and approved the manuscript, and ensure that this is the case.
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