Journal of Biotechnology 199 (2015) 47–54
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In vitro production and antifungal activity of peptide ABP-dHC-cecropin A Jiaxin Zhang, Ali Movahedi, Junjie Xu, Mengyang Wang, Xiaolong Wu, Chen Xu, Tongming Yin, Qiang Zhuge ∗ Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
a r t i c l e
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Article history: Received 5 January 2015 Received in revised form 1 February 2015 Accepted 4 February 2015 Available online 19 February 2015 Keywords: Antibacterial peptide Escherichia coli expression ABP-dHC-cecropin A SUMO Antifungal activity
a b s t r a c t The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, testing of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve expression of this peptide in E. coli, ABP-dHC-cecropin A was cloned into a pSUMO vector and transformed into E. coli, resulting in the production of a pSUMO-ABP-dHC-cecropin A fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography, yielding a total of 496-mg protein per liter of fermentation culture. The SUMO-ABP-dHC-cecropin A fusion protein was then cleaved using a SUMO protease and re-purified by Ni-IDA chromatography, yielding a total of 158mg recombinant ABP-dHC-cecropin A per liter of fermentation culture at a purity of ≥94%, the highest yield reported to date. Antifungal activity assays performed using this purified recombinant peptide revealed strong antifungal activity against both Candida albicans and Neurospora crassa, as well as Rhizopus, Fusarium, Alternaria, and Mucor species. Combined with previous analyses demonstrating strong antibacterial activity against a number of important bacterial pathogens, these results confirm the use of ABP-dHC-cecropin A as a broad-spectrum antimicrobial peptide, with significant therapeutic potential. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, antimicrobial peptides (AMPs) have become an important topic in antimicrobial research due to their broadspectrum activity against both bacterial and fungal pathogens (Keymanesh and Sardari, 2009). Important AMP classes identified to date include cecropins, histatins, defensins, and cathelicidins (Koczulla and Bals, 2003). While defensin-like proteins have been isolated from a wide range of organisms, cathelicidin production appears to be limited to mammalian species (Dorschner et al., 2001; Gallo et al., 1997). PR-39, which was the first AMP found in mammalian skin, was isolated from porcine wound fluid, consistent with its role in antimicrobial defense (Gallo et al., 1994). The histatins are more closely associated with antifungal responses, and have
Abbreviations: ABP-dHC-cecropin A, antibacterial peptide drury (Hyphantria cunea) Cecropin A; SUMO, small ubiquitin-related modifier; SEM, scanning electron microscopy; MALDI–TOF MS, matrix-assisted laser desorption ionization/time of flight mass spectrometry; EMSA, electrophoretic mobility shift assay. ∗ Corresponding author. Tel.: +86 02585428701. E-mail address: [email protected] (Q. Zhuge). http://dx.doi.org/10.1016/j.jbiotec.2015.02.018 0168-1656/© 2015 Elsevier B.V. All rights reserved.
been isolated from the saliva of humans and other higher primates (Helmerhorst et al., 1997). The cecropins are a group of positively charged peptides originally isolated from the hemolymph of giant silk moths (Hultmark et al., 1980). These small, cationic, C-terminally amidated peptides possess a helix–bend–helix structure, which is thought to confer broad-spectrum antibacterial activity against both Gramnegative and -positive bacteria (Boman et al., 1991; Cociancich et al., 1994). Cecropins are thought to function by forming specific amphipathic ␣-helices, allowing them to target non-polar lipid membranes. After binding to the membrane, these proteins go on to form ion-permeable channels, resulting in cell depolarization, irreversible cytolysis, and cell death (Boman, 2003). Against bacteria, cecropins act as quaternary detergents targeting the outer membrane, destroying the permeability barrier by forming an ionic channel in the bacterial membrane via interaction between the bacterial membrane and the hydrophobic side of these amphipathic peptides (Bonmatin et al., 1992; Holak et al., 1988; Merrifield et al., 1982). One member of the cecropin family, ABP-dHC-cecropin A, is a highly cationic 37-amino-acid peptide isolated from the fat bodies of drury moths (Hyphantria cunea). This peptide has been shown
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to confer potent antibacterial activity (Zhang et al., 2014) in vitro, suggesting a possible therapeutic use for this peptide. Due to their natural toxicity and relative sensitivity to proteolytic degradation, antimicrobial peptides are often produced as pro-peptides within their natural hosts, as a way to neutralize their innate cytotoxic effects while maintaining robust expression levels. This approach has also been exploited as a means of heterologous expression of many peptides, including the F4 fragment of PurF (Pyo et al., 2004), thioredoxin (Xu et al., 2006), green fluorescent protein (GFP) (Skosyrev et al., 2003), and protein PaP3.30 (Rao et al., 2004). Small ubiquitin-related modifier (SUMO) is an ubiquitin-related protein that functions by covalent attachment to other proteins. When fused to the N-terminus of other proteins, SUMO is able to act as a chaperone, protecting the target protein from degradation, making it a useful tag for heterologous expression. Specific advantages of SUMO conjugation include increased protein expression, decreased proteolytic degradation of the target protein, and improved folding and solubility, along with easier detection and purification of the protein (Sun et al., 2008). In this work, the ABP-dHC-cecropin A gene was fused to SUMO, followed by heterogeneous expression in E. coli. The SUMO-ABPdHC-cecropin A construct was then digested with hydroxylamine, allowing for specific cleavage between the amino acid residues Asn–Gly, resulting in a purified 37-amino-acid ABP-dHC-cecropin A peptide. This SUMO fusion and customized expression protocol allows for greater peptide synthesis, while dramatically reducing overall production costs, enabling sufficient ABP-dHC-cecropin A synthesis necessary for downstream applications. Increased production of this peptide has allowed us to expand upon our previous efforts demonstrating robust antibacterial activity across a number of important pathogens (Zhang et al., 2014). Here, we examined six species of fungi to determine their sensitivity to ABP-dHCcecropin A. Macro- and microscopic analyses revealed significant antifungal activity against all fungi tested, further demonstrating the potential of ABP-dHC-cecropin A as a potent broad-spectrum antimicrobial. 2. Materials and methods 2.1. Bacteria, fungi, vectors, and enzymes E. coli DH5␣ (maintained in our laboratory) was used for subcloning and plasmid amplification; E. coli BL21 (DE3) (Novagen, USA) was used as the expression host. Candida albicans (ATTC 753), Neurospora crassa (ATCC 13837), Rhizopus sp. (ATCC 20577), Fusarium sp. (ATTC 20789), Alternaria sp. (ATTC 20492), and Mucor sp. (ATCC 52915) were obtained from Institute of Microbiology Chinese Academy of Sciences (IMCAS). The linearized pSUMO vector containing StuI and HindIII restriction sites was purchased from Life Sensors (Life-Sensors, Malvern, PA). All restriction enzymes and the T4 DNA ligase were purchased from Roche (USA). 2.2. Construction of expression vectors The gene encoding ABP-dHC-cecropin A was amplified by PCR from the cloning vector PMD19-T/ABP-dHC-cecropin A, which contained the full-length dHC-cecropin A cDNA. PCR fragments were then separated by 1.5% gel electrophoresis, and purified with a DNA gel extraction kit (Qiagen, Germany). The resulting PCR product was digested with StuI and HindIII, and ligated into the pSUMO plasmid at the corresponding restriction sites. The ligation mixture was then transformed into E. coli DH5␣ cells, and sequenced to verify proper assembly.
2.3. Fusion protein expression The pSUMO-ABP-dHC-cecropin A plasmid was transformed into competent E. coli BL21 (DE3). Two colonies were selected and cultured in 3-mL LB medium with vigorous shaking (220 rpm) at 37 ◦ C to a density of ∼0.6 absorbance units. Isopropyl--d-1thiogalactopyranoside (IPTG) (0.5 mM) was then added to induce expression of the recombinant protein, followed by incubation at 37 ◦ C for 3 h. 2.4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting Proteins were separated on a 12% polyacrylamide gel under reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Malakhov et al., 2004). The membrane was soaked for 15 min in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3), and nonspecific protein binding was blocked by incubating the membrane in 5% MPBS (5% skim milk in phosphate-buffered saline (PBS), pH 7.4) for 1 h. After washing with PBST (PBS containing 0.05% Tween 20) for 15 min, a 1:1000 dilution of anti-His6 IgG mAb (Novagen) in MPBS was added, followed by incubation at 37 ◦ C for 2 h. The membrane was then washed three times for 10 min with PBST, incubated for 60 min with a 1:5000 dilution of goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology) in PBST, and subsequently washed three times for 5 min with PBST. Blotting was performed using the tetramethyl benzidine (TMB) chemiluminescence system (Promega). 2.5. Large-scale expression of recombinant protein by batch fermentation Fermentations were performed as described previously (Ma et al., 2006), with minor modifications. Briefly, a single colony of E. coli BL21 (DE3) harboring the pSUMO ABP-dHC-cecropin A vector was inoculated into 10-mL LB medium supplemented with 50 g/mL kanamycin in a 50-mL flask. The seed culture was incubated at 37 ◦ C and 220 rpm on a rotary shaker overnight. Secondary seed cultures were generated by inoculating 200-mL 29 YT medium (tryptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L, and 50 g/mL kanamycin) in a 500-mL flask, followed by incubation at 37 ◦ C and 220 rpm on a rotary shaker for an additional 4 h. Fermentation was performed in production medium (10 g/L yeast extract, 10 g/L tryptone, 10 g/L glucose, 4 g/L K2 HPO4 ·3H2 O, 7 g/L Na2 HPO4 ·12H2 O, 1.2 g/L (NH4 )2 SO4 , 2 g/L KH2 PO4 , and 3 mL/L 50% (v/v) antifoam) supplemented with a trace element solution (0.001 g/L MnSO4 ·5H2 O, 0.004 g/L CaCl2 ·6H2 O, 0.002 g/L Na2 MoO4 ·2H2 O, 0.002 g/L ZnCl2 , 0.002 g/L CuSO4 ·5H2 O, 0.0005 g/L H3 BO4 , 0.02 g/L FeSO4 ·7H2 O, 0.02 g/L CaCl2 ·2H2 O, and 0.3 g/L MgSO4 ·7H2 O). Secondary seed cultures were inoculated into production medium at an inoculum size of 5% (v/v), and maintained at 37 ◦ C with an initial agitation rate of 300 rpm in a 5-L fermenter (BioXin, Shanghai). Dissolved oxygen was maintained at 50% air saturation by an agitation cascade between 300 and 800 rpm. Cultivation pH was kept at 7.0 by the addition of NH4 OH or H2 SO4 . After 4 h of incubation, target protein expression was induced by addition of 0.5 mM IPTG. Cells were then incubated for an additional 3 h, and collected by centrifugation (6000 rpm, 10 min). The resulting cell pellet then was stored at −80 ◦ C until needed. 2.6. Purification of the SUMO fusion protein Cell pellets were resuspended in 15-mL binding buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, and 10 mM PMSF, pH 8.0) and lysed on ice by sonication at 400 W for 100 cycles
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(4 s working, 8 s free). Cells were then centrifuged at 12,000 × g for 20 min at 4 ◦ C, and the supernatant applied to a Ni2+ -chelating column. After extensive washing with binding buffer, the fusion protein was eluted with five volumes of elution buffer (20 mM Tris, 500 mM NaCl, and 250 mM imidazole, pH 8.0). The peak fractions containing the fusion protein were pooled and dialyzed overnight at 4 ◦ C against phosphate-buffered saline (PBS). 2.7. Purification of ABP-dHC-cecropin A Purified SUMO-ABP-dHC-cecropin A protein (50 g) was incubated in the presence of a SUMO protease (3 U) at 30 ◦ C for 1 h. Samples were then reapplied to the nickel column containing NiIDA resin to remove his-tagged SUMO and SUMO proteases. A majority of the cleaved ABP-dHC-cecropin A was eluted in the flowthrough (unbound) fractions; the remaining fraction was recovered by washing the resin with binding buffer. Eluates and washes exhibiting high UV values at OD280 were then pooled, and resolved on 4–20% gradient gels (Mini-R/PROTEAN TGX Gels, Bio-Rad). Purification by preparative reverse-phase high-performance liquid chromatography (RP-HPLC) yielded a final product deemed about 99% pure by analytical RP-HPLC. Final confirmation was performed using ESI-mass spectrometry and matrix-assisted laser desorption/ionization, time-of-flight (MALDI–TOF) mass spectrometry (MS).
Fig. 1. Schematic representation of the pSUMO-ABP-dHC-cecropin A expression vector. The SUMO-tag was fused to the N-terminus of ABP-dHC-cecropin A.
3. Results 3.1. Construction and expression of ABP-dHC-cecropin A fusion protein
Lyophilized ABP-dHC-cecropin A was dissolved in 0.9% mM NaCl to a final concentration of 4 mg/mL. Antifungal activity was tested using an agar well diffusion assay (Lehrer et al., 1991).
A schematic of the ABP-dHC-cecropin A expression construct is depicted in Fig. 1. Proper construction of the pSUMO-ABP-dHCcecropin A plasmid was verified by DNA sequencing, followed by transformation into E. coli BL21 (DE3). An obvious protein band was visible after IPTG induction (Fig. 2), confirming proper synthesis of the pSUMO-ABP-dHC-cecropin A protein.
2.9. Electrophoretic mobility shift assay of ABP-dHC-cecropin A bound to fungal genomic DNA
3.2. High-yield expression of pSUMO-ABP-dHC-cecropin A by fermentation
Genomic DNA of C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp. and Mucor sp. were extracted using a fungal DNA kit (Omega, USA). DNA was then added to increasing concentrations of ABP-dHC-cecropin A protein (50–300 ng). Samples were incubated for 30 min at 4 ◦ C, and visualized by 1% agarose gel electrophoresis for EMSA.
Fermentation of transformed E. coli was performed as described above. The fermenter batch yielded 46.8 g/L wet biomass at an OD600 of ∼22, over 12-fold higher than that achieved in flask culture with LB medium. From this, a total of 2675 mg/L soluble protein was recovered, confirming both the efficiency and solubility of this fusion construct.
2.10. Scanning electron microscopy of fungi treated with ABP-dHC-cecropin A
3.3. Purification of the SUMO-ABP-dHC-cecropin A fusion protein
2.8. Antifungal sensitivity assay
Following treatment with ABP-dHC-cecropin A by agar well diffusion, samples were taken from within the zone of inhibition and peripheral areas, and fixed in glutaraldehyde for 1 d. Samples were then washed in increasing concentrations of ethanol (30–100%, v/v) ethanol, dried, sputter-coated with gold, and observed under a scanning electron microscope (FEI Quanta 200, USA).
Purification of the SUMO-ABP-dHC-cecropin A fusion construct was performed using a Ni-IDA resin. His-tagged proteins were captured using a Ni-IDA resin, washed repeatedly in a buffer containing 20 mM imidazole, and eluted in a buffer containing 250 mM imidazole. Purified SUMO-ABP-dHC-cecropin A was isolated at a purity of >90%, and was analyzed using the Biorad Chemi Doc XRS + Molecular Imager’s lmage LabTM Software quantitative
Fig. 2. Analysis of the expressed fusion protein by SDS–PAGE. Lane M: molecular mass marker; lane 1: negative control; lanes 2–9: colonies 1–8, respectively, induced by 0.5 mM IPTG. The molecular weight of the target protein was about 24 kDa, and is indicated by the black arrow.
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Table 1 Isolation of recombinant ABP-dHC-cecropin A from pSUMO-ABP-dHC-cecropin A fusion protein. Purification step Sonicated supernatant SUMO-ABP-dHC-cecropin A after affinity chromatography Recombinant ABP-dHC-cecropin A after affinity chromatography Dialysis
Total protein (mg) a
Fusion protein (mg) b
Recombinant ABP-dHC-cecropin A (mg) c
Purity (%)d
2675 NA
684 496b
237 211c
NA 90
194a
NA
173c
>94
NA
NA
158a
>94
Estimations are based on 1 L of bacterial culture (about 46.8 g wet weight). NA, not applicable. a Determined using a Bradford assay. b Percent of SUMO-ABP-dHC-cecropin A fusion protein from total proteins was estimated by SDS gel scanning. c The amount of recombinant ABP-dHC-cecropin A was calculated as a fraction of SUMO-ABP-dHC-cecropin A fusion protein. d Purity of protein or peptide was estimated by SDS gels stained by Coomassie Blue.
tools (Fig. 3), at a concentration of ∼496 mg/L bacterial culture (Table 1).
3.4. Cleavage and purification of recombinant ABP-dHC-cecropin A Next, 50-g SUMO-ABP-dHC-cecropin A protein were cleaved with SUMO protease, with complete digestion confirmed by SDS–PAGE (Fig. 4). The cleaved sample was then reapplied to a Ni-IDA column to remove any residual His-tagged SUMO and SUMO protease, and resolved by SDS-PAGE, yielding a final ABPdHC-cecropin A peptide (∼4.06 kDa) at a purity of ≥94% (Fig. 4). Finally, the purified ABP-dHC-cecropin A was passed through a 0.22-m filter and stored at −80 ◦ C until needed (Fig. 5). The final yield of purified recombinant ABP-dHC-cecropin A was 158 mg/L (Table 1).
3.5. Antifungal activity assays The antifungal activity of ABP-dHC-cecropin A was determined using an agar well diffusion assay at a starting concentration of 4 mg/mL. All treatments were performed in triplicate (Fig. 6), with ring diameters measured using as automatic colony counter Scan1200 (Table 2). Significant antifungal activity was evident against all six species tested (C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp., and Mucor sp.), confirming the broadspectrum efficacy of this compound.
Table 2 Antifungal circle diameter measurement. Fungal name
No. 2 (mm)
No. 3 (mm)
No. 4 (mm)
Average (mm)
±SD
Canidia albicans Neurospora crassa Rhizopus sp. Fusarium sp. Alternaria sp. Mucor sp.
9.8 10.0 12.4 10.8 11.6 10.4
10.2 11.5 12.4 10.6 12.4 9.8
9.2 11.7 12.6 11.2 11.8 10.6
9.7 11.1 12.5 10.9 11.9 10.3
0.253 0.863 0.013 0.093 0.173 0.173
20-L (4 mg/mL final concentration) ABP-dHC-cecropin A was loaded in each lane.
3.6. EMSA of ABP-dHC-cecropin A bound to fungal genomic DNA The binding of ABP-dHC-cecropin A to fungal genomic DNA was determined by EMSA across a range of protein concentrations (50–300 ng). A significant, dose-dependent binding of ABP-dHCcecropin A to fungal DNA was evident across all concentrations tested (Fig. 7). Similar results were observed for all six fungi tested (C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp., and Mucor sp.) indicative of DNA maybe as a target of ABP-dHC-cecropin A activity. 3.7. Electron microscopy of fungi treated with ABP-dHC-cecropin A While the antifungal activity of ABP-dHC-cecropin A was clearly demonstrated using well-diffusion assays, the mechanism underlying this antimicrobial activity is not clearly understood.
Fig. 3. Purification of the SUMO-ABP-dHC-cecropin A fusion protein. (A) The eluted fusion protein exhibited ≥90% purity following resolution on a 12% SDS–PAGE gel. Lane M: molecular weight marker; lane 2: precipitate of cell lysate; lane 3: supernatant of cell lysate; lane 4: flow-through; lane 5: wash; lane 6: elution; lane 7: Western blot analysis of purified SUMO-ABP-dHC-cecropin A using a mAb against the His6 tag. (B) Ni-IDA affinity chromatography of the fusion protein using LP data view.
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Fig. 4. Analysis of SUMO-ABP-dHC-cecropin A fusion protein by SDS-PAGE. Analysis of SUMO-ABP-dHC-cecropin A fusion protein following cleavage with SUMO protease. Lane M: low molecular weight marker; lane 1: purified SUMO-ABP-dHC-cecropin A fusion protein; lane 2: SUMO-ABP-dHC-cecropin A cleavage products following SUMO digestion. Lane 3: purified recombinant ABP-dHC-cecropin A.
ABP-dHC-cecropin A is an amphipathic peptide with ␣-helical structures, making pore formation (Ludtke et al., 1996; Shai and Oren, 2001) or ion channel generation (Huang, 2000) the most likely mechanism of activity. Here, we observed fungal lysis as a result of transmembrane pore formation (Fig. 8). Fungi isolated from within the zone of inhibition exhibited both growth arrest and lysis (Fig. 8A, C, E, G, I, and K), while those isolated from the outer limits of this zone were observed in various states of cell decay, including significant atrophy of the fungal mycelium (Fig. 8B, D, F, H, J, and L) Taken together, these results indicate strong antifungal activity of ABP-dHC-cecropin A.
4. Discussion Insects have been shown to produce several kinds of antimicrobial products in response to infection or injury (Boman, 1987; Cociancich et al., 1994; Hultmark, 1993; Kanost et al., 1990; Dunn, 1986). Recent advances in heterologous expression technology may allow for many of these, including ABP-dHC-cecropin A, to be developed into new antimicrobial therapies. Previously, we had shown that ABP-dHC-cecropin A exhibited strong antibacterial activity, making this a promising candidate for further studies. However, the compound used in this study was
Fig. 5. (A)The RP-HPLC chromatogram of the purified peptide. The purified ABP-dHC-cecropin A was 99% pure. (B) The molecular mass of the recombinant ABP-dHC-cecropin A is 4060.94 Da.
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Fig. 6. Bioactivity of ABP-dHC-cecropin A. Bioactivity of ABP-dHC-cecropin A against (A) Candida albicans, (B) Neurospora crassa, (C) Rhizopus sp., (D) Fusarium sp., (E) Alternaria sp., and (F) Mucor sp. Lane 1: negative control. Lanes 2–4: ABP-dHC-cecropin A (4 mg/mL); a 20-L sample was loaded in each lane.
Fig. 7. EMSA analysis of ABP-dHC-cecropin A binding with fungal genomic DNA. EMSA analysis of ABP-dHC-cecropin A binding with genomic DNA of (A) Candida albicans, (B) Neurospora crassa, (C) Rhizopus sp., (D) Fusarium sp., (E) Alternaria sp., and (F) Mucor sp. Lane M: 15-kb DNA marker, lane 1: fungal genomic DNA; lanes 2–5: genomic DNA mixed with 50-, 100-, 200-, and 300-ng protein, respectively.
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Fig. 8. Scanning electron micrographs showing the effect of ABP-dHC-cecropin A on fungal surface morphology. Analysis of (A) Candida albicans (SEM, 4000×), (C) Neurospora crassa (SEM, 2400×), (E) Rhizopus sp. (SEM, 1200×), (G) Fusarium sp. (SEM, 1200×), (I) Alternaria sp. (SEM, 1200×), and (K) Mucor sp. (SEM, 600×), respectively, harvested from areas outside the zones of inhibition following disc diffusion assays. Analysis of (B) C. albicans (SEM, 80×), (D) N. crassa (SEM, 150×), (F) Rhizopus sp. (SEM, 150×), (H) Fusarium sp. (SEM, 80×), (J) Alternaria sp. (SEM, 150×), and (L) Mucor sp. (SEM, 50×), respectively, harvested from within the zones of inhibition (red ovals) following disc diffusion assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
produced through direct chemical synthesis, severely limiting the quantity of protein available for testing. One method for overcoming this limitation is through heterologous expression in hosts such as E. coli or Saccharomyces cerevisiae; however, the utility of such a method is limited in the case of native cationic antimicrobial peptides due to the inherent toxicity to the host organism, and the sensitivity of these recombinant peptides to intracellular proteases. Fortunately, many of these problems can be overcome by the addition of a fusion partner, as seen with the F4 fragment of PurF (Pyo SH, 2004), green fluorescent protein (GFP) (Skosyrev et al., 2003), and protein PaP3.30 (Rao et al., 2004). Significant variability in gene expression has been observed among many traditional fusion systems, along with other problems, such as inefficient cleavage of the fusion protein, or cleavage within the target protein, both of which can confound purification efforts. Recently, SUMO has emerged as a superior choice among fusion tag options in terms of both enhanced protein expression and solubility, with the added distinction of generating recombinant proteins with native conformations (Butt et al., 2005; Wu et al., 2009). The SUMO tag has advantages over the traditional fusion tag and enhanced both the expression and cleavage of the fusion protein. The effect of SUMO on enhancing protein solubility can be partly explained by its structure. SUMO has an external hydrophilic surface and inner hydrophobic core, which may have a detergentlike effect on otherwise insoluble proteins (Butt et al., 2005).
Additionally, the SUMO-specific protease Ulp1 efficiently cleaves the conserved Gly–Gly motif at the C-terminus of SUMO from the recombinant fusion protein under suitable conditions. Unlike EK or TEV protease, whose recognition sequences are short and degenerate, Ulp1 recognizes the tertiary sequence of SUMO. As a result, Ulp1 does not cleave within the fused protein of interest, so intact proteins of interest with little exogenous amino acid residue can be obtained. We hypothesized that the attachment of a highly stable and compact SUMO structure to the N-terminus of ABP-dHC-cecropin A would facilitate enhanced solubility and expression, while maintaining the native protein conformation. Here, the SUMO fusion protein was successfully expressed in E. coli, resulting in high expression of soluble fusion protein (Fig. 3a). Downstream processing was similarly successful, with efficient cleavage of the SUMO tag using SUMO protease (Fig. 4). The resulting ABP-dHC-cecropin A peptide (∼4.06 kDa) was recovered at a purity of ≥94%, yielding 158-mg protein per liter of fermentation culture. These results clearly demonstrate that the SUMO fusion system and customized expression and purification protocol described here have dramatically improved both the cost and efficiency of producing ABP-dHC-cecropin A. It is likely that the approach described here could be widely applied to the production of other cytotoxic cationic peptides in E. coli, which may accelerate the pace of discovery of antimicrobial peptides.
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In an antifungal activity assay, ABP-dHC-cecropin A exhibited strong antifungal activity against C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp., and Mucor sp. (Figs. 6–8). This activity appears to be mediated via the direct binding of ABP-dHC-cecropin A to fungal DNA (Fig. 7), likely due to the strong charge differences between the cationic ABP-dHC-cecropin A and the negatively charged phosphate groups of DNA. ABP-dHC-cecropin A has a net charge of +5 at neutral pH due to the presence of multiple Arg1,10,16 , Lys3,6,7,21,33,37 Glu9 , and Asp17 residues (Zhang et al., 2014). The secondary structure of this molecule is a typical amphipathic ␣-helix, which positions the positively charged groups on the outside of the helix, while negatively charged groups are found primarily on the inside. The pattern of ABP-dHC-cecropin A binding with fungal DNA may be as follow: ABP-dHC-cecropin A combines with phosphate groups of fungal DNA by electrostatic attraction to form ABP-dHC-cecropin A anf fungal DNA structures. ABP-dHC-cecropin A further combines with the DNA double helix grooves, becoming embedded or partially embedded in the fungal DNA (Kumar and Asuncion, 1993). In fungal models, ABP-dHC-cecropin A has additional effects on fungal cell membranes, and may act through cell membranes, binding with the internal cell structure (Kumar and Asuncion, 1993). Binding of ABP-dHC-cecropin A to fungal DNA blocks both the replication and transcription of DNA, which will be examined in detail in future studies. This led to growth arrest and cell lysis within the zone of inhibition, while those isolated from the outer limits of this zone were observed in various states of cell decay, including significant atrophy of the fungal mycelium. These results confirm the direct antifungal activity of ABP-dHC-cecropin A in vitro. When taken together, the data presented here suggest a multifaceted approach to fungal lysis, including disruption of the plasma membrane and incorporation into the nucleus, followed by direct binding to DNA, and possible inhibition of DNA replication and transcription. This model of antimicrobial activity is consistent with a very broad spectrum of activity, highlighting the potential of ABPdHC-cecropin A as an important antimicrobial candidate. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by the National 863 Program of China (No. 2013AA102703), the International Science & Technology Cooperation Program of China (2014DFG32440), the National Science Foundation of China (No. 31400567), the Graduate Innovative Project of Jiangsu Province (CXZZ13 0530), the Doctoral Degree Thesis Innovation Foundation of Nanjing Forestry University (No. 2013Y07), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Program for Innovative Research Team in University of Educational Department and Jiangsu Province, China. References Boman, H.G., 2003. Antibacterial peptides: basic facts and emerging concepts. J. Intern. Med. 254, 197–215. Boman, H.G., Faye, I., Gudmundsson, G.H., Lee, J.Y., Lidholm, D.A., 1991. Cellfree immunity in Cecropia. A model system for antibacterial proteins. Eur. J. Biochem./FEBS 201, 23–31. Boman HG, H.D., 1987. Cell-free immunity in insects. Ann. Rev. Microbiol. 41, 103–126.
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Pyo SH, L.J., Park, H.B., Cho, J.S., Kim, H.R., Han, B.H., Park, Y.S., 2004. Expression and purification of a recombinant buforin derivative from Escherichia coli. Process Biochem. 39, 1731–1736. Rao, X.C., Li, S., Hu, J.C., Jin, X.L., Hu, X.M., Huang, J.J., Chen, Z.J., Zhu, J.M., Hu, F.Q., 2004. A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli. Protein Expr. Purif. 36, 11–18. Shai, Y., Oren, Z., 2001. From “carpet” mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides. Peptides 22, 1629–1641. Skosyrev, V.S., Rudenko, N.V., Yakhnin, A.V., Zagranichny, V.E., Popova, L.I., Zakharov, M.V., Gorokhovatsky, A.Y., Vinokurov, L.M., 2003. EGFP as a fusion partner for the expression and organic extraction of small polypeptides. Protein Expr. Purif. 27, 55–62. Sun, Z., Xia, Z., Bi, F., Liu, J.N., 2008. Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli. Appl. 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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
In vitro production and antifungal activity of peptide ABP-dHC-cecropin A Jiaxin Zhang, Ali Movahedi, Junjie Xu, Mengyang Wang, Xiaolong Wu, Chen Xu, Tongming Yin, Qiang Zhuge ∗ Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
a r t i c l e
i n f o
Article history: Received 5 January 2015 Received in revised form 1 February 2015 Accepted 4 February 2015 Available online 19 February 2015 Keywords: Antibacterial peptide Escherichia coli expression ABP-dHC-cecropin A SUMO Antifungal activity
a b s t r a c t The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, testing of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve expression of this peptide in E. coli, ABP-dHC-cecropin A was cloned into a pSUMO vector and transformed into E. coli, resulting in the production of a pSUMO-ABP-dHC-cecropin A fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography, yielding a total of 496-mg protein per liter of fermentation culture. The SUMO-ABP-dHC-cecropin A fusion protein was then cleaved using a SUMO protease and re-purified by Ni-IDA chromatography, yielding a total of 158mg recombinant ABP-dHC-cecropin A per liter of fermentation culture at a purity of ≥94%, the highest yield reported to date. Antifungal activity assays performed using this purified recombinant peptide revealed strong antifungal activity against both Candida albicans and Neurospora crassa, as well as Rhizopus, Fusarium, Alternaria, and Mucor species. Combined with previous analyses demonstrating strong antibacterial activity against a number of important bacterial pathogens, these results confirm the use of ABP-dHC-cecropin A as a broad-spectrum antimicrobial peptide, with significant therapeutic potential. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, antimicrobial peptides (AMPs) have become an important topic in antimicrobial research due to their broadspectrum activity against both bacterial and fungal pathogens (Keymanesh and Sardari, 2009). Important AMP classes identified to date include cecropins, histatins, defensins, and cathelicidins (Koczulla and Bals, 2003). While defensin-like proteins have been isolated from a wide range of organisms, cathelicidin production appears to be limited to mammalian species (Dorschner et al., 2001; Gallo et al., 1997). PR-39, which was the first AMP found in mammalian skin, was isolated from porcine wound fluid, consistent with its role in antimicrobial defense (Gallo et al., 1994). The histatins are more closely associated with antifungal responses, and have
Abbreviations: ABP-dHC-cecropin A, antibacterial peptide drury (Hyphantria cunea) Cecropin A; SUMO, small ubiquitin-related modifier; SEM, scanning electron microscopy; MALDI–TOF MS, matrix-assisted laser desorption ionization/time of flight mass spectrometry; EMSA, electrophoretic mobility shift assay. ∗ Corresponding author. Tel.: +86 02585428701. E-mail address: [email protected] (Q. Zhuge). http://dx.doi.org/10.1016/j.jbiotec.2015.02.018 0168-1656/© 2015 Elsevier B.V. All rights reserved.
been isolated from the saliva of humans and other higher primates (Helmerhorst et al., 1997). The cecropins are a group of positively charged peptides originally isolated from the hemolymph of giant silk moths (Hultmark et al., 1980). These small, cationic, C-terminally amidated peptides possess a helix–bend–helix structure, which is thought to confer broad-spectrum antibacterial activity against both Gramnegative and -positive bacteria (Boman et al., 1991; Cociancich et al., 1994). Cecropins are thought to function by forming specific amphipathic ␣-helices, allowing them to target non-polar lipid membranes. After binding to the membrane, these proteins go on to form ion-permeable channels, resulting in cell depolarization, irreversible cytolysis, and cell death (Boman, 2003). Against bacteria, cecropins act as quaternary detergents targeting the outer membrane, destroying the permeability barrier by forming an ionic channel in the bacterial membrane via interaction between the bacterial membrane and the hydrophobic side of these amphipathic peptides (Bonmatin et al., 1992; Holak et al., 1988; Merrifield et al., 1982). One member of the cecropin family, ABP-dHC-cecropin A, is a highly cationic 37-amino-acid peptide isolated from the fat bodies of drury moths (Hyphantria cunea). This peptide has been shown
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to confer potent antibacterial activity (Zhang et al., 2014) in vitro, suggesting a possible therapeutic use for this peptide. Due to their natural toxicity and relative sensitivity to proteolytic degradation, antimicrobial peptides are often produced as pro-peptides within their natural hosts, as a way to neutralize their innate cytotoxic effects while maintaining robust expression levels. This approach has also been exploited as a means of heterologous expression of many peptides, including the F4 fragment of PurF (Pyo et al., 2004), thioredoxin (Xu et al., 2006), green fluorescent protein (GFP) (Skosyrev et al., 2003), and protein PaP3.30 (Rao et al., 2004). Small ubiquitin-related modifier (SUMO) is an ubiquitin-related protein that functions by covalent attachment to other proteins. When fused to the N-terminus of other proteins, SUMO is able to act as a chaperone, protecting the target protein from degradation, making it a useful tag for heterologous expression. Specific advantages of SUMO conjugation include increased protein expression, decreased proteolytic degradation of the target protein, and improved folding and solubility, along with easier detection and purification of the protein (Sun et al., 2008). In this work, the ABP-dHC-cecropin A gene was fused to SUMO, followed by heterogeneous expression in E. coli. The SUMO-ABPdHC-cecropin A construct was then digested with hydroxylamine, allowing for specific cleavage between the amino acid residues Asn–Gly, resulting in a purified 37-amino-acid ABP-dHC-cecropin A peptide. This SUMO fusion and customized expression protocol allows for greater peptide synthesis, while dramatically reducing overall production costs, enabling sufficient ABP-dHC-cecropin A synthesis necessary for downstream applications. Increased production of this peptide has allowed us to expand upon our previous efforts demonstrating robust antibacterial activity across a number of important pathogens (Zhang et al., 2014). Here, we examined six species of fungi to determine their sensitivity to ABP-dHCcecropin A. Macro- and microscopic analyses revealed significant antifungal activity against all fungi tested, further demonstrating the potential of ABP-dHC-cecropin A as a potent broad-spectrum antimicrobial. 2. Materials and methods 2.1. Bacteria, fungi, vectors, and enzymes E. coli DH5␣ (maintained in our laboratory) was used for subcloning and plasmid amplification; E. coli BL21 (DE3) (Novagen, USA) was used as the expression host. Candida albicans (ATTC 753), Neurospora crassa (ATCC 13837), Rhizopus sp. (ATCC 20577), Fusarium sp. (ATTC 20789), Alternaria sp. (ATTC 20492), and Mucor sp. (ATCC 52915) were obtained from Institute of Microbiology Chinese Academy of Sciences (IMCAS). The linearized pSUMO vector containing StuI and HindIII restriction sites was purchased from Life Sensors (Life-Sensors, Malvern, PA). All restriction enzymes and the T4 DNA ligase were purchased from Roche (USA). 2.2. Construction of expression vectors The gene encoding ABP-dHC-cecropin A was amplified by PCR from the cloning vector PMD19-T/ABP-dHC-cecropin A, which contained the full-length dHC-cecropin A cDNA. PCR fragments were then separated by 1.5% gel electrophoresis, and purified with a DNA gel extraction kit (Qiagen, Germany). The resulting PCR product was digested with StuI and HindIII, and ligated into the pSUMO plasmid at the corresponding restriction sites. The ligation mixture was then transformed into E. coli DH5␣ cells, and sequenced to verify proper assembly.
2.3. Fusion protein expression The pSUMO-ABP-dHC-cecropin A plasmid was transformed into competent E. coli BL21 (DE3). Two colonies were selected and cultured in 3-mL LB medium with vigorous shaking (220 rpm) at 37 ◦ C to a density of ∼0.6 absorbance units. Isopropyl--d-1thiogalactopyranoside (IPTG) (0.5 mM) was then added to induce expression of the recombinant protein, followed by incubation at 37 ◦ C for 3 h. 2.4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting Proteins were separated on a 12% polyacrylamide gel under reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Malakhov et al., 2004). The membrane was soaked for 15 min in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3), and nonspecific protein binding was blocked by incubating the membrane in 5% MPBS (5% skim milk in phosphate-buffered saline (PBS), pH 7.4) for 1 h. After washing with PBST (PBS containing 0.05% Tween 20) for 15 min, a 1:1000 dilution of anti-His6 IgG mAb (Novagen) in MPBS was added, followed by incubation at 37 ◦ C for 2 h. The membrane was then washed three times for 10 min with PBST, incubated for 60 min with a 1:5000 dilution of goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology) in PBST, and subsequently washed three times for 5 min with PBST. Blotting was performed using the tetramethyl benzidine (TMB) chemiluminescence system (Promega). 2.5. Large-scale expression of recombinant protein by batch fermentation Fermentations were performed as described previously (Ma et al., 2006), with minor modifications. Briefly, a single colony of E. coli BL21 (DE3) harboring the pSUMO ABP-dHC-cecropin A vector was inoculated into 10-mL LB medium supplemented with 50 g/mL kanamycin in a 50-mL flask. The seed culture was incubated at 37 ◦ C and 220 rpm on a rotary shaker overnight. Secondary seed cultures were generated by inoculating 200-mL 29 YT medium (tryptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L, and 50 g/mL kanamycin) in a 500-mL flask, followed by incubation at 37 ◦ C and 220 rpm on a rotary shaker for an additional 4 h. Fermentation was performed in production medium (10 g/L yeast extract, 10 g/L tryptone, 10 g/L glucose, 4 g/L K2 HPO4 ·3H2 O, 7 g/L Na2 HPO4 ·12H2 O, 1.2 g/L (NH4 )2 SO4 , 2 g/L KH2 PO4 , and 3 mL/L 50% (v/v) antifoam) supplemented with a trace element solution (0.001 g/L MnSO4 ·5H2 O, 0.004 g/L CaCl2 ·6H2 O, 0.002 g/L Na2 MoO4 ·2H2 O, 0.002 g/L ZnCl2 , 0.002 g/L CuSO4 ·5H2 O, 0.0005 g/L H3 BO4 , 0.02 g/L FeSO4 ·7H2 O, 0.02 g/L CaCl2 ·2H2 O, and 0.3 g/L MgSO4 ·7H2 O). Secondary seed cultures were inoculated into production medium at an inoculum size of 5% (v/v), and maintained at 37 ◦ C with an initial agitation rate of 300 rpm in a 5-L fermenter (BioXin, Shanghai). Dissolved oxygen was maintained at 50% air saturation by an agitation cascade between 300 and 800 rpm. Cultivation pH was kept at 7.0 by the addition of NH4 OH or H2 SO4 . After 4 h of incubation, target protein expression was induced by addition of 0.5 mM IPTG. Cells were then incubated for an additional 3 h, and collected by centrifugation (6000 rpm, 10 min). The resulting cell pellet then was stored at −80 ◦ C until needed. 2.6. Purification of the SUMO fusion protein Cell pellets were resuspended in 15-mL binding buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, and 10 mM PMSF, pH 8.0) and lysed on ice by sonication at 400 W for 100 cycles
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(4 s working, 8 s free). Cells were then centrifuged at 12,000 × g for 20 min at 4 ◦ C, and the supernatant applied to a Ni2+ -chelating column. After extensive washing with binding buffer, the fusion protein was eluted with five volumes of elution buffer (20 mM Tris, 500 mM NaCl, and 250 mM imidazole, pH 8.0). The peak fractions containing the fusion protein were pooled and dialyzed overnight at 4 ◦ C against phosphate-buffered saline (PBS). 2.7. Purification of ABP-dHC-cecropin A Purified SUMO-ABP-dHC-cecropin A protein (50 g) was incubated in the presence of a SUMO protease (3 U) at 30 ◦ C for 1 h. Samples were then reapplied to the nickel column containing NiIDA resin to remove his-tagged SUMO and SUMO proteases. A majority of the cleaved ABP-dHC-cecropin A was eluted in the flowthrough (unbound) fractions; the remaining fraction was recovered by washing the resin with binding buffer. Eluates and washes exhibiting high UV values at OD280 were then pooled, and resolved on 4–20% gradient gels (Mini-R/PROTEAN TGX Gels, Bio-Rad). Purification by preparative reverse-phase high-performance liquid chromatography (RP-HPLC) yielded a final product deemed about 99% pure by analytical RP-HPLC. Final confirmation was performed using ESI-mass spectrometry and matrix-assisted laser desorption/ionization, time-of-flight (MALDI–TOF) mass spectrometry (MS).
Fig. 1. Schematic representation of the pSUMO-ABP-dHC-cecropin A expression vector. The SUMO-tag was fused to the N-terminus of ABP-dHC-cecropin A.
3. Results 3.1. Construction and expression of ABP-dHC-cecropin A fusion protein
Lyophilized ABP-dHC-cecropin A was dissolved in 0.9% mM NaCl to a final concentration of 4 mg/mL. Antifungal activity was tested using an agar well diffusion assay (Lehrer et al., 1991).
A schematic of the ABP-dHC-cecropin A expression construct is depicted in Fig. 1. Proper construction of the pSUMO-ABP-dHCcecropin A plasmid was verified by DNA sequencing, followed by transformation into E. coli BL21 (DE3). An obvious protein band was visible after IPTG induction (Fig. 2), confirming proper synthesis of the pSUMO-ABP-dHC-cecropin A protein.
2.9. Electrophoretic mobility shift assay of ABP-dHC-cecropin A bound to fungal genomic DNA
3.2. High-yield expression of pSUMO-ABP-dHC-cecropin A by fermentation
Genomic DNA of C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp. and Mucor sp. were extracted using a fungal DNA kit (Omega, USA). DNA was then added to increasing concentrations of ABP-dHC-cecropin A protein (50–300 ng). Samples were incubated for 30 min at 4 ◦ C, and visualized by 1% agarose gel electrophoresis for EMSA.
Fermentation of transformed E. coli was performed as described above. The fermenter batch yielded 46.8 g/L wet biomass at an OD600 of ∼22, over 12-fold higher than that achieved in flask culture with LB medium. From this, a total of 2675 mg/L soluble protein was recovered, confirming both the efficiency and solubility of this fusion construct.
2.10. Scanning electron microscopy of fungi treated with ABP-dHC-cecropin A
3.3. Purification of the SUMO-ABP-dHC-cecropin A fusion protein
2.8. Antifungal sensitivity assay
Following treatment with ABP-dHC-cecropin A by agar well diffusion, samples were taken from within the zone of inhibition and peripheral areas, and fixed in glutaraldehyde for 1 d. Samples were then washed in increasing concentrations of ethanol (30–100%, v/v) ethanol, dried, sputter-coated with gold, and observed under a scanning electron microscope (FEI Quanta 200, USA).
Purification of the SUMO-ABP-dHC-cecropin A fusion construct was performed using a Ni-IDA resin. His-tagged proteins were captured using a Ni-IDA resin, washed repeatedly in a buffer containing 20 mM imidazole, and eluted in a buffer containing 250 mM imidazole. Purified SUMO-ABP-dHC-cecropin A was isolated at a purity of >90%, and was analyzed using the Biorad Chemi Doc XRS + Molecular Imager’s lmage LabTM Software quantitative
Fig. 2. Analysis of the expressed fusion protein by SDS–PAGE. Lane M: molecular mass marker; lane 1: negative control; lanes 2–9: colonies 1–8, respectively, induced by 0.5 mM IPTG. The molecular weight of the target protein was about 24 kDa, and is indicated by the black arrow.
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Table 1 Isolation of recombinant ABP-dHC-cecropin A from pSUMO-ABP-dHC-cecropin A fusion protein. Purification step Sonicated supernatant SUMO-ABP-dHC-cecropin A after affinity chromatography Recombinant ABP-dHC-cecropin A after affinity chromatography Dialysis
Total protein (mg) a
Fusion protein (mg) b
Recombinant ABP-dHC-cecropin A (mg) c
Purity (%)d
2675 NA
684 496b
237 211c
NA 90
194a
NA
173c
>94
NA
NA
158a
>94
Estimations are based on 1 L of bacterial culture (about 46.8 g wet weight). NA, not applicable. a Determined using a Bradford assay. b Percent of SUMO-ABP-dHC-cecropin A fusion protein from total proteins was estimated by SDS gel scanning. c The amount of recombinant ABP-dHC-cecropin A was calculated as a fraction of SUMO-ABP-dHC-cecropin A fusion protein. d Purity of protein or peptide was estimated by SDS gels stained by Coomassie Blue.
tools (Fig. 3), at a concentration of ∼496 mg/L bacterial culture (Table 1).
3.4. Cleavage and purification of recombinant ABP-dHC-cecropin A Next, 50-g SUMO-ABP-dHC-cecropin A protein were cleaved with SUMO protease, with complete digestion confirmed by SDS–PAGE (Fig. 4). The cleaved sample was then reapplied to a Ni-IDA column to remove any residual His-tagged SUMO and SUMO protease, and resolved by SDS-PAGE, yielding a final ABPdHC-cecropin A peptide (∼4.06 kDa) at a purity of ≥94% (Fig. 4). Finally, the purified ABP-dHC-cecropin A was passed through a 0.22-m filter and stored at −80 ◦ C until needed (Fig. 5). The final yield of purified recombinant ABP-dHC-cecropin A was 158 mg/L (Table 1).
3.5. Antifungal activity assays The antifungal activity of ABP-dHC-cecropin A was determined using an agar well diffusion assay at a starting concentration of 4 mg/mL. All treatments were performed in triplicate (Fig. 6), with ring diameters measured using as automatic colony counter Scan1200 (Table 2). Significant antifungal activity was evident against all six species tested (C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp., and Mucor sp.), confirming the broadspectrum efficacy of this compound.
Table 2 Antifungal circle diameter measurement. Fungal name
No. 2 (mm)
No. 3 (mm)
No. 4 (mm)
Average (mm)
±SD
Canidia albicans Neurospora crassa Rhizopus sp. Fusarium sp. Alternaria sp. Mucor sp.
9.8 10.0 12.4 10.8 11.6 10.4
10.2 11.5 12.4 10.6 12.4 9.8
9.2 11.7 12.6 11.2 11.8 10.6
9.7 11.1 12.5 10.9 11.9 10.3
0.253 0.863 0.013 0.093 0.173 0.173
20-L (4 mg/mL final concentration) ABP-dHC-cecropin A was loaded in each lane.
3.6. EMSA of ABP-dHC-cecropin A bound to fungal genomic DNA The binding of ABP-dHC-cecropin A to fungal genomic DNA was determined by EMSA across a range of protein concentrations (50–300 ng). A significant, dose-dependent binding of ABP-dHCcecropin A to fungal DNA was evident across all concentrations tested (Fig. 7). Similar results were observed for all six fungi tested (C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp., and Mucor sp.) indicative of DNA maybe as a target of ABP-dHC-cecropin A activity. 3.7. Electron microscopy of fungi treated with ABP-dHC-cecropin A While the antifungal activity of ABP-dHC-cecropin A was clearly demonstrated using well-diffusion assays, the mechanism underlying this antimicrobial activity is not clearly understood.
Fig. 3. Purification of the SUMO-ABP-dHC-cecropin A fusion protein. (A) The eluted fusion protein exhibited ≥90% purity following resolution on a 12% SDS–PAGE gel. Lane M: molecular weight marker; lane 2: precipitate of cell lysate; lane 3: supernatant of cell lysate; lane 4: flow-through; lane 5: wash; lane 6: elution; lane 7: Western blot analysis of purified SUMO-ABP-dHC-cecropin A using a mAb against the His6 tag. (B) Ni-IDA affinity chromatography of the fusion protein using LP data view.
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Fig. 4. Analysis of SUMO-ABP-dHC-cecropin A fusion protein by SDS-PAGE. Analysis of SUMO-ABP-dHC-cecropin A fusion protein following cleavage with SUMO protease. Lane M: low molecular weight marker; lane 1: purified SUMO-ABP-dHC-cecropin A fusion protein; lane 2: SUMO-ABP-dHC-cecropin A cleavage products following SUMO digestion. Lane 3: purified recombinant ABP-dHC-cecropin A.
ABP-dHC-cecropin A is an amphipathic peptide with ␣-helical structures, making pore formation (Ludtke et al., 1996; Shai and Oren, 2001) or ion channel generation (Huang, 2000) the most likely mechanism of activity. Here, we observed fungal lysis as a result of transmembrane pore formation (Fig. 8). Fungi isolated from within the zone of inhibition exhibited both growth arrest and lysis (Fig. 8A, C, E, G, I, and K), while those isolated from the outer limits of this zone were observed in various states of cell decay, including significant atrophy of the fungal mycelium (Fig. 8B, D, F, H, J, and L) Taken together, these results indicate strong antifungal activity of ABP-dHC-cecropin A.
4. Discussion Insects have been shown to produce several kinds of antimicrobial products in response to infection or injury (Boman, 1987; Cociancich et al., 1994; Hultmark, 1993; Kanost et al., 1990; Dunn, 1986). Recent advances in heterologous expression technology may allow for many of these, including ABP-dHC-cecropin A, to be developed into new antimicrobial therapies. Previously, we had shown that ABP-dHC-cecropin A exhibited strong antibacterial activity, making this a promising candidate for further studies. However, the compound used in this study was
Fig. 5. (A)The RP-HPLC chromatogram of the purified peptide. The purified ABP-dHC-cecropin A was 99% pure. (B) The molecular mass of the recombinant ABP-dHC-cecropin A is 4060.94 Da.
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Fig. 6. Bioactivity of ABP-dHC-cecropin A. Bioactivity of ABP-dHC-cecropin A against (A) Candida albicans, (B) Neurospora crassa, (C) Rhizopus sp., (D) Fusarium sp., (E) Alternaria sp., and (F) Mucor sp. Lane 1: negative control. Lanes 2–4: ABP-dHC-cecropin A (4 mg/mL); a 20-L sample was loaded in each lane.
Fig. 7. EMSA analysis of ABP-dHC-cecropin A binding with fungal genomic DNA. EMSA analysis of ABP-dHC-cecropin A binding with genomic DNA of (A) Candida albicans, (B) Neurospora crassa, (C) Rhizopus sp., (D) Fusarium sp., (E) Alternaria sp., and (F) Mucor sp. Lane M: 15-kb DNA marker, lane 1: fungal genomic DNA; lanes 2–5: genomic DNA mixed with 50-, 100-, 200-, and 300-ng protein, respectively.
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Fig. 8. Scanning electron micrographs showing the effect of ABP-dHC-cecropin A on fungal surface morphology. Analysis of (A) Candida albicans (SEM, 4000×), (C) Neurospora crassa (SEM, 2400×), (E) Rhizopus sp. (SEM, 1200×), (G) Fusarium sp. (SEM, 1200×), (I) Alternaria sp. (SEM, 1200×), and (K) Mucor sp. (SEM, 600×), respectively, harvested from areas outside the zones of inhibition following disc diffusion assays. Analysis of (B) C. albicans (SEM, 80×), (D) N. crassa (SEM, 150×), (F) Rhizopus sp. (SEM, 150×), (H) Fusarium sp. (SEM, 80×), (J) Alternaria sp. (SEM, 150×), and (L) Mucor sp. (SEM, 50×), respectively, harvested from within the zones of inhibition (red ovals) following disc diffusion assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
produced through direct chemical synthesis, severely limiting the quantity of protein available for testing. One method for overcoming this limitation is through heterologous expression in hosts such as E. coli or Saccharomyces cerevisiae; however, the utility of such a method is limited in the case of native cationic antimicrobial peptides due to the inherent toxicity to the host organism, and the sensitivity of these recombinant peptides to intracellular proteases. Fortunately, many of these problems can be overcome by the addition of a fusion partner, as seen with the F4 fragment of PurF (Pyo SH, 2004), green fluorescent protein (GFP) (Skosyrev et al., 2003), and protein PaP3.30 (Rao et al., 2004). Significant variability in gene expression has been observed among many traditional fusion systems, along with other problems, such as inefficient cleavage of the fusion protein, or cleavage within the target protein, both of which can confound purification efforts. Recently, SUMO has emerged as a superior choice among fusion tag options in terms of both enhanced protein expression and solubility, with the added distinction of generating recombinant proteins with native conformations (Butt et al., 2005; Wu et al., 2009). The SUMO tag has advantages over the traditional fusion tag and enhanced both the expression and cleavage of the fusion protein. The effect of SUMO on enhancing protein solubility can be partly explained by its structure. SUMO has an external hydrophilic surface and inner hydrophobic core, which may have a detergentlike effect on otherwise insoluble proteins (Butt et al., 2005).
Additionally, the SUMO-specific protease Ulp1 efficiently cleaves the conserved Gly–Gly motif at the C-terminus of SUMO from the recombinant fusion protein under suitable conditions. Unlike EK or TEV protease, whose recognition sequences are short and degenerate, Ulp1 recognizes the tertiary sequence of SUMO. As a result, Ulp1 does not cleave within the fused protein of interest, so intact proteins of interest with little exogenous amino acid residue can be obtained. We hypothesized that the attachment of a highly stable and compact SUMO structure to the N-terminus of ABP-dHC-cecropin A would facilitate enhanced solubility and expression, while maintaining the native protein conformation. Here, the SUMO fusion protein was successfully expressed in E. coli, resulting in high expression of soluble fusion protein (Fig. 3a). Downstream processing was similarly successful, with efficient cleavage of the SUMO tag using SUMO protease (Fig. 4). The resulting ABP-dHC-cecropin A peptide (∼4.06 kDa) was recovered at a purity of ≥94%, yielding 158-mg protein per liter of fermentation culture. These results clearly demonstrate that the SUMO fusion system and customized expression and purification protocol described here have dramatically improved both the cost and efficiency of producing ABP-dHC-cecropin A. It is likely that the approach described here could be widely applied to the production of other cytotoxic cationic peptides in E. coli, which may accelerate the pace of discovery of antimicrobial peptides.
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In an antifungal activity assay, ABP-dHC-cecropin A exhibited strong antifungal activity against C. albicans, N. crassa, Rhizopus sp., Fusarium sp., Alternaria sp., and Mucor sp. (Figs. 6–8). This activity appears to be mediated via the direct binding of ABP-dHC-cecropin A to fungal DNA (Fig. 7), likely due to the strong charge differences between the cationic ABP-dHC-cecropin A and the negatively charged phosphate groups of DNA. ABP-dHC-cecropin A has a net charge of +5 at neutral pH due to the presence of multiple Arg1,10,16 , Lys3,6,7,21,33,37 Glu9 , and Asp17 residues (Zhang et al., 2014). The secondary structure of this molecule is a typical amphipathic ␣-helix, which positions the positively charged groups on the outside of the helix, while negatively charged groups are found primarily on the inside. The pattern of ABP-dHC-cecropin A binding with fungal DNA may be as follow: ABP-dHC-cecropin A combines with phosphate groups of fungal DNA by electrostatic attraction to form ABP-dHC-cecropin A anf fungal DNA structures. ABP-dHC-cecropin A further combines with the DNA double helix grooves, becoming embedded or partially embedded in the fungal DNA (Kumar and Asuncion, 1993). In fungal models, ABP-dHC-cecropin A has additional effects on fungal cell membranes, and may act through cell membranes, binding with the internal cell structure (Kumar and Asuncion, 1993). Binding of ABP-dHC-cecropin A to fungal DNA blocks both the replication and transcription of DNA, which will be examined in detail in future studies. This led to growth arrest and cell lysis within the zone of inhibition, while those isolated from the outer limits of this zone were observed in various states of cell decay, including significant atrophy of the fungal mycelium. These results confirm the direct antifungal activity of ABP-dHC-cecropin A in vitro. When taken together, the data presented here suggest a multifaceted approach to fungal lysis, including disruption of the plasma membrane and incorporation into the nucleus, followed by direct binding to DNA, and possible inhibition of DNA replication and transcription. This model of antimicrobial activity is consistent with a very broad spectrum of activity, highlighting the potential of ABPdHC-cecropin A as an important antimicrobial candidate. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by the National 863 Program of China (No. 2013AA102703), the International Science & Technology Cooperation Program of China (2014DFG32440), the National Science Foundation of China (No. 31400567), the Graduate Innovative Project of Jiangsu Province (CXZZ13 0530), the Doctoral Degree Thesis Innovation Foundation of Nanjing Forestry University (No. 2013Y07), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Program for Innovative Research Team in University of Educational Department and Jiangsu Province, China. References Boman, H.G., 2003. Antibacterial peptides: basic facts and emerging concepts. J. Intern. Med. 254, 197–215. Boman, H.G., Faye, I., Gudmundsson, G.H., Lee, J.Y., Lidholm, D.A., 1991. Cellfree immunity in Cecropia. A model system for antibacterial proteins. Eur. J. Biochem./FEBS 201, 23–31. Boman HG, H.D., 1987. Cell-free immunity in insects. Ann. Rev. Microbiol. 41, 103–126.
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