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HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY Dafeng Song
a b
b
b
a
, Yue Chen , Xuan Li , Muyuan Zhu & Qing Gu
b
a
Key Laboratory for Cell and Gene Engineering of Zhejiang Province , College of Life Sciences, Zhejiang University , Hangzhou , China b
Key Laboratory for Food Microbial Technology of Zhejiang Province , Zhejiang Gongshang University , Hangzhou , China Accepted author version posted online: 03 Oct 2013.Published online: 05 Feb 2014.
To cite this article: Dafeng Song , Yue Chen , Xuan Li , Muyuan Zhu & Qing Gu (2014) HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY, Preparative Biochemistry and Biotechnology, 44:6, 598-607, DOI: 10.1080/10826068.2013.835735 To link to this article: http://dx.doi.org/10.1080/10826068.2013.835735
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Preparative Biochemistry & Biotechnology, 44:598–607, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.835735
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HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY
Dafeng Song,1,2 Yue Chen,2 Xuan Li,2 Muyuan Zhu,1 and Qing Gu2 1 Key Laboratory for Cell and Gene Engineering of Zhejiang Province, College of Life Sciences, Zhejiang University, Hangzhou, China 2 Key Laboratory for Food Microbial Technology of Zhejiang Province, Zhejiang Gongshang University, Hangzhou, China
& Heterologous expression of dermaseptin S4 (DS4), which has cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms, was examined in Escherichia coli. The plasmid pGEX-4T-1, encoding DS4 fused with glutathione S-transferase (GST), was constructed and cloned into the E. coli strain BL21 (DE3). The fusion protein was overexpressed in this strain after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified to homogeneity using GST affinity chromatography. To recover biologically active DS4, the purified fusion protein was cleaved using thrombin protease; the liberated DS4 was shown to be bactericidally active against an indicator strain. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express purify DS4 in E. coli using a GST fusion system. Keywords antimicrobial peptide, dermaseptin S4, Escherichia coli, GST, heterologous expression, pGEX-4T-1
INTRODUCTION An antimicrobial peptide is a peptide produced by the body’s defense system and is active against external pathogens.[1,2] It exists in a wide range of organisms from prokaryotes to humans. Since the Swedish scientist Boman found and isolated the first substance with an antibacterial effect (cecropin),[3] more than 2000 similar active substances have been identified.[4] Antimicrobial peptides were found to have broad-spectrum antiseptic effects against fungi, protozoa, viruses, and tumor cells.[5,6] Due to the antimicrobial nature of these peptides, they are potentially Address correspondence to Qing Gu, Key Laboratory for Food Microbial Technology of Zhejiang Province, Zhejiang Gongshang University, 149 Jiaogong laod, Hangzhou, China. E-mail: guqing2002@ hotmail.com
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useful as novel antibiotics and food-preserving agents.[7] However, industrial applications of antipeptides require a system that produces high levels of biologically active antimicrobial peptide. The antimicrobial peptide dermaseptin S4 (DS4) is an amphiphilic alpha-helical peptide comprised of 28 amino acids and found in the skin of the amphibian Pyllomedusa sauvagei.[1,8] It has broad-spectrum antimicrobial activity.[9] DS4 not only has an antibacterial effect but also antiviral activity. It is powerful against the herpes simplex virus (HSV) and human immunodeficiency virus (HIV).[2,8] DS4 exerts antiviral activity through its a-helical structure, which targets viral-cell-surface phospholipids preventing fusion of the virus particles to the host cell; thus, this antiviral activity can work in early stages of infection. Additionally, it can also act directly on virus particles, destroying the viral envelope and disrupting virus structural organization to expose nucleotide particles. Escherichia coli is an important prokaryotic host for cloning and expressing heterologous genes due to its extensive genetic characterization. Many of its biological processes are well understood and there is a wide range of genetic tools readily available for its manipulation. Thus, these bacteria have been used to recombine and express a variety of genes involved in the biosynthesis of many antimicrobial peptides.[10] Purified or partially purified antimicrobial peptides can be used as preservatives, or to reduce or eliminate certain pathogens. However, isolation and purification of antimicrobial peptides from native hosts is a time-consuming and laborious process. The present work examines heterologous expression of DS4 in E. coli. These results suggest that our production method will be useful in obtaining a large quantity of DS4 peptide for further studies.
MATERIALS AND METHODS Bacterial Strains and Culture Conditions Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5a (Clontech, Mountain View, CA) and E. coli BL21 (DE3) (Novagen, Madison, WI) were grown in LB medium at 37 C in a shaking incubator. Agar plates were prepared by adding 1.5% agar to broth media. Antibiotics (Sangon Biotech, Shanghai, China) were added as selective agents at appropriate concentrations (ampicillin, 100 mg=mL).
Gene Synthesis and DNA Amplification Based on the amino acid sequence of DS4, codon optimization of the DS4 gene for E. coli expression was performed. Using DNASTAR software
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TABLE 1
Bacterial Strains and Plasmids Used in This Study Relevant Characteristicsa
Strain and Plasmids Strains E. coli DH5a E. coli BL21 (DE3) Micrococcus luteus Plasmids pGEX-4T-1 pGEX-DS4
Reference or Source
supE44 DlacU169 (u80 dlacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA fhuA2 [lon] ompT gal [dcm] DhsdSkDE3 DS4 sensitive indicator
Novagen This laboratoryb
GST fusion vector, Ampr GST fusion vector, Ampr, DS4 coding region
Pharmacia This study
Gibco-BRL
Ampr, ampicillin resistance. Department of Biotechnology, Zhejiang Gongshang University, Hangzhou, China.
a
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(Madison, WI), two DNA primers were designed and synthesized. Upstream and downstream primers were designed to include cleavage sites for BglII and PstI, respectively. DS4 gene fragments and primers were synthesized by Sangon Biotech. The following primer sequences were used: DS4F: 50 -AATCAGATCTATGGCACTGTGGATG-30 (BglII); DS4R: 50 -TTACCTGCAGTCACCGCGTTCGCAC-30 (PstI). Amplification reactions were carried out in a Biometra T personal polymerase chain reaction (PCR) system (Go¨ettingen, Germany). Final reaction volume was 50 mL, and the final concentration of MgCl2 was 1.5 mM. Reagents were used at the following concentrations: dNTPs, 200 mM; primers, 1 mM; and Taq polymerase (Takara HiFi DNA polymerase, Takara Bio, Shiga, Japan), 1 unit per reaction. The thermal program included the following steps: (1) initial denaturation, 5 min at 94 C; (2) 35 cycles of denaturation (94 C, 30 s), annealing (55 C, 30 s), and extension (72 C, 1 min); and (3) final incubation at 72 C for 5 min to complete extension. Plasmid Construction To express DS4 protein with a glutathione S-transferase (GST) tag, the pGEX-4T-1 system was used (Table 1). Briefly, the DS4 coding sequence was amplified using primers DS4F and DS4R and cloned into the expression vector pGEX-4T-1, resulting in pGEX-DS4 (Table 1), which was used to express GST-DS4 in E. coli. All plasmids were transformed in E. coli DH5a, verified by DNA sequencing, and then transformed into E. coli BL21 (DE3) for protein expression. Expression of GST-Tagged Fused DS4 in E. coli An overnight culture of E. coli BL21 (pGEX-DS4) was inoculated into 100 mL of LB broth containing ampicillin (100 mg=mL) and cultured to
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an OD600 of 0.5–0.6. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the culture was incubated for an additional 4 hr. The culture was centrifuged and the cell pellet was stored at 20 C.
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GST Fusion Protein SDS-PAGE and Immunoblot To investigate the molecular mass of the target protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli[11] using a Mini-Protean electrophoresis cell following the manufacturer’s instructions (Bio-Rad, Hercules, CA). Proteins were separated on 15% SDS-PAGE gel at 150 V for 2 hr; the gel was stained using Coomassie brilliant blue R250 (Bio-Rad), washed with distilled and deionized H2O, and protein bands were examined. To confirm the presence of an antimicrobial peptide band, the gel was blotted onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) and anti-GST tag mouse monoclonal antibodies (Sangon, CN) were used as primary antibodies. Goat anti-mouse horseradish peroxidase-conjugated antibodies (Sangon, CN) were used as secondary antibodies. Chemiluminescent detection was used (Sangon, CN). Purification of Biologically Active DS4 Fusion proteins were isolated from bacterial lysates using affinity chromatography with GST Sepharose 4B (GE Healthcare, Little Chalfont, UK). The cells from 50 mL of IPTG-induced culture were harvested by centrifugation (8,000 g, 6 min, 4 C). For cell lysis, the cell pellet was thawed on ice for 15 min and resuspended in 4 mL phosphate-buffered saline (PBS). The mixture was incubated on ice for 30 min after addition of lysozyme (1 mg=mL) and sonicated for 10 min with 10-s pauses under native conditions. The lysate was centrifuged at 12,000 g for 15 min and the supernatant was retained. Next, 1 mL of 50% GST Sepharose beads was added to 4 mL of cleared lysate and gently mixed by shaking at 4 C under native conditions at room temperature and denaturing conditions for 60 min. The lysate–GST mixture was loaded onto a column and washed twice with 4 mL of PBS (pH 7.4). To recover biologically active DS4 from GST-fused DS4, the fusion protein was cleaved using thrombin protease according to the manufacturer’s instruction. Antimicrobial Peptide Activity Assay To examine the biological activity of purified proteins, a simplified version of the agar diffusion test was used. A 100-mL sample of purified
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recombinant protein was applied to a small hole in an LB soft agar overlayer seeded with the indicator strain Micrococcus luteus. Plates were incubated at 37 C for 16 hr, and antimicrobial activity was observed as the halo of inhibition in the bacterial lawn of the indicator strain formed around the sample. Antimicrobial peptide activity, expressed as arbitrary units (AU) per milliliter, was defined as the reciprocal of the highest twofold dilution showing inhibitory action against the indicator strain. RESULTS
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Expression of GST-Tagged Fused DS4 in E. coli To confirm the presence of recombinant DNA, pGEX-DS4, plasmid profiling after restriction enzyme treatment and nucleotide sequencing were performed. pGEX-DS4 was digested using BglII and PstI. The nucleotide sequence encoding the thrombin protease recognition site and the inserted mature DS4 sequence were found to be correctly ligated. The sizes of expressed proteins were investigated using SDS-PAGE (Figure 1). GST-tagged fused DS4 was overexpressed in E. coli BL21 (pGEX-DS4) after IPTG induction (Figure 1, lane 3), whereas expression in induced E. coli BL21 and E. coli BL21 pGEX-4T-1 controls was not observed. Protein bands were observed at approximately 29 kD, which corresponds to
FIGURE 1 SDS-PAGE and Western blot analysis of expressed proteins in recombinant E. coli BL21. Lane M: polypeptide molecular mass standards; Lane 1: E. coli whole-cell lysate with IPTG induction; Lane 2 E. coli (carrying plasmid pGEX-4T-1) whole-cell lysate with IPTG induction; Lane 3: E. coli (carrying plasmid pGEX-DS4) whole-cell lysate with IPTG induction; Lane 4: Western blot of SDS-PAGE products of E. coli carrying plasmid pGEX-DS4 after 3 hr of induction.
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the predicted molecular mass of recombinant proteins (GST 26 kD þ DS4 3 kD). Western blot analyses with polyclonal antibodies targeting the GST fusion partner were performed for E. coli pGEX-DS4 after 3 hr of induction. A band corresponding to the expected molecular mass of the fusion protein was detected at 29 kD (Figure 1, lane 4). Using densitometric analysis, expression level of the recombinant proteins was compared with the total cellular protein content. DS4 represented approximately 3% of the total cellular protein content. The yield of recombinant proteins was 1.69 mg=mL DS4 per 1 g of wet cells. No other contaminating protein bands were observed in analyzed samples (Figure 1). The total yield of recombinant DS4 was approximately 0.02 mg of purified protein per milliliter of culture. Purification of Recombinant DS4 The DS4 fusion peptide was purified using GST Sepharose 4B at a concentration of 0.6 mg=mL. The yields of recombinant proteins were 30 mg per 1 g of wet cells. Unlike His-tagged recombinant antimicrobial peptide produced in E. coli BL21 (pET system), which has been previously studied,[12–15] GST-tagged fused DS4 (29 kD) was only minimally toxic to the host cell and was stably expressed in E. coli BL21 (DE3). Because host toxicity is very important in producing large amounts of target protein in E. coli, GST-fused DS4 appears to be well suited for this purpose (Figure 2).
FIGURE 2 Growth model of E. coli BL21 (DE3) pGEX-DS4. &: BL21, .: BL21 (pGEX-DS4) non-induced culture, ~: BL21 (pGEX-DS4) induced culture with IPTG added after 8 hr of growth.
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TABLE 2
Purification Steps of DS4 From E. coli
Purification Step Bacterial lysate GST affinity chromatography
Volume (ml)
Total Protein (mg)
GST-DS4 (mg)
DS4 (mg)
Purity (%)
Yield (%)
50 5
657a 1.25
30 8.5
3.5b 1.0
NAc 80
100 29
a
Protein concentration was determined by Bradford protein assay. The amount of DS4 was calculated from the mass ratio compared to the determined amount of fusion protein. c NA, not applicable.
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b
Expressed GST-tagged fused DS4 was purified at high yield and purity when purified under denaturing conditions, whereas recovery was very low under native conditions (Table 2). These results indicate that most of the fusion protein exists as inclusion bodies in the E. coli cytoplasm. Thrombin Protease Treatment and Biological Activity of Recombinant DS4 Biological activity of recombinant DS4 was verified using an agar diffusion assay. Purified DS4 that had been treated with thrombin protease to clear the GST fusion partner showed bactericidal activity against M. luteus. This indicates that the biologically active DS4 was liberated from the fusion protein during thrombin protease treatment (Figure 3).
FIGURE 3 Agar diffusion test of antimicrobial activity of purified recombinant proteins on M. luteus. (A) Noninduced control (cell-free extract of E. coli BL21); (B) recombinant DS4. The same amount of protein (approximately 20 ng, corresponding to 200 AU of DS4) was applied to the hole in an LB soft agar overlayer seeded with the indicator strain M. luteus.
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DISCUSSION Antimicrobial peptide is a small polypeptide with broad-spectrum antimicrobial activity. Among these antimicrobial peptides, DS4 is a very promising candidate because it has not only an antibacterial effect but also antiviral activity. The highly efficient production of biologically active DS4, with large quantity and low cost, is an absolute need for both laboratory studies and potential clinical applications. Production and purification of the antimicrobial peptide DS4 has been reported previously by Mor et al.[16,17] Native DS4 was purified to homogeneity in three steps using molecular sieve filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. The amount of purified dermaseptin recovered was 40 mg starting from 1 g of dried Phyllomedusa sauvagii skin. Thus, isolation and purification of antimicrobial peptides from their native hosts is a time-consuming and laborious process. Industrial applications require large quantities of DS4; therefore, we sought a production method suitable for DS4 production. Heterologous expression may offer several advantages over native systems, such as facilitating control of antimicrobial peptide gene expression or increased production levels of pure and uniformly labeled protein.[18] In the current work, we constructed a highly efficient system for the expression and purification of DS4 in E. coli, using the pGEX-4T-1 expression vector. Escherichia coli has been used as a host for cloning a variety of genes involved in antimicrobial peptide biosynthesis. Several antimicrobial peptides (e.g., pediocin PA-1, divergicinV41, enterocin A, and enterocin P) have been expressed in E. coli.[12,14,19–21] GST is a soluble cytoplasmic protein that is well established as a recombinant production fusion partner of highly soluble and stable fusion proteins, and has been used in the expression of many biologically active proteins in E. coli. In this study, fusion expression was used to increase expression levels and decrease toxicity, as well as to provide purification tags for a heterologous expression strategy. The first step toward achieving high levels of DS4 production was to construct a genetically engineered plasmid carrying the DS4 gene with a GST-tag fusion at the N terminus. DS4 was highly overexpressed in E. coli BL21 (DE3) after IPTG induction as a full-length GSTtagged recombinant protein of 29 kD. To the best of our knowledge, this is the first report on the effective expression and purification of intact and bioactive DS4. Our strategy may provide a solution for production of unmodified antimicrobial peptides. The expression system and purification method of DS4 will assist laboratory studies. In the near future, the availability of purified DS4 could also enable further clinical, biochemical, and biological investigations.
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FUNDING This research was supported by the National Natural Science Foundation of China (31071513, 31271821), the Natural Science Foundation of Zhejiang Province (Z3110399), the China Agriculture Research System (CARS-5), the Zhejiang Province Program for Science and Technology (2007C12037), the Scientific Research Project for the Education Department of Zhejiang Province, China (Y201120078), and the key science and technology innovation team of Zhejiang Province (2010R50032).
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REFERENCES 1. Mor, A.; Hani, K.; Nicolas, P. The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms. J. Biol. Chem. 1994, 269, 31635–31641. 2. Krugliak, M.; Feder, R.; Zolotarev, V.Y.; Gaidukov, L.; Dagan, A.; Ginsburg, H.; Mor, A. Antimalarial Activities of Dermaseptin S4 Derivatives. Antimicrob. Agents Chemother. 2000, 44, 2442–2451. 3. Boman, H.G. Peptide Antibiotics and Their Role in Innate Immunity. Annu. Rev. Immunol. 1995, 13, 61–92. 4. Pasupuleti, M.; Schmidtchen, A.; Malmsten, M. Antimicrobial Peptides: Key Components of the Innate Immune System. Crit. Rev. Biotechnol. 2012, 32, 143–171. 5. Belaid, A.; Aouni, M.; Khelifa, R.; Trabelsi, A.; Jemmali, M.; Hani, K. In Vitro Antiviral Activity of Dermaseptins Against Herpes Simplex Virus Type 1. J. Med. Virol. 2002, 66, 229–234. 6. Lorin, C.; Saidi, H.; Belaid, A.; Zairi, A.; Baleux, F.; Hocini, H.; Be´lec, L.; Hani, K.; Tangy, F. The Antimicrobial Peptide Dermaseptin S4 Inhibits HIV-1 Infectivity In Vitro. Virology 2005, 334, 264–275. 7. Abee, T. Pore-Forming Bacteriocins of Gram-Positive Bacteria and Self-Protection Mechanisms of Producer Organisms. FEMS Microbiol. Lett. 1995, 129, 1–9. 8. Ammar, B.; Pe´rianin, A.; Mor, A.; Sarfati, G.; Tissot, M.; Nicolas, P.; Giroud, J.; Roch-Arveiller, M. Dermaseptin, a Peptide Antibiotic, Stimulates Microbicidal Activities of Polymorphonuclear Leukocytes. Biochem. Biophys. Res. Commun. 1998, 247, 870–875. 9. Feder, R.; Dagan, A.; Mor, A. Structure–Activity Relationship Study of Antimicrobial Dermaseptin S4 Showing the Consequences of Peptide Oligomerization on Selective Cytotoxicity. J. Biol. Chem. 2000, 275, 4230–4238. 10. Rodrı, A.X.; Guez, J.M.; Martı, A.X.; Nez, M.I.; Horn, N.; Dodd, H.M. Heterologous Production of Bacteriocins by Lactic Acid Bacteria. Int J Food Microbiol 2003, 80, 101–116. 11. Laemmli, U.K. Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature 1970, 227, 680–685. 12. Moon, G.; Pyun, Y.; Kim, W.J. Expression and Purification of a Fusion-Typed Pediocin PA-1 in Escherichia coli and Recovery of Biologically Active Pediocin PA-1. Int J Food Microbiol 2006, 108, 136–140. 13. Klocke, M.; Mundt, K.; Idler, F.; Jung, S.; Backhausen, J.E. Heterologous Expression of Enterocin A, a Bacteriocin From Enterococcus faecium, Fused to a Cellulose-Binding Domain in Escherichia coli Results in a Functional Protein With Inhibitory Activity Against Listeria. Appl Microbiol Biotechnol 2005, 67, 532–538. 14. Richard, C.; Drider, D.; Elmorjani, K.; Marion, D.; Prevost, H. Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli. J. Bacteriol. 2004, 186, 4276–4284. 15. Liu, S.N.; Han, Y.; Zhou, Z.J. Fusion Expression of pedA Gene to Obtain Biologically Active Pediocin PA-1 in Escherichia coli. J. Zhejiang Univ. Sci. B 2011, 12, 65–71. 16. Mor, A.; Nguyen, V.H.; Delfour, A.; Migliore-Samour, D.; Nicolas, P. Isolation, Amino Acid Sequence, and Synthesis of Dermaseptin, a Novel Antimicrobial Peptide of Amphibian Skin. Biochemistry 1991, 30, 8824–8830.
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17. Mor, A.; Amiche, M.; Nicolas, P. Structure, Synthesis, and Activity of Dermaseptin b, a Novel Vertebrate Defensive Peptide From Frog Skin: Relationship With Adenoregulin. Biochemistry 1994, 33, 6642–6650. 18. Ingham, A.B.; Moore, R.J. Recombinant Production of Antimicrobial Peptides in Heterologous Microbial Systems. Biotechnol. Appl. Biochem. 2007, 47, 1–9. 19. Gutierrez, J.; Criado, R.; Citti, R.; Martin, M.; Herranz, C.; Nes, I.F.; Cintas, L.M.; Hernandez, P.E. Cloning, Production and Functional Expression of Enterocin P, a sec-Dependent Bacteriocin Produced by Enterococcus faecium P13, in Escherichia coli. Int. J. Food Microbiol. 2005, 103, 239–250. 20. Kawai, Y.; Arakawa, K.; Itoh, A.; Saitoh, B.; Ishii, Y.; Nishimura, J.; Kitazawa, H.; Itoh, T.; Saito, T. Heterologous Expression of Gassericin A, a Bacteriocin Produced by Lactobacillus gasseri LA39. Animal Science Journal 2003, 74, 45–51. 21. Biet, F.; Berjeaud, J.M.; Worobo, R.W.; Cenatiempo, Y.; Fremaux, C. Heterologous Expression of the Bacteriocin Mesentericin Y105 Using the Dedicated Transport System and the General Secretion Pathway. Microbiologyþ 1998, 144, 2845–2854.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY Dafeng Song
a b
b
b
a
, Yue Chen , Xuan Li , Muyuan Zhu & Qing Gu
b
a
Key Laboratory for Cell and Gene Engineering of Zhejiang Province , College of Life Sciences, Zhejiang University , Hangzhou , China b
Key Laboratory for Food Microbial Technology of Zhejiang Province , Zhejiang Gongshang University , Hangzhou , China Accepted author version posted online: 03 Oct 2013.Published online: 05 Feb 2014.
To cite this article: Dafeng Song , Yue Chen , Xuan Li , Muyuan Zhu & Qing Gu (2014) HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY, Preparative Biochemistry and Biotechnology, 44:6, 598-607, DOI: 10.1080/10826068.2013.835735 To link to this article: http://dx.doi.org/10.1080/10826068.2013.835735
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Waikato] at 05:15 07 July 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:598–607, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.835735
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HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY
Dafeng Song,1,2 Yue Chen,2 Xuan Li,2 Muyuan Zhu,1 and Qing Gu2 1 Key Laboratory for Cell and Gene Engineering of Zhejiang Province, College of Life Sciences, Zhejiang University, Hangzhou, China 2 Key Laboratory for Food Microbial Technology of Zhejiang Province, Zhejiang Gongshang University, Hangzhou, China
& Heterologous expression of dermaseptin S4 (DS4), which has cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms, was examined in Escherichia coli. The plasmid pGEX-4T-1, encoding DS4 fused with glutathione S-transferase (GST), was constructed and cloned into the E. coli strain BL21 (DE3). The fusion protein was overexpressed in this strain after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified to homogeneity using GST affinity chromatography. To recover biologically active DS4, the purified fusion protein was cleaved using thrombin protease; the liberated DS4 was shown to be bactericidally active against an indicator strain. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express purify DS4 in E. coli using a GST fusion system. Keywords antimicrobial peptide, dermaseptin S4, Escherichia coli, GST, heterologous expression, pGEX-4T-1
INTRODUCTION An antimicrobial peptide is a peptide produced by the body’s defense system and is active against external pathogens.[1,2] It exists in a wide range of organisms from prokaryotes to humans. Since the Swedish scientist Boman found and isolated the first substance with an antibacterial effect (cecropin),[3] more than 2000 similar active substances have been identified.[4] Antimicrobial peptides were found to have broad-spectrum antiseptic effects against fungi, protozoa, viruses, and tumor cells.[5,6] Due to the antimicrobial nature of these peptides, they are potentially Address correspondence to Qing Gu, Key Laboratory for Food Microbial Technology of Zhejiang Province, Zhejiang Gongshang University, 149 Jiaogong laod, Hangzhou, China. E-mail: guqing2002@ hotmail.com
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useful as novel antibiotics and food-preserving agents.[7] However, industrial applications of antipeptides require a system that produces high levels of biologically active antimicrobial peptide. The antimicrobial peptide dermaseptin S4 (DS4) is an amphiphilic alpha-helical peptide comprised of 28 amino acids and found in the skin of the amphibian Pyllomedusa sauvagei.[1,8] It has broad-spectrum antimicrobial activity.[9] DS4 not only has an antibacterial effect but also antiviral activity. It is powerful against the herpes simplex virus (HSV) and human immunodeficiency virus (HIV).[2,8] DS4 exerts antiviral activity through its a-helical structure, which targets viral-cell-surface phospholipids preventing fusion of the virus particles to the host cell; thus, this antiviral activity can work in early stages of infection. Additionally, it can also act directly on virus particles, destroying the viral envelope and disrupting virus structural organization to expose nucleotide particles. Escherichia coli is an important prokaryotic host for cloning and expressing heterologous genes due to its extensive genetic characterization. Many of its biological processes are well understood and there is a wide range of genetic tools readily available for its manipulation. Thus, these bacteria have been used to recombine and express a variety of genes involved in the biosynthesis of many antimicrobial peptides.[10] Purified or partially purified antimicrobial peptides can be used as preservatives, or to reduce or eliminate certain pathogens. However, isolation and purification of antimicrobial peptides from native hosts is a time-consuming and laborious process. The present work examines heterologous expression of DS4 in E. coli. These results suggest that our production method will be useful in obtaining a large quantity of DS4 peptide for further studies.
MATERIALS AND METHODS Bacterial Strains and Culture Conditions Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5a (Clontech, Mountain View, CA) and E. coli BL21 (DE3) (Novagen, Madison, WI) were grown in LB medium at 37 C in a shaking incubator. Agar plates were prepared by adding 1.5% agar to broth media. Antibiotics (Sangon Biotech, Shanghai, China) were added as selective agents at appropriate concentrations (ampicillin, 100 mg=mL).
Gene Synthesis and DNA Amplification Based on the amino acid sequence of DS4, codon optimization of the DS4 gene for E. coli expression was performed. Using DNASTAR software
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TABLE 1
Bacterial Strains and Plasmids Used in This Study Relevant Characteristicsa
Strain and Plasmids Strains E. coli DH5a E. coli BL21 (DE3) Micrococcus luteus Plasmids pGEX-4T-1 pGEX-DS4
Reference or Source
supE44 DlacU169 (u80 dlacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA fhuA2 [lon] ompT gal [dcm] DhsdSkDE3 DS4 sensitive indicator
Novagen This laboratoryb
GST fusion vector, Ampr GST fusion vector, Ampr, DS4 coding region
Pharmacia This study
Gibco-BRL
Ampr, ampicillin resistance. Department of Biotechnology, Zhejiang Gongshang University, Hangzhou, China.
a
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(Madison, WI), two DNA primers were designed and synthesized. Upstream and downstream primers were designed to include cleavage sites for BglII and PstI, respectively. DS4 gene fragments and primers were synthesized by Sangon Biotech. The following primer sequences were used: DS4F: 50 -AATCAGATCTATGGCACTGTGGATG-30 (BglII); DS4R: 50 -TTACCTGCAGTCACCGCGTTCGCAC-30 (PstI). Amplification reactions were carried out in a Biometra T personal polymerase chain reaction (PCR) system (Go¨ettingen, Germany). Final reaction volume was 50 mL, and the final concentration of MgCl2 was 1.5 mM. Reagents were used at the following concentrations: dNTPs, 200 mM; primers, 1 mM; and Taq polymerase (Takara HiFi DNA polymerase, Takara Bio, Shiga, Japan), 1 unit per reaction. The thermal program included the following steps: (1) initial denaturation, 5 min at 94 C; (2) 35 cycles of denaturation (94 C, 30 s), annealing (55 C, 30 s), and extension (72 C, 1 min); and (3) final incubation at 72 C for 5 min to complete extension. Plasmid Construction To express DS4 protein with a glutathione S-transferase (GST) tag, the pGEX-4T-1 system was used (Table 1). Briefly, the DS4 coding sequence was amplified using primers DS4F and DS4R and cloned into the expression vector pGEX-4T-1, resulting in pGEX-DS4 (Table 1), which was used to express GST-DS4 in E. coli. All plasmids were transformed in E. coli DH5a, verified by DNA sequencing, and then transformed into E. coli BL21 (DE3) for protein expression. Expression of GST-Tagged Fused DS4 in E. coli An overnight culture of E. coli BL21 (pGEX-DS4) was inoculated into 100 mL of LB broth containing ampicillin (100 mg=mL) and cultured to
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an OD600 of 0.5–0.6. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the culture was incubated for an additional 4 hr. The culture was centrifuged and the cell pellet was stored at 20 C.
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GST Fusion Protein SDS-PAGE and Immunoblot To investigate the molecular mass of the target protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli[11] using a Mini-Protean electrophoresis cell following the manufacturer’s instructions (Bio-Rad, Hercules, CA). Proteins were separated on 15% SDS-PAGE gel at 150 V for 2 hr; the gel was stained using Coomassie brilliant blue R250 (Bio-Rad), washed with distilled and deionized H2O, and protein bands were examined. To confirm the presence of an antimicrobial peptide band, the gel was blotted onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) and anti-GST tag mouse monoclonal antibodies (Sangon, CN) were used as primary antibodies. Goat anti-mouse horseradish peroxidase-conjugated antibodies (Sangon, CN) were used as secondary antibodies. Chemiluminescent detection was used (Sangon, CN). Purification of Biologically Active DS4 Fusion proteins were isolated from bacterial lysates using affinity chromatography with GST Sepharose 4B (GE Healthcare, Little Chalfont, UK). The cells from 50 mL of IPTG-induced culture were harvested by centrifugation (8,000 g, 6 min, 4 C). For cell lysis, the cell pellet was thawed on ice for 15 min and resuspended in 4 mL phosphate-buffered saline (PBS). The mixture was incubated on ice for 30 min after addition of lysozyme (1 mg=mL) and sonicated for 10 min with 10-s pauses under native conditions. The lysate was centrifuged at 12,000 g for 15 min and the supernatant was retained. Next, 1 mL of 50% GST Sepharose beads was added to 4 mL of cleared lysate and gently mixed by shaking at 4 C under native conditions at room temperature and denaturing conditions for 60 min. The lysate–GST mixture was loaded onto a column and washed twice with 4 mL of PBS (pH 7.4). To recover biologically active DS4 from GST-fused DS4, the fusion protein was cleaved using thrombin protease according to the manufacturer’s instruction. Antimicrobial Peptide Activity Assay To examine the biological activity of purified proteins, a simplified version of the agar diffusion test was used. A 100-mL sample of purified
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recombinant protein was applied to a small hole in an LB soft agar overlayer seeded with the indicator strain Micrococcus luteus. Plates were incubated at 37 C for 16 hr, and antimicrobial activity was observed as the halo of inhibition in the bacterial lawn of the indicator strain formed around the sample. Antimicrobial peptide activity, expressed as arbitrary units (AU) per milliliter, was defined as the reciprocal of the highest twofold dilution showing inhibitory action against the indicator strain. RESULTS
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Expression of GST-Tagged Fused DS4 in E. coli To confirm the presence of recombinant DNA, pGEX-DS4, plasmid profiling after restriction enzyme treatment and nucleotide sequencing were performed. pGEX-DS4 was digested using BglII and PstI. The nucleotide sequence encoding the thrombin protease recognition site and the inserted mature DS4 sequence were found to be correctly ligated. The sizes of expressed proteins were investigated using SDS-PAGE (Figure 1). GST-tagged fused DS4 was overexpressed in E. coli BL21 (pGEX-DS4) after IPTG induction (Figure 1, lane 3), whereas expression in induced E. coli BL21 and E. coli BL21 pGEX-4T-1 controls was not observed. Protein bands were observed at approximately 29 kD, which corresponds to
FIGURE 1 SDS-PAGE and Western blot analysis of expressed proteins in recombinant E. coli BL21. Lane M: polypeptide molecular mass standards; Lane 1: E. coli whole-cell lysate with IPTG induction; Lane 2 E. coli (carrying plasmid pGEX-4T-1) whole-cell lysate with IPTG induction; Lane 3: E. coli (carrying plasmid pGEX-DS4) whole-cell lysate with IPTG induction; Lane 4: Western blot of SDS-PAGE products of E. coli carrying plasmid pGEX-DS4 after 3 hr of induction.
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the predicted molecular mass of recombinant proteins (GST 26 kD þ DS4 3 kD). Western blot analyses with polyclonal antibodies targeting the GST fusion partner were performed for E. coli pGEX-DS4 after 3 hr of induction. A band corresponding to the expected molecular mass of the fusion protein was detected at 29 kD (Figure 1, lane 4). Using densitometric analysis, expression level of the recombinant proteins was compared with the total cellular protein content. DS4 represented approximately 3% of the total cellular protein content. The yield of recombinant proteins was 1.69 mg=mL DS4 per 1 g of wet cells. No other contaminating protein bands were observed in analyzed samples (Figure 1). The total yield of recombinant DS4 was approximately 0.02 mg of purified protein per milliliter of culture. Purification of Recombinant DS4 The DS4 fusion peptide was purified using GST Sepharose 4B at a concentration of 0.6 mg=mL. The yields of recombinant proteins were 30 mg per 1 g of wet cells. Unlike His-tagged recombinant antimicrobial peptide produced in E. coli BL21 (pET system), which has been previously studied,[12–15] GST-tagged fused DS4 (29 kD) was only minimally toxic to the host cell and was stably expressed in E. coli BL21 (DE3). Because host toxicity is very important in producing large amounts of target protein in E. coli, GST-fused DS4 appears to be well suited for this purpose (Figure 2).
FIGURE 2 Growth model of E. coli BL21 (DE3) pGEX-DS4. &: BL21, .: BL21 (pGEX-DS4) non-induced culture, ~: BL21 (pGEX-DS4) induced culture with IPTG added after 8 hr of growth.
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TABLE 2
Purification Steps of DS4 From E. coli
Purification Step Bacterial lysate GST affinity chromatography
Volume (ml)
Total Protein (mg)
GST-DS4 (mg)
DS4 (mg)
Purity (%)
Yield (%)
50 5
657a 1.25
30 8.5
3.5b 1.0
NAc 80
100 29
a
Protein concentration was determined by Bradford protein assay. The amount of DS4 was calculated from the mass ratio compared to the determined amount of fusion protein. c NA, not applicable.
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b
Expressed GST-tagged fused DS4 was purified at high yield and purity when purified under denaturing conditions, whereas recovery was very low under native conditions (Table 2). These results indicate that most of the fusion protein exists as inclusion bodies in the E. coli cytoplasm. Thrombin Protease Treatment and Biological Activity of Recombinant DS4 Biological activity of recombinant DS4 was verified using an agar diffusion assay. Purified DS4 that had been treated with thrombin protease to clear the GST fusion partner showed bactericidal activity against M. luteus. This indicates that the biologically active DS4 was liberated from the fusion protein during thrombin protease treatment (Figure 3).
FIGURE 3 Agar diffusion test of antimicrobial activity of purified recombinant proteins on M. luteus. (A) Noninduced control (cell-free extract of E. coli BL21); (B) recombinant DS4. The same amount of protein (approximately 20 ng, corresponding to 200 AU of DS4) was applied to the hole in an LB soft agar overlayer seeded with the indicator strain M. luteus.
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DISCUSSION Antimicrobial peptide is a small polypeptide with broad-spectrum antimicrobial activity. Among these antimicrobial peptides, DS4 is a very promising candidate because it has not only an antibacterial effect but also antiviral activity. The highly efficient production of biologically active DS4, with large quantity and low cost, is an absolute need for both laboratory studies and potential clinical applications. Production and purification of the antimicrobial peptide DS4 has been reported previously by Mor et al.[16,17] Native DS4 was purified to homogeneity in three steps using molecular sieve filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. The amount of purified dermaseptin recovered was 40 mg starting from 1 g of dried Phyllomedusa sauvagii skin. Thus, isolation and purification of antimicrobial peptides from their native hosts is a time-consuming and laborious process. Industrial applications require large quantities of DS4; therefore, we sought a production method suitable for DS4 production. Heterologous expression may offer several advantages over native systems, such as facilitating control of antimicrobial peptide gene expression or increased production levels of pure and uniformly labeled protein.[18] In the current work, we constructed a highly efficient system for the expression and purification of DS4 in E. coli, using the pGEX-4T-1 expression vector. Escherichia coli has been used as a host for cloning a variety of genes involved in antimicrobial peptide biosynthesis. Several antimicrobial peptides (e.g., pediocin PA-1, divergicinV41, enterocin A, and enterocin P) have been expressed in E. coli.[12,14,19–21] GST is a soluble cytoplasmic protein that is well established as a recombinant production fusion partner of highly soluble and stable fusion proteins, and has been used in the expression of many biologically active proteins in E. coli. In this study, fusion expression was used to increase expression levels and decrease toxicity, as well as to provide purification tags for a heterologous expression strategy. The first step toward achieving high levels of DS4 production was to construct a genetically engineered plasmid carrying the DS4 gene with a GST-tag fusion at the N terminus. DS4 was highly overexpressed in E. coli BL21 (DE3) after IPTG induction as a full-length GSTtagged recombinant protein of 29 kD. To the best of our knowledge, this is the first report on the effective expression and purification of intact and bioactive DS4. Our strategy may provide a solution for production of unmodified antimicrobial peptides. The expression system and purification method of DS4 will assist laboratory studies. In the near future, the availability of purified DS4 could also enable further clinical, biochemical, and biological investigations.
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FUNDING This research was supported by the National Natural Science Foundation of China (31071513, 31271821), the Natural Science Foundation of Zhejiang Province (Z3110399), the China Agriculture Research System (CARS-5), the Zhejiang Province Program for Science and Technology (2007C12037), the Scientific Research Project for the Education Department of Zhejiang Province, China (Y201120078), and the key science and technology innovation team of Zhejiang Province (2010R50032).
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REFERENCES 1. Mor, A.; Hani, K.; Nicolas, P. The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms. J. Biol. Chem. 1994, 269, 31635–31641. 2. Krugliak, M.; Feder, R.; Zolotarev, V.Y.; Gaidukov, L.; Dagan, A.; Ginsburg, H.; Mor, A. Antimalarial Activities of Dermaseptin S4 Derivatives. Antimicrob. Agents Chemother. 2000, 44, 2442–2451. 3. Boman, H.G. Peptide Antibiotics and Their Role in Innate Immunity. Annu. Rev. Immunol. 1995, 13, 61–92. 4. Pasupuleti, M.; Schmidtchen, A.; Malmsten, M. Antimicrobial Peptides: Key Components of the Innate Immune System. Crit. Rev. Biotechnol. 2012, 32, 143–171. 5. Belaid, A.; Aouni, M.; Khelifa, R.; Trabelsi, A.; Jemmali, M.; Hani, K. In Vitro Antiviral Activity of Dermaseptins Against Herpes Simplex Virus Type 1. J. Med. Virol. 2002, 66, 229–234. 6. Lorin, C.; Saidi, H.; Belaid, A.; Zairi, A.; Baleux, F.; Hocini, H.; Be´lec, L.; Hani, K.; Tangy, F. The Antimicrobial Peptide Dermaseptin S4 Inhibits HIV-1 Infectivity In Vitro. Virology 2005, 334, 264–275. 7. Abee, T. Pore-Forming Bacteriocins of Gram-Positive Bacteria and Self-Protection Mechanisms of Producer Organisms. FEMS Microbiol. Lett. 1995, 129, 1–9. 8. Ammar, B.; Pe´rianin, A.; Mor, A.; Sarfati, G.; Tissot, M.; Nicolas, P.; Giroud, J.; Roch-Arveiller, M. Dermaseptin, a Peptide Antibiotic, Stimulates Microbicidal Activities of Polymorphonuclear Leukocytes. Biochem. Biophys. Res. Commun. 1998, 247, 870–875. 9. Feder, R.; Dagan, A.; Mor, A. Structure–Activity Relationship Study of Antimicrobial Dermaseptin S4 Showing the Consequences of Peptide Oligomerization on Selective Cytotoxicity. J. Biol. Chem. 2000, 275, 4230–4238. 10. Rodrı, A.X.; Guez, J.M.; Martı, A.X.; Nez, M.I.; Horn, N.; Dodd, H.M. Heterologous Production of Bacteriocins by Lactic Acid Bacteria. Int J Food Microbiol 2003, 80, 101–116. 11. Laemmli, U.K. Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature 1970, 227, 680–685. 12. Moon, G.; Pyun, Y.; Kim, W.J. Expression and Purification of a Fusion-Typed Pediocin PA-1 in Escherichia coli and Recovery of Biologically Active Pediocin PA-1. Int J Food Microbiol 2006, 108, 136–140. 13. Klocke, M.; Mundt, K.; Idler, F.; Jung, S.; Backhausen, J.E. Heterologous Expression of Enterocin A, a Bacteriocin From Enterococcus faecium, Fused to a Cellulose-Binding Domain in Escherichia coli Results in a Functional Protein With Inhibitory Activity Against Listeria. Appl Microbiol Biotechnol 2005, 67, 532–538. 14. Richard, C.; Drider, D.; Elmorjani, K.; Marion, D.; Prevost, H. Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli. J. Bacteriol. 2004, 186, 4276–4284. 15. Liu, S.N.; Han, Y.; Zhou, Z.J. Fusion Expression of pedA Gene to Obtain Biologically Active Pediocin PA-1 in Escherichia coli. J. Zhejiang Univ. Sci. B 2011, 12, 65–71. 16. Mor, A.; Nguyen, V.H.; Delfour, A.; Migliore-Samour, D.; Nicolas, P. Isolation, Amino Acid Sequence, and Synthesis of Dermaseptin, a Novel Antimicrobial Peptide of Amphibian Skin. Biochemistry 1991, 30, 8824–8830.
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