Protein Expression and Purification 73 (2010) 147–151

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Expression and purification of two different antimicrobial peptides, PR-39 and Protegrin-1 in Escherichia coli Fu Fan, Yueming Wu *, Jianxin Liu College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China

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Article history: Received 5 March 2010 and in revised form 19 May 2010 Available online 23 May 2010 Keywords: Antimicrobial peptide PR-39 Prokaryotic expression Protegrin-1 Purification

a b s t r a c t To implement coexpression of antimicrobial peptides PR-39 and Protegrin-1 (PG-1) in prokaryotic expression system, a tandem gene fragment encoding PR-39 and PG-1 has been synthesized chemically. The cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced between PR-39 and PG-1. The fragment was inserted into vector pGEX-4T-1 and expressed in Escherichia coli. The fusions of single peptides to GST were created at the same time. The fusion protein GST–PR-39–PG-1, purified by affinity chromatography, was cleaved first by hydroxylamine hydrochloride to release recombinant PG-1 and then by enterokinase to release PR-39. Purification of recombinant PR-39 and PG-1 was achieved. About 1.9 mg/l recombinant PR-39 and 1.1 mg/l PG-1 were obtained. The recombinant antimicrobial peptides showed antibacterial activities that were similar to those released from fusions of single peptides to GST. Ó 2010 Published by Elsevier Inc.

Introduction The effectiveness of antibiotics in controlling infectious bacterial disease, and increased availability due to successful mass production, has led to widespread and often inappropriate use. This results in the rise of antibiotic resistance and an associated increase in the prevalence of infections caused by antibiotic-resistant bacteria [1]. To address this problem, alternative antimicrobials are being sought. The antimicrobial peptides are one potential source of novel antibiotics [2]. PR-39 is a linear proline–arginine-rich peptide with 39 amino acid residues which was first found in the pig intestinal tissue and shortly thereafter in the neutrophil granules, and has played a multifunction role in host innate immunity [3–5]. PR-39 shows a broad spectrum of antimicrobial activity [6–7], and is chemotactic for neutrophils [8–9] and is capable of regulating vascular cell–cell interaction [10]. It can inhibit invasion and metastasis of cancer cells [11–12] and apoptosis of hypoxic endothelial cells [13]. There is a significant interest in developing this peptide for pharmaceutical applications. Protegrin-1 (PG-1)1 is a b-hairpin antimicrobial peptide of 18 amino acids that was first discovered in porcine leukocytes [14].

It kills microorganism by forming ion channels in cellular membranes [15]. Due to the broad range of antimicrobial activity of PG-1, it is considered as a potential pharmaceutical agent [16]. There is no report on the heterologous expression of mature antimicrobial peptides PR-39 and PG-1. For pharmaceutical applications, a large quantity of antimicrobial peptides is required. Preparative isolation of antimicrobial peptides from natural sources and chemical synthesis is not efficient and economical [17]. Now numerous biological expression systems have been introduced for the economical production of antimicrobial peptides [18]. Escherichia coli is one of the major systems used for producing recombinant antimicrobial peptides and accounts for more than 80% of all cases [19]. However, in former reports, only one kind of antimicrobial peptide was produced by biological engineering in one case. In this study, tandem antimicrobial peptides PR-39 and PG-1 were expressed in E. coli by employing a glutathione-S-transferase (GST) fusion system. The recombinant antimicrobial peptides were purified and their antimicrobial activity was confirmed.

Materials and methods Bacterial strains, vectors and enzymes

* Corresponding author. Address: College of Animal Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou 310029, PR China. Fax: +86 571 86971930. E-mail address: [email protected] (Y. Wu). 1 Abbreviations used: PG-1, Protegrin-1; GST, glutathione-S-transferase; LB, Luria– Bertani; IPTG, isopropyl b-D-thiogalactoside; PVDF, polyvinylidene difluoride; HRP, horseradish peroxide; MIC, minimum inhibitory concentration; BSA, bovine serum albumin. 1046-5928/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.pep.2010.05.012

E. coli DH5a was used for plasmid amplification and E. coli BL21 (DE3) was used for the expression of fusion protein. Plasmid pGEX4T-1, a vector for producing fusion protein with GST, was used as the expression plasmid. The Glutathione Sepharose 4B was used for the purification of fusion protein. Restriction endonucleases,

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BamHI and XhoI, Ex Taq and T4 DNA ligase were purchased from Takara Biotech Co., Ltd. (Dalian, China). The antibodies used for Western blot analysis were purchased from GenScript. Enterokinase and hydroxylamine hydrochloride were used for cleaving the antimicrobial peptides and GST tag. All oligonucleotides were ordered from Takara. Luria–Bertani (LB) medium was used for general culture. All other chemical reagents were made in China and were of analytical grade. Synthesis of PR-39 and PG-1 tandem gene by PCR Based on the primary amino acid sequences of the mature peptide and according to codon preference of E. coli, the PR-39 and PG1 tandem genes were synthesized with oligonucleotides 1–4 (Fig. 1). Sixteen bases between every two consecutive oligonucleotides were complementary. The sequence of enterokinase cleavage site (DDDDK) was added upstream of the PR-39 codon sequence and the cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced between PR-39 and PG-1. BamHI and XhoI endonuclease sites were incorporated at the 50 end of the oligonucleotides 1 and 4, respectively. The PCR mixture consists of oligonucleotides 1–4 (10 lM) 5, 0.2, 0.2, and 5 ll, respectively, EX Taq buffer 10 ll, dNTP mixture 8 ll and 0.5 U Taq DNA Polymerase in a total volume of 100 ll. The chain extension reaction was carried out as follows: 94 °C for 5 min, 35 cycles of (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s), and a final extension of 72 °C for 5 min. At the same time, the single peptides genes of PR-39 and PG-1 were obtained, respectively, with the same method, and the sequence of enterokinase cleavage site was added upstream of them.

Fig. 2. Schematic representation of the expression vector pGEX–PR-39–PG-1. EN, the cleavage site of enterokinase; Asn-Gly, the cleavage site of hydroxylamine hydrochloride.

10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). After having freezed at 80 °C for 1 h and thawed on ice, the cells were broken by brief pulses of sonication on ice and a final concentration of 1 mg/ml of lysozyme, 0.5 mM PMSF was added. The cell debris was removed by centrifugation at 12,000g for 10 min at 4 °C. The presence of fusion proteins in the supernatant was analysed by 12% sodium lauryl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).

Construction of the fusion expression plasmids

Western blot analysis

The resulting PCR products were digested with BamHI and XhoI, and ligated into the pGEX-4T-1 plasmid that was digested with the same restriction enzymes to generate GST fusion vector pGEX–PR39–PG-1 (Fig. 2), pGEX–PR-39 and pGEX–PG-1. The ligation mixture was transformed into E. coli DH5a competent cells for propagation of the recombinant plasmids. The recombinant plasmids were confirmed by the digestion of restriction endonucleases and DNA sequencing analysis.

The cell lysates in the supernatant were subjected to 12% SDS– PAGE and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was immersed in blocking solution (5% skim milk in TBST) for 2 h, then immunoblotted with primary antibodies (monoclonal mouse anti-GST at 1:5000 dilutions) for 2 h at room temperature. The membrane was washed with TBST buffer (0.3% Tris, 0.8% NaCl, 0.02% KCl, 0.1% Tween 20) for three times and followed by 2 h incubation with horseradish peroxide (HRP)-conjugated secondary antimouse IgG from goat (1:3000 dilution). After three washes, the bound antibodies were visualized using TMB-stabilized substrate for HRP (Promega).

Expression of fusion proteins The confirmed recombinants containing fusion genes were transformed into competent E. coli BL21 (DE3) cells, and the transformed cells were grown in 1 l Luria–Bertani (LB) medium with 100 lg ampicillin/ml at 210 rpm at 37 °C. Overnight cultures were diluted 1:100 into fresh LB medium. When the cell density reached about 0.6 OD600, the expression of fusion proteins was initiated by adding isopropyl b-D-thiogalactoside (IPTG) to 0.6 mM. After 4 h induction at 210 rpm at 28 °C, the cells were collected by centrifugation at 8000g at 4 °C for 10 min. The cell pellets were washed and resuspended in 50 ml of icecold phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl,

Purification of fusion proteins The supernatant containing the fusion proteins GST–PR-39–PG1 (from construct pGEX–PR-39–PG-1), GST–PR-39 (from construct pGEX–PR-39) or GST–PG-1 (from construct pGEX–PG-1) was loaded onto a Glutathione Sepharose 4B affinity chromatography column equilibrated with PBS to purify the GST fusion protein. Then the column was washed with 20 bed volumes of ice-cold PBS to remove contaminating proteins. The fusion proteins were

Fig. 1. Design of the tandem gene fragment encoding PR-39 and PG-1. 1–4, sequences of oligos for the tandem gene fragment.

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eluted with 10 bed volumes of freshly made 10 mM reduced glutathione elution buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris–HCl, pH 8.0). The fusion proteins containing fractions were pooled and dialyzed against 100 bed volumes of PBS with three changes within 24 h. After dialysis, the fusion proteins were concentrated using Centricon Microconcentrators. Protein concentration was determined by the method of Bradford using BSA as a protein standard. Cleavage and purification of recombinant PG-1 and PR-39 The fusion protein GST–PR-39–PG-1 was incubated in hydroxylamine cleavage buffer (2 M hydroxylamine hydrochloride, 0.2 M Tris/HCl, pH 9.0) for 4 h at 45 °C first. The reaction was terminated by lowering the temperature and adjusting the pH with HCl to about six. The cleavage mixture was passed through Glutathione Sepharose 4B column with the flow rate maintained at 1 ml/min at 4 °C. The flowthrough which contained recombinant PG-1 was pooled, dialyzed, concentrated using Centricon Microconcentrators and further analyzed by 16.5% Tricine/SDS–PAGE. The Glutathione Sepharose 4B column was rinsed with 10 mM reduced glutathione elution buffer and the eluant containing fusion protein GST–PR-39 was collected and concentrated. The fusion protein GST–PR-39 was dialyzed against enterokinase buffer (150 mM Tris–HCl, pH 7.4, 150 mM NaCl and 2.5 mM CaCl2) with three changes within 24 h at 4 °C. To get rid of GST, enterokinase was added to the fusion protein at 10 U/mg protein, and the digestion reaction was performed at 21 °C for 16 h with stirring. To remove GST, the reaction mixture was passed through Glutathione Sepharose 4B column. The eluant containing recombinant PR-39 was pooled, dialyzed, concentrated and analyzed by 16.5% Tricine/SDS–PAGE. The fusion proteins GST–PR-39 from construct pGEX–PR-39 and GST–PG-1 from construct pGEX–PG-1 were purified and cleaved by enterokinase. The recombinant PR-39 and PG-1 were pooled, dialyzed, concentrated and analyzed by 16.5% Tricine/SDS–PAGE at last.

Fig. 3. (A) The PCR products of target genes. M, DNA marker; lane 1, the PCR fragment of PG-1; lane 2, the PCR fragment of PR-39; lane 3, the PCR fragment of PR-39–PG-1; lane 4, negative control. (B) The digestion of expression vectors by BamHI and XhoI. M, DNA marker; lane 1, the digestion of pGEX–PG-1 recombinant; lane 2, the digestion of pGEX–PR-39 recombinant; lane 3, the digestion of pGEX–PR39–PG-1 recombinant.

digestion and agarose gel electrophoresis (Fig. 3B). These results apparently suggested that the expression plasmids were constructed successfully. Expression of fusion proteins E. coli BL21 (DE3) was transformed with recombinant plasmids and was induced with IPTG. Theoretically, the molecular weights of the target products of BL21 (DE3)/pGEX–PR-39–PG-1, BL21 (DE3)/ pGEX–PR-39 and BL21 (DE3)/pGEX–PG-1 should be 33.6, 31.3 and 28.7 kDa, respectively. SDS–PAGE analysis clearly indicated that the target proteins were all expressed (Fig. 4A). Western blot analysis showed that three strong bands were observed at corresponding molecular weight of the expected proteins, confirming that three kinds of fusion proteins were expressed as expected (Fig. 4B). It was also found that most of the expressed protein existed in the supernatant of the cell lysate by gel run on the pellet (data not show).

Antimicrobial activity assay

Purification of fusion proteins, recombinant PG-1 and PR-39

The antibacterial activity of recombinant PR-39 and PG-1 was tested by agar well diffusion assay using E. coli ATCC25922. The minimum inhibitory concentration (MIC) of the purified PR-39 and PG-1 was determined against E. coli ATCC25922, Staphylococcus aureus ATCC25923, Salmonella gallinarum, Aspergillus niger and Penicillium chrysogenum by a liquid growth-inhibition assay [20]. Series of 2-fold dilutions of recombinant peptides ranging from 640 to 1.25 lg/ml were made in 0.2% bovine serum albumin (BSA), 0.01% acetic acid buffer. Ten microliters from each dilution were distributed in a 96-well polypropylene microtiter plate, and each well was inoculated with 90 ll of a suspension of mid-log microorganisms. Cultures were grown in LB-broth for 24 h with vigorous shaking at 37 °C. The antibacterial effect was evaluated by measuring the culture OD600 using a microtiter reader. The MIC was taken as the concentration at which greater than 50% of growth inhibition was observed.

Fig. 5 showed that intense bands corresponding to the molecular weights of the expected proteins, an approximately 33.6 kDa band for GST–PR-39–PG-1 (lane 1), 31.3 kDa for GST–PR-39 (lane 2), 28.7 kDa for GST–PG-1 (lane 3), and about 4.8 kDa band for PR-39 (lanes 4 and 6) and about 2.2 kDa for PG-1 (lanes 5 and 7)

Results Construction of an expression vectors The tandem DNA fragment (PR-39–PG-1) and those of single peptide (PR-39 and PG-1) were successfully obtained by PCR and verified by the agarose gel electrophoresis (Fig. 3A) and sequencing. The generated recombinant expression vectors pGEX–PR-39– PG-1, pGEX–PR-39 and pGEX–PG-1 were verified by enzyme

Fig. 4. (A) SDS–PAGE analysis of fusion proteins expressed in E. coli BL (DE3). M, protein molecular weight marker; lane 1, total protein before induction of E. coli/ pGEX4T–PR-39–PG-1; lane 2, total protein of induced E. coli/pGEX4T–PR-39–PG-1 with IPTG; lane 3, induced E. coli/pGEX4T–PR-39; lane 4, induced E. coli/pGEX4T– PG-1; lane 5, induced E. coli/pGEX4T-1. (B) Western blot analysis of fusion proteins expressed in E. coli BL (DE3). M, protein molecular weight marker; lane 1, E. coli/ pGEX4T–PR-39–PG-1 induced with IPTG; lane 2, E. coli/pGEX4T–PR-39 induced with IPTG; lane 3, E. coli/pGEX4T–PG-1 induced with IPTG; lane 4, E. coli/pGEX4T-1 induced with IPTG.

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Fig. 5. Tricine/SDS–PAGE analysis of fusion proteins and recombinant PR-39 and PG-1. M, protein molecular weight marker; lane 1, GST–PR-39–PG-1 fusion protein; lane 2, GST–PR-39 fusion protein; lane 3, GST–PG-1 fusion protein; lane 4, recombinant PR-39 released from GST–PR-39–PG-1 fusion protein; lane 5, recombinant PG-1 released from GST–PR-39–PG-1 fusion protein; lane 6, recombinant PR-39 released from GST–PR-39 fusion protein; lane 7, recombinant PG-1 released from GST–PG-1 fusion protein.

Fig. 6. The antimicrobial activity of recombinant PR-39 (A) and PG-1 (B) against E. coli ATCC25922. 1, control; 2, treatment with recombinant peptides (10 lg) released from GST–PR-39–PG-1 fusion protein; 3, treatment with recombinant peptides (10 lg) released from GST–PR-39 or GST–PG-1 fusion proteins.

Table 2 The MIC of recombinant PR-39 and PG-1 against microorganisms (lg/ml). Microorganisms

Recombinant PR-39 a

were observed. From 1 l of bacterial culture, about 1.9 mg recombinant PR-39 and 1.1 mg PG-1 were obtained (Table 1). The yields of the recombinant peptides released from GST–PR-39–PG-1 were a little higher than those released from fusions of single peptides to GST. Antimicrobial activity assay of recombinant PR-39 and PG-1 a

Antimicrobial activity assaying was performed to determine the function of the purified recombinant PR-39 and PG-1. Fig. 6 shows that there was obvious inhibition zone around the treated well but not around the control. The MICs of the purified recombinant PR-39 and PG-1 against several microorganisms are shown in Table 2. These approaches demonstrated that the recombinant peptides were functional and active after separation from the protein complex and the recombinant peptides that released from fusion proteins GST–PR-39–PG-1 have similar antimicrobial properties to those released from fusions of single peptides to GST. Discussion Now, antimicrobial peptides have been studied extensively because of their possible clinical applications as pharmaceutical agents. Among these antimicrobial peptides, PR-39 and PG-1 are promising candidates. The efficient production of biologically active antimicrobial peptides in large quantities and low cost is in absolute need for potential clinical applications. Due to the need for large quantity of antimicrobial peptides, we sought a production method utilizing E. coli. To express antimicrobial peptides in Table 1 Approximate yield and purity of total proteins after purification with affinity chromatography and ultrafiltration. Proteins

Total protein GST–PR-39–PG-1 GST–PR-39 GST–PG-1 PG-1 PR-39

E. coli/pGEX–PR39–PG-1

E. coli/pGEX–PR39

E. coli/pGEX– PG-1

Yield (mg)a

Purity (%)b

Yield (mg)a

Purity (%)b

Yield (mg)a

Purity (%)b

378.5 32 21 NA 1.1 1.9

NA 90 90 NA 99 95

346 NA 19 NA NA 1.3

NA NA 90 NA NA 99

361 NA NA 17 1.0 NA

NA NA NA 90 99 NA

NA, not applicable. a Based on 1 l bacterial culture. Protein concentration was determined by Bradford protein assay. b Purity of protein was estimated by SDS gel stained by Coomassie blue.

b

Recombinant PG-1 G-PG-1a

PG-1b

4 >64 8

0.5 2 4

0.5 2 4

16 8

8 8

8 8

PR-39-N

PR-39

Bacteria E. coli ATCC25922 S. aureus ATCC25923 Salmonella gallinarum

4 >64 8

Fungi A. niger P. chrysogenum

16 8

b

Recombinant peptides released from GST–PR-39–PG-1. Recombinant peptides released from GST–PR-39 or GST–PG-1.

E. coli, many carrier proteins are used to neutralize their innate toxic activity to host bacterial cells and to increase their expression levels [21]. The GST fusion system is versatile for the expression, purification and detection of fusion proteins [22]. In this study, we constructed pGEX–PR-39–PG-1 fusion plasmid and successfully expressed GST–PR-39–PG-1 fusion protein as soluble portion in E. coli BL21 (DE3). The fusion protein was purified using affinity chromatography directly and easily. It is reported that the native N-terminal segment is a prerequisite for maintaining the activity of AMPs against microbes [23]. The recombinant AMPs need to be released from carrier proteins by the site-specific cleavage tools, which consist of site-specific proteases and chemical reagents. Enterokinase is a highly specific protease that has a pentapeptide recognition sequence (DDDDK) and cleaves at the C-terminal side of the recognition site. There are no leaving additional residues at N-terminal of the cleaved product after enterokinase digestion. In this work, we used enterokinase to cleave the carrier protein. To express antimicrobial peptides PR-39 and PG-1 in prokaryotic expression system at the same time, a tandem gene fragment encoding PR-39 and PG-1, between which the cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced, has been synthesized chemically. The enterokinase cleavage site was added at the N-terminal of the PR-39. The recombinant PG-1 was released by incubation of the fusion protein with hydroxylamine hydrochloride first. The recombinant PR-39 was released by incubation of the fusion protein GST–PR-39 with enterokinase. After cleavage, there is one additional amino acid residue (Gly) that attaches on the N-terminus of PG-1, and another additional amino acid residue (Asn) on the C-terminus of PR-39. Experiments (Table 2) showed that the additional amino acid residues have little effect on the antibacterial characteristics of recombinant peptides. Glycine is the simplest amino acid and is asymmetric, and the side-chain of it, a hydrogen atom, has the least conformational space of any other amino acid residue. Therefore, glycine does not affect the conformations of the other amino acids of the peptides significantly [24]. Maybe, the first 26 amino acid residues of the

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NH2 terminus are the functional antibacterial domain of PR-39 [6], so the additional Asn on the C-terminus has little effect on antibacterial characteristics of PR-39. Although the recombinant peptides showed obvious antibacterial activity, the effect of additional amino acid residues on the biological activity of recombinant peptides, such as immunogenicity, needs to be investigated further. In summary, we constructed an efficient system for the expression and purification of PR-39 and PG-1 in E. coli. The recombinant strain can potentially be adapted for large-scale production of biologically active antimicrobial peptide PR-39 and PG-1 at the same time. Acknowledgments This research was financially supported by Zhejiang Department of Science and Technology (No. 2006C22045) and Proj. of Hangzhou Science and Technology Innovation Fund, China (No. 20051322B34). References [1] A.J. Alanis, Resistance to antibiotics: are we in the post-antibiotic era?, Arch Med. Res. 36 (2005) 697–705. [2] M.R. Yeaman, N.Y. Yount, Unifying themes in host defence effector polypeptides, Nat. Rev. Microbiol. 5 (2007) 727–740. [3] H.G. Boman, Peptide antibiotics and their role in innate immunity, Annu. Rev. Immunol. 13 (1995) 61–92. [4] Y. Sang, F. Blecha, Porcine host defense peptides: expanding repertoire and functions, Dev. Comp. Immunol. 33 (2009) 334–343. [5] M. Zanetti, Cathelicidins, multifunctional peptides of the innate immunity, J. Leukoc. Biol. 75 (2004) 39–48. [6] J.S. Shi, C.R. Ross, M.M. Chengappa, et al., Antibacterial activity of a synthetic peptide (PR-26) derived from PR-39, a proline–arginine-rich neutrophil antimicrobial peptide, Antimicrob. Agents Chemother. 40 (1996) 115–121. [7] C.M.A. Linde, S.E. Hoffner, E. Refai, et al., In vitro activity of PR-39, a proline– arginine-rich peptide against susceptible and multi-drug-resistant Mycobacterium tuberculosis, J. Antimicrob. Chemother. 47 (2001) 575–580.

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