Expression of a novel bacteriocin—the plantaricin Pln1—in Escherichia coli and its functional analysis

Accepted Manuscript Expression of a novel bacteriocin—the plantaricin Pln1—in Escherichia coli and its functional analysis Fanqiang Meng, Haizhen Zhao, Chong Zhang, Fengxia Lu, Xiaomei Bie, Zhaoxin Lu PII:

S1046-5928(15)30100-5

DOI:

10.1016/j.pep.2015.11.008

Reference:

YPREP 4825

To appear in:

Protein Expression and Purification

Received Date: 27 September 2015 Revised Date:

3 November 2015

Accepted Date: 9 November 2015

Please cite this article as: F. Meng, H. Zhao, C. Zhang, F. Lu, X. Bie, Z. Lu, Expression of a novel bacteriocin—the plantaricin Pln1—in Escherichia coli and its functional analysis, Protein Expression and Purification (2015), doi: 10.1016/j.pep.2015.11.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Expression of a novel bacteriocin—the plantaricin Pln1—in Escherichia coli and its functional analysis

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Fanqiang Meng, Haizhen Zhao, Chong Zhang, Fengxia Lu, Xiaomei Bie, Zhaoxin Lu*

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Fanqiang Meng [email protected] Haizhen Zhao, [email protected] Chong Zhang, [email protected] Fengxia Lu, [email protected] Xiaomei Bie, [email protected] Zhaoxin Lu* [email protected]

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College of Food Science and Technology, Nanjing Agriculture University Nanjing, China, 210095

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Abstract A potential bacteriocin gene was isolated from 18575 ORFs by bioinformatics methods. It was named pln1, and cloned into pET32a. Then, it was expressed as a thioredoxin-Pln1 fusion protein in Escherichia coli BL21 (DE3). The fusion protein was purified by Ni-NTA, and thioredoxin was removed by enterokinase. Finally, Pln1was purified using a cation affinity column. The yields of fused and cleaved Pln1 peptides were 100~110 mg/l and 9~11 mg/l, respectively. Pln1 was stable in an acidic environment and at temperatures below 60°C, but was easily degraded under alkaline conditions and by protease treatment. The cleaved and purified Pln1 showed strong antimicrobial activity against gram-positive bacteria such as Micrococcus luteus CMCC 63202, Staphylococcus epidermidis, Lactococcus lactis NZ3900, Lactobacillus paracasei CICC 20241, and Listeria innocua CICC 10417. In particular, Pln1 had a better activity against methicillin-resistant S. epidermidis (MRSE) than nisin, thereby offering an attractive approach to counter bacterial antibiotic resistance.

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Keywords: plantaricin, heterologous expression, antimicrobial activity, MRSE

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1. Introduction Plantaricins are a group of peptides or small proteins produced by L. plantarum, which are able to inhibit other competing bacterial species as well as strains of the same species [1]. Bacteriocins, including plantaricin from lactic acid bacteria, have attracted considerable interest for their potential use as natural and nontoxic food preservatives. Currently, the identified plantaricins include plantaricin A, C, D, E, F, J, K, S, T, Y, 423, 163, 149, 35D, BN, SA6, LC74, KW30, ZJ008, LD1, UG1, NC8, LP84, C11, and NA [2-10]. However, plantaricin yields are low and purification is difficult. For example, the yield of plantaricin LR14 [11] is only 59.21 µg/l, while the highest yield of plantaricin is 3.5 mg/l in L. plantarum A-1 [12]. Thus, it is difficult to achieve a desirable yield of 1 mg/l in its native host culture [13, 14]. This is a great bottleneck in the application of plantaricin. In an attempt to solve this problem, a number of different expression systems have been developed, beginning in the 1990s. In the first system, carnobacteriocin B2 and brochocin-C were expressed in Pichia pastoris [15, 16]. Then, colicin V was expressed in lactic acid bacteria and secreted by signal peptides [17]. Later, piscicolin 126 [18], divercin AS7 [19], enterocin A [20], enterolysin A [21], epidermicin NI01 [22], gassericin A [23], glycinecin A [24], lactococcin 972 [25], and lactococcin K [26] were expressed in E. coli, and all showed antimicrobial activity. Enterocin A [27], enterocin P [28], hiracin JM79 [29], pediocin PA-1 [30], and sakacin A [31] were expressed in P. pastoris. In addition, a number of other bacteriocins were expressed in lactic acid bacteria [31-33], Methylobacterium extorquens [34], Saccharomyces cerevisiae [35], Leuconostoc mesenteroides [36], and Carnobacterium piscicola [15]. The success of these systems has promoted the research and application of bacteriocins. Many plantaricins have been identified from various strains of L. plantarum, but there has been little research on the heterologous expression of plantaricins. One of the two-peptide bacteriocins, plantaricin F, was expressed separately from plnE and showed activity against L. viridescens NCDO1655. This technique made it easier to isolate and purify plnF for subsequent structural and functional analyses [37]. Plantaricin E and F were successfully expressed in prokaryote cells [38]. plnE, F, J, and K were amplified from a soil metagenome and expressed in a pET32a vector in E. coli BL21 (DE3). These peptides had activity against L. innocua NRRL B33314, M. luteus MTCC 106, and lactic acid bacteria. The IC50 range was 0.09~1.5 µg/ml [6]. Plantaricin 423 was cloned into a shuttle vector under the control of a yeast promoter and the heterologous product was expressed in Saccharomyces cerevisiae. This protein has high antimicrobial activity against many gram-positive foodborne pathogens and spoilage bacteria [35]. All plantaricins that were expressed in a heterologous host were isolated and identified from their native hosts. In the current study, the plantaricin gene, pln1, was obtained by sequence analysis of DNA and amino acids. Further research proved that antibacterial substances were isolated and identified in L. plantarum 163 cultures; however, plantaricin Pln1 could not be detected by reverse phase high performance liquid chromatography (RP-HPLC). The purpose of this study was to establish an

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2. Material and Methods 2.1 Microbial strains and culture media E. coli DH5α and E. coli BL21 (DE3) were grown in Luria-Bertani (LB) medium at 37°C. The indicator strains used in the bacteriocin assays were Listeria innocua CICC 10417, Micrococcus luteus MTCC106, Staphylococcus epidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis (MRSE), S. aureus ATCC29213, E. coli ATCC35218/ATCC25922, Pseudomonas fluorescens AS 3.6452, Klebsiella pneumoniae CICC 21519, Bacillus cereus AS 1.1846, methicillin-resistant Staphylococcus aureus (MRSA), Lactococcus lactis NZ3900, and Lactobacillus paracasei CICC 20241. Listeria was grown in brain–heart infusion medium, and lactic acid bacteria were grown in de Man, Rogosa and Sharpe (MRS) medium; all other indicator strains were grown in LB medium at 37°C. The following were obtained from Vazyme Biotech (Nanjing, China): recombinase exnase II; E. coli strain DH5α, BL21 (DE3). Super Pfu DNA polymerase and DNA marker were purchased from DongSheng Biotech (Guangzhou, China). A low range protein ladder was purchased from Thermo Scientific (Massachusetts, USA). BamHI restriction endonuclease was purchased from Takara Biotechnology (Ostu, Japan). Ni-NTA columns and HiTrap SP FF columns were purchased from GE Healthcare (Pittsburgh, USA). Polyetherimide (PEI) and other chemical reagents were purchased from Aladdin Industrial Corporation (Shanghai China). All primers were synthesized by Shanghai Generay Biotech Co. (Shanghai, China).

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Escherichia coli-based expression system for the small putative plantaricin, which was identified from mining of the bacteriocin gene from the genome of L. plantarum 163. The pln1 gene was cloned into a pET32a expression vector. In addition, the physical and biological properties of Pln1 were investigated.

2.2 Bioinformatics analyses of the plantaricin gene The whole genome of L. plantarum 163 was sequenced by Illumina Hiseq 2000. To determine the presence of potential bacteriocin genes in the genome, the data were analyzed by EMBOSS (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::getorf) to obtain ORFs ranging from 60 bp to 200 bp. The SignalP4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) was used to identify ORFs with predicted signal peptides. The prediction tool in the Collection of Antimicrobial Peptides (CAMP) (http://www.camp.bicnirrh.res.in/predict/) was used to identify ORFs with potential antimicrobial activities. Finally, the ORFs were subjected to BLAST analysis against antimicrobial peptide databases (PepBank, http://pepbank.mgh.harvard.edu/search/basic; BACTIBASE, http://bactibase.pfba-lab-tun.org/main.php; APD, http://aps.unmc.edu/AP/), and the secondary structure was predicted by SWISS MODEL (http://www.swissmodel.expasy.org/). Analysis of the physical and chemical properties such as hydrophobicity, amino acid composition, and isoelectric point was performed using BioEdit.

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2.3 pln1 gene cloning from the L. plantarum 163 genome The L. plantarum 163 genome was extracted as described previously [37]. Recombinant primers were as follows (lowercase letters are recombinant sequence within the pET32a vector):pln1-F, acggagctcgaattcggatccATTTGGCAATGGATTGTGG; pln1-R, gccatggctgatatcgga tccCTAGTAGCCATAACCTTTTTTCTTGC. The pln1 gene was cloned from L. plantarum 163 using a high-fidelity Super Pfu enzyme. PCR (ABI Veriti Thermal Cycler, California, U.S.) was performed under the following conditions: initial denaturation at 95°C for 3 min; 30 cycles each of 95°C for 30 s, 45°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 7 min. The band for the pln1 gene PCR product was obtained by gel electrophoresis using a 3% agarose gel. The band was excised from the gel and purified with an E.Z.N.A. Gel Extraction Kit (Omega Bio-Tek, Norcross, Georgia, U.S.). The expression vector pET32a was digested with BamHI at 37°C for 8 h and the linearized product was recovered by 1% agarose electrophoresis. The reaction mix was prepared on ice and comprised 4 µl CEII Buffer, 140 ng linearized pET32a vector, 8 ng pln1 gene, and 2 µl ExnaseII recombinase, with a final volume of 20 µl made up using ddH2O. Samples were briefly centrifuged and incubated at 37°C for 30 min in a water bath; then, they were immediately transferred to an ice-water bath for 5 min. The reaction product was transformed into E. coli DH5α as described previously” [39]. The plasmid was extracted from positive clones and transformed into E. coli BL21 (DE3). The positive clones were then cultured and induced at initial conditions; when OD630 reached 0.6, 0.5 mmol/l IPTG was added and the clones were further cultured for 8 h at 25°C. The fusion protein was tested by western blot as described previously [40], using a horseradish peroxidase-conjugated anti-His antibody and 3,3 N-diaminobenzidine tetrahydrochloride (DAB) for direct detection. Induction conditions were optimized by varying the optical density (OD630, 0.5, 0.6, 0.7 and 0.8), time (4, 6, 8, and 10 h), temperature (20°C, 25°C, 30°C, and 37°C), and IPTG concentration (0.5, 1, 2, and 4 mM). Finally, protein expression was detected by SDS-PAGE as described previously [39]), and images were analyzed by the GelAnalyzer gel image software.

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2.4 Expression and purification of the recombinant Pln1 For expression of the recombinant protein, 1 liter of culture was induced under optimal conditions as described above. Cells were harvested by centrifugation (Heraeus Kendro D-37520, California, US) at 8,000 g for 10 min at 4°C. The pellet was resuspended in 50 ml PBS (with 0.1 M NaCl) and disrupted by sonication (SCIENJZ-IID, Ningbo, China) using 40% amplitude for 30 min. The cell-free supernatant was collected by centrifugation at 12,000 g for 10 min at 4°C. Then, 0.3% polyetherimide (PEI) was added to remove nucleic acids and neutral and basic proteins and to reduce the viscosity of the sample as described previously [41]. Next, Ni-NTA resin (GE Healthcare, Pittsburgh, US) was equilibrated with three column volumes of binding buffer (20 mM Na3PO4, 500 mM NaCl. pH 7.8). The sample was loaded onto the column with a flow rate of 1 ml/min, and the column was

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washed with six volumes of binding buffer, followed by four volumes of binding buffer containing 50 mM imidazole to remove unbound proteins. Finally, the binding protein was eluted with six volumes of binding buffer containing 200 mM imidazole; the UV peak was used to determine the purification efficiency by SDS-PAGE.

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2.5 Removal of the fusion protein and purification of Pln1 The imidazole and salt were removed by ultrafiltration (3KD) (Millipore Massachusetts US). Samples were incubated with 50 U enterokinase at 37°C for 16 h to cleave the fusion protein. The product was analyzed by Tricine-SDS-PAGE. Then, the cleaved fraction was centrifuged at 12,000 g for 20 min at 4°C, and the supernatant was purified using a HiTrap SP FF column in AKTA purifier UPC10 (GE Healthcare, Pittsburgh, US). Purification conditions were as follows: equilibration buffer, PBS (pH 8.0); flow rate, 5 ml/min; and pressure <0.3 Mpa. After the sample was loaded, the column was washed with five volumes of PBS (pH 8.0) and Pln1 was eluted with a linear gradient of sodium chloride (concentration ranging from 0.2 M to 1.5 M). The peak was used to test the antimicrobial activity. The concentration of Pln1 protein was determined using the Bradford assay. For quality control of purification, 20 µl of active eluted solution was applied to an AICHROMBond C18 (250 mm × 4.6 mm) column incorporated into the Ultimate 2000 HPLC system (Dionex, California, USA) and eluted for 20 min at a flow rate of 0.3 ml/min, using the following as the mobile phase: a mixture of 50% water containing 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile containing 0.1% TFA. The wavelength of the UV detector was 214 nm.

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2.6 Bacterial inhibitory activity of Pln1 An agar diffusion assay was used to detect potential inhibitory activity of Pln1. Indicator bacteria were grown in their respective media, and a 100-µl aliquot of an overnight culture was mixed with 10 ml of soft agar and poured onto a base agar. Wells (4.5 mm) were punched into the plate, 40 µl of Pln1 was added each well, and plates were incubated overnight at 37°C. Presence of an inhibition zone was indicative of antimicrobial activity. Pln1 activity was quantified using a 96-well microtiter plate assay. Indicator bacteria were grown as described above and centrifuged at 5,000 g for 5 min at 4°C. Pellets were resuspended in the appropriate media, and the concentration was adjusted to 2×106CFU/ml. A 50 µl aliquot of indicator bacteria and 50 µl serial dilutions of Pln1 were added to 96-well plates. Nisin (50 µl; 200 mg/ml) was used as a positive control, and 50 µl PBS (pH 7.4) was used as a negative control. The MIC was determined as the lowest concentration of Pln1 that inhibited visible growth (OD630 values showed no significant change compared to initial values) after overnight incubation at 37°C. All tests were carried out in triplicate. All data were significant as analyzed by IBM SPSS Statistics (Duncan correction).

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2.7 Stability of Pln1 under different enzyme, pH and temperature conditions A 100-µl aliquot of overnight culture (indicator bacterium, M. luteus CMCC 63202)

ACCEPTED MANUSCRIPT was mixed with 10 ml of soft agar and poured onto a base agar. Wells were punched into the agar and serial dilutions of Pln1 were added to the wells. The relationship between inhibition zone diameter and Pln1 concentration was analyzed to establish a fitting equation using Origin 9.0. The concentration of Pln1 was 1 mg/ml. Pln1 was treated with different proteases (proteinase E, proteinase K, pepsin, or papain) in their appropriate buffer (at a final concentration of 1 mg/ml) and at the appropriate temperature for 1 h. To calculate the relative potency, the pH was adjusted to 4.0 and the diameter of the inhibition zone was measured. The pH of Pln1 (1 mg/ml) was adjusted to 1.0, 2.0, 3.0, 12.0, 13.0, or 14.0 for 2 h; then, the pH was adjusted back to 4.0 to calculate the relative potency as described above. Pln1 was treated at 60°C, 80°C, and 100°C for 30 min to calculate the relative potency as described above.

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3. Results 3.1 Sequence analysis of pln1 The length of most antimicrobial peptides ranges from 7 to 60 amino acids, and the DNA length of bacteriocins ranges from 60 bp to 200 bp (including signal peptides). EMBOSS data showed that 18,575 ORFs were within this range, and SignalP 4.1 predicted that 321 of these ORFs had signal peptides. Antimicrobial activities were predicted by CAMP, and 58 ORFs showed potential activity. These potential ORFs were subjected to BLAST analysis, and 12 had high scores and similarities in antimicrobial peptide databases. Furthermore, the secondary structures and hydrophobicities were predicted for 6 ORFs that had α-helix and amphipathic structures. Finally, the relative environment of the ORFs in the genome was determined. The results showed that one ORF was present in the plantaricin locus and the others in integrases, prophage protein, or hypothetical proteins. It is likely that the gene within the pln locus is a potential bacteriocin. The gene in the plantaricin locus was named pln1; the locus is shown in Fig. 1. The composition of the plantaricin locus in L. plantarum 163 is similar to that of other L. plantarum strains, such as WCSF1, C11, and J51 [42]. pln1 is a previously unidentified gene; its signal peptide (MKNINNFQALQKNELSKVNGG) has a typical double-glycine leader- LSX2ELX2IXGG, which is a conserved sequence between other plantaricins and bacteriocins (Fig. 2), and this sequence is related to the secretion of bacteriocins [1]. The mature sequence of Pln1 is IWQWIVGGLGFLAGDAWSHSDQISSGIKKRKKKGYGY. The similarity was 48% compared to Enterocin Xbeta. Figure 1 Figure 2 3.2. Construction of recombinant plasmid and optimization of induction conditions Total genomic DNA from L. plantarum 163 was used for PCR amplification of the mature pln1 gene (Fig. 3); the PCR amplicon was of the expected length (145 bp). Sequencing data showed there was no base mutation in pln1 compared to the expected sequence. Figure 3

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The pln1 gene was cloned into the pET32a plasmid using the ExnaseII recombinase (Fig. 4), and transformed into E. coli DH5α. The recombinant pln1-pET32a plasmid was extracted and transformed into E. coli BL21 (DE3). Transformants were selected on LB plates containing ampicillin (100 µg/ml) and individual clones were further examined for recombinant protein expression. The western blot result is shown in Fig. 5. Figure 4 Induction conditions were optimized using different optical densities (OD630 reached 0.5, 0.6, 0.7, and 0.8), time (4, 6, 8, and 10 h), temperatures (20°C, 25°C, 30°C, and 37°C), and concentrations of IPTG (0.5 mM, 1 mM, 2 mM, and 4 mM). Expression of recombinant proteins was detected by SDS-PAGE and analyzed by GelAnalyzer. The results showed that all conditions tested above led to significant differences. When induction time was less than 6 h and OD630 was below 0.5, a low level of protein was expressed in the cytosol. The solubility of the recombinant protein was obviously reduced when induced at 37°C. The effect of IPTG concentration had less of an impact compared to other conditions (Fig. 6). In summary, the optimal induction conditions were an OD630 of 0.6, addition of 1 mM IPTG, and further growth for 10 h at 30°C. Figure 5 Figure 6 3.4 Expression and purification of Pln1 Pln1 was expressed in E. coli after IPTG induction as a full-length recombinant protein (21 kDa in size) with the fusion protein. The sonicated supernatant showed a significant band of the expected size on a gel. This suggested that the recombinant Pln1 protein could be soluble in PBS. Purification with PEI and ammonium sulfate showed that part of the protein had been removed, and the recombinant protein was concentrated. The recombinant protein was purified by Ni-NTA affinity chromatography, where most of the contaminating proteins did not bind to Ni-NTA and were washed away by the binding buffer with 50 mM imidazole. When the recombinant protein was treated with enterokinase, it was cleaved into two parts. The size of Pln1 was approximately 4 KD, and the fusion protein (TrxA) was removed using a HiTrap SP FF column. Tricine-SDS-PAGE was used to determine the efficiency of purification (Fig. 7). The yields of fused protein and cleaved peptides in the culture broth were in the range of 100~110 mg/l and 9~11 mg/l, respectively (Table 1). The results of RP-HPLC showed that Pln1 purified by the HiTrap SP FF column contained a low level of protein impurities. The highest peak appeared at 9.36 min (Fig. 8). Analysis of the peak area showed that more than 88% of the recovered protein was Pln1. Figure7

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Figure 8 3.5 Inhibitory activities of Pln1 The results of the current study showed that Pln1 had antimicrobial activity against gram-positive bacteria (Table 2) such as M. luteus CMCC 63202 and MSSE. Interestingly, it also showed activity against MRSE, which could be valuable in helping to solve the problem of antibiotic resistance. It also exhibited some activity against S. aureus ATCC29213, but the effect was not significant. Pln1 had antimicrobial activity against lactic acid bacteria, such as L. lactis NZ3900 and L. paracasei CICC 20241 and against L. innocua CICC 10417. Pln1 had no activity against gram-negative bacteria, such as E. coli ATCC35218/ATCC25922, P fluorescens AS 3.6452, and K. pneumoniae CICC 21519. Pln1 also showed no effect on some gram-positive bacteria, such as B. cereus AS 1.1846 and MRSA. Compared to nisin, Pln1 had better activity against M. luteus, MSSE, and MRSE; had the same activity against S. aureus; and had poor activity against lactic acid bacteria, E. coli, and B. cereus. Table 2. Antimicrobial spectrum and MIC of Pln1 and nisin Indicator bacteria Source Pln1 MIC (µg/ml) Nisin MIC (µg/ml) M. luteus CMCC 63202 100 ± 10a 210 ± 20b S. aureus ATCC 29213 475 ± 25a 500 ± 25a S. epidermidis JSPH 90 ± 15a 190 ± 20b Methicillin-resistant JSPH 180 ± 20a 330 ± 20b S. epidermidis (MRSE) LaboratoryL. lactis 55 ± 5a 7 ± 2b isolated L. paracasei CICC 20241 110 ± 10a 15 ± 2b L. innocua CICC 10417 40 ± 5a 15 ± 3b E. coli ATCC 35218 — 4500 ± 100 E. coli ATCC 25922 — 4300 ± 150 P. fluorescens AS 3.6452 — — K. pneumoniae CICC 21519 — — B. cereus AS 1.1846 — 800 ± 50

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Table 1. Purification of Pln1 by a series of steps

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3.6 Pln1 stability under different enzyme, pH, and temperature conditions Pln1 had antimicrobial activity and good stability in acidic conditions. There was no significant difference in stability on treatment at pH 1.0, 2.0, or 3.0 for 1 h. However, Pln1 was unstable under alkaline conditions. When treated at pH 12.0, 13.0, or 14.0 for 2 h, Pln1 lost most of its antimicrobial activity; for example, only 23% activity remained when on treatment at pH 14.0. Heat treatment progressively reduced the inhibitory activities of Pln1 at temperatures above 80°C. Only 66% activity remained after processing at 80°C for 1 h. However, Pln1 was stable below 60°C (Fig. 9). Pln1 was inactivated by proteolytic enzymes (proteinase E, proteinase K, pepsin, or papain). Figure 9

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4. Discussion In this study, a novel plantaricin gene, pln1, was identified from whole-genome sequencing data of L. plantarum 163 after a series of DNA and ORF analyses. Pln1 is a putative bacteriocin and could not be identified in the fermentation broth of L. plantarum 163 by RP-HPLC and MALDI-TOF-MS/MS. Therefore, heterologous expression was used in an attempt to solve this problem and obtain bioactive Pln1. The antimicrobial activity of Pln1 was predicted by CAMP, and all algorithms showed a strong possibility of antimicrobial activity. The spatial structure of Pln1 was predicted by SWISS MODEL, which showed that the C-terminus is an α helix and the N-terminus is a random coil. According to hydrophobicity results, the antimicrobial mechanism could be as follows: the C-terminus has more hydrophobic amino acids and forms an α helix which could be partitioning or binding to the cell membrane. The N-terminus has more hydrophilic amino acids and no fixed structure outside the cell membrane. This would create a force in the membrane to disrupt the membrane bilayer and associate and form pores in the membrane [43], resulting in leakage of cellular contents, thus limiting bacterial growth. Thioredoxin is often used to increase protein expression levels and solubility [44]. In this study, thioredoxin was chosen as a fusion partner since Pln1 is a small peptide and is susceptible to hydrolysis by proteases. Thioredoxin may also promote the formation of the correct structure of the fusion protein and reduce the toxic effects of recombinant peptides on the host cell [45]. Pln1 was expressed as a fusion protein (with thioredoxin) in E. coli BL21 (DE3). The fusion protein did not exhibit inhibitory activity on the indicator strains after purification using a Ni-NTA column.

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Methicillin-resistant JSPH — — S. aureus (MRSA) —, no inhibition. ATCC, American Type Culture Collection; CMCC, China Center of Medicine Culture Collection; AS, China General Microbiological Culture Collection Center; JSPH, Jiangsu Province Hospital. Lowercase letters indicate different statistical significances (overall P < 0.05, Duncan correction). Values in the same line with different letters indicate significant differences in antimicrobial activity between Pln1 and nisin.

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The thioredoxin was removed by enterokinase. Our results showed that the cleaved protein mixture and pure Pln1, which was purified using a HiTrap SP FF column, had antimicrobial activity against most gram-positive bacteria. This result represents a novel approach for building recombinant plasmids for small proteins and polypeptides using heterologous expression. Over the past few decades, bacteriocins have received considerable attention due to their potential applications in the food industry. However, isolation and purification of bacteriocins from their native hosts were time-consuming and difficult. A possible way to overcome these obstacles is to express the protein in a heterologous host. To date, all plantaricins that were expressed in a heterologous host were isolated and identified from their native hosts. In the current study, Pln1 was identified as a putative plantaricin that was analyzed at the genetic level and expressed in E. coli, and showed antimicrobial activity similar to that of nisin. The yield of Pln1 was 9~11 mg/l, which was a 7~9-fold increase as compared to the findings of Pal and others [6], indicating that the heterologous expression of bacteriocin could enhance the production yield while lowering the production cost. The activities of other plantaricins (E, F, J, K, SIK83, TF711, OL15) [46-49] isolated from their native hosts were monitored in arbitrary units (AU). Since there are various antimicrobial substances in any culture (such as plantaricins and other lactic acid bacteria), AU may not represent the accurate amount of plantaricin, thus making it difficult to determine which bacteriocin has antimicrobial activity. In this study, Pln1 was expressed and purified, its activity was clearly defined as protein concentration (µg/ml), and it was easy to compare its activity to other bacteriocins and in a range of indicator bacteria. The potential to use purified bacteriocins as food biopreservatives could also lead to synthetic chemical preservatives being replaced by natural antimicrobial peptides. Nisin is currently the only bacteriocin officially approved in the European Union as a food additive for protection against bacterial pathogens. In the current study, the activity of Pln1 against lactic acid bacteria was lower than that of nisin. However, Pln1 had greater activity (P < 0.05) in M. luteus and S. epidermidis. In addition, Pln1 showed strong antimicrobial activity in MRSE, a finding that has not been obtained for other plantaricins. Currently, staphylococcal infections account for a significant proportion of hospital-acquired infections, with S. epidermidis being the most common cause of bacteremia related to foreign bodies and indwelling medical devices. Nosocomial staphylococcal infections are associated with considerable morbidity and mortality, prolonging the duration of hospitalization and increasing costs. Data from the current study suggest that using Pln1 may represent a potential approach to solve the problem of antibiotic resistance of bacteria in hospitals. 5. Conclusions A putative plantaricin gene Pln1 was identified by a series of bioinformatics analyses. Pln1 was expressed abundantly in E. coli as a fusion protein, making it easier to isolate and purify the protein for subsequent structural and functional analyses. The yield of Pln1 was up to 9~11 mg/l, around ten-times higher than that for

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ACCEPTED MANUSCRIPT other plantaricins when they are expressed in heterologous hosts. In general, Pln1 exhibited activity similar to that of nisin, but Pln1 showed better antimicrobial activity against M. luteus, S. epidermidis, and methicillin-resistant S. epidermidis (P < 0.05). This technique therefore offers an attractive approach for future application in the food production industry.

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Acknowledgements The project was supported by the “Food biotechnology and enzyme engineering laboratory”. We thank all teachers and classmates for their supports and suggestions. We thank “Editage” for its manuscript language editing.

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[41] R.R. Burgess, M.P. Deutscher, Guide to protein purification,

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Fig. 2. Double-glycine leader of PLN1 compared with other bacteriocins Signal peptides of bacteriocins and plantaricins were subjected to BLAST using ClustalX. The signal peptide of PLN1 was same as the signal peptides of ORF3 in L. plantarum J51; however, the mature sequences were different.

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Fig. 1. Physical and genetic map of the pln locus of L. plantarum 163 The pln genes are shown by arrows corresponding to the approximate length of the genes. The pln locus in L. plantarum 163 contains five operons: plnLR, plnABD, plnEFI, plnSHSTUVWXY, and pln123. pln123 is a novel operon that is different from the operons of other strains of L. plantarum and it includes the putative plantaricin gene pln1.

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Fig. 3. Amplification of the pln1 gene Spectra multicolor low-range protein ladder was used as the marker; lane 1, pln1 amplified from genomic DNA of L. plantarum 163; lane 2, pln1 amplified from a positive clone of E. coli DH5α; lane 3, pln1 amplified from a positive clone of E. coli BL21 (DE3).

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Fig. 4. Construction of the recombinant plasmid The pln1 gene was cloned by PCR from L. plantarum 163 by using recombinant primers. The gray region of pln1 is the sequence homologous to pET32a. pln1 was cloned directly into pET32a by using ExnaseII.

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Fig. 5. Western blot of the fusion protein Pierce unstained protein MW marker was used for the blot. Lanes 1 and 2 contained sonicated supernatants after induction; lanes 3 and 4 contained fusion proteins purified by Ni-NTA.

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Fig. 6. Optimization of induction conditions PageRuler prestained protein ladder was used as the marker. Lane 1, total protein of E. coli BL21 (DE3) with recombinant plasmid before induction. A: Lanes 2-5, induction for different periods (4, 6, 8, and 10 h). Lanes 6-9, induction with different optical densities (OD630 was 0.5, 0.6, 0.7, and 0.8). B: Lanes 2-5, induction at different temperatures (20, 25, 30, and 37°C). Lanes 6-9, induction with different concentrations of IPTG (0.5, 1, 2, and 4 mM). The results are expressed as the relative yield of recombinant protein, as analyzed by GelAnalyzer. Fig. 7. Purification of Pln1 Spectra multicolor low-range protein ladder was used as the marker. Lane 1, sonicated supernatant; lane 2, sonicated supernatant purified by PEI and ammonium sulfate; lane 3, recombinant protein purified by Ni-NTA column; lane 4, recombinant protein treated with enterokinase; lane 5, enterokinase-treated protein purified by HiTrap SP

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Fig. 8. Quality control of purification by RP-HPLC The second peak that appeared at 9.36 min was PLN1, which was tested with indicator bacteria. Analysis of the peak area showed that more than 88% of the protein was PLN1.

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Fig. 9. Protein stability under different enzyme, pH, and temperature conditions All assays were carried out against Micrococcus luteus CMCC 63202. The activity of all positive controls was delimited to 100%. Titer equation: Y = 0.392X + 1.489 ; r2 = 0.994, Y is log(PLN1), and the concentration unit is µg/ml. X is the ring diameter of the inhibition zone.

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A novel bacteriocin gene, pln1, was identified using whole-genome sequencing data. The putative bacteriocin was successfully overexpressed in Escherichia coli. A yield of 9~11 mg/l was obtained for PLN1. Antimicrobial activity of PLN1 and its value as a food biopreservative were assessed. PLN1 had antimicrobial activity against some gram-positive and lactic acid bacteria.

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