Construction, Expression and Purification of Recombinant Pre-mature Peptide of Plantaricin F From Lactobacillus Plantarum S34 in Escherichia Coli

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F's leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.


INTRODUCTION
Lactic acid bacteria (LAB), a group of gram-positive bacteria, have received special attention to date because of their ability to produce bacteriocins that can be applied as a natural preservative agent (da-Silva et al. 2014). The usages of LAB and their metabolic products as anti-food contaminations were due to the GRAS (Generally Regarded as Safe) state received (Zacharof and Lovitt 2012). Furthermore, LAB have been isolated from various kinds of traditional Indonesian fermented foods such as shrimp paste terasi , fermented fish and meat bekasam , and soy mash tempe habitated with Tetragenococcus halophilus, Tetragenococcus muricatus, and Lactobacillus plantarum (Roling et al. 1996;Kobayashi et al. 2003;Candra et al. 2007;Mustopa et al. 2010).
Class IIb bacteriocins produced by LAB are mostly applied in food to prevent microbial contamination due to their strong antibacterial activity (Abee et al. 1995;Cleveland et al. 2000). These unique peptides have a conserved N-terminal, named GxxxG motifs in its mature peptide form (Fimland et al. 2008). These are such as plantaricins EF (PlnEF) and plantaricins JK (PlnJK) that belong to a large group of small, heatstable, cationic, non-lantibiotic peptides (Pal and Sheela 2014) and produced by different strains of L. plantarum (Anderssen et al. 1998).
The conventional procedures to collect native plantaricin peptides directly from L. plantarum using ammonium sulphate precipitation followed by chromatography-based purifications have recently been established with excellent biological activity and high in protein purity obtained (Atrih et al. 2001;Gong et al. 2010;Hata et al. 2010;Zhu et al. 2014). However, the procedures are hard to be applied to produce native plantaricins directly isolated from natural producers with high yield (Tiwari and Srivastava 2008).
These conventional methods if applied in industrial process for large scale production would be contraproductive because of time consuming, laborious process, high cost and difficulty in the purification steps (Li and O Sullivan 2002). To overcome the constraints, efforts had been done for example to heterologously produce plantaricins in quite different cells, such as Escherichia coli (Fang et al. 2010) that offer more rapid, high yield and economical production of recombinant proteins (Sivashanmugam et al. 2009).
Contrary with another type of class II bacteriocins such as lactation, enterocin, mesentericin, nisin, divercin and sakacin which had been well established expressed heterologously in any type of cells (Richard et al. 2004;Fimland et al. 2008;Rogne et al. 2009;Basanta et al. 2010;Fang et al. 2010;Borrero et al. 2012). In attempt to increase the yield of bacteriocin peptides, their heterologous expression in procaryotic species have been widely studied (Richard et al. 2004;Kloke et al. 2005). But until now, little study is known to report heterologous expression of plantaricin especially in the form of their pre-mature peptide state (Fimland et al. 2008;Fang et al. 2010). The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from L. plantarum S34 in E. coli.

Construction of Recombinant Vector
A plantaricin F gene isolated from bekasam-derived L. plantarum S34 was subcloned into pGEM-T (pGEM-T-pln EF) then the pln F gene was amplified from pGEM-T-pln F by PCR using a primer pair 5'-GACTGGATC CATGAAAAAATTTCTA GTTTT-3' as sense primer and 5'-GATCAAGCTTCTATCCGTGGAT GAATCC TC-3' as anti-sense primer. The primers contained upstream BamHI and downstream HindIII restriction site (underlined nucleotides). To prevent protein fusion between the recombinant pln F with Cterminal His tag protein, strong stop codons TGA were incorporated at 3-end of the anti-sense primer. All DNA manipulations were carried out according to the procedures described by Sambrook et al. (2001). PCR profile was 94°C, 1 minute of denaturation; 63°C, 1 minute of annealing; extension 72°C for 30 seconds and repeated for 35 cycles. The PCR product and vector pET32 a (Novagen) were cut with BamHI and HindIII enzymes. After purification with a Qiaquick ® PCR purification kit (Qiagen, ICI Americas Inc), the digested pln F gene and vector were ligated using T4 DNA ligase (Takara) and the ligation mixtures were transformed into competent E. coli BL21 (DE3) pLysS (Novagen). Recombinant plasmid-containing bacteria were initially selected on LB agar with ampicillin (100 mg mL -1 ) then the recombinant bacteria growth on the selection medium was subjected to PCR colony using the same primer as described above. The recombinant pET32a-pln F from PCR colony test was sequenced to confirm the inserted pln F gene nucleotide sequence.
Five hours after IPTG induction, recombinant bacteria were harvested by centrifugation at 8000 g, 4°C for 10 minutes. The pellets from recombinant bacteria were resuspended with lysis buffer pH 8 containing 20 mM Tris-HCl, 500 mM NaCl and 20 mM imidazole directed to sonication (0.5 cycles of 30 outputs, 15 s pulse on and 60 s pulse off). The lysate was then centrifuged at 18,000 g, 4°C for 30 minutes. After that, the supernatant was collected and the pellet was resuspended in the same lysis buffer. Pellet and supernatan fractions were separated using 12% SDS polyacrylamide gel electrophoresis (SDS PAGE).

Purification of Recombinant pln F
The purification method was done according to a previous study by Utama et al. (2000) and Li et al. (2010) with modification. Supernatant fraction was resuspended with resin Co 2+ (TALON) and bound on a rotator for 3 hours at 4°C. After that, the sample was centrifuged at 7000 g, 4°C for 7 minutes. The resulting supernatant was discarded, and the resin was resuspended in wash buffer (Tris HCl 20 mM pH 8, 500 mM NaCl, 60 mM imidazole). Subsequently, the supernatant was centrifuged at 7000 g, 4°C for 5 minutes. The washing process was repeated two times. The resin was then resuspended with elution buffer (Tris HCl 20 mM pH 8, 500 mM NaCl, 250 mM imidazole) and bound on a rotator at 4°C for 12 hours. Subsequently, the fraction was centrifuged at 7000 g, 4°C for 5 minutes. The supernatant was taken and then dialyzed using dialyzed buffer (500 mM Tris HCl, pH 8,10 mM CaCl 2 , 1% Tween-20 (v/v)).

SDS-PAGE
This method was used to determine whether recombinant pln F was expressed or not. The fractions from expression and purification of recombinant pln F were collected, then the mixture was dissolved with loading buffer (50 mm Tris HCl, pH 6.8, 2% SDS, 10% glycerol, 1% mercapto ethanol, 12.5 M EDTA, 0.02% Bromophenol blue, H 2 O). The fraction was boiled for 15 minutes, then separated by electrophoresis using 12% SDS-PAGE of a vertical electrophoresis (Mupid-eXu, Advance Japan). The gel was then stained with coomassie blue staining solution overnight distained with staining buffer for 30 minutes, three times. The gel picture was then taken using a digital scanner (Canon MP258, Canon).

Expression of Recombinant pln F in E. coli
pET32a containing Trx-plnF fusion protein for the expression was shown in Figure 1. A pre-mature pln F fragment containing BamHI and HindIII restriction sites with 159 bp in size was obtained by PCR amplification (Fig. 1a). Then the PCR product was sub-cloned into the pET32a expression vector, and the insertion was confirmed by double digestion using BamHI and HindIII restriction enzymes. Recombinant DNA sequence inserted was verified by DNA sequencing.
We had constructed and expressed pln F which consisted of leader and mature peptide (Fig. 1b) as protein fusion with thioredoxin-(His) 6tag at N-terminus in E.coli with a high degree of solubility as seen in the soluble fraction of supernatant obtained from lysed-recombinant E.coli. Fimland et al. (2008) had produced recombinant mature pln F fused to a fusion partner protein called GB1 using pGEV2 with 1 mM IPTG for induction, but the recombinant protein obtained was forming inclusion body. This contrary result with our study indicated that the difference in fusion partner protein, vector expression and IPTG used would impact on the degree of solubility of recombinant pln F produced. Interestingly, because of recombinant pln F in our study expressed as premature peptide, we predicted that the leader peptide itself may also have function as a guide to chaperon like-protein on translocation machinery thus helping proper folding of pln F (Mathiesen et al. 2008).
After heat shock treatment to deliver pET32a-pln F into the E. coli BL21 (DE3) pLysS, the targeted recombinant protein of pln F appeared as two dominant bands. The first band was +24 kDa which composed of recombinant pre-mature peptide of pln F and thioredoxin-(His) 6tag fusion protein and the second band was ±20 kDa contained only leader peptide. The fusion protein indicated that some of mature peptides of pln F have been proteolytically cleaved from its pre-mature state by a system in E.coli (Fig. 2a) and those two protein bands were not appeared in recombinant E.coli which was only transformed by pET32a(+) with no insert of premature pln F gene. Expression of recombinant protein fused with partner protein such thioredoxin-(His) 6tag would give several advantages compared to the unfused recombinant protein. As described by Cao et al. (2005), Khow and Suntrarachun (2012), and Tao et al. (2014), those benefits included (1) helping to increase solubility and proper folding of recombinant protein expressed, (2) protecting them from being degraded by intracellular protease and (3) simplifying the purification of recombinant protein. To obtain maximum production of recombinant pln F we tested different values of temperature incubation, IPTG concentration and incubation time. As shown in Figure 2 the recombinant pln F expression was temperature dependent with a protein level reached maximum value at 22°C (Fig. 2b, lane 6). For IPTG optimization, the yield of recombinant pln F protein was highest when 0.5 mM IPTG was added into the medium (Fig. 3a, lane 5). Meanwhile, 5 hours culture of recombinant E.coli containing pET32a-pln F at 22°C after 0.5 mM IPTG addition gave the best protein recombinant expression as pointed out in Figure 3b. The optimum condition obtained (induction temperature was 22°C, induction time was 5 hours, and IPTG concentration was 0.5 mM) then applied for production and purification of fused recombinant pln F.
In attempt to obtain the optimum production of recombinant pre-mature pln F, here we also had optimized critical parameters that contribute directly to the successfulness of protein expression in E.coli such as induction temperature, induction time and IPTG concentration addition (Sambrook and Russell 2001;Sivashanmugam et al. 2009). As we expected, the induction temperature where recombinant pln F was produced by recombinant E. coli had a prominent effect on protein expression yield and solubility. Induction in 37°C resulted in very thin recombinant protein band on SDS-PAGE result, meanwhile lowering the induction temperature to 22°C (IPTG was 0.5 mM and induction time was 5 hours) resulted in a thicker recombinant protein band. Recombinant mature pln F obtained from Fimland et al. (2008) was produced as inclusion body due to inappropiate induction temperature applied (37°C). Another study conducted by Rogne et al. (2009) also obtained similar result for recombinant pln J and K collected from 37°C incubated-recombinant E.coli. Tao et al. (2014) stated that lowering the induction temperature during IPTG added-medium culturing would help to increase solubility of recombinant protein expressed as much as +80%, although it would also impacte directly on the capability of recombinant cells to proliferate of being slow down compared to high temperature (Chen et al. 2012).
Construction and heterologous expression of mature pln F had well been established by other authors with excellent results (Fimland et al. 2008;Fang et al. 2010), yet no information was provided related to its heterologous expression as pre-mature peptide of pln F until today. We assumed that the expression of pln F as its pre-mature state, if any, would give essential information regarding biological function of homologous leader peptide in heterologous cells. For examples some leader peptides had been proven being acting as a molecular guide in properly directing of pre-mature peptides to the secretory machinery system, the process is called Fig. 3. The effects of IPTG and incubation time on recombinant pln F expression at induction temperature of 22°C, (a) lanes 1 to 3 were supernatants from the lysed-recombinant E. coli; lanes 4 to 5 were elution fractions of TALON-treated supernatant fraction after E. coli induction with IPTG at 0.1, 0.5 and 1 mM, (b) the effect of different incubation times. Lanes 1 to 5 were the supernatants of lysed-recombinant E.coli expressed for 1, 2, 3, 4, and 5 hours after addition of 0.5 mM IPTG; Lane 6 was pET32a with not insert pln F gene; lanes 7 to 11 were elution fractions of TALON-treated supernatant fraction expressed for 1, 2, 3, 4, and 5 hours after addition of 0.5 mM IPTG. M was molecular marker mass peptide (BIO-RAD). protein maturation and secretion (Belkum et al. 1997;Mathiesen et al. 2008). As explained above, pre-mature peptide of pln F had been successfully constructed in pET32a vector and expressed in E.coli BL21 (DE3) pLysS. The functionality of leader sequence within premature recombinant pln F had a size + 20 kDa bellowed from fused pre-mature of recombinant pln F band (Fig. 3a). We predicted this additional protein band was a union of thioredoxin-(His) 6tag fusion partner protein with S-tag and enterokinase cleavage site (total size was 18.15 kDa) followed by leader peptide sequence (size was 1.98 kDa) without recombinant mature of pln F. We predict that recombinant mature pln F may be secreted from intracellular part of the E.coli into the culture medium. According to Ennahar et al. (1996) and Kelly et al. (1996), the function of leader sequences of bacteriocins was not only as an inhibitor of antibacterial activity of the bacteriocins against their producer host cells, but also provided a recognition signal to the ABC transporter in maturation and secretion processes. The incomplete proteolytic digestion of a fused recombinant premature peptide of pln F in our study indicated the lack of recognition of pre-peptide of bacteriocin by domain of ABC transporter in heterologous cells (Belkum et al.1997).

Purified Recombinant pln F
To purify the recombinant pln F, the soluble fraction in each case was subjected to Co 2+ affinity coloum purification. The bound proteins were eluted in 200 mM of imidazole. At this optimum concentration of imidazole, the purity of target protein could be recovered significantly. As shown in Fig. 4 the fused recombinant pln F had been purified, in two forms of dominant protein bands, from the supernatant of intracellular part of lysed cells although some of unspecific bands were also co-purified in our procedure. This result highlighted that both forms of fused recombinant pln F had succeeded to be purified using this method.
Pre-mature plantaricin F peptide fused with 6× histidine theriodoxin has been successfully expressed and purified in E. coli BL21 (DE3) pLysS by using pET32a as an expression vector. In this study, the expression and purification of recombinant pln F in E. coli BL21 (DE3) pLysS was investigated. The expressed recombinant pln F has a molecular mass of 24 kDa when separated on SDS-PAGE. The recombinant pln F produced was temperature dependent and the level of recombinant plnF production could be enhanced when the host was induced by 0.5 mM IPTG and 25°C incubation and reached optimal at 22°C incubation.

CONCLUSION
Recombinant pre-mature pln F peptide was successfully expressed in Escherichia coli BL21 (DE3) pLysS by using pET32a as an expression vector. The optimum condition of expression was 0.5 mM of IPTG for 5 hours at 22°C. This recombinant peptide was successfully purified from E. coli cell.