KR100705776B1 - 1 Recombinant Pediocin PA-1 Having Antimicrobial Activity and Producing Method thereof - Google Patents

Abstract

The present invention provides a recombinant pediosin PA-1 and a method for producing the same, wherein the amino acid sequence comprising six consecutive histidine residues at the amino terminus exhibits antibacterial activity. According to the present invention, it can be stably expressed in E. coli, easy to purify, and does not require a separate cutting and purification process for active recovery, so the industrial effect is very large.

Recombinant Pediosin PA-1

Description

Recombinant Pediocin PA-1 Having Antimicrobial Activity and Producing Method

1 shows a block diagram of a recombinant vector pQEPED comprising a gene encoding recombinant pediosin PA-1 according to the present invention.

Figure 2 shows the base sequence of the recombinant vector pQEPED comprising a gene encoding a recombinant pediosin PA-1 and coding region of the recombinant pediosin PA-1.

Figure 3a is a result of performing a staining experiment (Bio-Safe ™ Coomassie, Bio-Rad, USA) after performing electrophoresis using recombinant pediosin PA-1 according to the present invention obtained by in vitro expression.

Figure 3b is the result of performing an active staining experiment after electrophoresis using the recombinant pediosin PA-1 according to the present invention obtained by in vitro expression.

M, standard protein; Lane 1, negative control; Lane 2, positive control (32 kDa protein made); Lane 3, using the vector (pQE-30 Xa, Qiagen, Germany) itself as a template; Lane 4, using recombinant DNA as a template; Lane 5, using recombinant DNA as a template and adding the transcription inducer isopropyl-β-D-thiogalactopyranoside (IPTG).

Figure 4a is a result of performing a staining experiment (Bio-Safe ™ Coomassie) by electrophoresis of the sample obtained by purification after purification of recombinant pediosin PA-1 according to the present invention by expression in E. coli by affinity chromatography .

Figure 4b is a result of performing an active staining experiment by electrophoresis of the sample obtained by purification after the recombinant pediosin PA-1 according to the present invention expressed in E. coli by affinity chromatography.

M, standard protein; CL, cell disruption supernatant; FT, flow through; W, wash; E, eluate.

The present invention relates to a recombinant pediosin PA-1 having an antimicrobial activity and a method for producing the same, more specifically, it can be stably expressed in E. coli, easy to purify, and separate cutting and purification processes for active recovery. The present invention relates to a recombinant pediosin PA-1 and a method for producing the same, which have a great industrial effect since it does not need to go through.

Pediocin PA-1 (pediocin PA-1) is a bacteriocin that has a wide range of antibacterial activity and particularly has a high antibacterial activity against Listeria .

To date, studies on heterologous expression of bacteriocins in other lactic acid bacteria have been carried out intermittently. However, expression studies in Escherichia coli for mass production are very poor.

In general, bacteriocin is unstable in E. coli due to cytotoxicity and small molecular weight, it is very difficult to express in the form of peptides. For this reason, all of the examples expressing bacteriocin in E. coli for the purpose of mass production have been limited to the expression of fusion proteins.

As such, in order to express bacteriocin in Escherichia coli, stability of bacteriocin is a particular problem, so it is expressed in the form of a fusion protein with other proteins, purified in the form of a fusion protein, and then separately cleaved and purified to obtain an active bacteriocin. The problem to be addressed is pointed out.

The present invention is presented to solve the problems of the prior art as described above,

An object of the present invention is to provide a recombinant pediosin PA-1 that can be stably expressed in E. coli, easy to purify, and does not require a separate cleavage and purification process for active recovery.

Another object of the present invention is to provide a method for producing recombinant pediosin PA-1 which can be stably expressed in E. coli, is easy to purify, and does not require a separate cleavage and purification process for active recovery.

The present invention to achieve the above object,

An amino acid sequence comprising a plurality of consecutive histidine residues at the amino terminus is combined to provide a recombinant pediosin PA-1 exhibiting antimicrobial activity.

According to the present invention, there is provided a recombinant pediosin PA-1 characterized in that six histidine residues in the amino acid sequence bonded to the amino terminus.

According to the present invention, the amino acid sequence that binds to the amino terminal provides recombinant pediosin PA-1, characterized in that consisting of 21 amino acids.

In another aspect, the present invention provides a recombinant pediosin PA-1 of SEQ ID NO: 2 that exhibits antimicrobial activity by binding to the amino terminal amino acid sequence comprising six consecutive histidine residues.

The present invention also provides a gene encoding recombinant pediosin PA-1 exhibiting antimicrobial activity by binding to an amino acid sequence comprising six consecutive histidine residues.

The present invention also provides a gene encoding recombinant pediosin PA-1 exhibiting antimicrobial activity by binding to an amino acid sequence comprising six consecutive histidine residues.

The present invention also provides an expression vector comprising a gene encoding a recombinant pediosin PA-1 exhibiting an antimicrobial activity by binding an amino acid sequence comprising a plurality of consecutive histidine residues at the amino terminus, thereby producing the recombinant vector. It provides a method for producing recombinant pediosin PA-1 having antibacterial activity by expression in E. coli.

According to the present invention, there is provided a method for producing recombinant pediosin PA-1, characterized in that the histidine residues in the amino acid sequence binding to the amino terminus are composed of six consecutive.

According to the present invention, the recombinant pediosin PA-1 provides a method for producing recombinant pediosin PA-1, characterized in that it has an amino acid sequence represented by SEQ ID NO: 2.

Hereinafter, the content of the present invention in more detail as follows.

The present invention provides a recombinant pediosin PA-1 capable of recovering pediosin PA-1 with a higher degree of purification in the purification process after expression in E. coli while having the same level of antimicrobial activity as before.

The recombinant pediosin PA-1 according to the present invention has a form in which an amino acid sequence (or peptide) containing a plurality of consecutive histidine residues is bound to the amino terminus of mature pediosin PA-1. The plurality of histidine residues provide high purity in the purification step using affinity chromatography after expression in E. coli.

According to the present invention, preferably, the contiguous histidine residue is about 6, and the amino acid sequence including the same is added to the amino terminus without losing the antibacterial activity of mature pediosin PA-1. According to a preferred embodiment of the present invention, the amino acid sequence containing a histidine residue and bonded to the amino terminus of mature pediosin PA-1 is composed of approximately 15 to 30 amino acids, more preferably in SEQ ID NO: 1 The amino acid sequence of 1-21 shown is included.

1 shows the structure of a recombinant vector containing recombinant pediosin PA-1 as an embodiment according to the present invention.

The recombination vector is a colony T5 promoter (PT5), lock operator (lac O), ribosomal binding site (RBS), start codon (ATG), 6 histidine residues (6x His), factor Xa protease recognition site (FXa recognition site, Ile-Glu-Gly-Arg), pQE-30 Xa vector (Qiagen) with multicloning sites (MCS) and stop codons with restriction enzyme recognition sites at both ends of Stu I and Hind III It was used and prepared by inserting the mature pediosin PA-1 gene ( pedA ) in the Stu I and Hind III site of the cloning site.

As the mature pediosin PA-1 gene, amplified by PCR using a known plasmid related to pediosin PA-1 as a template can be used. At this time, the primer for the amplification reversely put the restriction enzyme Hind III recognition site for cloning, PCR amplification products can be purified by treatment with Hind III restriction enzyme and then used as an insertion gene.

Figure 2 shows the amino acid sequence of the gene structure and protein of the recombinant vector containing the recombinant pediosin PA-1.

Six consecutive histidine residues are located at 178-195 and the mature PedA gene is located at 229-360. The sequence of mature pediosin PA-1 is as described in SEQ ID NO: 3 (or the peptide sequence is SEQ ID NO: 4), and the peptide sequence linked to the amino terminus is composed of 21 amino acids of N-MRGSHHHHHHGSGSGSGIEGR-C. Can be. These sequences confirmed that the antimicrobial activity of the mature pediosin PA-1 did not affect the experimental results of the present invention, the six consecutive histidine residues in the inside facilitate the purification of recombinant pediosin PA-1.

The present invention also provides a gene encoding recombinant pediosin PA-1 exhibiting antimicrobial activity by binding to an amino acid sequence comprising six consecutive histidine residues. These genes may be various combinations of genes encoding for expression of the polypeptide according to codon usage provided according to the type of host to be used for transformation. According to an embodiment of the present invention the gene comprises the gene sequence of SEQ ID NO: 2.

The present invention also provides a method for producing an expression vector containing the recombinant pediosin PA-1 gene, and transforming it into E. coli to produce a recombinant pediosin PA-1 gene. The process of transforming E. coli using a specific expression vector is a technique that is widely known. In the present invention, by introducing a recombinant vector into E. coli according to a known technique, the recombinant pediosin PA-1 is expressed therein, and then crushed and subjected to histidine affinity chromatography from the supernatant after centrifugation. Recombinant pediosin PA-1 can be recovered.

All parts (promoter, regulatory sequence, stabilization sequence, signal sequence or termination sequence, etc.) of the gene construct of the suitable recombinant vector of the present invention can be mutated to change their regulatory properties using known techniques, if necessary.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

<Example 1>

PQE-30 Xa (Qiagen) expression vector was used to express histidine-labeled recombinant pediosin PA-1 in Escherichia coli. The expression vector was simultaneously digested with restriction enzymes Stu I and Hind III to introduce a structural gene for pediosin PA-1.

The structural gene of pediosin PA-1 was amplified by PCR using only the nucleotide sequence corresponding to the active pediosin PA-1 as a template for the pedicine PA-1 production-related plasmid. In the reverse primer for amplification, a restriction enzyme Hind III recognition site was artificially inserted for cloning. PCR amplification products were purified by Hind III restriction enzyme and used as insertion gene. The recombinant vector pQEPED as shown in FIGS. 1 and 2 was confirmed by transforming Escherichia coli by connecting the vector treated with the restriction enzyme and the insert gene.

Primer set

pedSF: AAA TAC TAC GGT AAT GGG GTT AC (SEQ ID NO: 5)

pedSR-Hind III: CCC AAG CTT GGG CTA GCA TTT ATG ATT ACC TTG ATG TCC (SEQ ID NO: 6)

In vitro transcription & translation experiments were performed using recombinant DNA (pQEPED; pQE-30 Xa :: mature pedA ) shown in FIG. 2.

The experiment was performed using a kit from Qiagen (EasyXpress ™ protein synthesis mini kit). After adding the reaction buffer solution to the E. coli cell extract, 0.5 ㎍ of recombinant DNA (pQEPED) was added, adjusted to 50 ㎕ of the final volume with water, and then reacted at 37 ° C for 1 hour. After the reaction, the reaction was stopped by standing on ice.

Electrophoresis was performed using the results of in vitro transcription and translation experiments, and staining with staining solution (Bio-Safe ™ Coomassie, Bio-Rad, USA) showed that the desired peptide could not be identified (Fig. 3a), it was confirmed that the inhibitory ring was formed at the position of about 6.5 kDa in the simultaneous active staining (lanes 4 and 5 of FIG. 3b).

These results indicate that recombinant pediosin PA-1 was produced from the recombinant DNA prepared according to the present invention, indicating that the produced recombinant pediosin PA-1 has antibacterial activity.

<Example 2>

E. coli transformation using recombinant DNA used a known heat-shock method. E. coli JM109 strain purchased from Yeastern Biotech Co., Ltd. (Taipei, Taiwan) was mixed with 1-2 μl of recombinant DNA, left on ice for 5 minutes, left at 42 ° C for 45 seconds and then antibiotics (Ampicillin, 100 ㎍ / ml ) Plated onto LB plate media. Colonies grown after overnight incubation were inoculated to select E. coli transformed with the recombinant DNA as described above.

After expressing recombinant pediosin PA-1 in Escherichia coli transformed with recombinant DNA (pQEPED), a cell disruption supernatant was prepared under denatured conditions, and subjected to affinity chromatography (nickel-nitrilotriacetic acid metal-affinity chromatography). ), It was confirmed that recombinant pediosin PA-1 having antimicrobial activity was obtained in a high yield (Fig. 4a, b and Table 1).

TABLE 1

Fraction Capacity (ml) Protein concentration (mg / ml) Active (AU / ml) Total activity (AU) Relative Activity (AU / mg) Purification degree yield(%) Total protein amount (mg) cell 100 Cell disrupted supernatant 3.8 2.9 3,000 11,400 1,034 One 100 11.02 Purified liquid 2 0.24 5,000 10,000 20,833 20 87.7 0.48

According to the present invention, it can be stably expressed in E. coli, can be easily purified, and can provide a recombinant pediosin PA-1 having a great industrial effect because it does not need to undergo a separate cutting and purification process for active recovery. If the safety of recombinant pediosin PA-1 is secured, it is expected that it can be used in various ways as a natural antibacterial agent in food.

Attach sequence list electronic file  

Claims (9)

delete delete Recombinant pediocin PA-1 exhibiting antimicrobial activity wherein the amino acid sequence of MRGSHHHHHHHGSGSGSGIEGR is bound to the amino terminus of SEQ ID NO: 4. A gene encoding the recombinant pediosin PA-1 of SEQ ID NO: 2, which exerts antimicrobial activity in which the amino acid sequence of MRGSHHHHHHHGSGSGSGIEGR is bound to its amino terminal. delete delete An expression vector comprising a gene encoding a recombinant pediosin PA-1 exhibiting antimicrobial activity, in which the amino acid sequence of MRGSHHHHHHHGGSGSGSGIEGR is bound to the amino terminal of SEQ ID NO: 4, was prepared. How to produce pediosin PA-1. delete delete

Images