Promoter polynucleotides, signal polypeptides, and uses thereof

Abstract

A novel signal polypeptide is provided that can express and secrete a foreign product at a high level in lactic acid bacteria. The present invention provides a signal polypeptide having a specific amino acid sequence, a polynucleotide encoding the signal polypeptide, a vector and host cell containing the polynucleotide, and a method for producing a foreign protein by culturing the host cell.

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2017-0184819, filed with the Korean Intellectual Property Office on December 29, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a promoter polynucleotide, a signal polypeptide, and uses thereof. [Background technology]

[0003] Microorganisms such as bacteria, yeasts and molds are becoming increasingly important as hosts for recombinant expression. Bacteria such as Lactobacillus sp. or Streptococcus sp. are useful as delivery vehicles. Additionally, generally recognized as safe (GRAS) microorganisms may be administered to humans or animals.

[0004] In order to achieve high levels of foreign product expression in lactic acid bacteria, there is a need for new promoters and signal polypeptides isolated from lactic acid bacteria that are capable of expressing and secreting foreign products, particularly proteins, at high levels. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect provides an isolated promoter. Another aspect provides a recombinant polynucleotide comprising the promoter. Another aspect provides a host cell containing the recombinant polynucleotide. Another aspect provides a method of producing a product using the host cell. Another aspect provides isolated signal polypeptides, and polynucleotides encoding same. Another aspect provides a recombinant polynucleotide comprising a polynucleotide encoding the isolated signal polypeptide. Another aspect provides a host cell containing a recombinant polynucleotide encoding the isolated signal polypeptide. Another aspect provides a method for producing a protein using a host cell containing a recombinant polynucleotide encoding the isolated signal polypeptide. [Means for solving the problem]

[0006] One aspect provides an isolated promoter comprising a polynucleotide having 85% or greater sequence identity to the nucleotide sequence of SEQ ID NO:1 (hereinafter also referred to as the "PR4 promoter").

[0007] Another aspect provides a recombinant polynucleotide comprising the promoter. As used herein, "promoter" refers to a region on a nucleic acid molecule, particularly a DNA molecule, to which RNA polymerase binds and initiates transcription. A promoter is generally located upstream, i.e., 5', of a transcribed sequence that is regulated by the promoter. The promoter may also be a constitutive promoter. The promoter may also have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence of SEQ ID NO:1. The promoter may also have the nucleotide sequence of SEQ ID NO:1.

[0008] The recombinant polynucleotide is also a vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. The vector includes not only a vector inserted into the genome of a host cell into which it is introduced, but also a vector acting as a self-replicating nucleic acid structure. An expression vector refers to a vector capable of directing the expression of a nucleic acid operably linked thereto. The vector may be a plasmid or a vector derived from a virus.

[0009] The vector may be a cloning vector or an expression vector. The expression vector may comprise a nucleotide sequence encoding a protein operably linked to the promoter.

[0010] The expression vector also includes the promoter and a first polynucleotide comprising a nucleotide sequence encoding a product, the first polynucleotide being operably linked to the promoter. The product includes anything that can be produced by expression of the first polynucleotide. The product can be a polypeptide or a nucleic acid. The polypeptide can be a cytokine, such as IL-10, or an enzyme, such as amylase. The nucleic acid can be DNA or RNA.

[0011] As used herein, the term "operably linked" refers to a linkage that allows transcription or translation to occur and produces a functional transcription or translation product.

[0012] The vector may further include one or more selected from the group consisting of a ribosomal binding site (RBS), a cloning site, a selection marker gene, a transcription terminator, and a translation initiation factor. The cloning site is also operably linked to the promoter. The cloning site is also a multiple cloning site.

[0013] The recombinant polynucleotide also has a second polynucleotide (hereinafter also referred to as "SP4 signal polypeptide") operably linked between the promoter and the first polynucleotide, the second polynucleotide encoding a signal polypeptide comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:2. In this case, the first polynucleotide also encodes a polypeptide.

[0014] As used herein, the term "signal polypeptide" refers to a sequence present at the N-terminus of a secretory protein precursor, but not present in a naturally occurring mature protein. The signal polypeptide may be cleaved from the protein precursor. In general, a signal polypeptide is cleaved by a protease when secreted outside a cell. The protease is also generally referred to as a signal peptidase. The signal polypeptide may have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity with the amino acid sequence of SEQ ID NO: 2. The signal polypeptide has an activity of secreting an expression product of a gene fused in frame with a nucleotide sequence encoding the signal polypeptide outside a cell.

[0015] As used herein, "secretion" of a protein or polypeptide molecule includes the transport of the protein or polypeptide molecule to the outside of the bacterial cell, the protein or polypeptide molecule being present in a completely free form in the culture medium, only a portion of the protein or polypeptide molecule being present outside the bacterial cell, and the protein or polypeptide molecule being present on the surface layer of the bacterial cell.

[0016] The signal polypeptide may be derived from Lactobacillus paracasei and may have secretion promoting ability. The second polynucleotide may have the nucleotide sequence of SEQ ID NO:3. Another aspect provides a host cell comprising the recombinant polynucleotide. The host cell may be a bacterial cell. The bacterial cell may be a Gram-positive bacterium. The bacterial cell may be a Lactobacillus or Escherichia genus. The Lactobacillus may be from the genera Lactobacillus, Lactococcus, Bifidobacteria, Streptococcus, Leuconostoc, Weissella, Pediococcus or Enterococcus.

[0017] The recombinant polynucleotide may be introduced into the host cell by any conventional nucleic acid transfer method, including electroporation, transformation, transduction or transfection.

[0018] Another aspect provides a method for producing a product or a metabolite thereof, comprising culturing the host cell in a medium to produce a product, and isolating the product or a metabolite thereof from the culture. The product may be any product produced by expression of a first polynucleotide. The product may be a polypeptide or a nucleic acid. The polypeptide may be a cytokine, such as IL-10, or an enzyme, such as amylase. The nucleic acid may be DNA or RNA. The metabolic product may be a substance produced by the product exerting its activity in a cell. For example, if the product is an enzyme, the metabolic product may be a direct product produced by the exertion of the enzymatic activity, or a substance produced by a metabolic pathway to which the enzyme belongs.

[0019] In the method, the culturing step may be performed by a general method known in the art depending on the host cell selected. The medium used for the culturing may contain, individually or as a mixture, carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, as a sugar source; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil; fatty acids such as palmitic acid, stearic acid, linoleic acid; organic acids such as glycerol, acetic acid. The medium may contain, individually or as a mixture, peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal and urea, as a nitrogen source, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate. The medium may contain, individually or as a mixture, a phosphorus source, such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salt. The medium may contain, for example, metal salts necessary for growth, such as magnesium sulfate or ferrous sulfate. In addition, essential growth substances such as amino acids and vitamins, or appropriate precursors, are added to the culture during the cultivation process in an appropriate manner, for example, batchwise or continuously.

[0020] The cultivation may be carried out under aerobic conditions, microaerobic conditions, anaerobic conditions, or a combination thereof.

[0021] The method may further include a step of isolating the product from the culture. The isolation may be performed by selecting an appropriate method depending on the type of product selected. When the product is a protein, the isolation may include centrifuging the culture medium to remove cells and isolating the protein from the supernatant, or harvesting and disrupting the cells to isolate the protein. The isolation may involve one or more of salting out, precipitation, chromatography, centrifugation, and filtration. The chromatography may be one or more of anion exchange, cation exchange, size exclusion, and affinity chromatography.

[0022] Another aspect provides an isolated signal polypeptide comprising an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 2. The signal polypeptide may also have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0023] Another aspect provides a polynucleotide encoding the signal polypeptide. The polynucleotide encoding the signal polypeptide may have the nucleotide sequence of SEQ ID NO:3.

[0024] Another aspect provides an expression vector comprising a second polynucleotide encoding the signal polypeptide and a first polynucleotide encoding a protein, wherein the second polynucleotide is operably linked to the promoter and the first polynucleotide is fused in-frame with the second polynucleotide.

[0025] Another aspect provides a host cell comprising the expression vector. The host cell may be a bacterial cell. The bacterial cell may be a Gram-positive bacterium. The bacterial cell may be a Lactobacillus or Escherichia genus. The Lactobacillus may be a Lactobacillus, Lactococcus, Bifidobacteria, Streptococcus, Leuconostoc, Weissella, Pediococcus or Enterococcus genus.

[0026] The recombinant polynucleotide can be introduced into the host cell by conventional nucleic acid transfer methods, including electroporation, transformation, transduction or transfection.

[0027] Another aspect provides a method of producing a protein comprising culturing the host cell in a culture medium to produce the protein, and isolating the protein from the culture.

[0028] In the method, the culturing step may be performed by a general method known in the art depending on the host cell selected. The medium used for the culturing may contain, individually or as a mixture, carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch as a sugar source; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil; fatty acids such as palmitic acid, stearic acid, linoleic acid; organic acids such as glycerol, acetic acid. The medium may contain, individually or as a mixture, peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal and urea as a nitrogen source, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate. The medium may contain, individually or as a mixture, a phosphorus source, such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salt. The medium may contain, for example, a metal salt necessary for growth, such as magnesium sulfate or ferrous sulfate. In addition, essential growth substances such as amino acids and vitamins, or appropriate precursors, are added to the culture during the cultivation process in an appropriate manner, for example, batchwise or continuously.

[0029] The cultivation may be carried out under aerobic conditions, microaerobic conditions, anaerobic conditions, or a combination thereof.

[0030] The method may further comprise isolating the protein from the culture. The isolation may involve centrifuging the culture to remove the cells and isolating the protein from the supernatant, or alternatively harvesting and disrupting the cells and isolating the protein. The isolation may involve one or more of salting out, precipitation, chromatography, centrifugation and filtration. The chromatography may be one or more of anion exchange, cation exchange, size exclusion and affinity chromatography. Effect of the Invention

[0031] The isolated promoter according to one aspect, and recombinant polynucleotides containing the same, can be used for efficient expression of foreign genes.

[0032] According to another aspect, the host cell containing the recombinant polynucleotide is capable of efficiently expressing a foreign gene.

[0033] According to another aspect, the method of producing a product using the host cell can be used to efficiently produce the product.

[0034] According to another aspect, the isolated signal polypeptide, the polynucleotide encoding the same, and the recombinant polynucleotide comprising the same can also be used to secrete a foreign protein outside a cell. A host cell containing a recombinant polynucleotide that includes a polynucleotide encoding the isolated signal polypeptide according to another aspect can efficiently secrete the product of a foreign gene outside the cell.

[0035] According to another aspect of the present invention, a method for producing a protein using the host cell can efficiently produce the protein. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 shows the structure of pMT48, a shuttle vector between E. coli and lactic acid bacteria. [Diagram 2] FIG. 1 shows the structure of the pMT54-PR4-IL10-SP4 vector. [Diagram 3] FIG. 1 shows the results of transforming three types of lactic acid bacteria with the pMT54-PR4-IL10-SP4 vector and examining the level of extracellular expression. [Figure 4] FIG. 1 shows the results of transforming the LMT1-21 strain with the pMT54-PR4-amylase-SP4 vector and examining the level of extracellular expression. [Diagram 5]13 is a graph showing the measurement of IL-10 mRNA levels in the pMT54-P11-IL10-SP4 vector and the pMT54-PR4-IL10-SP4 vector for comparison of promoter strength. [Figure 6] 1 is a graph showing the measurement of IL-10 secreted from LMT1-21 transformed with pMT54-PR4-IL10-SP4 and pMT54-PR4-IL10-USP45 for comparison of signal peptide strength. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0037] The present invention will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples. Example 1: Cloning of promoter and signal polypeptide and confirmation of their effects 1. Cloning of promoter and signal polypeptide The promoter and the nucleotide sequence encoding the signal polypeptide were amplified by PCR. Specifically, a 593 kb-sized amplification product was obtained by PCR using the genome of Lactobacillus paracasei LMT1-21 (Accession No. KCTC 13422BP) as a template and primers. The primers used were PS4_F / R (SEQ ID NO: 4 and 5).

[0038] The amplified product was ligated to pMT54 vector cleaved with EcoRV and SalI via infusion cloning (Clontech). The vector was then transformed into E. coli Top10 strain (Invitrogen) by the method of Sambrook et al. (Sambrook et al. Molecular cloning: A laboratory Manual, 2nd edition, 1989). The transformed E. coli was then smeared on an LB plate containing 10 μg / ml chloramphenicol to obtain colonies. The pMT54 vector was recovered from the obtained colonies and analyzed for sequence. As a result, it was confirmed that it contained a nucleotide sequence (SEQ ID NO: 3) encoding PR4 (SEQ ID NO: 1). Hereinafter, the vector is referred to as MT54-PR4-SP4 vector.

[0039] The pMT54 vector is a vector in which a multiple cloning site (SEQ ID NO: 6) is introduced into the HindIII enzyme and XhoI enzyme sites of the pMT48 vector. The multiple cloning site is not only a recognition site for multiple restriction enzymes, but is also tagged with human influenza hemagglutinin (HA) to confirm the expression of a target protein. The pMT48 vector is a vector in which the Rep gene (SEQ ID NO: 7), which is the replication origin sequence of the plasmid pLMT1-74, is introduced into the EcoRI site of pUC19 (New England Biolabs). The pMT48 vector was constructed as follows.

[0040] First, a provisional plasmid pLMT1-74 was isolated from the LMT1-74 strain (Leuconostoc mesenteroides KCTC 13164BP) isolated from kimchi using a Plasmid midi kit (Qiagen, Inc., Valencia, CA). The Rep gene (SEQ ID NO: 7), which is the replication origin sequence of the plasmid pLMT1-74, was amplified by PCR using the plasmid pLMT1-74 as a template and the oligonucleotides of SEQ ID NO: 8 and 9 as primers. The amplified product was digested with EcoRI and ligated to pUC19 digested with the same enzyme to obtain the pMT48 vector. The polynucleotide of SEQ ID NO: 7 was also chemically synthesized. The vector pUC19 has the nucleotide sequence of SEQ ID NO: 10.

[0041] 1 is a diagram showing the structure of pMT48, a shuttle vector between E. coli and lactic acid bacteria. In the vector, Rep has the nucleotide sequence of SEQ ID NO: 7, is a partial sequence of the Rep origin sequence, which is the replication origin of pLMT1-74, a broad-spectrum lactic acid bacteria host vector, and confers replication ability to lactic acid bacteria. E. coli ori is the DNA replication origin of E. coli, and has the nucleotide sequence of pUC19 ori, i.e., SEQ ID NO: 11. CM is a gene encoding chloramphenicol acetyltransferase and is a chloramphenicol resistance gene.

[0042] 2. Cloning of target protein IL-10 (1) Experimental group vector preparation: pMT54-PR4-IL10-SP4 vector The IL-10 gene (SEQ ID NO: 12) was synthesized by an outside supplier (Macrogen Inc., Korea). The synthesized gene fragment and the pMT54-PR4-SP4 vector were treated with restriction enzymes SalI and XhoI to cleave the cloning site of the vector. The cleaved product was purified using a Gel purification kit (Bioneer) and then dephosphorylated using alkaline phosphatase. 5.5 μl of distilled water was added to a mixture of 1 μl of the vector DNA, 3 μl of the gene (IL-10), 0.5 μl of T4 DNA ligase (Takara) and 1 μl of buffer solution thus prepared to prepare a total reaction mixture of 10 μl. The reaction mixture was incubated at 16° C. for 12 hours, and the gene was ligated to the cloning site of the vector. The resulting ligation product was transformed into E. coli Top10 strain in the same manner as described above, and its sequence was confirmed. As a result, it was confirmed that the gene had been introduced, and the vector was named pMT54-PR4-IL10-SP4 vector. FIG. 2 is a diagram showing the structure of the pMT54-PR4-IL10-SP4 vector. In FIG. 2, the promoter, signal peptide, and target gene represent PR4, SP4, and IL-10, respectively. The vector is a shuttle vector between E. coli and Lactobacillus bacteria, and contains an E. coli replication origin, a Lactobacillus replication origin (rep gene), and a chloramphenicol resistance gene. A promoter, a signal peptide, a target gene, an HA tag, and a His tag are linked to the multiple cloning site.

[0043] (2) Preparation of control group 1 vector: pMT54-PR4-IL10-USP45 vector A vector was constructed in the same manner as the experimental vector, except that the polynucleotide SP4 encoding the signal polypeptide was replaced with the USP45 polynucleotide, in order to confirm the effect of other signal polypeptides on the extracellular secretion of IL-10 protein in the same promoter.

[0044] Specifically, the portion of the vector from which SP4 was removed was amplified by PCR using the pMT54-PR4-IL10-SP4 vector as a template and the oligonucleotides of SEQ ID NOs: 13 and 14 as primers. A polynucleotide encoding USP45 (SEQ ID NO: 15) was synthesized (Macrogen Inc., Korea). The amplified product and the polynucleotide encoding USP45 were ligated by infusion cloning and introduced into E. coli to clone the pMT54-PR4-IL10-USP45 vector. USP45 is a signal polypeptide from Lactococcus lactis that is known to play a role in secreting protein products such as homologous proteinase (PrtP) and alpha-amylase from Bacillus stearothermophilus (van Asseldonk M1, et al. Mol Gen Genet. 1993 Sep; 240 (3): 428-34).

[0045] (3) Preparation of control group 2 vector: pMT54-P11-IL10-SP4 vector A vector was constructed in the same manner as the experimental vector, except that the promoter PR4 was replaced with the P11 promoter. This is to confirm the effect of other promoters on the expression of IL-10 protein in the signal polypeptide. P11 is a synthetic promoter with strong transcription initiation activity in Lactobacillus plantarum (Lars Axelsson, Microbiology (2006), 152, 1011-019).

[0046] Specifically, the portion of the vector from which PR4 had been removed was amplified by PCR using the pMT54-PR4-IL10-SP4 vector as a template and the oligonucleotides of SEQ ID NO: 16 and 17 as primers. The P11 promoter (SEQ ID NO: 18) was synthesized (Macrogen Inc., Korea). The amplified product was ligated to the P11 promoter by infusion cloning and introduced into E. coli to clone the pMT54-P11-IL10-SP4 vector.

[0047] 3. Transformation and IL-10 Protein Expression (1) Confirmation of IL-10 protein expression by pMT54-PR4-IL10-SP4 vector The pMT54-PR4-IL10-SP4 vector and the pMT54-P11-IL10-SP4 vector were transformed into three strains of lactic acid bacteria, Lactobacillus paracasei KCTC 13422BP, Lactobacillus plantarum KCTC 13421BP, and Lactobacillus brevis KCTC 13423BP, all of which were isolated from kimchi. These strains are also called LMT1-21, LMT1-9, and LMT1-46, respectively.

[0048] Each strain was cultured at OD 600 After culturing until the .mu.m. of the culture medium reached 0.5, the medium was centrifuged at 7,000 rpm for 10 min at 4°C, and the cell pellet was washed twice with 25 mL of ice-cold EPS (EPS: 1 mM K2HPO4, 1 mM KH2PO4, pH 7.4, 1 mM MgCl2, and 0.5 M sucrose).

[0049] After washing, the cells were resuspended in 1 mL cold EPS to prepare competent cells for electroporation and stored in a -80°C deep freezer. 40 μl of competent cells and 1 μl of each vector DNA (1 μg / μl) were placed in a cuvette and left on ice for 5 minutes. After applying an electric pulse under conditions of 25 μF, 8 kV / cm, and 400 ohms, the cells were immediately added to 1 mL MRS liquid medium and cultured at 37°C for 1 hour. The cultured cells were then smeared on MRS medium containing 10 μg / ml chloramphenicol and cultured at 37°C for 48 hours.

[0050] Figure 3 shows the results of transforming three types of lactic acid bacteria with the pMT54-PR4-IL10-SP4 vector and examining the degree of extracellular expression. In Figure 3, the control group is a vector in which pMT54-P11-IL10-USP45 was used instead of pMT54-PR4-IL10-SP4.

[0051] As shown in Figure 3, the pMT54-PR4-IL10-SP4 vector expressed IL-10 protein extracellularly in the three Lactobacillus subtilis strains, but the control strain did not express IL-10 protein, indicating that the PR4 promoter was activated to express the gene and the SP4 signal peptide secreted the expressed protein extracellularly.

[0052] (2) Expression of mRNA level: Confirmation of promoter strength The pMT54-PR4-IL10-SP4 vector and the pMT54-P11-IL10-SP4 vector were transformed into Lactobacillus paracasei KCTC 13422BP (LMT1-21) lactic acid bacteria by the same method as in (1).

[0053] The strain thus obtained, into which the vector was introduced, was statically cultured in MRS liquid medium at 37°C for 16 hours. 1 ml of the culture was centrifuged at 7,000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was taken. From the culture, mRNA was extracted using an RNA prep kit (Macherey-Nagel, cat. no. 740955.50) according to the manufacturer's protocol. cDNA was synthesized using 100 ng of mRNA as a template. cDNA synthesis was performed using Rocketscript cycle RT premix from Bioneer. Real-time (RT)-PCR was performed using the synthesized cDNA as a template and oligonucleotides of SEQ ID NO: 20 and SEQ ID NO: 21 as primers. RT-PCR was performed using SYBR premix (Takara, RR820B) according to the manufacturer's protocol.

[0054] Figure 5 shows RT-PCR using cDNA derived from transformed cells as a template. As shown in Figure 5, the IL-10 mRNA level was significantly higher in the strain transformed with the pMT54-PR4-IL10-SP4 vector than in the L. Paracasei KCTC 13422BP strain transformed with the pMT54-P11-IL10-SP4 vector, i.e., the control vector. This indicates that the PR4 promoter drives transcription more strongly than the P11 promoter. The "2△△CT" on the Y axis of Figure 5 is the result of analyzing the relative transcription level, and indicates the increased transcription level compared to the control group.

[0055] (3) Protein expression: Confirmation of signal peptide strength The pMT54-PR4-IL10-SP4 vector and the pMT54-PR4-IL10-USP45 vector were transformed into Lactobacillus paracasei KCTC 13422BP (LMT1-21) lactic acid bacteria in the same manner as in (1).

[0056] The strains thus obtained, into which each vector had been introduced, were statically cultured in MRS liquid medium at 37°C for 16 hours. The cultures were inoculated into MRS liquid medium to a concentration of 3 (v / v)%, and then statically cultured at the same temperature for 8 hours. 1 ml of the cultures was centrifuged at 7,000 rpm for 5 minutes, and the supernatant was taken. 1 ml of the supernatant was added with 100 μl of trichloroacetic acid, and the mixture was allowed to stand at 4°C for 1 hour to concentrate the culture components. The reaction mixture was centrifuged at 13,000 rpm at 4°C for 10 minutes, after which the pellet was washed once with 1 ml of cold acetone, dried at room temperature for 10 minutes, and then eluted with 100 μl of Tris-HCl buffer (pH 8.8).

[0057] The eluate was added with 4x loading buffer (Thermo) and 10x reducing agent (Thermo), and then electrophoresed on SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane using a Trans blot semi-dry cell (bio-rad), and Western blotting was performed. Specifically, the membrane was blocked with TBST buffer containing 1% skim milk for 1 hour, reacted with anti-HA antibody (Santa Cruz) at room temperature for 2 hours, washed three times for 5 minutes each with TBST, and then detected by ECL. In the pMT54-PR4-IL10-SP4 vector, the IL-10 gene is operably linked to the HA gene at its 3' end, and expressed in an HA-tagged state.

[0058] Figure 6 shows the amount of IL-10 secreted from LMT1-21 transformed with pMT54-PR4-IL10-SP4 and pMT54-PR4-IL10-USP45 to compare the strength of the signal peptides. As shown in Figure 6, the SP4 signal peptide caused the expressed protein to be secreted in greater amounts than the USP45 signal peptide.

[0059] Example 2: Expression of amylase gene using PR4 promoter and SP4 sequence The pMT54-PR4-amylase-SP4 vector was prepared by the same process as in Example 1 (2) and (3), except that the alpha-amylase gene (SEQ ID NO: 19) and primers F / R (SEQ ID NO: 22 and 23) were used instead of the IL-10 gene, and the vector was transformed into L. Paracasei LMT1-21 to confirm the degree to which alpha-amylase was expressed extracellularly. The genomic DNA of Lactobacillus amylovorus (KCTC 3597) was used to amplify the alpha-amylase gene.

[0060] The amylase activity of the transformed LMT1-21 strain was measured using an iodine test. First, the LMT1-21 strain into which the pMT54-PR4-amylase-SP4 vector had been introduced was statically cultured in MRS liquid medium at 37°C for 12 hours. The culture was then spread in small dots on an MRS plate containing 0.5% soluble potato starch and 10mg / l chloramphenicol. It was then statically cultured at 37°C for 12 hours to allow amylase to fully decompose the potato starch. Then, Lugol's iodine solution (iodine-potassium iodide solution) was evenly spread on the MRS plate to react with the undecomposed potato starch. The lower the amylase activity, the greater the amount of remaining potato starch, and the stronger the iodine-potato starch reaction, turning the cells purple. On the other hand, when the amylase activity is high, the amount of remaining potato starch around the cells is small, resulting in a transparent ring.

[0061] Figure 4 shows the results of transforming the pMT54-PR4-amylase-SP4 vector into the LMT1-21 strain and examining the extracellular expression level. In Figure 4, the control vector is the same as the pMT54-PR4-amylase-SP4 vector, except that the P11 promoter was used instead of PR4 and USP45 was used instead of SP4.

[0062] As shown in Figure 4, the experimental group using the pMT54-PR4-amylase-SP4 vector expressed alpha-amylase extracellularly in the LMT1-21 strain, forming a large transparent ring, while the control group showed no expression and formed a small ring, indicating that PR4 was activated to express the amylase gene and enhance the ability of SP4 to secrete it extracellularly.

[0063] It should be understood that the embodiments described herein are to be considered for illustrative purposes only, and not for limiting purposes, and the description of a feature or aspect within each embodiment should typically be considered as applicable to other similar features or aspects of other embodiments.

[0064] Although one or more embodiments have been described with reference to the drawings, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the disclosure as defined in the following claims.

Claims

1. An isolated signal polypeptide having the amino acid sequence of SEQ ID NO:

2.

2. 10. The method of claim 1, further comprising: The second polypeptide is not peptidase C51; Recombinant polypeptides.

3. A recombinant polynucleotide comprising a polynucleotide encoding the isolated signal polypeptide of claim 1.

4. 4. The recombinant polynucleotide of claim 3, wherein the polynucleotide encoding the isolated signal polypeptide of claim 1 is operably linked to a polynucleotide encoding a protein.

5. 5. The recombinant polynucleotide of claim 4, further comprising a promoter, said promoter being operably linked to a polynucleotide encoding the isolated signal polypeptide of claim 1.

6. 4. The recombinant polynucleotide of claim 3, further comprising a promoter, said promoter being operably linked to a polynucleotide encoding the isolated signal polypeptide of claim 1.

7. 7. A recombinant polynucleotide according to any one of claims 3 to 6, which is a vector.

8. A host cell comprising a recombinant polynucleotide according to any one of claims 3 to 6.

9. 10. A method for producing a protein, comprising culturing the host cell of claim 8 in a medium and isolating the protein from the culture.

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