Patent Application
20200339637
This application claims the benefit of Korean Patent Application No. 10-2017-0184819, filed on Dec. 29, 2017, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
The present disclosure relates to a promoter polynucleotide, a signal polypeptide, and use thereof.
Microorganisms such as bacteria, yeast, and fungi are becoming increasingly important as hosts for recombinant expression.
Bacteria such as Lactobacillus or Streptococcus sp. may be useful as delivery vehicles. In addition, generally recognized as safe (GRAS) microorganisms may be administered to humans or animals.
To achieve a high expression level of a foreign product in lactic acid bacteria, there is a demand for novel promoter and signal polypeptides that may be isolated from lactic acid bacteria and may express and secrete the foreign product, in particular a protein, at a high level.
An aspect provides an isolated promoter.
Another aspect provides a recombinant polynucleotide including the promoter.
Still another aspect provides a host cell including the recombinant polynucleotide.
Still another aspect provides a method of producing a product using the host cell.
Still another aspect provides an isolated signal polypeptide and a polynucleotide encoding the same.
Still another aspect provides a recombinant polynucleotide including the polynucleotide encoding the isolated signal polypeptide.
Still another aspect provides a host cell including the recombinant polynucleotide encoding the isolated signal polypeptide.
Still another aspect provides a method of producing a protein using the host cell including the recombinant polynucleotide encoding the isolated signal polypeptide.
An aspect provides an isolated promoter including a polynucleotide having a sequence identity of 85% or more with a nucleotide sequence of SEQ ID NO: 1 (hereinafter, referred to as ‘PR4 promoter’).
Another aspect provides a recombinant polynucleotide including the promoter. As used herein, the term “promoter” refers to a nucleic acid molecule, particularly, a region on a DNA molecule, to which an RNA polymerase binds to initiate transcription. The promoter is generally located upstream, i.e., 5′ of a sequence to be transcribed, which is regulated by the promoter. The promoter may be a constitutive promoter. The promoter may have a sequence identity of 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% with the nucleotide sequence of SEQ ID NO: 1. The promoter may have the nucleotide sequence of SEQ ID NO: 1.
The recombinant polynucleotide may be a vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of propagate another nucleic acid linked thereto. The vector may include a vector as a self-replicating nucleic acid structure as well as a vector incorporated into the genome of a host cell into which it has been introduced. An expression vector refers to a vector that directs expression of a nucleic acid operably linked thereto. The vector may be a plasmid or a vector derived from a virus.
The vector may be a cloning vector or an expression vector. The expression vector may include a nucleotide sequence encoding a protein, the nucleotide sequence being operably linked to the promoter.
The expression vector may include the promoter, and a first polynucleotide including a nucleotide sequence encoding a product, the first polynucleotide being operably linked to the promoter. The product may include any product which may be produced by expression of the 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.
As used herein, the term “operably linked” means a linkage that allows transcription or translation to produce a functional transcription or translation product.
The vector may further include one or more selected from the group consisting of a ribosome binding site (RBS), a cloning site, a selection marker gene, a transcription terminator, and a translation initiator factor. The cloning site may be operably linked to the promoter. The cloning site may be a multiple cloning site.
The recombinant polynucleotide may be a recombinant polynucleotide wherein a second polynucleotide encoding a signal polypeptide including an amino acid sequence having a sequence identity of 85% or more with an amino acid sequence of SEQ ID NO: 2 (hereinafter, referred to as “SP4 signal polypeptide”) is operably linked between the promoter and the first polynucleotide. In this case, the first polynucleotide may encode a polypeptide.
As used herein, the term “signal polypeptide” refers to a sequence that is present at the N-terminus of a secreted protein precursor but not present in a naturally existing mature protein. The signal polypeptide may be cleaved off from the protein precursor. In general, the signal polypeptide may be cleaved by a protease when extracellularly secreted. The protease may be generally called signal peptidase. The signal polypeptide may have a sequence identity of 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% with the amino acid sequence of SEQ ID NO: 2. The signal polypeptide has activity to allow extracellular secretion of an expression product of a gene which is fused in frame to the nucleotide sequence encoding the signal polypeptide.
As used herein, the term “secretion” of a protein or a polypeptide molecule may include transport of the protein or the polypeptide molecule outside of a bacterial cell, presence of the protein or the polypeptide molecule in a completely free form in a medium, presence of only part of the protein or the polypeptide molecule outside the bacterial cell, and presence of the protein or the polypeptide molecule on the surface of the bacterial cell.
The signal polypeptide may be derived from L. paracasei and may have a secretion-promoting ability. The second polynucleotide may have a nucleotide sequence of SEQ ID NO: 3.
Another aspect provides a host cell including 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 lactic acid bacterium or may belong to the genus Escherichia. The lactic acid bacterium may be the genus Lactobacillus, Lactococcus, Bifidobacteria, Streptococcus, Leuconostoc, Weissella, Pediococcus, or Enterococcus.
The recombinant polynucleotide may be introduced into the host cell by a common nucleic acid introduction method. The nucleic acid introduction method may include electroporation, transformation, transduction, or transfection.
Still another aspect provides a method of producing a product or a metabolite thereof, the method including producing the product by culturing the host cell in a medium; and isolating the product or the metabolite thereof from the culture. The product may include any product that may be produced by expression of the 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 metabolite may be a substance produced when the product exerts its activity in a cell. For example, when the product is an enzyme, the metabolite may be a direct product produced when the enzyme exerts its enzymatic activity, or a substance produced from a metabolic pathway in which the enzyme is involved.
In the method, the culturing may be performed by a common method known in the art according to a host cell selected. The medium used in the culturing may include, as a sugar source, for example, carbohydrate e.g., glucose, saccharose, lactose, fructose, maltose, and starch, oil and fat, e.g., soybean oil, sunflower oil, castor oil, coconut oil, etc., a fatty acid, e.g., palmitic acid, stearic acid, and linolenic acid, glycerol, and an organic acid, e.g., acetic acid, singly or in a mixture. The medium may include, as a nitrogen source, for example, peptone, a yeast extract, a meat extract, a malt extract, corn steep liquor, soy meal, urea, or an inorganic compound, e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, singly or in a mixture. The medium may include, as a phosphorous source, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt thereof. The medium may include, for example, a metal salt, e.g., magnesium sulfate or iron sulfate, which is required for growth. Also, in the culturing, substances required for growth, such as amino acids and vitamins, or suitable precursors may be added to the culture. Those components may be added to the culture in a proper manner, for example, in a batch or continuous manner during the culturing.
The culturing may be performed under aerobic conditions, microareobic, unaerobic conditions, or a combination thereof.
The method may further include isolating the product from the culture. The isolating may be performed by an appropriate method according to a kind of a product to be selected. When the product is a protein, the isolating may include isolating the protein from a supernatant after removing cells by centrifugation of the culture, or isolating the protein by cell disruption after recovering the cells. The isolating may be subjected to one or more processes of salting-out, precipitation, chromatography, centrifugation, and filtration. The chromatography may be one or more of anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, and affinity chromatography.
Still another aspect provides an isolated signal polypeptide including an amino acid sequence having sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 2. The signal polypeptide may have sequence identity of 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% with the amino acid sequence of SEQ ID NO: 2.
Still another aspect provides a polynucleotide encoding the signal polypeptide. The polynucleotide encoding the signal polypeptide may have a nucleotide of SEQ ID NO: 3.
Still another aspect provides an expression vector including the second polynucleotide encoding the signal polypeptide and the first polynucleotide encoding the protein, wherein the second polynucleotide is operably linked to the promoter and the first polynucleotide is fused in frame to the second polynucleotide.
Still another aspect provides a host cell including 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 lactic acid bacterium or may belong to the genus Escherichia. The lactic acid bacterium may be the genus Lactobacillus, Lactococcus, Bifidobacteria, Streptococcus, Leuconostoc, Weissella, Pediococcus, or Enterococcus.
The recombinant polynucleotide may be introduced into the host cell by a common nucleic acid introduction method. The nucleic acid introduction method may include electroporation, transformation, transduction, or transfection.
Still another aspect provides a method of producing a protein, the method including producing the protein by culturing the host cell in a medium; and isolating the protein from the culture.
In the method, the culturing may be performed by a common method known in the art according to a host cell to be selected. The medium used in the culturing may include, as a sugar source, for example, carbohydrate e.g., glucose, saccharose, lactose, fructose, maltose, and starch, oil and fat, e.g., soybean oil, sunflower oil, castor oil, coconut oil, etc., a fatty acid, e.g., palmitic acid, stearic acid, and linolenic acid, glycerol, and an organic acid, e.g., acetic acid, singly or in a mixture. The medium may include, as a nitrogen source, for example, peptone, a yeast extract, a meat extract, a malt extract, corn steep liquor, soy meal, urea, or an inorganic compound, e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, singly or in a mixture. The medium may include, as a phosphorous source, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt thereof. The medium may include, for example, a metal salt, e.g., magnesium sulfate or iron sulfate, which is required for growth. Also, in the culturing, substances required for growth, such as amino acids and vitamins, or suitable precursors may be added to the culture. Those components may be added to the culture in a proper manner, for example, in a batch or continuous manner during the culturing.
The culturing may be performed under aerobic conditions, micro aerobic conditions, unaerobic conditions, or a combination thereof.
The method may further include isolating the protein from the culture. The isolating may include isolating the protein from a supernatant after removing cells by centrifugation of the culture, or isolating the protein by cell disruption after recovering the cells. The isolating may be subjected to one or more processes of salting-out, precipitation, chromatography, centrifugation, and filtration. The chromatography may be one or more of anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, and affinity chromatography.
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description.
An isolated promoter and a recombinant polynucleotide including the same according to an aspect may be used in efficiently expressing a foreign gene.
A host cell including the recombinant polynucleotide according to another aspect may be used in efficiently expressing a foreign gene.
A method of producing a product using the host cell according to still another aspect may be used to efficiently produce the product.
An isolated signal polypeptide, a polynucleotide encoding the signal polypeptide, and a recombinant polynucleotide including the polynucleotide according to still another aspect may be used in extracellularly secreting a foreign protein.
A host cell including the recombinant polynucleotide including the polynucleotide encoding the isolated signal polypeptide according to still another aspect may efficiently secrete a product of a foreign gene out of the cell.
A method of producing a protein using the host cell according to still another aspect may be used to efficiently produce the protein.
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:
Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.
1. Cloning of Promoter and Signal Polypeptide
A promoter and a nucleotide sequence encoding a signal polypeptide were amplified by PCR. In detail, PCR was performed using a genome of Lactobacillus paracasei LMT1-21 (Accession No: KCTC 13422BP) as a template and primers to obtain an amplification product of 593 kb. The used primers were PS4_F/R (SEQ ID NOS: 4 and 5).
The amplification product was ligated to pMT54 vector which had been digested with EcoRV and SalI by Infusion cloning (Clontech). Thereafter, the vector was transformed into E. coli Top 10 strain (Invitrogen) by a method of Sambrook et al. (Sambrook et al. Molecular cloning: A laboratory Manual, 2nd edition, 1989). Thereafter, the transformed E. coli was spread on an LB plate supplemented with 10 μg/ml chloramphenicol to obtain colonies. The pMT54 vector was recovered from the obtained colonies, followed by sequencing analysis. As a result, the vector was confirmed to include a PR4 (SEQ ID NO: 1)-SP4-econding nucleotide sequence (SEQ ID NO: 3). Hereinafter, this vector is referred to as a pMT54-PR4-SP4 vector.
The pMT54 vector was a vector in which a multiple cloning site (SEQ ID NO: 6) was introduced into HindIII and XhoI restriction sites of pMT48 vector. The multiple cloning site has multiple restriction enzyme recognition sites and is tagged with human influenza hemagglutinin (HA) to confirm expression of a target protein. The pMT48 vector was a vector in which Rep gene (SEQ ID NO: 7) which is an origin of replication of a plasmid pLMT1-74 was introduced into the EcoRI site of pUC19 (New England Biolabs). The pMT48 vector was constructed as follows.
First, a tentative plasmid pLMT1-74 was isolated from a LMT1-74 strain (Leuconostoc mesenteroides KCTC 13164BP) which had been isolated from kimchi using a plasmid midi kit (Qiagen, Inc., Valencia, Calif.). PCR was performed using the plasmid pLMT1-74 as a template and oligonucleotides of SEQ ID NOS: 8 and 9 as primers to amplify Rep gene (SEQ ID NO: 7) which is an origin of replication of plasmid pLMT1-74. The amplified product was digested with EcoRI, and ligated to pUC19 which had been digested with the same enzyme to obtain the pMT48 vector. The polynucleotide of SEQ ID NO: 7 may be also chemically synthesized. The vector pUC19 has a nucleotide sequence of SEQ ID NO: 10.
2. Cloning of Target Protein IL-10
(1) Construction of Experimental Vector: pMT54-PR4-IL10-SP4 Vector
Synthesis of IL-10 gene (SEQ ID NO: 12) was conducted by Macrogen Inc. (Korea). The synthesized gene fragment and the pMT54-PR4-SP4 vector were treated with restriction enzymes SalI and XhoI to cleave cloning sites of the vector. The cleaved product was purified using a Gel purification kit (Bioneer), and dephosphorylated using alkaline phosphatase. To a mixture of 1 μl of the prepared vector DNA, 3 μl of the gene (IL-10), 0.5 μl of T4 DNA ligase (Takara), and 1 μl of buffer, 5.5 μl of distilled water was added to prepare total 10 μl of a reaction mix. This reaction mix was incubated at 16° C. for 12 hours to ligate the gene into the cloning site of the vector. The obtained ligation product was transformed into E. coli Top 10 strain in the same manner as above, followed by sequencing. As a result, introduction of the gene was confirmed, and this product was designated as a pMT54-PR4-IL10-SP4 vector.
(2) Construction of Control Vector 1: pMT54-PR4-IL10-USP45 Vector
A vector was constructed in the same manner as in the experimental vector, except that the polynucleotide SP4 encoding the signal polypeptide was replaced by USP45 polynucleotide. This is to examine an effect of a different signal polypeptide on extracellular secretion of IL-10 protein when the same promoter was used.
In detail, PCR was performed using the pMT54-PR4-IL10-SP4 vector as a template and oligonucleotides of SEQ ID NOS: 13 and 14 as primers to amplify the vector from which SP4 was excluded. A USP45-encoding polynucleotide (SEQ ID NO: 15) was synthesized (Macrogen, Korea). The amplified product and the USP45-encoding polynucleotide were ligated by an infusion cloning method, and introduced into E. coli to clone a pMT54-PR4-IL10-USP45 vector. USP45 is a signal polypeptide derived from Lactococcus lactis and is known to play a role in secreting protein products such as homologous proteinase (PrtP) and Bacillus stearothermophilus-derived alpha-amylase (van Asseldonk M1, et al. Mol Gen Genet. 1993 September; 240(3):428-34).
(3) Construction of Control Vector 2: pMT54-P11-IL10-SP4 Vector
A vector was constructed in the same manner as in the experimental vector, except that the promoter PR4 was replaced by P11 promoter. This is to examine an effect of a different promoter on expression of IL-10 protein when the same signal polypeptide was used. P11 is a synthetic promoter having a strong transcription initiation activity in Lactobacillus plantarum (Lars Axelsson, Microbiology (2006), 152, 1011-019).
In detail, PCR was performed using the pMT54-PR4-IL10-SP4 vector as a template and oligonucleotides of SEQ ID NOS: 16 and 17 as primers to amplify the vector from which PR4 was excluded. P11 promoter (SEQ ID NO: 18) was synthesized (Macrogen, Korea). The amplified product and the P11 promoter were ligated by an infusion cloning method, and introduced into E. coli to clone a pMT54-P11-IL10-SP4 vector.
3. Transformation and Expression of IL-10 Protein
(1) Expression of IL-10 Protein by pMT54-PR4-IL10-SP4 Vector
The pMT54-PR4-IL10-SP4 vector and the pMT54-P11-IL10-SP4 vector were transformed into three different kinds of lactic acid bacteria, respectively. The three different kinds of lactic acid bacteria were Lactobacillus paracasei KCTC 13422BP, Lactobacillus plantarum KCTC 13421BP, and Lactobacillus brevis KCTC 13423BP, all separated from kimchi. These strains are also called LMT1-21, LMT1-9, and LMT1-46, respectively.
Each of the strains was cultured in 50 mL of MRS medium (Difco Co., USA) until OD600 reached 0.5, and then centrifuged at 4° C. and 7,000 rpm for 10 minutes. Cell pellets were washed twice with 25 mL of ice-cold EPS (EPS: 1 mM K2HPO4, 1 mM KH2PO4, pH 7.4, 1 m MgCl2, and 0.5 M sucrose).
After washing, cells were re-suspended in 1 mL of ice-cold EPS, and competent cells to be used in electroporation were prepared, and stored in a deep freezer at −80° C. 40 μl of competent cells and each 1 μl of vector DNA (1 μg/μl) were put in a cuvette and left on ice for 5 minutes. Electric field pulse was applied thereto under conditions of 25 μF, 8 kV/cm, 400 ohms, and the cells were immediately added to 1 mL of MRS liquid medium, followed by incubation at 37° C. for 1 hour. Thereafter, the incubated cells were spread on MRS medium containing 10 μg/ml of chloramphenicol, followed by incubation at 37° C. for 48 hours.
As shown in
(2) Expression at mRNA Level: Examination of Promoter Strength
Each of the pMT54-PR4-IL10-SP4 vector and the pMT54-P11-IL10-SP4 vector was transformed into Lactobacillus paracasei KCTC 13422BP (LMT1-21) lactic acid bacterium in the same manner as in (1).
The strain introduced with each of the vector was subjected to stationary culture in MRS medium at 37° C. for 16 hours. 1 ml of the culture was centrifuged at 7,000 rpm for 5 minutes, and then a supernatant was discarded, and a cell pellet was obtained. mRNA was extracted therefrom using an RNA prep kit (Macherey-nagel, cat. no 740955.50) in accordance with the manufacturer's protocol. 100 ng of mRNA was used as a template to synthesize cDNA. cDNA synthesis was performed using a Roketscript cycle RT premix of Bioneer. The synthesized cDNA was used as a template and oligonucleotides of SEQ ID NO: 20 and SEQ ID NO: 21 were used as primers to perform real-time (RT) PCR. RT-PCR was performed using a SYBR premix (takara, RR820B) in accordance with the manufacturer's protocol.
(3) Expression at Protein Level: Examination of Signal Peptide Strength
Each of the pMT54-PR4-IL10-SP4 vector and the pMT54-PR4-IL10-USP45 vector was transformed into Lactobacillus paracasei KCTC 13422BP (LMT1-21) lactic acid bacterium in the same manner as in (1).
The strain introduced with each of the vector was subjected to stationary culture in MRS medium at 37° C. for 16 hours. The culture was seeded in MRS liquid medium at 3 (v/v) %, and then subjected to stationary culture at the same temperature for 8 hours. 1 ml of the culture was centrifuged at 7,000 rpm for 5 minutes, and a supernatant was obtained. 100 μl of trichloroacetic acid was added to 1 ml of the supernatant, which was left at 4° C. for 1 hour to concentrate components of the culture. The resultant was centrifuged at 4° C. and 13,000 rpm for 10 minutes, and a pellet was washed with 1 ml of cold acetone once, dried at room temperature for 10 minutes, and eluted with 100 μl of Tris-HCl buffer (pH 8.8).
A 4× loading buffer (Thermo) and a 10× reducing agent (Thermo) were added to the eluate, followed by electrophoresis on SDS-PAGE gel. This gel was transferred onto a nitrocellulose membrane using a Trans blot semi-dry cell (bio-rad), followed by Western blotting. In detail, the membrane was blocked with a TBST buffer containing 1% skim milk for 1 hour, and reacted with anti-HA antibody (santa cruz) at room temperature for 2 hours. The membrane was washed with TBST for 5 minutes three times, and detected using an ECL. In the pMT54-PR4-IL10-SP4 vector, HA gene was operably linked to IL-10 gene at the 3′-terminus thereof, and thus the HA-tagged gene was expressed.
A pMT54-PR4-amylase-SP4 vector was constructed in the same manner as in 2 and 3 of Example 1, except that alpha-amylase gene (SEQ ID NO: 19) instead of IL-10 gene and primers F/R(SEQ ID NOS: 22 and 23) were used, and this vector was transformed into a lactic acid bacterium L. paracasei LMT1-21 to examine extracellular expression of alpha-amylase. Amplification of the alpha-amylase gene was performed using genomic DNA of Lactobacillus amylovorus (KCTC3597).
Amylase activity of the transformed LMT1-21 strain was examined by an iodine test. First, the LMT1-21 strain introduced with the pMT54-PR4-amylase-SP4 vector was subjected to stationary culture in MRS liquid medium at 37° C. for 12 hours. Thereafter, the culture was applied in small dots to an MRS plate containing 0.5% soluble starch and 10 mg/l of chloramphenicol, and subjected to stationary culture at 37° C. for 12 hours to allow amylase to sufficiently degrade starch. Thereafter, a Lugol's iodine solution (iodine/potassium iodide solution) was evenly applied onto the MRS plate to allow reaction with undegraded starch. As the amylase activity is lower, the amount of remaining starch is larger, and as a result, a strong iodine-starch reaction occurs to show a purple color. On the contrary, as the amylase activity is higher, the amount of starch remaining around cells is smaller, and as a result, a transparent circle is formed.
As shown in
It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.
While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0184819 | Dec 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/013520 | 11/8/2018 | WO | 00 |
