Cell surface overexpression technology using the PrsA expression system for the extracellular lipoprotein

Abstract

The present invention relates to a vector system for cell surface expression of a target protein, which comprises PrsA, an outer membrane protein of lactic acid bacteria having a cell surface expression anchor motif function, a sequence encoding said PrsA, and a strong promoter located upstream of the PrsA gene.

Description

[Technical Field]

[0001] The present invention relates to lipoprotein PrsA and its derivatives present in the outer cell membrane of lactic acid bacteria for cell surface expression of target proteins, as well as to a cell surface expression vector comprising a PrsA promoter that induces overexpression of PrsA and a base sequence encoding the PrsA. [Background technology]

[0002] Cell surface display systems express proteins or peptides on the cell surface and expose them outside the cell, allowing the proteins or peptides displayed on the cell surface to be freely exposed to the extracellular space. Displaying foreign proteins on the cell surface is considered important in biotechnology and industrial applications such as vaccine development, whole-cell biocatalysis, bioabsorbents, and biosensors.

[0003] To express a protein on the cell surface, the protein must have a secretion signal in its primary sequence that allows the protein synthesized within the cell to pass through the cell membrane. Furthermore, different surface expression systems are used depending on the type of cell in which the target protein, enzyme, or peptide is to be expressed on the surface. In bacteria, proteins synthesized in the cytoplasm or near the cell membrane must have a signal sequence to enter the cell membrane and be secreted. Gram-negative bacteria, in particular, must pass through the intracellular and extracellular membrane spaces, insert into and attach to the extracellular membrane, and be retained so that they can protrude from the membrane. To achieve this, they require a secretion signal present in the extracellular membrane protein or the enzyme or toxic protein secreted outside the cell, as well as a targeting signal that allows them to be retained on the cell surface.

[0004] There are four types of anchor proteins that can anchor foreign proteins to the thick cell walls of Gram-positive bacteria such as lactic acid bacteria: (i) transmembrane anchor, (ii) lipoprotein anchor, (iii) LPXTG anchor, and (iv) LysM-repeat anchor (Boekhorst et al. Microbiology, 2006, 152:3175-3183; Michon et al. Microb Cell Fact. 2016, 15:70).

[0005] Transmembrane-anchored proteins, a common type of membrane protein found in Gram-positive and Gram-negative bacteria, form an anchor by inserting 20–30 amino acids at the N- or C-terminus, composed of hydrophobic amino acids, into the cell membrane in an alpha-helical structure. However, their overexpression in the cell membrane significantly impacts membrane rigidity, limiting their feasibility. The second type, lipoprotein anchors, exist on the cell surface by covalently linking the -SH group of a cysteine ​​residue immediately following the secretion signal to the carbon atom in the glycerol head of a lipid, a cell membrane component. Unlike the previously described transmembrane anchors, these lipoprotein anchors exist suspended from the lipid bilayer without affecting membrane rigidity by penetrating the cell membrane, offering the significant advantage of being able to be overexpressed on the cell surface.

[0006] In most cases, such as Gram-positive and Gram-negative bacteria or yeast, the biggest obstacle to commercializing vaccine and biocatalyst development using microbial surface expression systems is the difficulty of expressing target proteins on the cell surface in large quantities. In particular, over the past 50 years of molecular biology research has led to the development of large-scale expression systems using promoter sequence changes or inducers that bind to repressors within the expression system, as well as the development of microbial hosts capable of stable intracellular expression of target proteins through specific gene manipulation or deletion within the chromosome of specific bacteria used as vectors. However, with the exception of certain industrial strains such as E. coli, there is currently no artificial technology capable of expressing a specific target protein at more than 1% of the total protein content per microorganism. Furthermore, there are virtually no systems capable of expressing a specific target protein on the cell membrane or extracellular membrane rather than in the cytoplasm. The most important key factors in commercializing cell surface expression technology for biocatalysts for bioconversion processes are the amount and stability of the enzyme expressed on the surface. In particular, in vaccine development, the amount of bacterial cells that can be administered into the body is limited due to side effects such as inflammation, and if the total amount of antigens that can be presented relative to the amount of bacterial cells administered is limited, the induced immune response may be insufficient to protect against disease, so the cell surface expression level of the target antigen is a very important key issue.

[0007] Lactic acid bacteria have been widely used for a long time in fermented foods containing lactic acid bacteria worldwide and are recognized as safe as GRAS (Generally Recognized As Safe) microorganisms. Therefore, various research is being conducted using GRAS microorganisms, which have almost no toxicity or side effects and can relatively minimize the possibility of safety issues.

[0008] Korean Patent Publication No. 2019-0037481 discloses a "method for cell surface expression of a target protein using a cell anchoring motif derived from Corynebacterium," Korean Patent Publication No. 2002-0010428 discloses a "novel cell wall adhesion-mediating protein isolated from yeast, its gene, and a cell surface expression system using the same," using four GPI adhesion protein (glycosyl phosphatidyl inositol anchor) genes, including HpSED1, HpGAS1, HpTIP1, and HpCWP1, isolated from the methanol-magnetic yeast Hansenula polymorpha, and Korean Patent Publication No. 2004-0032824 discloses a "surface expression vector using a poly-gamma glutamate synthesis gene derived from a Bacillus strain isolated from Cheonggukjang, and a method for microbial surface expression of a protein using the same." However, there is no description of a "cell surface overexpression technique using an outer membrane lipoprotein PrsA expression system" using PrsA, the lactic acid bacteria surface protein of the present invention, or a mutant thereof, and the PrsA promoter. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Republic of Korea Patent Publication No. 2019-0037481 [Patent Document 2] Republic of Korea Patent Publication No. 2002-0010428 [Patent Document 3] Republic of Korea Patent Publication No. 2004-0032824 [Patent Document 4] Republic of Korea Patent Registration No. 10-0469800 [Non-patent literature]

[0010] [Non-Patent Document 1] Boekhorst et al.Microbiology,2006,152:3175~3183;Michon et al.Microb Cell Fact.2016,15:70 [Non-patent document 2] Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) [Non-patent document 3] Conjugation;Heinze et al.BMC microbiology 2018,18:56 [Non-patent document 4] Bonnie L.Elder et al.,J.Clin.Microbiol.1982,16:141-144;Albritton et al.,PLOS One 2017,12(8):e0183101 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention has been made in response to the above-mentioned needs. The inventors isolated and cultivated GRAS microorganisms, lactic acid bacteria, from kakkimchi, a traditional fermented food, and selected PrsA, an extracellular membrane lipoprotein that is constantly and abundantly expressed on the surface of the isolated GRAS lactic acid bacteria, and its promoter, using a proteolytic surface-shaving method.

[0012] The structure of the selected PrsA protein was analyzed, and cell surface expression vectors were constructed in which the coding sequence of a PrsA wild-type, PrsA serine-rich domain-deleted mutant, PrsA hinge region-deleted mutant, or PrsA hinge region and serine-rich domain-deleted mutant was operably linked under the control of the PrsA promoter. A reporter gene was cloned into the vector, which was then transformed into lactic acid bacteria to analyze the cell surface expression of the reporter protein. As a result, it was observed that the reporter protein was stably expressed on the cell surface in all lactic acid bacteria transformed with vectors containing the coding sequence of the PrsA protein or its mutants. This confirmed that the PrsA protein and its mutants of the present invention can function as a surface expression anchor motif, thereby completing the present invention. [Means for solving the problem]

[0013] To solve the above problems, the present invention provides a recombinant vector for cell surface expression of a target protein, characterized in that a polynucleotide encoding PrsA or a variant thereof consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding the target protein are sequentially linked downstream of a PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0014] The present invention also provides a microorganism transformed with the recombinant vector.

[0015] The present invention also provides a method for expressing a target protein on the surface of a microorganism, which comprises transforming a microorganism with the recombinant vector.

[0016] The present invention also provides a method for producing a microorganism in which a target protein is expressed on its cell surface, comprising the steps of: culturing a microorganism transformed with the recombinant vector to express a target protein on the cell surface; and recovering the microorganism in which the target protein is expressed on its cell surface.

[0017] The present invention also provides a microorganism in which the target protein produced by the above method is expressed on the cell surface. The present invention also provides an injectable preparation containing, as an active ingredient, a microorganism in which the target protein is expressed on the cell surface.

[0018] The present invention also provides an oral preparation containing, as an active ingredient, a microorganism in which the target protein is expressed on the cell surface.

[0019] The present invention also provides a composition for inducing immunity in vertebrates other than humans, which contains the microorganism as an active ingredient.

[0020] The present invention also provides a method for producing a protein array, which comprises the step of immobilizing, on the surface of a substrate, a microorganism produced by the method of the present invention and expressing a target protein on its surface.

[0021] The present invention also provides a method for inducing immunity in a vertebrate, comprising the step of administering to the vertebrate a microorganism produced by the method of the present invention and expressing an antigen on its surface. [Effects of the Invention]

[0022] The present invention uses proteins isolated from lactic acid bacteria, which are GRAS bacteria, and by providing a method for stably expressing a foreign protein on the surface of lactic acid bacteria, it can be used as a cell surface expression method for foreign proteins that have previously been difficult to stably express on the cell surface or to express in large quantities, and is therefore expected to be highly applicable industrially.

[0023] Furthermore, when the expression system of the present invention is used to express a ligand protein, receptor protein, or enzyme protein involved in in vivo signal transduction of general cells, including self-proteins, it can be used as a therapeutic agent for metabolic diseases; when a ligand or receptor of an immune cell is expressed, it can be used as an immunotherapeutic agent; and when a bacterium or bacterial antigen is expressed, it can be used as a preventive or therapeutic vaccine. Therefore, the expression system of the present invention can be useful in the pharmaceutical industry. [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows the MS / MS analysis results of the five confirmed Lys-C cleavage peptides, including the representative peptide with the sequence from amino acid 255 to 264 (SEQ ID NO: 6). [Figure 2] The nucleotide and amino acid sequences of the PrsA protein from Lactobacillus sakei are shown. The hinge region (dotted underline, bold) and serine-rich domain (solid underline, bold) that were deleted during the production of the PrsA protein derivative are also shown. [Figure 3] pGOSTalpha:PrsA vector map containing the PrsA promoter sequence and PrsA anchor motif coding sequence. [Figure 4] Figure 3 shows the expression of L. sakei PrsA in L. paracasei transformed with the pGOSTα:PrsA vector. (A) is a CBB-stained gel image of total protein, and (B) is a Western blot image of total protein transferred to a PVDF membrane using an anti-PrsA antibody. (C) is a CBB-stained gel image of the cytoplasmic and plasma membrane proteins separated by high-speed centrifugation. The position of the PrsA-anchored membrane protein (theoretical molecular weight, 31.4 kDa) is indicated by a red arrow. Figure 4A, lane B, lane M: protein size marker (ExcelBang 3-color Broad Range Protein Marker, #PM2700 Thermo Scientific); lane 1: untransformed L. paracasei; lane 2: transformed L. paracasei. Figure 4C, lane M: protein size marker, lane 1: total protein, lane 2: cytoplasmic protein fraction, lane 3: plasma membrane protein fraction. [Figure 5] FIG. 4 shows a vector map of pGOSTa:PrsA-sfGFP, in which the reporter gene sfGFP was cloned into the vector of FIG. 3. [Figure 6] Lactobacillus paracasei was transformed with recombinant vectors containing PrsA, a PrsA serine-rich domain-deleted mutant, a PrsA hinge region-deleted mutant, or a PrsA serine-rich domain and hinge region-deleted mutant gene fused to the sfGFP gene at the 3' end (GOSTa:PrsA-sfGFP, GOSTa:PrsA DS-sfGFP, GOSTa:PrsA DH-sfGFP, and GOSTa:PrsA WD-sfGFP, respectively), and then cultured to analyze protein expression levels. (A) is a photograph of a CBB-stained gel, and (B) is the result of Western blot analysis using an anti-GFP antibody. [Figure 7] Lactobacillus paracasei was transformed with recombinant vectors in which the sfGFP gene was fused to the 3' end of PrsA, PrsA serine-rich domain-deficient mutant, PrsA hinge region-deficient mutant, or PrsA serine-rich domain and hinge region-deficient mutant gene (GOSTa:PrsA-sfGFP, GOSTa:PrsA DS-sfGFP, GOSTa:PrsA DH-sfGFP, and GOSTa:PrsA WD-sfGFP, respectively) and then cultured. sfGFP expressed on the cell surface of the Lactobacillus was analyzed by whole-cell ELISA using an anti-GFP antibody, and the amount of sfGFP expressed on the outer surface of the cell membrane was compared. [Figure 8] The nucleotide sequence (A) and amino acid sequence (B) of the protein in which the mouse B7-H1 gene was fused to the 3' end of the PrsA gene are shown. The nucleotide and amino acid sequences of PrsA are shown in normal bold, while the mouse B7-H1 sequence is shown in bold. [Figure 9]After culturing Lactobacillus paracasei transformed with a vector containing the coding sequence for a fusion protein in which the mouse B7-H1 gene was fused to the 3' end of the PrsA gene, total proteins were stained with CBB (A) and Western blot results using an anti-PrsA antibody (B). M: protein size marker; 1: untransformed Lactobacillus paracasei; 2: GOSTaPrsA-transformed Lactobacillus paracasei control; 3: GOSTa:PrsA-mB7H1-transformed Lactobacillus paracasei; a: PrsA protein size position; b: PrsA-mB7H1 fusion protein size position. [Figure 10] Killed Lactobacillus paracasei cells transformed with the GOSTa:PrsA-mB7H1 vector were injected intramuscularly into mice twice at two-week intervals, and anti-B7H1 antibody levels were measured in serum four weeks later. (A) shows the animal experiment stages, indicating the time of intramuscular injection and serum collection time. (B) shows the results of ELISA analysis of anti-B7H1 antibodies in six mice administered GOSTa:PrsA-mB7H1 / Lactobacillus paracasei. Results of t-test analysis: * indicates p<0.05, ** indicates p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0025] To achieve the objectives of the present invention, the present invention provides a recombinant vector for cell surface expression of a target protein, characterized in that a polynucleotide encoding PrsA or its variant consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding the target protein are sequentially linked downstream of a PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0026] The PrsA protein according to the present invention may consist of the amino acid sequence of SEQ ID NO: 2 derived from Lactobacillus sakei, but is not limited thereto.

[0027] The scope of PrsA according to the present invention includes a protein having the amino acid sequence set forth in SEQ ID NO: 2 and functional equivalents of said protein. "Functional equivalents" refer to proteins that have at least 30% or more, preferably 40% or more, and more preferably 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 as a result of amino acid addition, substitution, or deletion, and that exhibit substantially the same physiological activity as the protein set forth in SEQ ID NO: 2. "Substantially the same physiological activity" refers to the activity of expressing a target protein on the cell surface.

[0028] Furthermore, in the recombinant vector of the present invention, the PrsA mutant may be one in which the 162nd to 166th residues in the amino acid sequence of SEQ ID NO: 2 are deleted, one in which the 281st to 303rd residues in the amino acid sequence of SEQ ID NO: 2 are deleted, or one in which the 162nd to 166th residues and the 281st to 303rd residues in the amino acid sequence of SEQ ID NO: 2 are deleted, but is not limited thereto.

[0029] In the present invention, residues 162 to 166 in the amino acid sequence of SEQ ID NO: 2 constitute the hinge region of the PrsA protein, and residues 281 to 303 in the amino acid sequence of SEQ ID NO: 2 constitute the serine-rich domain.

[0030] In one embodiment of the present invention, the polynucleotide encoding PrsA having the amino acid sequence of SEQ ID NO: 2 may have the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. Homologs of this nucleotide sequence are also included within the scope of the present invention. The "percent sequence identity" for a polynucleotide is determined by comparing the comparison region with two optimally aligned sequences. A portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0031] As used herein, the term "recombinant" refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, heterologous peptide, or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, in either sense or antisense form. Recombinant cells can also express genes found in the cell in its native state, but where the gene has been altered and reintroduced into the cell by artificial means.

[0032] The term "vector" is also used to refer to a DNA fragment or nucleic acid molecule that is transferred into a cell. A vector allows DNA to replicate and can be reproduced independently in a host cell. The term "vector" is often used interchangeably with "vector." The term "expression vector" refers to a recombinant DNA molecule that contains a target coding sequence and the appropriate nucleic acid sequences necessary to express the operably linked coding sequence in a particular host organism.

[0033] In the present invention, the polynucleotide encoding PrsA or its mutant and the gene sequence encoding the target protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Generally, any plasmid or vector can be used as long as it can replicate and be stabilized in a host. Important characteristics of the expression vector include a replication origin, a promoter, a marker gene, and a translation control element.

[0034] An expression vector containing a polynucleotide encoding the PrsA or its variant of the present invention, a gene sequence encoding a target protein, and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector may also contain a ribosome binding site as a translation initiation site and a transcription terminator.

[0035] As used herein, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or greater, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned variant.

[0036] In addition, in a recombinant vector according to one embodiment of the present invention, the polynucleotide encoding PrsA or its variant may be operably linked downstream of a PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

[0037] As used herein, the term "operably linked" refers to a nucleic acid fragment being bound to another nucleic acid fragment such that its function or expression is affected by the other nucleic acid fragment. That is, the polynucleotide encoding PrsA or a variant thereof can be linked to the PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3 so that its expression can be regulated by the PrsA promoter.

[0038] As used herein, the term "target protein" refers to a protein that can be expressed on the cell surface of a microorganism transformed by inserting a polynucleotide encoding the protein into a recombinant vector.

[0039] In the recombinant vector according to the present invention, the target protein may be any one selected from the group consisting of a ligand protein, a receptor protein, an enzyme protein, and a protein derived from a virus or a bacterium, but is not limited thereto. Proteins such as hormones, hormone analogs, enzyme inhibitors, antibodies or fragments thereof, toxin proteins, cytokines, transcriptional regulators, and blood coagulation factors may also be included in the scope of the target protein according to the present invention.

[0040] The recombinant vector according to the present invention is capable of expressing a target protein on the cell surface and is characterized by being an E. coli-Lactobacillus shuttle vector.

[0041] The present invention also provides a microorganism transformed with a recombinant vector for cell surface expression of the target protein of the present invention.

[0042] In the present invention, the microorganism may preferably be a lactic acid bacterium, more preferably a Lactobacillus lactic acid bacterium, but is not limited thereto.

[0043] The present invention also provides a method for expressing a target protein on the surface of a microorganism, which comprises the step of transforming a microorganism with a recombinant vector for cell surface expression of the target protein of the present invention.

[0044] In the method for expressing a target protein on the surface of a microorganism according to the present invention, the recombinant vector for cell surface expression, the target protein, and the microorganism are the same as those described above.

[0045] Methods for transporting the recombinant vector of the present invention into a microorganism, i.e., for transformation, can be carried out by, but are not limited to, the CaCl2 method, the Hanahan method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580), conjugation (Heinze et al. BMC microbiology 2018, 18:56), and electroporation.

[0046] The present invention also provides a method for producing a microorganism in which a target protein is expressed on its cell surface, the method comprising the steps of: culturing a microorganism transformed with a recombinant vector for cell surface expression of a target protein to express the target protein on the cell surface; and recovering the microorganism in which the target protein is expressed on its cell surface; and a microorganism in which the target protein is expressed on its cell surface produced by the method.

[0047] In the method for producing a microorganism in which a target protein is expressed on the cell surface according to the present invention, the recombinant vector for cell surface expression, the target protein, and the microorganism are the same as those described above.

[0048] The transformed microorganism can be cultured in a medium suitable for the production of the target protein using known techniques. Suitable culture media can be commercially available or prepared using ingredients and composition ratios described in publications such as the American Type Culture Collection catalog, but are not limited thereto.

[0049] In accordance with one embodiment of the present invention, the microorganism expressing a target protein on its cell surface may be, but is not limited to, a lactic acid bacterium expressing B7-H1 (B7 homolog 1, also known as programmed death-ligand 1 or cluster of differentiation 274) or a fragment thereof on its cell surface. B7-H1 (or PD-L1) is a type of immune checkpoint inhibitor and is a factor used as a major target in research and development of immune anticancer drugs that activate immune cells to attack cancer cells.

[0050] The present invention provides an injectable or oral preparation containing, as an active ingredient, a microorganism that expresses B7-H1 or a fragment thereof as a target protein on the cell surface by using a bacterium belonging to the genus Lactobacillus, which is a GRAS microorganism, as an antigen carrier.

[0051] The term "injectable formulation" as used herein refers to a formulation suitable for injection into humans and / or vertebrates, whether intradermal, subcutaneous, intramuscular, or intravenous. Such formulations are sterile, pyrogen-free, and have a physiologically acceptable pH. The pH of an injectable formulation is important, particularly in relation to the safety and comfort of injection, especially when the formulation is provided as a liquid formulation. Suitable formulations may contain preservatives, such as sodium benzoate, methylparaben, and propylparaben, and may have a pH of 6.8 to 8.0 at 25°C. The pH is preferably maintained by a buffer.

[0052] The oral preparation may be in the form of powder, granules, tablets, capsules, lozenges, suspensions, emulsions, syrups, or aerosols. The solid preparation for oral administration may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0053] The present invention also provides a composition for inducing immunity in vertebrates other than humans, which contains the microorganism as an active ingredient.

[0054] The composition for inducing immunity according to the present invention comprises, as an active ingredient, lactic acid bacteria that express an antigen as a target protein on the cell surface using a recombinant vector for cell surface expression of a target protein according to the present invention, and the lactic acid bacteria can be administered to a vertebrate animal other than a human to induce an immune response. The vertebrate animal may be, preferably, but not limited to, a mammal other than a human.

[0055] The composition for inducing immunity of the present invention has antigens expressed on the cell surface of lactic acid bacteria, which are recognized as safe as GRAS microorganisms, and can successfully induce immunity through a common administration method, thereby having efficient and excellent immunity-inducing effects and, as a result, having disease treatment effects through immune induction.

[0056] The present invention also provides a method for producing a protein array, which comprises the step of immobilizing, on the surface of a substrate, a microorganism that is produced by the method for producing a microorganism of the present invention and expresses a target protein on its surface.

[0057] Protein arrays, like DNA arrays or DNA chips, provide a means for analyzing the presence or absence and level of expression of desired target proteins in specific cells by arraying various proteins, particularly antibodies, on a solid surface. To manufacture a protein array, proteins to be arrayed must be secured and immobilized on a solid surface. Analysis using a protein array involves various treatments, such as high temperature, salt concentration, and pH changes, to bind to the immobilized proteins and wash away unbound proteins. This requires the immobilization of stable proteins that can withstand such harsh environments. However, cloning thousands to tens of thousands of protein genes into expression vectors, expressing and isolating them, and then immobilizing them on a solid surface requires numerous repeated steps. Therefore, a simpler and faster method for performing these steps is needed.

[0058] The protein array of the present invention can be produced by any method commonly used in the art. The protein array produced by the method of the present invention can be used in diagnostic kits, gene expression analysis, protein-protein, protein-ligand, and antigen-antibody interaction analysis, metabolic process analysis, discovery of new or improved enzymes, combinatorial biochemical synthesis, and biosensors.

[0059] Solid substrates that can be used in the present invention include glass (e.g., glass with exposed functional groups), Si, Ge, GaAs, GaP, SiO, SiN, modified silicon nitrocellulose, polyvinylidene fluoride, polystyrene, polytetrafluoroethylene, polycarbonate, nylon, fiber, or a combination thereof. Linker molecules may be attached to the above substrates for protein immobilization, and the remaining unspotted areas are preferably blocked. The amount of surface-expressing cells of the present invention applied to each spot (or address) is determined by the array format. Interaction between a sample and the surface-expressed protein of the present invention immobilized on a solid substrate can be detected using the intrinsic properties of the protein (e.g., immunoreactivity), or by binding a suitable label (e.g., fluorescent substance, luminescent substance, radioactive substance, epitope) to the surface-expressed protein and detecting a change in the signal of the label. Analysis of the final results from the protein array of the present invention can be performed using automated devices known in the art as "scanners" or "readers."

[0060] The present invention also provides a method for inducing immunity in a vertebrate, comprising the step of administering to the vertebrate a microorganism that is produced by the method for producing a microorganism of the present invention and that expresses an antigen on its surface.

[0061] In the method for inducing immunity in a vertebrate according to the present invention, the vertebrate may be preferably a mammal other than a human, but is not limited thereto.

[0062] As used herein, when any part "comprises" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified. Also, as used herein, the term "combinations thereof" included in a Markush expression means a mixture or combination of one or more elements selected from the group consisting of the elements set forth in the Markush expression, and means that it includes one or more elements selected from the group consisting of the elements.

[0063] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0064] Example 1. Isolation and selection of lactic acid bacteria from traditional fermented kimchi To isolate GRAS lactic acid bacteria from kak kimchi (mustard greens kimchi) and Chinese cabbage kimchi made using traditional methods in Changwon, Gyeongnam, South Korea, 1 mL of kimchi juice was inoculated into 100 mL of MRS (phosphate-yeast-peptone-sodium nitrate; K2HPO46g / L, KH2PO42g / L, Bactotripton 5g / L, NaNO3 2.55g / L, and yeast extract 10g / L) liquid medium and enriched for 24 hours at 30°C under anaerobic conditions. The resulting enrichment cultures were diluted and plated on MRS agar medium. After 48 hours of anaerobically incubation at 30°C, grown colonies were selected and subjected to 16S rDNA sequencing analysis. As shown in Table 1 below, a variety of microorganisms were isolated from each sample, with 16S rDNA sequence identity of over 99.6%.

[0065] [Table 1]

[0066] Example 2. Screening of overexpressed cell membrane proteins from Lactobacillus sakei The strain used in the experiment (Sample #: FT007, L. sakei) was cultured in MRS medium (Difco TMLactobacilli were cultured in anaerobic conditions in MRS Broth (BD) for 24 hours, washed three times with 0.85% saline, suspended in 50 mM ammonium bicarbonate (NH₄HCO₃, pH 8.0) buffer, and treated with lysyl endopeptidase (Lys-C, Cat# NC9223464, Wako) for 2 hours. The cells were then centrifuged at 4,500 × g for 20 minutes to remove lactic acid bacteria. The supernatant, which contained Lys-C-digested peptide fragments derived from proteins present in the cell outer membrane, was purified using a C18 SepPak cartridge (Sep-Pak C18 1 cc Vac, WAT054955, Waters). The Lys-C peptide fragments were then collected in 80% acetonitrile and lyophilized at -80°C for 24 hours. The dried peptide pool was dissolved in 20 μL of ammonium bicarbonate buffer, and 5 μL of the solution was separated using a nano-LC (EASY-nLC1000, Thermo Fisher) liquid chromatography system equipped with a C18 column. The resulting solution was subjected to electrospray ionization, and each peptide was sequenced using a mass spectrometer (LTQ Orbitrap Velos LC-MS / MS, Thermo). The amino acid sequences of the identified peptides, separated sequentially using a C18 hydrophobic column with a 2% to 30% acetonitrile gradient, were then subjected to a MASCOT database search (Matrix Science) to identify extracellular membrane proteins, confirming the presence of the PrsA lipid membrane protein, which is abundant in the extracellular membrane. Figure 1 shows the MS / MS results for the representative peptide N-WANDQTVMAK-C (SEQ ID NO: 6), consisting of amino acids 255 to 264, from the five Lys-C cleavage peptide sequences identified as PrsA lipid membrane proteins.

[0067] Example 3. Cloning of the PrsA promoter and the gene encoding the PrsA protein from Lactobacillus sakei Based on the strain information and identified protein information obtained in Examples 1 and 2, it can be assumed that the PrsA protein is a membrane protein that is highly likely to be a cell surface expression anchor protein. Since it is expressed in large quantities on the outer cell wall, the promoter that induces the expression of the PrsA protein is a very strong promoter. Therefore, the KEGG database (https: / / www.kegg.jp) was used to confirm the PrsA gene information of L. sakei and the promoter information that regulates PrsA mRNA synthesis. The 300 base sequence from before the ATG start codon of the PrsA gene in the 5' upstream direction was considered to be the PrsA promoter, and the primer set [5'-end promoter of PrsA of sFT007:5'-aa a ct gca g ga aat caa aac aac agc tg-3' (underlined: PstI recognition site, SEQ ID NO: 7) and 3'-end of PrsA of sFT007: 5'-ttt- tct-ata The 1.22 kb gene was amplified by PCR using chromosomal DNA of L. sakei isolated and purified using the primers [-tta tta gga tcc ttt tga tga tga ttt gac-3' (underlined: XbaI recognition site, SEQ ID NO: 8)] as a template, and then cloned into the lactic acid bacteria-Escherichia coli shuttle vector pFT003 (Korean Patent Registration No. 10-0469800, identical to pHCE1LB:BCA) using PstI and XbaI to prepare pGOSTa:PrsA (Figure 3).

[0068] Example 4. Induction of PrsA expression in the outer membrane of Lactobacillus paracasei pGOSTa:PrsA prepared in Example 3 was electroporated into the L. paracasei FT003 strain listed in Table 1. The transformed L. paracasei colonies were inoculated into 15 mL of MRS liquid medium and cultured at 30°C for 24 hours under anaerobic conditions. Five mL of the culture medium was harvested, washed three times with 0.85% saline, and disrupted using a bead beater. 20 μg of the recovered total protein was electrophoresed on a 12.5% ​​SDS-PAGE gel and stained with Coomassie Brilliant Blue (CBB) (Figure 4A). Protein bands separated by size on SDS-PAGE were transferred to a PVDF membrane and then Western blotted using an anti-PrsA antibody. The exact location and size of the PrsA protein, as well as the amount of PrsA protein expressed relative to the total protein, were confirmed (Figure 4B).

[0069] Generally, there are few methods for stable, over-expression of target proteins in lactic acid bacteria through genetic engineering. However, as shown in Figure 4, the L. sakei PrsA promoter does not compete with the PrsA promoter present on the L. paracasei chromosome, enabling stable, over-expression of L. sakei PrsA in L. paracasei host cells. This was confirmed by Western blot analysis using a PrsA-specific antibody along with CBB staining (Figures 4A and 4B). In particular, an anti-PrsA polyclonal antibody (KOMA#24462) prepared using purified L. sakei PrsA protein did not cross-react with L. paracasei PrsA (54% amino acid sequence identity and 71% similarity between the L. sakei and L. praracsei PrsA proteins). Furthermore, the L. sakei PrsA protein synthesized in the pGOSTa:PrsA surface expression system accounted for more than 3% of the total protein expressed in L. praracsei.

[0070] Next, to confirm whether the overexpressed L. sakei PrsA protein was present in the L. paracasei cell membrane, 0.5 mL of a 1.5 mg / mL solution of total protein disrupted with a bead beater was centrifuged at 20,000 × g for 4 hours at 4°C. The supernatant was transferred to a new tube, and the pellet was redissolved in 0.5 mL of PBS. Eight μl of each protein solution containing total protein was mixed with 2 μl of 5X SDS-PAGE sample buffer, heated at 97°C for 5 minutes, and then electrophoresed on a 10% SDS-PAGE gel followed by CBB staining. As shown in Figure 4C, the 31.4 kDa L. sakei PrsA protein present in lane 1 of the total protein sample was not detected in lane 2 of the cytoplasmic solution, but was entirely detected in lane 3 of the cell membrane protein solution. This confirms that the majority of the PrsA protein overexpressed by the GOSTa:PrsA vector is present in the L. paracasei cell membrane.

[0071] Example 5. Preparation of PrsA-sfGFP fusion protein expression vector (pGOSTa:PrsA-sfGFP) The sfGFP gene cloned into the pQI30 vector was ligated with forward primer sfGFP BamHI [5'-aaa a gg atc c at gag caa agg aga ag-3' (underlined: BamHI recognition site, SEQ ID NO: 9)] and the reverse primer sfGFP KpnI [5'-tct t gg tac c tt tgt aga gct cat cca-3' (underlined: KpnI recognition site, SEQ ID NO: 10)] was amplified by PCR, followed by treatment with BamHI and KpnI. The pGOSTa:PrsA vector prepared in Example 3 was then treated with the same restriction enzymes, and the two DNA fragments were ligated to prepare pGOSTa:PrsA-sfGFP (Figure 5).

[0072] Example 6. PrsA structural analysis, PrsA anchor motif improvement, and construction of an sfGFP fusion surface expression system The PrsA protein is firmly anchored to the cell membrane by a covalent bond between the 21st amino acid cysteine ​​at its N-terminus and a carbon atom in the glycerol molecule, a lipid that is a major component of the cell membrane. The C-terminus of PrsA is composed of an NC domain located close to the N-terminus. PrsA proteins have a single dimer structure, with the N- and C-termini of each protein closely adjacent to each other, forming a cyclic dimer. The serine-rich domain at the C-terminus of each PrsA protein is believed to play an important role in the formation of the NC domain structure and is thought to interact with the peptidoglycan layer, a component of the cell wall of Gram-positive bacteria, maintaining structural stability within the cell wall.

[0073] The key to surface expression in lactic acid bacteria is to maximize the expression of a specific protein outside the outer membrane, rather than inside the peptidoglycan layer. The present inventors determined that deleting 23 amino acid residues (DLSDILSSYGVNAKKSSAKSSSK, SEQ ID NO: 4, Figure 2) in the serine-rich domain of PrsA would enable the delivery of foreign proteins to the outside of the cell wall more efficiently than wild-type PrsA. To delete the serine-rich domain of the PrsA protein, the pGOSTa:PrsA vector prepared in Example 3 was used as a template, and the 5'-end promoter of PrsA of sFT007 primer (SEQ ID NO: 7) and the delete C-term serine-rich domain of PrsA primer [5'-ggg ggg tac ctt atc a gg atc c PCR was performed using the following sequence: TT TAT CCT TGA TTG TTA CGT CGG C-3' (underlined: BamHI recognition site, SEQ ID NO: 11), and the amplified 1.4 kb DNA fragment was recovered by digestion with the restriction enzymes PstI and BamHI. The restriction enzyme-treated DNA fragment and pGOSTa:PrsA-sfGFP prepared in Example 5 were treated with the same restriction enzymes PstI and BamHI, and the recovered 7.25 kb vector DNA fragment was ligated to obtain pGOSTa:PrsA DS-sfGFP.

[0074] As previously described, PrsA has a curved structure with its N-terminus and C-terminus closely spaced. In the present invention, we confirmed the hinge region, located in the middle of the PrsA protein sequence, through structural analysis. We then determined that deleting the 'KDNST' amino acid sequence (SEQ ID NO: 5, Figure 2), which is presumed to play the most crucial role in the hinge region, would enable efficient delivery of a specific protein fused to PrsA to the outside of the cell wall. To delete the five amino acids in the hinge region, we used the pGOSTa:PrsA-sfGFP vector obtained in Example 5 as a template and a forward-direction hinge deletion primer [5'-ccc c ct cga g aa gaa gta ctc aac aga t-3' (underlined: XhoI recognition site, SEQ ID NO: 12)] and a hinge deletion reverse primer [5'-ccc c ct cga g PCR was performed using the following fragment: ct taa gtt cag aaa taa c-3' (underlined: XhoI recognition site, SEQ ID NO: 13), and an 8.45-kb PCR product was recovered. The recovered PCR product was digested with the restriction enzyme XhoI and then self-ligated to obtain pGOSTa:PrsA DH-sfGFP.

[0075] The PrsA WD mutant, in which both the PrsA serine-rich domain and the hinge region were deleted, was generated using the pGOST:PrsADS-sfGFP plasmid as a template and the hinge deletion full sequence forward primer [5'-ccc c ct cga g aa gaa gta ctc aac aga t-3' (underlined: XhoI recognition site, SEQ ID NO: 14)] and a hinge deletion full sequence reverse primer [5'-ccc c ct cga gPCR was performed using the following fragment: ct taa gtt cag aaa taa c-3' (underlined: XhoI recognition site, SEQ ID NO: 15). The amplified 7.27-kb DNA fragment was digested with XhoI and then self-ligated to obtain pGOST:PrsA WD-sfGFP.

[0076] Example 7. Confirmation of lactic acid bacteria cell surface expression of sfGFP fused with PrsA improved anchor motif The four isolated plasmids, pGOST:PrsA-sfGFP, pGOST:PrsA DS-sfGFP, pGOST:PrsA DH-sfGFP, and pGOST:PrsA WD-sfGFP, were electroporated into L. paracasei isolated from kakkimchi. The four transformed L. paracasei strains were cultured in MRS liquid medium under anaerobic conditions for 24 hours. Five milliliters of each L. paracasei culture was harvested, washed three times with 0.85% saline, and disrupted using a bead beater to recover total protein. Total protein content in each L. paracasei strain was quantified using the bicinchoninic acid (BCA) method. 20 μg of total protein was then electrophoresed on two 12.5% ​​SDS-PAGE gels to separate the proteins by size. After electrophoresis, one PAGE gel was stained with CBB, and the remaining gel was transferred to a PVDF membrane and then Western blotted using an anti-sfGFP antibody (Santa Cruz Biotechnology, Cat# sc-9996).

[0077] As shown in Figure 6, the CBB image shows that the five sample proteins, including the control and L. paracasei, were uniformly distributed on SDS-PAGE. The Western blot images confirm the stable expression of sfGFP fused to PrsA and PrsA mutants in L. paracasei. Furthermore, each fusion protein showed slight differences in the mutants at the same position as the theoretical molecular weight. The PrsA hinge region-deleted anchor, PrsA hinge region and serine-rich domain-deleted anchor, and PrsA serine-rich domain-deleted anchor showed slight differences in surface expression compared to the PrsA wild-type anchor protein. These results confirm the stable expression of the target protein sfGFP in L. paracasei using four plasmids containing PrsA and PrsA-modified anchor motifs: pGOST:PrsA-sfGFP, pGOST:PrsA DS-sfGFP, pGOST:PrsA DH-sfGFP, and pGOST:PrsA WD-sfGFP.

[0078] Next, we used a whole-cell ELISA method (Bonnie L. Elder et al., J. Clin. Microbiol. 1982, 16:141-144; Albritton et al., PLOS One 2017, 12(8):e0183101) to compare and analyze whether sfGFP expressed in the four plasmids fused with PrsA or the PrsA-improved anchor motif was effectively expressed on the outer surface of L. paracasei cells.

[0079] As shown in Figure 7, the level of sfGFP expressed on the cell surface was determined in the following order: PrsA hinge region-extracted anchor (PrsA DH), PrsA hinge region and serine-rich domain-extracted anchor (PrsA WD), PrsA serine-rich domain-extracted anchor (PrsA DS), and PrsA wild-type anchor (PrsA) motif. These results demonstrate that PrsA wild-type and PrsA improved anchor motifs can effectively express target proteins on the cell surface of lactic acid bacteria, and that the PrsA improved anchor motif is more effective at expressing target proteins on the cell surface than PrsA wild-type.

[0080] Example 8. Preparation and surface expression of surface expression vector pGOSTa:PrsA-mB7-H1 The PD-L1 (CD274) (NM_021893) Mouse Tagged ORF clone (ORiGENE, Cat. # MR203953) containing the mouse B7-H1 gene was used as a template, and the mouse B7-H1 forward primer [5'-AAA GGA TCC GAC TTG TAC GTG GTG GAG-3' (underlined: BamHI recognition site, SEQ ID NO: 16)] and mouse B7-H1 reverse primer [5'-GGG TCT AGA The mouse B7-H3 gene was amplified by PCR using the following sequence: ACT AGT GTC GAC TTA GTT GAT TTT GCG GTA TGG GGC ATT-3' (underlined: XbaI recognition site, SEQ ID NO: 17). The PCR product was then digested with the restriction enzymes BamHI and XbaI to recover a DNA fragment of approximately 360 bp. The recovered 360 bp DNA fragment and the vector prepared in Example 3 were digested with BamHI and XbaI, respectively, and the recovered 360 bp DNA fragment and 7.7 kb DNA fragment were ligated to construct pGOSTa:PrsA-mB7-H1. The nucleotide sequence of the entire gene encoding the PrsA-mB7-H1 fusion protein and the amino acid sequence of the fusion protein are shown in Figure 8.

[0081] The isolated pGOSTa:PrsA-mB7-H1 plasmid was electroporated into the L. paracasei FT003 strain isolated in Example 1, and the transformed lactic acid bacteria colonies were cultured as described in Example 4. Five milliliters of culture medium was harvested, washed three times with 0.85% saline, and disrupted using a bead beater. 20 μg of the recovered total protein was electrophoresed on a 12.5% ​​SDS-PAGE gel and stained with CBB (Figure 9A). Protein bands separated by size on SDS-PAGE were transferred to a PVDF membrane and Western blotted using an anti-B7-H1 antibody (R&D Systems Cat #AF1019) (Figure 9B). The large-scale expression and size of the PrsA-mB7-H1 fusion protein were confirmed. Furthermore, the expression level of the PrsA-mB7-H1 fusion protein relative to the total protein was confirmed, and it was confirmed that the expression level of the PrsA-mB7-H1 fusion protein was the most stable and abundant, as confirmed by SDS-PAGE CBB staining.

[0082] Example 9. Antibody-inducing effect of lactic acid bacteria expressing mouse B7-H1 protein on their surface To examine the antigenicity of the surface protein of the L. paracasei FT003 strain transformed with pGOSTa:PrsA-mB7-H1 prepared in Example 8, we examined whether neutralizing antibodies were formed. L. paracasei strains expressing the antigen on their surface were killed using ethanol, washed three times with 0.85% sodium chloride solution, and then transferred to six 6-week-old male BALB / c mice at 5 weeks of age and allowed to stand for one week. 1 x 10 7Killed cells were administered intramuscularly twice at two-week intervals. Six mice were used for each of the 0.85% sodium chloride control group and the L. paracasei FT003-only experimental group. Serum from each experimental group was collected four weeks after the second intramuscular injection, and neutralizing antibody titers against each antigen were measured and compared using ELISA. As a result, as shown in Figure 10 (B), the anti-B7-H1 antibody titer in the pGOSTa:PrsA-mB7-H1-transformed L. paracasei-treated group was higher than that in the 0.85% sodium chloride control group and the L. paracasei FT003-transfectant-only group, confirming statistical significance.

[0083] Based on the above results, it was found that the pGOSTa:PrsA vector of the present invention can be used as a platform technology that can express various target proteins on the cell surface of microorganisms by using PrsA as a cell membrane anchor, and it was confirmed that when microbial cells transformed with "pGOSTa:PrsA-antigen" are administered to animals in order to use the surface-expressed target protein as an antigen, antibodies against the antigen can be induced.

Claims

1. A polynucleotide encoding PrsA or a variant thereof consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding a target protein are sequentially linked downstream of a PrsA promoter consisting of the base sequence of SEQ ID NO:

3. A recombinant vector for cell surface expression of a target protein, characterized in that:

2. The PrsA mutant lacks residues 162 to 166 in the amino acid sequence of SEQ ID NO:

2. A recombinant vector for cell surface expression of the target protein according to claim 1.

3. The PrsA mutant lacks residues 281 to 303 in the amino acid sequence of SEQ ID NO:

2. A recombinant vector for cell surface expression of the target protein according to claim 1.

4. The PrsA mutant lacks residues 162 to 166 and residues 281 to 303 in the amino acid sequence of SEQ ID NO:

2. A recombinant vector for cell surface expression of the target protein according to claim 1.

5. The target protein is any one selected from the group consisting of a ligand protein, a receptor protein, an enzyme protein, and a protein derived from a virus or a bacterium. A recombinant vector for cell surface expression of the target protein according to claim 1.

6. Transformed with the recombinant vector according to any one of claims 1 to 5 A microorganism characterized by:

7. The microorganism is a lactic acid bacterium The microorganism described in claim 6.

8. 6. A method for producing a recombinant vector comprising the steps of: transforming a microorganism with a recombinant vector according to any one of claims 1 to 5; A method for expressing a target protein on the surface of a microorganism, comprising:

9. Culturing the transformed microorganism of claim 6 to express the target protein on the cell surface; and recovering the microorganisms in which the target protein is expressed on the cell surface. A method for producing a microorganism in which a target protein is expressed on the cell surface, characterized in that

10. The target protein is any one selected from the group consisting of a ligand protein, a receptor protein, an enzyme protein, and a protein derived from a virus or a bacterium. The method of claim 9.

11. Produced by the method of claim 9 A microorganism in which a target protein characterized by the above-mentioned is expressed on the cell surface.

12. The target protein is B7-H1 (B7 homolog 1) or a fragment thereof. A microorganism in which the target protein according to claim 11 is expressed on the cell surface.

13. A method for treating a disease comprising administering to a patient a microorganism according to claim 12 as an active ingredient.

1. An injectable preparation comprising:

14. A method for treating a disease comprising administering to a patient a microorganism according to claim 12 as an active ingredient. An oral preparation characterized by:

15. The method according to claim 9, further comprising the step of immobilizing a microorganism on the surface of a substrate, the microorganism having a target protein expressed on its surface. A method for producing a protein array, comprising:

16. 10. A method for producing a microorganism having an antigen expressed on its surface, comprising administering the microorganism to a vertebrate. A method for inducing immunity in a vertebrate.

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