Cell surface high-volume expression technology using an extracellular membrane lipoprotein PrsA expression system

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 derivatives thereof present on the outer membrane of lactic acid bacteria for cell surface expression of target proteins, and a cell surface expression vector containing a PrsA promoter that induces mass expression of PrsA and a nucleotide sequence encoding the PrsA.

Background Art

[0002] A cell surface display system expresses proteins and peptides on the cell surface and exposes them outside the cell. This system freely exposes the proteins and peptides displayed on the cell surface to the extracellular space. Displaying foreign proteins on the cell surface is considered important in biotechnological and industrial application fields such as vaccine development, whole-cell biocatalysis, bioabsorbents, and biosensors.

[0003] When attempting to express a protein on the surface of a cell, the protein synthesized intracellularly must have a secretion signal that can pass through the cell membrane on the primary sequence of the protein. In addition, there are differences in the cell surface expression system depending on the type of cell in which the target protein, enzyme, peptide, etc. are to be surface-expressed. In the case of bacteria, proteins synthesized in the cytoplasm or near the cell membrane must have a signal sequence in order to enter the cell membrane or be secreted outside the cell. In particular, in the case of Gram-negative bacteria, they must be retained so that they can pass through the inner cell membrane and the periplasmic space and be inserted and attached to the outer cell membrane and protrude outside the membrane. For this purpose, a secretion signal present in outer membrane proteins, enzymes or toxin proteins secreted outside the cell, and a targeting signal to be retained on the cell surface are required.

[0004] Four types of anchor proteins exist that allow foreign proteins to be fixed to the thick cell walls of Gram-positive bacteria such as lactic acid bacteria. These types are (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 anchor proteins, which belong to the general membrane protein types of Gram-positive and Gram-negative bacteria, are composed of hydrophobic amino acids, with 20-30 amino acids at the N-terminus or C-terminus inserted into the cell membrane in an alpha-helix structure to form an anchor. However, when expressed in large quantities on the cell membrane, they significantly affect membrane rigidity, thus limiting their expression. The second type of lipoprotein anchor exists on the cell surface through a mechanism in which the -SH group of a cysteine ​​residue, present immediately after the secretory signal, is covalently linked to the carbon of the glycerol head of a lipid, a component of the cell membrane, thus anchoring it to the cell membrane. Unlike the transmembrane anchors described above, such lipoprotein anchors do not penetrate the cell membrane and affect membrane rigidity, and exist in a form that hangs from the lipid bilayer, thus offering the significant advantage of being expressible in large quantities on the cell surface.

[0006] In most cases, such as Gram-positive and Gram-negative bacteria or yeast, the biggest obstacle to commercialization of vaccine and biocatalyst development using microbial surface expression systems is the difficulty in expressing target proteins in large quantities on the cell surface. In particular, over the past 50 years of molecular biology research, progress has been made in developing high-expression systems using base sequence changes of promoters within the expression system or derivatives (inducers) that bind to inhibitors (repressors), and in developing microbial hosts that can stably express target proteins within cells through specific gene manipulation or removal within the chromosomes of specific bacteria used as carriers. However, to date, with the exception of certain industrialized strains such as E. coli, it can be said that there is no artificial technology that can express a single specific target protein at a concentration of 1% or more of the total protein per microorganism. Furthermore, there are virtually no systems that can express specific target proteins in large quantities on the cell membrane or extracellular membrane, rather than in the cytoplasm. In the commercialization of cell surface expression technology for biocatalysts for biotransformation processes, the most important core factors are the amount of enzyme expressed on the surface and the stability of that enzyme. In particular, in vaccine development, the amount of bacteria that can be administered into the body is limited due to side effects such as inflammation. When the total amount of antigen that can be presented relative to the amount of microorganisms administered is limited, the induced immune response may be insufficient to protect against disease. Therefore, the cell surface expression level of the target antigen is a crucial factor.

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

[0008] Furthermore, Korean Published Patent No. 2019-0037481 discloses "a method for cell surface expression of a target protein using a cell-fixed motif derived from Corynebacterium," Korean Published Patent 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 inositolanchor protein) genes, including HpSED1, HpGAS1, HpTIP1, and HpCWP1, isolated from the methanol-magnetized yeast Hansenula polymorpha, and Korean Published Patent No. 2004-0032824 discloses "a surface expression vector using a polygamma-glutamic acid synthesis gene derived from a Bacillus strain isolated from Cheonggukjang, and a method for microbial surface expression of a protein using the same." However, the "technology for high-volume cell surface expression using an extracellular membrane lipoprotein PrsA expression system" using PrsA or its variants, which are lactic acid bacteria surface proteins of the present invention, and the PrsA promoter is not described. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Republic of Korea Published Patent No. 2019-0037481 [Patent Document 2] Republic of Korea Published Patent No. 2002-0010428 [Patent Document 3] Republic of Korea Published Patent No. 2004-0032824 [Patent Document 4] Registered Patent Gazette No. 10-0469800 of the Republic of Korea [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 [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention was derived from the aforementioned requirements. The inventors isolated and cultured lactic acid bacteria, which are GRAS microorganisms, from kakkimchi, a traditional fermented food, and selected PrsA, an extracellular membrane lipoprotein that is constitutively expressed in large quantities on the surface of the isolated GRAS lactic acid bacteria through a proteolytic surface-shaving method, and its promoter.

[0012] The structure of selected PrsA proteins was analyzed, and cell surface expression vectors were constructed in which PrsA wild-type, PrsA serine-rich domain-removed mutant, PrsA hinge-region-removed mutant, or PrsA hinge-region and serine-rich domain-removed mutant coding sequences were operably linked under the control of the PrsA promoter. After cloning the reporter gene into the vectors, lactic acid bacteria were transformed, and the presence or absence of cell surface expression of the reporter protein was analyzed. 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 PrsA protein or its mutant coding sequences. Through this, the present invention was completed by confirming that the PrsA protein and its mutants can function as surface expression anchor motifs. [Means for solving the problem]

[0013] To solve the aforementioned 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 a target protein are sequentially linked downstream of a PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3.

[0014] Furthermore, the present invention provides microorganisms transformed with the recombinant vector.

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

[0016] Furthermore, the present invention provides a method for producing microorganisms expressing a target protein on the cell surface, comprising the steps of: culturing microorganisms transformed with the recombinant vector to express the target protein on the cell surface; and recovering the microorganisms expressing the target protein on the 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, containing the microorganism as an active ingredient.

[0020] The present invention also provides a method for manufacturing a protein array, including the step of immobilizing a microorganism produced by the method of the present invention and having a target protein expressed on its surface onto the surface of a substrate.

[0021] The present invention also provides a method for inducing immunity in vertebrates, including the step of administering a microorganism produced by the method of the present invention and having an antigen expressed on its surface to a vertebrate.

Advantages of the Invention

[0022] The present invention uses a protein 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 utilized in a method for cell surface expression of foreign proteins that have been difficult to stably express on the cell surface or to express in large quantities conventionally, and thus is expected to have a high industrial utilization rate.

[0023] Also, when expressing a ligand protein, receptor protein, or enzyme protein involved in in vivo signal transduction of general cells containing self-proteins using the expression system of the present invention, it can be utilized as a therapeutic agent for metabolic diseases. When expressing ligands or receptors of immune cells, it can be used as an immunotherapeutic agent, and when expressing bacteria or bacterial antigens, it can be used as a preventive or therapeutic vaccine. Therefore, the expression system of the present invention can be usefully used in the pharmaceutical industry.

Brief Description of the Drawings

[0024] [Figure 1] This is the MS / MS analysis result for the sequence from amino acid 255 to 264 (SEQ ID NO: 6), which is the representative peptide among the five Lys-C cleaved peptides identified. [Figure 2] This document shows the nucleotide and amino acid sequence information for the PrsA protein of Lactobacillus sakei. 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 indicated. [Figure 3] This is a pGOSTalpha:PrsA vector map containing the PrsA promoter sequence and the PrsA anchor motif coding sequence. [Figure 4] Figure 3 shows the results of confirming the expression of PrsA derived from L.sakei in Lactobacillus paracasei transformed with the pGOSTα:PrsA vector. (A) is a CBB-stained gel image of the total protein, (B) is the result of Western blotting using an anti-PrsA antibody after transferring the total protein to a PVDF membrane, and (C) is a gel image after separating cytoplasmic proteins and cell membrane proteins by high-speed centrifugation of the total protein and then staining each fraction with CBB. The position of the PrsA-anchored membrane protein (theoretical molecular weight, 31.4 kDa) is indicated by a red arrow. Figure 4A, B lane M: protein size marker (ExcelBang 3-color Broad Range Protein Marker, #PM2700 Thermo), lane 1: untransformed Lactobacillus paracasei, lane 2: transformed Lactobacillus paracasei. Figure 4C: lane M: protein size marker, lane 1: total protein, lane 2: cytoplasmic protein fraction, lane 3: cell membrane protein fraction. [Figure 5] Figure 3 shows the vector map of pGOSTa:PrsA-sfGFP, in which the reporter gene sfGFP was cloned into the vector. [Figure 6] The following are the results of analyzing protein expression levels after transforming Lactobacillus paracasei with recombinant vectors in which the sfGFP gene was fused to the 3' end of PrsA, PrsA serine-rich domain-deficient mutants, PrsA hinge region-deficient mutants, or PrsA serine-rich domain and hinge region-deficient mutant genes (GOSTa:PrsA-sfGFP, GOSTa:PrsA DS-sfGFP, GOSTa:PrsA DH-sfGFP, and GOSTa:PrsA WD-sfGFP, respectively), and culturing the cells. (A) is a CBB-stained gel photograph, and (B) is a Western blot result 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 either the PrsA, PrsA serine-rich domain-deficient mutant, PrsA hinge region-deficient mutant, or PrsA serine-rich domain and hinge region-deficient mutant genes (GOSTa:PrsA-sfGFP, GOSTa:PrsA DS-sfGFP, GOSTa:PrsA DH-sfGFP, and GOSTa:PrsA WD-sfGFP, respectively), and then cultured. The amount of sfGFP expressed on the surface of the lactic acid bacteria cells was then analyzed using an anti-GFP antibody via whole-cell ELISA to compare the amount of sfGFP expressed on the outside of the cell membrane. [Figure 8] The nucleotide sequence (A) and amino acid sequence (B) of a protein formed by fusing the mouse B7-H1 gene to the 3' end of the PrsA gene are shown. The nucleotide and amino acid sequences of PrsA are shown in standard thickness, while the sequence of mouse B7-H1 is shown in bold. [Figure 9]The images show gel photographs (A) and Western blot results (B) obtained by staining all proteins with CBB after culturing Lactobacillus paracasei transformed with a vector containing a fusion protein coding sequence in which the mouse B7-H1 gene is fused to the 3' end of the PrsA gene. M: protein size marker, 1: untransformed Lactobacillus paracasei, 2: GOSTaPrsA-transformed Lactobacillus paracasei control group, 3: GOSTa:PrsA-mB7H1-transformed Lactobacillus paracasei, a: PrsA protein size position, b: PrsA-mB7H1 fusion protein size position. [Figure 10] This shows the results of measuring anti-B7H1 antibody levels in serum four weeks after two intramuscular injections of dead Lactobacillus paracasei transformed with the GOSTa:PrsA-mB7H1 vector into mice at two 2-week intervals. (A) shows the stage of the animal experiment, indicating the timing of intramuscular injection administration and serum collection, and (B) shows the results of ELISA analysis of anti-B7H1 antibodies in six mice administered GOSTa:PrsA-mB7H1 / Lactobacillus paracasei. In the t-test analysis results, * indicates efficacy of p<0.05, and ** indicates efficacy of p<0.01. [Modes for carrying out 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 a variant thereof, consisting of the amino acid sequence of SEQ ID NO: 2, and a gene encoding a target protein are sequentially ligated downstream of a PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3.

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

[0027] The scope of PrsA according to the present invention includes proteins having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of said proteins. A "functional equivalent" is a protein that, as a result of the addition, substitution, or deletion of amino acids, has at least 30%, preferably 40%, and more preferably 50% sequence homology to the amino acid sequence shown in SEQ ID NO: 2, and exhibits substantially the same physiological activity as the protein shown in SEQ ID NO: 2. "Substantially the same physiological activity" means the activity that causes the target protein to be expressed on the cell surface.

[0028] Furthermore, in the recombinant vector according to the present invention, the PrsA mutant may be, but is not limited to, a mutant in which residues 162 to 166 are missing in the amino acid sequence of SEQ ID NO: 2, residues 281 to 303 are missing in the amino acid sequence of SEQ ID NO: 2, or residues 162 to 166 and 281 to 303 are missing in the amino acid sequence of SEQ ID NO: 2.

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

[0030] In one embodiment of the present invention, the polynucleotide encoding PrsA, consisting of the amino acid sequence of SEQ ID NO: 2, may, but is not limited to, the base sequence of SEQ ID NO: 1. Furthermore, homologs of the base sequence are included within the scope of the present invention. The "percentage of sequence homology" for a polynucleotide is determined by comparing two optimally sequenced sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) to the optimal sequences of the two sequences.

[0031] In this specification, the term “recombinant” refers to a cell that replicates or expresses a different nucleic acid, or a peptide, a different peptide, or a protein encoded by a different nucleic acid. Recombinant cells can express genes or gene sections not found in the cell’s native form, in either sense or antisense form. Recombinant cells can also express genes found in cells in their native state, however, such genes are modified and have been artificially reintroduced into the cell.

[0032] The term "vector" is also used to refer to DNA fragments or nucleic acid molecules that are transmitted within a cell. Vectors can replicate DNA and be independently reproduced in the host cell. The term "transmitter" is frequently used interchangeably with "vector." The term "expression vector" refers to a recombinant DNA molecule containing a target coding sequence and the appropriate nucleic acid sequences essential for expressing the coding sequence operably linked in a particular host organism.

[0033] In the present invention, the polynucleotide encoding PrsA or its variant and the gene sequence encoding the target protein may be inserted into a recombinant expression vector. The term “recombinant expression vector” means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Generally, any plasmid and vector that can replicate and stabilize in a host can be used. The key characteristics of the expression vector are that it has an origin of replication, a promoter, a marker gene, and a translation control element.

[0034] An expression vector comprising a polynucleotide encoding PrsA or a variant thereof, a gene sequence encoding a target protein, and an appropriate transcription / translation regulatory signal can be constructed by methods well known to those skilled in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques. The DNA sequence can be effectively ligated to an appropriate promoter within the expression vector to guide mRNA synthesis. The expression vector may also include a ribosome binding site as a translation initiation site and a transcription terminator.

[0035] In this specification, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently linked in a long chain, and is a DNA or RNA chain of a certain length or longer, and more specifically, a polynucleotide fragment that encodes the variant.

[0036] Furthermore, in a recombinant vector according to one embodiment of the present invention, the polynucleotide encoding PrsA or a variant thereof may, but is not limited to, being operably ligated downstream of a PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0037] In this specification, "operably linked" means that one nucleic acid fragment is bound to another nucleic acid fragment so that its function or expression is influenced by the other nucleic acid fragment. That is, the polynucleotide encoding PrsA or a variant thereof can be linked so that its expression is regulated by the PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0038] In this specification, the term "target protein" means 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, as described by a person skilled in the art.

[0039] In the recombinant vector according to the present invention, the target protein may be any one selected from the group consisting of ligand proteins, receptor proteins, enzyme proteins, and viral and bacterial-derived proteins, but is not limited thereto. Proteins such as hormones, hormone analogs, enzyme inhibitors, antibodies or fragments thereof, toxin proteins, cytokines, transcription regulators or 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 characterized by its ability to express target proteins on the cell surface (cell surface display) and by being an E. coli-Lactobacillus shuttle vector.

[0041] The present invention also provides microorganisms 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, and 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, comprising the step of transforming the 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 delivering the recombinant vector of the present invention into microorganisms, i.e., transformation, can be carried out by 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, but are not limited thereto.

[0046] The present invention also provides a method for producing a microorganism expressing a target protein on its cell surface, 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 expressing the target protein on its cell surface; and a microorganism expressing the target protein on its cell surface produced by the said method.

[0047] In the method for producing a microorganism expressing a target protein on its 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] Furthermore, the culture of the transformed microorganisms may be carried out in a medium suitable for the production of the target protein using known techniques. Suitable culture media may be commercially available or prepared according to the components and compositional ratios described in publications such as the American Type Culture Collection catalog, but are not limited thereto.

[0049] In a microorganism expressing a target protein on its cell surface according to one embodiment of the present invention, the microorganism 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 the development of immunosuppressant cancer drugs that activate immune cells to attack cancer cells.

[0050] The present invention provides an injectable or oral formulation containing, as an active ingredient, a microorganism that expresses B7-H1 or fragments thereof as a target protein on the surface of a cell, by using bacteria belonging to the genus Lactobacillus, which are GRAS microorganisms, as antigen carriers.

[0051] As used in this invention, the term "injectable formulation" means a formulation suitable for injection into humans and / or vertebrates, where the injection is intradermal, subcutaneous, intramuscular, or intravenous. Such formulations are sterile, pyrogenic, and have a physiologically acceptable pH. The pH of an injectable formulation is important, particularly in relation to safety and comfort during injection, and especially when the formulation is supplied as a liquid formulation. A suitable formulation may contain preservatives, such as sodium benzoate, methylparaben, and propylparaben, and its pH may be 6.8 to 8.0 at 25°C. The pH is preferably maintained by a buffer.

[0052] Furthermore, the oral formulation may be a powder, granules, tablet, capsule, lozenge, suspension, emulsion, syrup, or aerosol. The solid formulation for oral administration may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose, or 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 an immune induction composition for vertebrates other than humans, comprising the aforementioned microorganism as an active ingredient.

[0054] The immune-inducing composition according to the present invention contains lactic acid bacteria as an active ingredient, which 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 an immune response can be induced by administering the lactic acid bacteria to vertebrate individuals other than humans. The vertebrate is preferably a mammal other than a human, but is not limited thereto.

[0055] The immune-inducing composition of the present invention is a lactic acid bacterium whose safety has been recognized as GRAS (Generally Recognized as Safe) microorganisms, on which an antigen is expressed on the cell surface. By enabling successful immune induction through a general administration method, it has an efficient and excellent immune-inducing effect, and consequently, can have a disease-treating effect through immune induction.

[0056] The present invention also provides a method for producing a protein array, which includes the step of immobilizing a microorganism produced by the method for producing microorganisms of the present invention, on which a target protein is expressed on the surface, onto the surface of a substrate.

[0057] Protein arrays, like DNA arrays or DNA chips, provide a means to analyze the presence 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, the proteins to be arrayed must be secured and immobilized on a solid surface. The analysis process using protein arrays involves various treatments, such as changes in temperature, salt concentration, and pH, to bind immobilized proteins and wash away unbound proteins. This requires the immobilization of stabilized 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 a great deal of repetition. Therefore, there is a need to perform these operations more simply and quickly.

[0058] The manufacturing process for the protein array of the present invention may be subject to manufacturing methods commonly used in the industry. Protein arrays manufactured by the method of the present invention may be used in diagnostic kits, gene expression analysis, protein-protein, protein-ligand, and antigen-antibody interaction analysis, metabolic process analysis, novel or improved enzyme discovery, combination 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, SiN4, modified silicon nitrocellulose, polyvinylidene fluoride, polystyrene, polytetrafluoroethylene, polycarbonate, nylon, fiber, or combinations thereof. Linker molecules may be attached to the above substrates for protein immobilization, and it is preferable that the remaining unspotted areas are blocked. The amount of surface-expressed cells of the present invention applied to each spot (or address) is determined by the array configuration. The interaction between the surface-expressed proteins of the present invention immobilized on the solid substrate and the sample can be detected using the intrinsic properties of the protein (e.g., immunoreactivity), or by binding a labeling substance suitable for the surface-expressed protein (e.g., fluorescent substance, luminescent substance, radioactive substance, epitope) and detecting the signal change of the labeling substance. Analysis of the final results from the protein array of the present invention can be performed using automated equipment known in the industry as a "scanner" or "reader".

[0060] The present invention also provides a method for inducing immunization in vertebrates, comprising the step of administering to vertebrates microorganisms produced by the method for producing microorganisms of the present invention, on which an antigen is expressed on the surface.

[0061] In the method for inducing immunity in vertebrates according to the present invention, the vertebrates are preferably mammals other than humans, but are not limited thereto.

[0062] In this specification, when any part is said to "contain" a certain component, this does not mean that it excludes other components, but rather that it may further contain other components, unless otherwise stated. Also in this specification, the term "these combinations" in a Markush expression means one or more mixtures or combinations selected from the group of components described in the Markush expression, and means that it includes one or more selected from the group of components.

[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 (Generally Recognized as Safe) lactic acid bacteria from traditionally produced mustard greens kimchi and napa cabbage kimchi in Changwon, Gyeongsangnam-do, South Korea, 1 mL of kimchi juice was inoculated into 100 mL of MRS (phosphate-yeast-peptone-sodium nitrate; K2HPO4 6 g / L, KH2PO4 2 g / L, Bactotripton 5 g / L, NaNO3 2.55 g / L, and yeast extract 10 g / L) liquid medium, and enriched at 30°C under anaerobic conditions for 24 hours. Each enriched culture was diluted and spread onto MRS agar, then cultured at 30°C under anaerobic conditions for 48 hours. After culturing, the grown colonies were selected and subjected to 16S rDNA sequencing analysis. As shown in Table 1 below, a variety of microorganisms with 16S rDNA sequence homology of 99.6% or higher were isolated from each sample.

[0065] [Table 1]

[0066] Example 2. Selection of cell membrane proteins overexpressing Lactobacillus sakei. The bacterial strain used in the experiment (Sample #: FT007, L.sakei) was prepared in MRS medium (Difco TMLactobacilli (MRS Broth, BD) was cultured under anaerobic conditions 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) enzyme for 2 hours. Next, lactic acid bacteria were removed by centrifugation at 4,500 × g for 20 minutes, and the peptide fragments degraded by Lys-C from proteins present in the extracellular membrane, contained in the supernatant buffer solution, were purified using a C18 SepPak cartridge (Sep-Pak C18 1 cc Vac, WAT054955, Waters). The Lys-C peptide fragments were then recovered in 80% acetonitrile solution and freeze-dried at -80°C for 24 hours. The dried peptide pool was dissolved in 20 μl of ammonium bicarbonate buffer, and 5 μl was separated using nano LC (EASY-nLC1000, Thermo Fisher) liquid chromatography with a C18 column to separate the peptides. The separated solution was then electrospray ionized, and the sequence of each peptide was analyzed using a mass spectrometer (LTQ Orbitrap Velos LC-MS / MS, Thermo). The amino acid sequences of the peptides identified by sequential separation via a 2% to 30% acetonitrile concentration gradient using a C18 hydrophobic column were identified as extracellular membrane proteins through a MASCOT database search (Matrix Science), confirming the presence of PrsA lipid membrane protein, which is abundant in the extracellular membrane. Figure 1 shows the MS / MS results for N-WANDQTVMAK-C (SEQ ID NO: 6), a representative peptide from amino acids 255 to 264 of the five Lys-C cleavage MS / MS analysis peptide sequences identified as PrsA lipid membrane protein.

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

[0068] Example 4. Induction of PrsA expression in the extracellular membrane of Lactobacillus paracasei. The pGOSTa:PrsA prepared in Example 3 was introduced into the L. paracasei FT003 strain shown in Table 1 by electroporation. The transformed lactic acid bacteria colonies were inoculated into 15 mL of MRS liquid medium and cultured at 30°C for 24 hours under anaerobic conditions. After collecting 5 mL of the culture solution, it was washed three times with 0.85% saline, then disrupted with a bead beater. 20 μg of the recovered total protein was subjected to electrophoresis on 12.5% ​​SDS-PAGE and stained with CBB (coomassie brilliant blue) (Figure 4A). The protein bands separated by size by SDS-PAGE were transferred to a PVDF membrane, and Western blotting was performed using an anti-PrsA antibody. As a result, the precise location and size of the PrsA protein could be confirmed, and the expression level of PrsA protein in the total protein could be confirmed (Figure 4B).

[0069] Generally, there are few methods to stably and rapidly express target proteins in lactic acid bacteria through genetic manipulation. However, as can be seen in Figure 4, in L. paracasei host cells, the PrsA promoter derived from L. saucei does not compete with the PrsA promoter present on the L. paracasei chromosome, and it was confirmed by Western blotting using a PrsA-specific antibody along with CBB staining that L. saucei-derived PrsA can be stably expressed in large quantities (Figures 4A and 4B). In particular, it was confirmed that the anti-PrsA polyclonal antibody (KOMA#24462) produced using pure isolated and purified L. saucei PrsA protein does not cross-react with L. paracasei-derived PrsA (54% homology and 71% similarity of amino acid sequences between L. saucei and L. praracsei-derived PrsA proteins), and it was confirmed that the L. saucei-derived PrsA protein biosynthesized in the pGOSTa:PrsA surface expression system accounts for more than 3% of the total protein expressed in L. praracsei.

[0070] Next, to confirm whether the L.sakei-derived PrsA protein, which was observed to be highly expressed, was present in the L.paracasei cell membrane, 0.5 mL of a 1.5 mg / mL total protein solution, which had been 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 precipitate was redissolved in 0.5 mL of PBS. 8 μl of each protein solution containing the total protein was taken and mixed with 2 μl of 5X SDS-PAGE sample buffer. The mixture was then heat-treated at 97°C for 5 minutes, followed by electrophoresis using 10% SDS-PAGE and CBB staining. As confirmed in Figure 4C, the 31.4 kDa L.sakei-derived PrsA protein, present in the total protein sample of lane 1, was not found in the cytoplasmic solution of lane 2, but was found entirely in the cell membrane protein solution of lane 3. Therefore, it was confirmed that the majority of the PrsA protein highly expressed with the GOSTa:PrsA vector was present in the L.paracasei cell membrane.

[0071] Example 5. Preparation of a PrsA-sfGFP fusion protein expression vector (pGOSTa:PrsA-sfGFP) The sfGFP gene cloned into the pQI30 vector is placed under 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 reverse primer sfGFP KpnI[5'-tct t gg tac c After PCR amplification using tt tgt aga gct cat cca-3' (underlined: KpnI recognition site, SEQ ID NO: 10), the DNA was treated with BamHI and KpnI. The pGOSTa:PrsA vector prepared in Example 3 was then treated with the same restriction enzyme, and the two DNA sections were ligated to produce pGOSTa:PrsA-sfGFP (Figure 5).

[0072] Example 6. PrsA structural analysis and PrsA anchor motif improvement and construction of an sfGFP fusion surface expression system The PrsA protein has a structure in which the 21st amino acid, cysteine, at the N-terminus is covalently linked to the intracellular carbon of glycerol, a lipid that is a major component of the cell membrane, thus firmly fixing it to the cell membrane. The C-terminus of PrsA consists of an NC domain structure located close to the N-terminus. Furthermore, the PrsA protein has a single dimer structure, with each N-terminus and C-terminus being in close proximity to each other, and the dimer structure is cyclic. In addition, the serine-rich domain present at the C-terminus of each PrsA is thought to play an important role in the formation of the NC domain structure and is presumed to interact with the peptidoglycan layer, a component of the cell wall of Gram-positive bacteria, and play a role in maintaining structural stability within the cell wall.

[0073] The core of lactic acid bacteria surface expression lies in enabling the maximum expression of specific proteins outside the outer membrane rather than inside the peptidoglycan layer. The inventors determined that removing 23 amino acid residues (DLSDILSSYGVNAKKSSAKSSSK, SEQ ID NO: 4, Figure 2) from the serine-rich domain of PrsA would allow for significantly more efficient delivery of foreign proteins to the outside of the cell wall than wild-type PrsA. To remove the serine-rich domain of the PrsA protein, the pGOSTa:PrsA vector prepared in Example 3 was used as a template, along with 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 TT TAT CCT TGA TTG TTA CGT CGG C-3' (underlined: BamHI recognition site, SEQ ID NO: 11), and the amplified 1.4kb DNA section was recovered by treating it with restriction enzyme PstI and BamHI. The restriction enzyme-treated DNA section and the pGOSTa:PrsA-sfGFP prepared in Example 5 were treated with the same restriction enzyme PstI and BamHI, and then the recovered 7.25kb vector DNA section was ligated to secure pGOSTa:PrsA DS-sfGFP.

[0074] As previously described regarding the structure of PrsA, PrsA is known to have a curved structure with its N-terminus and C-terminus in close proximity. In this invention, after confirming the hinge region located in the middle of the PrsA protein sequence through structural analysis, it was determined that by removing the "KDNST" amino acid sequence (SEQ ID NO: 5, Figure 2), which is presumed to play the most crucial role in the hinge region, the specific protein fused to PrsA can be efficiently delivered to the outside of the cell wall. To remove the five amino acids in the hinge region, the pGOSTa:PrsA-sfGFP vector secured in Example 5 was used as a template, and a hinge deletion forward primer [5'-ccc c ct ccar g aa gaa gta ctc aac aga t-3' (underlined: XhoI recognition site, SEQ ID NO: 12) and hinge deletion reverse primer [5'-ccc c ct ccar g After performing PCR using ct taa gtt cag aaa taa c-3' (underlined: XhoI recognition site, SEQ ID NO: 13), an 8.45kb PCR product was recovered. The recovered PCR product was treated with restriction enzyme XhoI and then self-ligated to secure pGOSTa:PrsA DH-sfGFP.

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

[0076] Example 7. Confirmation of sfGFP fused with a modified PrsA anchor motif on the surface of Lactobacillus cells. Four plasmids, pGOST:PrsA-sfGFP, pGOST:PrsA DS-sfGFP, pGOST:PrsA DH-sfGFP, and pGOST:PrsA WD-sfGFP, were introduced into L. paracasei isolated from kakimchi by electroporation. The four transformed L. paracasei strains were then cultured anaerobically in MRS liquid medium for 24 hours. After collecting 5 mL of each L. paracasei culture, they were washed three times with 0.85% saline and then disrupted with a bead beater to recover the total protein. The total protein of each L. paracasei strain was quantified using the BCA (bicinchoninic acid) method, and 20 μg of the total protein was subjected to electrophoresis on two pre-prepared 12.5% ​​SDS-PAGEs to separate the proteins by size. After electrophoresis was completed, one PAGE gel was stained with CBB, and the remaining gel was used to transfer the bands using a PVDF membrane, followed by Western blotting with anti-sfGFP antibody (Santa Cruz Biotechnology, Cat# sc-9996).

[0077] As a result, as shown in Figure 6, the CBB image shows that the five sample proteins, including the control group and L. paracasei, were evenly spread on SDS-PAGE, and the Western blot image confirms that sfGFP fused with PrsA and PrsA mutants is stably expressed in L. paracasei. Furthermore, each fusion protein shows slight differences from mutant to mutant at the same position as the theoretical molecular weight, and the surface expression levels differ gradually in the order of PrsA wild-type anchor protein, PrsA hinge region removed anchor, PrsA hinge region and serine-rich domain removed anchor, and PrsA serine-rich domain removed anchor. From the above, it was confirmed that the target protein sfGFP is stably expressed in Lactobacillus using four plasmids containing PrsA and PrsA improved anchor motifs: pGOST:PrsA-sfGFP, pGOST:PrsA DS-sfGFP, pGOST:PrsA DH-sfGFP, and pGOST:PrsA WD-sfGFP.

[0078] Next, using the 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), we compared and analyzed whether sfGFP, expressed by fusing PrsA or a modified PrsA anchor motif with the four plasmids mentioned above, was effectively expressed outside the surface of L.paracasei cells.

[0079] As a result, as can be seen in Figure 7, the levels of sfGFP expressed on the cell surface were confirmed in the following order: PrsA hinge region removed anchor (PrsA DH), PrsA hinge region and serine-rich domain removed anchor (PrsA WD), PrsA serine-rich domain removed anchor (PrsA DS), and PrsA wild-type anchor (PrsA) motif. From these results, it was found that PrsA wild-type and PrsA modified anchor motifs can effectively express target proteins on the cell surface of Lactobacillus, and that PrsA modified anchor motifs are even superior to PrsA wild-type in their effect on the cell surface expression of target proteins.

[0080] Example 8. Preparation and surface expression of the surface expression vector pGOSTa:PrsA-mB7-H1 Using the PD-L1(CD274)(NM_021893) Mouse Tagged ORF clone (ORiGENE, Cat # MR203953) containing the mouse B7-H1 gene as a template, mouse B7-H1 positive primer [5'-AAA GGA TCC GAC TTG TAC GTG GTG GAG-3' (underlined: BamHI recognition site, sequence number 16) and mouse B7-H1 reverse primer [5'-GGG TCT AGA The mouse B7-H3 gene was amplified by PCR using ACT AGT GTC GAC TTA GTT GAT TTT GCG GTA TGG GGC ATT-3' (underlined: XbaI recognition site, SEQ ID NO: 17), and then treated with restriction enzymes BamHI and XbaI to recover approximately 360 bp DNA sections. The recovered 360 bp DNA sections and the vector prepared in Example 3 were ligated with 360 bp DNA sections and 7.7 kb DNA sections recovered by treating with BamHI and XbaI, respectively, 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 secured pGOSTa:PrsA-mB7-H1 plasmid was introduced into L. paracasei FT003 strain isolated from Example 1 by electroporation, and the transformed lactic acid bacteria colonies were cultured using the method described in Example 4. After collecting 5 mL of culture medium, it was washed three times with 0.85% saline, then disrupted with a bead beater, and 20 μg of the recovered total protein was subjected to electrophoresis on 12.5% ​​SDS-PAGE and stained with CBB (Figure 9A). Furthermore, the protein bands separated by size on SDS-PAGE were transferred to a PVDF membrane, and Western blotting was performed using an anti-B7-H1 antibody (R&D Systems Cat #AF1019) (Figure 9B). As a result, the high expression and size of the PrsA-mB7-H1 fusion protein were confirmed. Furthermore, we were able to confirm the expression level of the PrsA-mB7-H1 fusion protein, which accounts for the total protein expression, and confirmed that the PrsA-mB7-H1 fusion protein is the most stable and highly expressed protein, as evidenced by high expression levels observed in SDS-PAGE CBB staining.

[0082] Example 9. Antibody induction effect of lactic acid bacteria expressing mouse B7-H1 protein on the surface. To investigate the surface protein antigenicity of the L. paracasei FT003 strain transformed with pGOSTa:PrsA-mB7-H1, which was prepared in Example 8 described above, we determined whether or not neutralizing antibodies were formed. After the L. paracasei expressing the antigen on its surface underwent ethanol-based inactivation, it was washed three times with a 0.85% sodium chloride solution, stored at 5 weeks of age, and subjected to a 1-week purification period. Six 6-week-old male BALB / c mice were each given 1 × 10⁶ of the antigen. 7Dead cells were administered by intramuscular injection twice at two-week intervals. Six mice each were used in the control groups: a 0.85% sodium chloride group and a L. paracasei monotherapy group. Serum from each group was collected four weeks after the second intramuscular injection, and neutralizing antibody titers against each antigen were measured and compared using ELISA. As shown in Figure 10(B), the anti-B7-H1 antibody titer in the L. paracasei-treated group transformed with pGOSTa:PrsA-mB7-H1 was higher than in the control groups (0.85% sodium chloride group and L. paracasei FT003 lactic acid bacteria transduction group), confirming statistical effectiveness in each case.

[0083] Through these results, it was found that the pGOSTa:PrsA vector of the present invention can be utilized in platform technologies that enable the expression of various target proteins on the cell surface of microorganisms using PrsA as a cell membrane anchor. Furthermore, it was confirmed that when microbial cells transformed with "pGOSTa:PrsA-antigen" are administered to animals for the purpose of using the surface-expressed target protein as an antigen, antibodies against the antigen can be induced.

Claims

1. Downstream of the PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3, 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 ligated. The aforementioned PrsA mutant has a missing residue between positions 162 and 166 in the amino acid sequence of SEQ ID NO:

2. A recombinant vector for cell surface expression of a target protein, characterized by the following features.

2. Downstream of the PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3, 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 ligated. The aforementioned PrsA mutant has a missing residue between positions 281 and 303 in the amino acid sequence of SEQ ID NO:

2. A recombinant vector for cell surface expression of a target protein, characterized by the following features.

3. Downstream of the PrsA promoter consisting of the nucleotide sequence of SEQ ID NO: 3, 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 ligated. The aforementioned PrsA mutant lacks residues 162-166 and 281-303 in the amino acid sequence of SEQ ID NO:

2. A recombinant vector for cell surface expression of a target protein, characterized by the following features.

4. The target protein is one selected from the group consisting of ligand proteins, receptor proteins, enzyme proteins, and proteins derived from viruses and bacteria. A recombinant vector for cell surface expression of a target protein according to claim 1.

5. A transformed with the recombinant vector according to any one of claims 1 to 4 A microorganism characterized by the following features.

6. The aforementioned microorganism is a lactic acid bacterium. The microorganism described in claim 5.

7. Includes the step of transforming a microorganism with the recombinant vector according to any one of claims 1 to 4. A method for expressing a target protein on the surface of a microorganism, characterized by the following:

8. The step of culturing the transformed microorganism according to claim 5 and expressing the target protein on the surface of the cells; This includes the step of recovering microorganisms on which the target protein has been expressed on the cell surface; A method for producing microorganisms in which a target protein is expressed on the cell surface.

9. The target protein is one selected from the group consisting of ligand proteins, receptor proteins, enzyme proteins, and proteins derived from viruses and bacteria. The manufacturing method according to claim 8.

10. A product manufactured by the method described in Claim 8 A microorganism in which a target protein characterized by the following is expressed on the cell surface.

11. The target protein is B7-H1 (B7 homolog 1) or a fragment thereof. A microorganism expressing the target protein described in claim 10 on its cell surface.

12. Contains the microorganism described in Claim 11 as an active ingredient An injectable preparation characterized by the following features.

13. Contains the microorganism described in Claim 11 as an active ingredient An oral formulation characterized by the following features.

14. A method produced by the method of Claim 8, comprising the step of immobilizing a microorganism on which a target protein is expressed on its surface onto the surface of a substrate. A method for producing a protein array characterized by the following:

15. A method comprising administering a microorganism produced by the method of Claim 8, on which an antigen is expressed on its surface, to a vertebrate (excluding humans). A method for inducing immunity in vertebrates, characterized by the following features.

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