Method for expression of protein

Abstract

To provide a highly versatile technique that enables a simple enhancement of the expression amount of a target protein upon expression by a protein expression system.SOLUTION: The foregoing problem is solved by expressing a tagged protein in which a peptide tag consisting of a specific amino acid sequence is linked to the N-terminal side.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a protein expression method, and more particularly to a novel expression method that enables an increase in the amount of protein expressed, and uses thereof. [Background technology]

[0002] Advances in recombinant DNA technology have led to the development of various protein expression systems, including E. coli, yeast, insect cells, animal cells, and cell-free systems. When expressing a target protein using an E. coli expression system, expression levels can be low depending on the protein. To improve protein expression levels, various techniques, such as promoter and vector selection, codon optimization of the gene encoding the target protein, and optimization of culture conditions, are commonly used, but all of these require significant labor. Cell-free protein synthesis systems, on the other hand, generally synthesize proteins by adding DNA or mRNA encoding the target protein sequence to a solution containing factors for the intracellular translation process. This technique is also capable of synthesizing cytotoxic proteins that cannot be produced in living cells. However, due to their higher cost compared to other expression systems, further improvements in protein synthesis efficiency are needed.

[0003] In Patent Document 1, an improvement in expression level is achieved by expressing a target protein as a tagged protein in which a peptide tag consisting of the amino acid sequence SK, SKX, SKXX, AKXX, or KKXX (wherein X represents any amino acid residue) is linked to the N-terminus using an Escherichia coli expression system or a yeast expression system. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2016 / 204198 issue Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a highly versatile technology that can easily increase the expression level when expressing a target protein using a protein expression system. [Means for solving the problem]

[0006] The present inventors discovered that the expression level of a target protein can be increased by expressing the target protein as a tagged protein in which a tag peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is added to the N-terminus of the target protein, and thus completed the present invention.

[0007] That is, the present invention encompasses the following aspects [1] to

[10] . [1] A tagged protein in which a tag peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is added to the N-terminus of a target protein. [2] The tagged protein according to [1], wherein a signal peptide is further added to the N-terminus or C-terminus of the tag peptide. [3] The tagged protein according to [1] or [2], wherein the target protein is an immunoglobulin or an Fc-binding protein. [4] A polynucleotide encoding the tagged protein according to any one of [1] to [3]. [5] An expression vector comprising the polynucleotide described in [4]. [6] A method for expressing a target protein, characterized in that the target protein is expressed as a tagged protein in which a tag peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is added to the N-terminus of the target protein. [7] The method described in [6], which uses an expression system utilizing Escherichia coli. [8] The method according to [6] or [7], which uses a cell-free protein synthesis system. [9] The method according to any one of [6] to [8], wherein the target protein is an immunoglobulin or an Fc-binding protein. [Effects of the Invention]

[0008] The expression level of a target protein can be improved by expressing the target protein as a tagged protein in which a tag peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 has been added to the N-terminus of the target protein. This method is useful for producing proteins in various protein expression systems, such as E. coli, yeast, insect cells, animal cells, and cell-free systems. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the difference in the expression level of an Fc-binding protein depending on whether or not a tag peptide is added to the N-terminus of the protein. [Figure 2] FIG. 1 shows the difference in the amount of synthesized antibody capable of binding to an antigen depending on whether or not a tag peptide is added to the N-terminus of the antibody, and depending on the type of tag peptide added. DETAILED DESCRIPTION OF THE INVENTION

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the preferred embodiments. However, the present invention is not limited to the following embodiments and can be practiced in various modified forms within the scope of the present invention.

[0011] The tagged protein of the present invention has the tag peptide shown in SEQ ID NO: 1 bound to the N-terminus of the target protein.

[0012] The peptide tag may be bound directly to the N-terminus of the target protein, or may be bound via one or several sequences.

[0013] Examples of the sequence via one or several sequences include a protease recognition sequence and a sequence without a specific function, such as a sortase recognition sequence, an HRV3C recognition sequence, or a TEV protease recognition sequence.

[0014] The peptide tag may be linked immediately after the amino acid corresponding to the start codon.

[0015] A signal peptide may be further added to the N-terminus or C-terminus of the tag peptide. Examples of signal peptides include signal peptides that cause protein secretion into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Application Laid-Open No. 2011-097898). When yeast is used as a host, examples of such signal peptides include an invertase secretion signal, a P3 secretion signal, and an α-factor secretion signal. Furthermore, in order to express the protein in a specific cellular compartment, a transport signal peptide such as an endoplasmic reticulum retention signal peptide or a vacuolar targeting signal peptide may be added.

[0016] The tagged protein of the present invention may further be tagged with a tag for detection or purification, such as a His tag, a FLAG tag, a C tag, a GST tag, or a PA tag.

[0017] The target protein constituting the tagged protein of the present invention is not particularly limited, but examples include growth factors, hormones, cytokines, viral proteins, blood proteins, enzymes, antigens, antibodies (immunoglobulins), transcription factors, receptors, fluorescent proteins, or partial peptides or mutants thereof.

[0018] Growth factors include EGF (Epidermal growth factor), IGF (Insulin-like growth factor), TGF (Transforming growth factor), bFGF or FGF2 (Basic fibroblast growth factor), NGF (Nerve growth factor), BDNF (Brain-derived neurotrophic factor), VEGF (Vesicular endothelial growth factor), G-CSF (Granulocyte-colony stimulating factor), GM-CSF (Granulocyte-macrophage-colony stimulating factor), PDGF (Platelet-derived growth factor), and These include platelet-derived growth factor (EPO), erythropoietin (Erythropoietin), thrombopoietin (TPO), hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF).

[0019] Hormones include insulin, glucagon, somatostatin, growth hormone, prolactin, leptin, and calcitonin.

[0020] Cytokines include interferon, interleukin (IL)-1, and IL-6.

[0021] Viral proteins include constituent proteins of virus-like particles (VLPs), including influenza virus, human immunodeficiency virus (HIV), human hepatitis C virus (HCV), human hepatitis B virus (HBV), adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), retrovirus, and coronavirus.

[0022] Blood proteins include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

[0023] Examples of enzymes include lipase, protease, steroid synthesis enzyme, kinase, phosphatase, luciferase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase, and chitinase.

[0024] Examples of antibodies (immunoglobulins) include complete antibodies, Fab, F(ab'), F(ab')2, Fc, Fc fusion proteins, heavy chains (H chains), light chains (L chains), single-chain Fvs (scFvs), sc(Fv)2, disulfide-linked Fvs (sdFvs), diabodies, VHH antibodies, chimeric antibodies, monoclonal antibodies, and albumin-binding proteins.

[0025] An example of a monoclonal antibody is anti-EGFR (Epidermal growth factor receptor).

[0026] Examples of receptors include adrenergic receptors, adenosine receptors, rhodopsin, nicotinic acetylcholine receptors, insulin receptors, LDL receptors, transferrin receptors, Fc receptors, and virus receptors.

[0027] Fluorescent proteins include jellyfish green fluorescent protein (GFP), mNeonGreen, and mCherry.

[0028] A mutant may refer to a protein containing an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted, or added at least one or more positions in the amino acid sequence of a native target protein. "Substitution of one or more amino acid residues" includes not only the amino acid substitution at the specific positions described above, but also conservative substitutions in which amino acids with similar physical and / or chemical properties are substituted. Those skilled in the art know that conservative substitutions generally maintain protein function between substituted and unsubstituted versions. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). Another example of such amino acid substitutions is a substitution for monomerizing the AAV binding protein of the present invention. Specifically, an amino acid substitution such as substituting a cysteine ​​residue, which is likely to form a higher-order structure, with a serine or methionine residue is used. The term "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of the target protein and the type of amino acid residue, but as an example, it means any of 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0029] A mutant may also refer to a protein containing an amino acid sequence that is 70% or more identical to the entire amino acid sequence of a native target protein. "Amino acid sequence identity" refers to identity with respect to the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of amino acid residues in those amino acid sequences that have the same type of amino acid (Experimental Medicine, 31(3), Yodosha). Amino acid sequence identity can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA. Amino acid sequence identity should be 70% or more, but may also be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more).

[0030] Furthermore, the mutant may retain the function of the native target protein.

[0031] For example, the mutant may specifically be a protein selected from the following (i) to (iii): (i) a protein comprising the amino acid sequence from isoleucine at position 26 to methionine at position 124 of SEQ ID NO: 2; (ii) A protein having an amino acid sequence from isoleucine at position 26 to methionine at position 124 of SEQ ID NO: 2, which contains one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, with the proviso that specific mutations are maintained, and which has antibody-binding activity; (iii) A protein having an amino acid sequence that is 70% or more identical to the amino acid sequence from isoleucine at position 26 to methionine at position 124 of SEQ ID NO: 2, with specific mutations maintained, and that has antibody binding activity.

[0032] The term "one or several" means, for example, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0033] The amino acid sequence identity may be 70% or more, and may be higher (for example, 80% or more, 85% or more, 90% or more, or 95% or more).

[0034] The target protein may be a naturally occurring protein that does not contain the amino acid sequence consisting of MKAIK at the N-terminus of its amino acid sequence. This is because when the amino acid sequence of a naturally occurring protein contains the amino acid sequence of MKAIK at the N-terminus of the protein, the protein already benefits from increased expression, and therefore addition of the tag peptide of the present invention is not expected to have the effect of increasing expression level.

[0035] The method for expressing the protein of the present invention will be described in detail below.

[0036] The protein expression system used in the protein expression method of the present invention is not particularly limited as long as it can express the target protein, and examples thereof include microbial expression systems (e.g., expression systems using bacteria as hosts or yeast expression systems), plant cell expression systems, insect cell expression systems (e.g., baculovirus expression systems), animal cell expression systems (e.g., mammalian cell expression systems), and cell-free protein synthesis systems.

[0037] In expression systems using bacteria as hosts, examples of the bacteria include bacteria belonging to the Enterobacteriaceae family, coryneform bacteria, and Bacillus bacteria.

[0038] Examples of bacteria belonging to the Enterobacteriaceae family include bacteria belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified as Enterobacteriaceae according to the classification used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Examples of bacteria belonging to the genus Escherichia include, but are not limited to, bacteria classified as Escherichia according to classifications known to microbiologists. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, pp. 2460-2488. Table 1. In F.D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of Escherichia bacteria include Escherichia coli. Examples of Escherichia coli include Escherichia coli B strains such as the BL21(DE3) strain; Escherichia coli K-12 strains such as the W3110 strain (ATCC 27325) and the MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); and derivative strains thereof.Examples of bacteria of the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. Examples of bacteria of the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of bacteria of the genus Erwinia include Erwinia amylovora and Erwinia carotovora. Examples of bacteria of the genus Klebsiella include Klebsiella planticola.

[0039] The genus Corynebacterium includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0040] Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus. Specific examples of Bacillus subtilis include Bacillus subtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79 strain (Plasmid, 1984, 12, 1-9). Specific examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC 23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

[0041] Examples of yeasts used in yeast expression systems include yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae; Candida, such as Candida utilis; Pichia, such as Pichia pastoris; Hansenula, such as Hansenula polymorpha; and Schizosaccharomyces, such as Schizosaccharomyces pombe.

[0042] These strains can be obtained, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain has a corresponding accession number, which can be used for distribution (see http: / / www.atcc.org / ). The accession numbers for each strain are listed in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depository institution where they were deposited.

[0043] In the animal cell expression system, animal cells include fibroblasts, CHO cells, Vero cells, COS cells, NSO cells, HeLa cells, myeloma cells, BHK cells, HEK293 cells, and human cells.

[0044] Introduction of a gene into a host can be achieved by introducing the gene into the host's chromosome. Introduction of a gene into a chromosome can be achieved, for example, by homologous recombination (Miller, JH, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods that utilize homologous recombination include methods using linear DNA, such as Red-driven integration (Datsenko, K. A., and Wanner, BL, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), methods using plasmids containing a temperature-sensitive replication origin, methods using conjugatively transferable plasmids, methods using suicide vectors lacking a replication origin that functions in the host, and transduction methods using phages. Only one copy of a gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence that exists in multiple copies on the chromosome. Examples of sequences present in multiple copies on chromosomes include repetitive DNA sequences and inverted repeats at both ends of transposons. Homologous recombination can also be performed by targeting appropriate chromosomal sequences, such as genes not required for carbonic anhydrase production. Genes can also be randomly introduced into chromosomes using transposons or Mini-Mu (see JP-A-2-109985, US Pat. No. 5,882,888, and EP 805867 B1).

[0045] The introduction of the target gene into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0046] Introduction of a gene into a host can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the target gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to introduce the gene into the host. A host transformed with an expression vector is also called a transformant. A DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template.

[0047] The vector may be a vector capable of autonomous replication in host cells. The vector may be a multicopy vector. The vector may also have a marker, such as an antibiotic resistance gene, to select transformants. The vector may also have a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria, such as Escherichia coli, include pUC19, pUC18, pHSG299, pHSG398, pBR322, pSTV29, pCold-series vectors (all available from Takara Bio Inc.), pACYC177, pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pET-series vectors (Merck), and pQE-series vectors (Qiagen).

[0048] When a gene is introduced, it is sufficient that the gene can be expressed by the host. Specifically, the gene is maintained so that it is expressed under the control of a promoter that functions in the host. A "promoter that functions in the host" may refer to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the gene to be introduced or a promoter of another gene. Specific examples of promoters include the T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Bifidobacterium-derived Pm1 promoter, PR promoter, PL promoter, P4 promoter, and P8 promoter.

[0049] A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the gene to be introduced or a terminator from another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0050] A target protein can be expressed by culturing a host having a gene encoding the target protein. For example, the target protein may be expressed by transforming a host with an expression vector containing the gene for the target protein and culturing the resulting transformant.

[0051] The medium used is not particularly limited as long as it allows the host to grow and the target protein to be expressed. For example, a conventional medium used for culturing microorganisms such as bacteria and yeast can be used as the medium. The medium may contain medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as needed. The types and concentrations of medium components may be appropriately determined depending on various conditions such as the type of host.

[0052] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. The form of use of plant-derived materials is not particularly limited, and they can be used in any form, such as raw products, squeezed juice, pulverized products, or purified products. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Because hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses. Xylose may also be supplied by converting hexoses such as glucose into xylose, for example, by providing the host with a pathway for converting hexoses to xylose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0053] The concentration of the carbon source in the medium is not particularly limited as long as the host can grow and the target protein can be expressed. The concentration of the carbon source in the medium may be as high as possible, for example, within a range that does not inhibit the production of the target protein. The initial concentration of the carbon source in the medium may be, for example, usually 5 to 30 w / v%, preferably 10 to 20 w / v%. Additionally, the carbon source may be additionally supplied to the medium as appropriate. For example, the carbon source may be additionally supplied in response to a decrease or depletion of the carbon source as the culture progresses. While the carbon source may be temporarily depleted as long as the target protein is ultimately produced, it may be preferable to carry out the culture so that the carbon source does not become depleted or does not continue to be depleted.

[0054] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or aqueous ammonia, which are used for pH adjustment, may also be used as a nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0055] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0056] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0057] Specific examples of other various organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and soy protein hydrolysate. As other various organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0058] When an auxotrophic mutant strain that requires nutrients such as amino acids for growth is used, it is preferable to supplement the medium with such required nutrients.

[0059] The culture conditions are not particularly limited as long as the host can grow and the target protein can be expressed. The culture can be carried out under normal conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions can be appropriately set depending on various conditions such as the type of host. Furthermore, the expression of the gene encoding the target protein can be induced as necessary.

[0060] Culturing can be carried out using a liquid medium. For example, the host may be cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the host may be seed cultured in a liquid medium and then inoculated into the liquid medium for main culture. That is, the culture may be divided into a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may or may not be the same. The target protein only needs to be expressed in the main culture. The amount of the host contained in the medium at the start of culture is not particularly limited. For example, a seed culture solution with an OD660 of 4 to 100 may be added at the start of culture in an amount of 0.1% by mass to 100% by mass, preferably 1% by mass to 50% by mass, relative to the medium for main culture.

[0061] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination of these. The medium at the start of cultivation is also called the "initial medium." The medium supplied to a cultivation system (e.g., a fermenter) in fed-batch or continuous cultivation is also called the "fed-batch medium." Supplying a fed-batch medium to a cultivation system in fed-batch or continuous cultivation is also called "fed-batch." When cultivation is carried out separately into a seed culture and a main culture, the cultivation forms of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out by batch culture, or the seed culture may be carried out by batch culture and the main culture may be carried out by fed-batch or continuous culture.

[0062] Various components such as a carbon source may be contained in the initial medium, the feed medium, or both. That is, various components such as a carbon source may be additionally supplied to the medium during the culture process, either alone or in any combination. These components may be supplied once, multiple times, or continuously. The types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium. Furthermore, the concentrations of each component contained in the initial medium may or may not be the same as the concentrations of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are performed intermittently, the types and / or concentrations of components contained in each feed medium may or may not be the same.

[0063] The culture can be carried out under aerobic conditions, for example. "Aerobic conditions" may mean that the dissolved oxygen concentration in the medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the dissolved oxygen concentration may be controlled to, for example, 1 to 100% of the saturated oxygen concentration, preferably about 20 to 100%. The culture can be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During culture, the pH of the medium can be adjusted as needed. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or an aqueous phosphoric acid solution. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the activity of the host is lost.

[0064] By culturing the host in this manner, a culture containing the target protein can be obtained. The mutant carbonic anhydrase can be accumulated, for example, within the host's bacterial cells. The term "bacterial cells" may be appropriately interpreted as "cells" depending on the type of host. Depending on the host used and / or the genetic design of the target protein, it may be possible to accumulate the target protein in the periplasm or to secrete and produce the target protein outside the bacterial cells.

[0065] The target protein may be recovered as it is contained in the culture (specifically, the medium or bacterial cells), or may be recovered from the culture (specifically, the medium or bacterial cells). The target protein may be purified during the process of recovering it from the culture. Purification can be carried out to the desired extent. The target protein may be recovered in the form of a purified enzyme, in the form of a fraction thereof (i.e., in the form contained in such a fraction), or in a combination thereof. Such fractions are not particularly limited, as long as the target protein is contained in such a form that it can act on its substrate. Such fractions include cultures of hosts containing the target protein (i.e., hosts containing the target protein), bacterial cells recovered from such cultures, culture supernatants recovered from such cultures, processed products thereof (e.g., bacterial cell disruption, bacterial cell lysate, bacterial cell extract, and other processed bacterial products such as those described below), partially purified products thereof (i.e., crude products), and combinations thereof. Note that the term "purified target protein" may also include crude products. These fractions can be recovered individually or in appropriate combinations.

[0066] For example, the target protein may be recovered in a form contained in bacterial cells. The method for recovering bacterial cells from the culture medium is not particularly limited, and known methods can be used, for example. Such methods include, for example, natural sedimentation, centrifugation, and filtration. A flocculant may also be used. These methods can be used alone or in appropriate combination. The recovered bacterial cells can be washed appropriately using an appropriate medium. The recovered bacterial cells can also be resuspended appropriately using an appropriate medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

[0067] As another example, the target protein may be recovered from the form contained in the bacterial cells by subjecting the bacterial cells to appropriate treatment. Examples of bacterial cell treatment include immobilization on a carrier such as acrylamide or carrageenan, freeze-thaw treatment, treatment to increase membrane permeability, and physical disruption using ultrasonic disruption or a pressure homogenizer. Membrane permeability can be increased, for example, by using a surfactant or organic solvent. These treatments can be used alone or in appropriate combination.

[0068] A cell-free protein synthesis system is a method for synthesizing proteins in vitro from nucleic acid templates using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. Cell-free protein synthesis systems typically use cell extracts obtained by purifying cell lysates as needed. Cell extracts generally contain the ribosomes, factors such as initiation factors, and enzymes necessary for protein synthesis, such as tRNA. When synthesizing proteins, other substances necessary for protein synthesis, such as various amino acids, energy sources such as ATP and GTP, and creatine phosphate, are added to the cell extract. Of course, separately prepared ribosomes, factors, and / or enzymes may also be added during protein synthesis, as needed.

[0069] A synthetic system constructed by isolating and purifying the factors required for peptide translation and reconstituting them in vitro may also be used (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001).

[0070] Examples of cell-free protein synthesis systems include an Escherichia coli S30 extract system, a wheat germ extract system, and a rabbit reticulocyte lysate system.

[0071] E. coli 30S extract is prepared by collecting, disrupting, and purifying E. coli cells. Preparation of the E. coli 30S fraction and cell-free coupled transcription-translation reaction can be carried out by known methods or methods equivalent thereto. (Pratt, JM: Chapter 7, in "Transcription and Translation: A Practical Approach," ed. by BD Hames & SJ Higgins, pp. 179-209, IRL Press, New York, 1984; Ellman, J. et al.: Methods Enzymol., 202, 301-336, 1991) Commercially available cell extracts for protein synthesis include those used in the RYTS Kit (Protein Express) and the RTS 500 Rapid Translation System (Roche).

[0072] A highly efficient and stable synthesis system using wheat germ extract can be constructed by preparing an extract from wheat germ after washing to remove the endosperm components (Madin, K. et al.: Proc. Natl. Acad. Sci. USA, 97: 559-564, 2000).

[0073] A wheat germ extract can be obtained by grinding wheat germ, centrifuging the mixture, and then separating the supernatant by gel filtration. The wheat germ extract can be prepared by the methods described in WO 00 / 68412 A1, JP 2006-042601 A, JP 2007-097438 A, JP 2008-029203 A, etc. A commercially available cell extract for protein synthesis includes a cell-free protein synthesis kit (NUProtein).

[0074] Rabbit reticulocyte lysate is prepared by injecting phenylhydrazine intravenously into rabbits for several days to induce anemia, collecting blood after a predetermined period (e.g., on the 8th day), and then subjecting the hemolyzed blood to ultracentrifugation or other procedures. The preparation method for rabbit reticulocyte lysate is described in Jackson, RJ and Hunt, This can be carried out by the method described in [Illegible Text], J. T.: Methods Enzymol., 96, 50-74 (1983). Commercially available cell extracts for protein synthesis include Rabbit Reticulocyte Lysate System (Promega).

[0075] The cell-free protein synthesis systems that can be used in carrying out the present invention are not limited to those described above. For example, extracts from bacteria other than Escherichia coli or plants other than wheat, extracts derived from insect cells (Swerdel et al., Comp Biochem Physiol B 93: 803-806 (1989)), extracts derived from animal cells, or systems constructed based on genome information may also be used.

[0076] Examples of animal cells include mammalian cells. Examples of mammalian cells include mouse L-cells, HeLa cells, and CHO cells. An example of a commercially available cell extract for protein synthesis is the Human Cell-Free Protein Expression System (manufactured by Takara Bio Inc.).

[0077] Expression systems that utilize E. coli include expression systems that use E. coli as a host (E. coli expression systems) and cell-free protein synthesis systems that use components derived from E. coli. [Example]

[0078] The present invention will be described in more detail with reference to the following examples and comparative examples, but the scope of the present invention is not limited to these examples.

[0079] Example 1 Expression of a soluble tag-fused human fetal Fc receptor (1) Escherichia coli BL21(DE3) strain was transformed with an expression vector containing a polynucleotide (SEQ ID NO: 3) encoding an Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 and an inducible promoter to prepare a recombinant Escherichia coli capable of expressing the protein (hereinafter also referred to as FcRn_m10a (KAIK-added)-expressing Escherichia coli). The N-terminal amino acid residue is a methionine (M) for translation initiation. The second lysine (K) to the fifth lysine (K) are the KAIK tag (SEQ ID NO: 1), The sequence from the 6th lysine (K) to the 25th alanine (A) is the OmpA signal peptide for expression in E. coli (the 2nd to 21st amino acid residues of UniProt No. P0A910), the region from isoleucine (I) at position 26 to methionine (M) at position 124 represents the β2 microglobulin region of the native human neonatal Fc receptor (human FcRn) β chain (corresponding to the region from positions 21 to 119 of SEQ ID NO: 5 (UniProt No. P61769)); The portion from glycine (G) at position 125 to serine (S) at position 149 is a linker. the region from alanine (A) at position 150 to serine (S) at position 423 represents the extracellular region of the native human FcRn α chain (corresponding to the region from positions 24 to 297 of SEQ ID NO: 4 (UniProt No. P55899)); The residues from cysteine ​​(C) at position 424 to glycine (G) at position 430 are a tag for immobilization on an insoluble carrier, and the extracellular domain contains the following 10 amino acid substitutions: Cysteine ​​(C) at position 71 of SEQ ID NO: 4 (corresponding to position 197 of SEQ ID NO: 2) is substituted with arginine (R) Asparagine (N) at position 78 of SEQ ID NO: 4 (corresponding to position 204 of SEQ ID NO: 2) is substituted with aspartic acid (D) Valine (V) at position 80 of SEQ ID NO: 4 (corresponding to position 206 of SEQ ID NO: 2) is substituted with aspartic acid (D) Lysine (K) at position 96 of SEQ ID NO: 4 (corresponding to position 222 of SEQ ID NO: 2) is substituted with glutamic acid (E) Asparagine (N) at position 172 of SEQ ID NO: 4 (corresponding to position 298 of SEQ ID NO: 2) is substituted with aspartic acid (D) Arginine (R) at position 192 of SEQ ID NO: 4 (corresponding to position 318 of SEQ ID NO: 2) is replaced with leucine (L) Asparagine (N) at position 196 of SEQ ID NO: 4 (corresponding to position 322 of SEQ ID NO: 2) is substituted with aspartic acid (D) Glutamine (Q) at position 232 of SEQ ID NO: 4 (corresponding to position 358 of SEQ ID NO: 2) is substituted with leucine (L) Cysteine ​​(C) at position 274 of SEQ ID NO: 4 (corresponding to position 400 of SEQ ID NO: 2) is substituted with serine (S) Lysine (K) at position 295 of SEQ ID NO: 4 (corresponding to position 421 of SEQ ID NO: 2) is substituted with glutamic acid (E).

[0080] (2) Escherichia coli expressing FcRn_m10a (KAIK-attached) was inoculated into 2xYT medium (phytone peptone: 16 g / L, yeast extract: 10 g / L, sodium chloride: 5 g / L, kanamycin sulfate: 50 mg / L) and pre-cultured for 16 hours at 30°C and 130 rpm.

[0081] (3) The preculture solution from (2) was added to TB medium (Difco Soytone (Thermo Fisher Scientific): 12 g / L, yeast extract: 24 g / L, glycerol: 10 g / L, dipotassium hydrogen phosphate: 9.4 g / L, potassium dihydrogen phosphate: 2.2 g / L) until the absorbance at 600 nm reached 0.02.

[0082] (4) After culturing for 4 hours at 30°C and 130 rpm, IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 0.05 mM, and the culture was continued for an additional 24 hours at 25°C and 130 rpm. After the culture was completed, 100 mL of the culture medium was centrifuged to collect the bacterial cells, and a pellet was obtained.

[0083] (5) 14 mL of a cell disruption buffer (50 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane buffer (pH 10) containing 150 mM sodium chloride, 2.4 mM magnesium sulfate, 3 kU / L Benzonase Nuclease, 60 mg / L lysozyme, and 6 g / L Triton X-100 (trade name)) was added to the cell pellet obtained in (4), and the mixture was shaken at 25°C and 150 rpm for 2 hours.

[0084] (6) The solution obtained in (5) was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was sterilized by filtration using a PVDF (polyvinylidene fluoride) membrane with a pore size of 0.22 μm, and collected as a bacterial cell extract.

[0085] (7) The extract of the cells obtained in (6) was purified by the IgG affinity chromatography method described below.

[0086] (7-1) 2 M phosphoric acid was added to the bacterial cell extract recovered in (6) and the pH was adjusted to 6.5 to prepare a sample to be applied to IgG affinity chromatography.

[0087] (7-2) An empty chromatography column was filled with IgG Sepharose (Cytiva, product number: 17096901) to prepare an IgG column.

[0088] (7-3) The prepared IgG column was equilibrated by passing an equilibration solution (50 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane buffer (pH 6.5) containing 150 mM sodium chloride) in an amount five times the amount of IgG Sepharose.

[0089] (7-4) The sample prepared in (7-1) was applied to the equilibrated IgG column, and the Fc-binding protein was adsorbed onto the IgG Sepharose.

[0090] (7-5) After the sample was applied, an equilibration solution in an amount 10 times the amount of IgG Sepharose was passed through the IgG column to wash away unadsorbed proteins.

[0091] (7-6) After washing, an eluate (50 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (pH 8.5) containing 150 mM sodium chloride) was passed through the IgG column in an amount six times that of the IgG Sepharose, and the eluate was collected as the IgG affinity purified eluate.

[0092] (8) The absorbance at 280 nm of the IgG affinity purified eluate recovered in (7) was measured, and the expression level of Fc-binding protein was calculated.

[0093] Comparative Example 1 Expression of human fetal Fc receptor unfused to a soluble tag The recombinant E. coli capable of expressing an Fc-binding protein was cultured in the same manner as in Example 1, except that E. coli capable of expressing an Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 (a polynucleotide encoding the Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 is set forth in SEQ ID NO: 7), the Fc-binding protein was purified from the culture medium, and its expression level was calculated. The amino acid sequence set forth in SEQ ID NO: 6 is the same as the amino acid sequence set forth in SEQ ID NO: 2, except that the KAIK tag (the region from the second to fifth positions in SEQ ID NO: 2) is not added.

[0094] The results of Example 1 and Comparative Example 1 are shown in Figure 1. It can be seen that even for Fc-binding proteins, which have a structure completely different from that of immunoglobulins (antibodies), the protein expression level is improved by adding a KAIK tag (SEQ ID NO: 1) to the N-terminus.

[0095] Example 2 Preparation of antibody genes Antibody genes were prepared for antibody synthesis in a cell-free protein synthesis system. Four types of anti-EGFR (Epidermal growth factor receptor) Fab antibody genes (clone names: Panitumumab, Necitumumab, GC1118A, and DL11) were used to synthesize the antibody genes shown below (a) to (f). (a) Anti-EGFR heavy chain gene without tag peptide (b) Anti-EGFR heavy chain gene tagged with KAIK (SEQ ID NO: 1) on the N-terminus (c) Anti-EGFR H chain gene tagged with SKIK (SEQ ID NO: 8) on the N-terminus (d) Anti-EGFR-L chain (light chain) gene without tag peptide (e) Anti-EGFR-L chain gene tagged with KAIK (SEQ ID NO: 1) on the N-terminus (f) Anti-EGFR-L chain gene tagged with SKIK (SEQ ID NO: 8) on the N-terminus (1) Synthesis of H chain gene ((a) to (c)) (1-1) Each H-chain gene of an anti-EGFR Fab antibody artificially synthesized by FASMAC (SEQ ID NOs: 9, 10, 11, and 12) was used as template DNA, and a combination of oligonucleotides consisting of any of the SEQ ID NOs shown in Table 1 was used as a primer set (forward / reverse) to prepare a reaction solution with the composition shown in Table 2, followed by PCR. Specifically, the PCR was carried out by subjecting the reaction solution to 35 cycles of reaction, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 15 seconds, and a third step at 72°C for 30 seconds.

[0096] [Table 1]

[0097] [Table 2]

[0098] (1-2) Using the PCR product from (1-1) as template DNA and oligonucleotides consisting of the sequences set forth in SEQ ID NOS: 29 and 30 as a primer set (forward / reverse), a reaction solution having the composition shown in Table 3 was prepared, and PCR was then performed in the same manner as in (1-1). Note that (Streptavidin-6H) DNA (SEQ ID NOS: 31) in Table 3 is a polynucleotide (artificially synthesized by FASMAC) encoding a polypeptide in which a purification tag consisting of six histidine residues (hereinafter also referred to as "6H") has been added to the C-terminus of a streptavidin-derived peptide.

[0099] [Table 3]

[0100] (1-3) The PCR product obtained in (1-2) was purified using a QIAquick PCR Purification Kit (Qiagen) to prepare the H chain gene.

[0101] (2) Synthesis of light chain genes ((e) to (h)) (2-1) PCR was performed in the same manner as in (1-1), except that the L chain genes of anti-EGFR Fab antibodies artificially synthesized by FASMAC (SEQ ID NOs: 32, 33, 34, and 35) were used as template DNA, and a combination of oligonucleotides consisting of any of the SEQ ID NOs shown in Table 4 was used as a primer set (forward / reverse).

[0102] [Table 4]

[0103] (2-2) PCR was performed in the same manner as in (1-1), except that the PCR product in (2-1) was used as template DNA and oligonucleotides consisting of the sequences set forth in SEQ ID NOs: 29 and 48 were used as a primer set (forward / reverse).

[0104] (2-3) The PCR product obtained in (2-2) was purified using a QIAquick PCR Purification Kit (Qiagen) to prepare the L chain gene.

[0105] Example 3: Antibody synthesis using a cell-free protein synthesis system The antibody was synthesized from the antibody gene prepared in Example 2 using a cell-free protein synthesis system using components derived from Escherichia coli.

[0106] (1) PUREfrex2.1 (Gene Frontier) was used as a cell-free protein synthesis system using components derived from Escherichia coli. A reaction solution with the composition shown in Table 5 was prepared and added to a 0.2 mL tube. In Table 5, the H-chain gene / L-chain gene combination without tag peptide is the combination of Examples 2(a) and (d), with KAIK tag (SEQ ID NO: 1) is the combination of Examples 2(b) and (e), and with SKIK tag (SEQ ID NO: 8) is the combination of Examples 2(c) and (f).

[0107] [Table 5]

[0108] (2) After incubation at 30°C for 6 hours, the mixture was centrifuged at 15,000 G for 30 minutes, and the soluble fraction of the supernatant was collected.

[0109] Example 4 Evaluation of antibody synthesis amount by Western blotting Using a fully automated simple western system (Protein Simple) and an HRP-labeled anti-6-His tag antibody (BETHYL), 6H linked to the H chain contained in the soluble fraction collected in Example 3 was detected, and the amount of antibody synthesis was calculated from the signal intensity.

[0110] The results are shown in Table 6. The amount of antibody synthesis is shown as a relative value to the amount of antibody synthesis without the tag peptide. In all antibody genes (clones), the addition of the KAIK tag (SEQ ID NO: 1) to the N-terminus improved the amount of antibody synthesis compared to when no tag peptide was used or when the SKIK tag (SEQ ID NO: 8) was used. This indicates that the effect of adding the KAIK tag does not depend on the antibody gene (clone).

[0111] [Table 6]

[0112] Example 5 Evaluation of binding activity by ELISA (Enzyme-linked immunosorbent assay) The binding activity of the anti-EGFR antibody contained in the soluble fraction collected in Example 3 to its antigen, EGFR, was evaluated by the following ELISA.

[0113] (1) EGFR (LSBio) was prepared at 0.5 μg / mL in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and 100 μL / well of the solution was added to a 96-well microplate (Thermo Fisher Scientific) for immobilization (4°C, 18 hours).

[0114] (2) After immobilization, the cells were blocked with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w / v) BSA (Bovine Serum Albumin) (Sigma-Aldrich) and 150 mM sodium chloride (30°C, 2 hours), and then washed with washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20 (trade name) and 150 mM sodium chloride).

[0115] (3) The soluble fractions of the antibody without tag peptide, the antibody with KAIK tag (SEQ ID NO: 1), and the antibody with SKIK tag (SEQ ID NO: 8) contained in the soluble fraction collected in Example 3 were diluted 20-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and the diluted solution was added at 100 μL / well to react the EGFR with the anti-EGFR antibody (30°C, 1 hour). After the reaction was completed, the plate was washed with the wash buffer.

[0116] (4) HRP-labeled biotin (Origen) was diluted 2000-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and the diluted solution was added at 100 μL / well and reacted at 30° C. for 1 hour. After the reaction was completed, the plate was washed with the washing buffer.

[0117] (5) After adding 50 μL / well of TMB Substrate (KPL), color development was stopped by adding 50 μL / well of 1 M phosphoric acid solution, and the absorbance at 450 nm was measured using a microplate reader (Tecan).

[0118] The results are shown in Figure 2. The vertical axis in Figure 2 represents the relative value, obtained by dividing the absorbance in the presence of antigen by the absorbance in the absence of antigen (so-called blank). For all antibody genes (clones), the addition of the KAIK tag (SEQ ID NO: 1) to the N-terminus showed higher values ​​than when no tag peptide was present or when the SKIK tag (SEQ ID NO: 8) was added. This indicates that the amount of synthesized antibody capable of binding to the antigen is improved regardless of the antibody gene (clone).

Claims

1. A tagged protein in which a tag peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is added to the N-terminus of a target protein.

2. The tagged protein according to claim 1 , further comprising a signal peptide added to the N-terminal or C-terminal side of the tag peptide.

3. 3. The tagged protein of claim 1, wherein the target protein is an immunoglobulin or an Fc-binding protein.

4. A polynucleotide encoding the tagged protein of claim 1 or 2.

5. An expression vector comprising the polynucleotide of claim 4.

6. A method for expressing a target protein, characterized in that the target protein is expressed as a tagged protein in which a tag peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is added to the N-terminus of the target protein.

7. The method according to claim 6, wherein an expression system using Escherichia coli is used.

8. The method according to claim 6, wherein a cell-free protein synthesis system is used.

9. 9. The method of claim 6, wherein the target protein is an immunoglobulin or an Fc-binding protein.

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