HK1128308B - Taurine transporter gene - Google Patents

Description

Taurine transporter gene

Technical Field

The present invention relates to a hamster taurine transporter, a gene encoding the hamster taurine transporter, and a method for producing a polypeptide using a cell highly expressing the taurine transporter.

Background

When producing proteins useful as medicines by using genetic recombination techniques, animal cells are often used as host cells for recombinant protein production in order to enable complicated post-translational modifications or folding that cannot be performed by prokaryotic cells.

In recent years, many biopharmaceuticals such as antibodies and physiologically active proteins have been put on the market, and the technology for producing recombinant proteins more efficiently in animal cells has been associated with cost reduction of biopharmaceuticals, and has restricted stable supply to patients.

Therefore, a method for producing a protein with higher production efficiency is desired.

Taurine is an amino acid rich in aquatic products and mollusks, and is a nutrient very important for the growth of mammals. Although not used for protein synthesis, it has effects in normalizing hypercholesterolemia, lowering blood pressure, removing toxic substance, maintaining immunity, stabilizing biological membrane, regulating nervous excitability, and resisting oxidation. It is known that cultured cells contribute to osmotic pressure regulation and stabilization of cell membranes (non-patent document 1). However, even if taurine is added to a medium for primary cultured astrocyte cells in which a taurine transporter functions, addition of taurine alone to the medium is not sufficient because taurine is not added to the cells (non-patent document 2).

On the one hand, it is unknown whether the incorporation of taurine or other amino acids into cultured cells by means of taurine transporter proteins contributes to the enhancement of the production of the desired recombinant protein in the cultured cells.

Although it has been known that several taurine transporters (human: non-patent document 3, mouse: non-patent document 4, rat: non-patent document 5) and other taurine transporters are involved in incorporation of amino acids such as taurine and beta-alanine into cells (non-patent document 6), it is not clear whether or not taurine transporters involved in hamsters, including the taurine transporters, exist.

Non-patent document 1: ian Henry Lambert, Neurochemical Research (2004)29(1), 27-63

Non-patent document 2: journal of Neurochemistry (2000), 75(3), 919-

Non-patent document 3: uchida, S.et.al, Proc.Natl.Acad.Sci.U.S.A (1992)89(17), 8230-8234

Non-patent document 4: liu, Q.R.et.al., Proc.Natl.Acad.Sci.U.S.A (1992)89(24), 12145-

Non-patent document 5: smith, K.E.et.al., mol.Pharmacol. (1992)42(4), 563-

Non-patent document 6: ryo Shioda. et. al., investigational ophthalmology & Visual Science (2002)43(9), 2916

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a method capable of producing a natural protein or a recombinant protein at low cost.

Means for solving the problems

The present inventors have made intensive efforts to solve the above problems, and as a result, have found that the production of a desired polypeptide can be increased by using a cell highly expressing a taurine transporter, and the present invention has been completed.

The main contents of the present invention are as follows.

(1) A method for producing a polypeptide, which comprises culturing a cell which highly expresses a taurine transporter and into which a DNA encoding a desired polypeptide has been introduced, and thereby producing the desired polypeptide.

(2) The process according to (1), wherein the cell highly expressing a taurine transporter is a cell into which a DNA encoding the taurine transporter has been introduced.

(3) The process according to (2), wherein the cell is a Chinese hamster ovary cell.

(4) The production method of any one of (1) to (3), wherein the desired polypeptide is an antibody.

(5) The method of any one of (2) to (4), wherein the DNA encoding a taurine transporter is any one of the following (a) to (e):

(a) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8,

(b) a DNA encoding a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or/and inserted in the amino acid sequence of SEQ ID NO. 2, 4, 6, or 8, and having a taurine transporter activity,

(c) DNA encoding a polypeptide having a homology of 70% or more with the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8 and a taurine transporter activity,

(d) DNA having the base sequence of SEQ ID NO. 1, 3, 5 or 7,

(e) a DNA that hybridizes under stringent conditions to a DNA complementary to the DNA having the base sequence of SEQ ID NO. 1, 3, 5 or 7 and encodes a polypeptide having taurine transporter activity.

(6) The method according to any one of (1) to (5), which comprises culturing in a medium containing taurine at a concentration of 0g/L to 100 g/L.

(7) A method for producing a pharmaceutical product, wherein the pharmaceutical product contains the polypeptide produced by the method according to any one of (1) to (6).

(8) A taurine transporter-encoding DNA (excluding DNAs having the base sequences of SEQ ID Nos. 3, 5 and 7) of any one of the following (a) to (e):

(a) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO. 2,

(b) a DNA encoding a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or/and inserted in the amino acid sequence of SEQ ID NO. 2 and having a taurine transporter activity,

(c) DNA encoding a polypeptide having a homology of 97% or more with the amino acid sequence of SEQ ID NO. 2 and having a taurine transporter activity,

(d) a DNA having the base sequence of SEQ ID NO. 1,

(e) a DNA that hybridizes under stringent conditions to a DNA complementary to the DNA having the base sequence of SEQ ID NO. 1 and encodes a polypeptide having a taurine transporter activity.

(9) Any one of the following (A) to (E) (excluding polypeptides having amino acid sequences of SEQ ID Nos. 4, 6 and 8):

(A) a polypeptide having the amino acid sequence of SEQ ID NO. 2,

(B) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or/and inserted in the amino acid sequence of SEQ ID NO. 2, and having a taurine transporter activity,

(C) a polypeptide having a homology of 97% or more with the amino acid sequence of SEQ ID NO. 2 and having a taurine transporter activity,

(D) a polypeptide encoded by a DNA having the base sequence of SEQ ID NO. 1,

(E) a polypeptide encoded by a DNA that hybridizes under stringent conditions to a DNA complementary to the DNA having the base sequence of SEQ ID NO. 1 and encodes a polypeptide having a taurine transporter activity.

(10) A recombinant vector comprising the DNA according to (8).

(11) A cell into which the DNA according to (8) has been introduced.

(12) A cell which highly expresses a taurine transporter and into which a DNA encoding a desired polypeptide has been introduced.

(13) A cell into which a DNA encoding a taurine transporter has been introduced.

ADVANTAGEOUS EFFECTS OF INVENTION

The desired polypeptide can be produced inexpensively by the present invention.

The present specification includes the contents described in the specification and/or drawings of japanese patent application, patent application 2006-110467, which is the basis of priority of the present application.

Brief description of the drawings

FIG. 1 shows the nucleotide sequence and amino acid sequence of a hamster taurine transporter gene derived from a newly cloned CHO cell.

FIG. 2 taurine transporter membrane topographs prepared with reference to FIG.5 of Proc, Natl.Acad.Sci.USAVol.89, pp.8230-8234, September1992, Shinichi Uchida et al, based on the transmembrane region and orientation predicted by the TMpred program from the amino acid sequence of hamster TauT from newly cloned CHO cells. Is a hamster TauT-specific amino acid residue, and has a plurality of amino acids different from human TauT in the 2 nd loop (EX: extracellular domain), 12 transmembrane domains (TM), and the C-terminal (IC: intracellular domain).

FIG. 3 shows a plasmid for expressing hamster TauT (622 amino acids).

FIG. 4 is a graph of viable cell density at day 7 of batch culture in a 50ml shake flask. The viable cell density of pHyg/TauT-introduced cells was predominant over that of pHyg-introduced cells (P < 0.05).

FIG.5 is a graph of lactic acid production at day 7 in a 50ml shake flask batch culture (n-7). pHyg/TauT-introduced cells produced low amounts of lactate, and were predominant over pHyg-introduced cells (P <0.05 in t-test).

Fig. 6 is a graph of anti-glypican (protein) glycan-3 antibody production at day 7 in a 50ml shake flask batch culture (n-7). 4 of the 7 pHyg/TauT cells had an antibody production yield of not less than the maximum value of pHyg cells.

FIG. 7 is a graph of anti-glypican (protein) glycan-3 antibody production at day 7 of fed-batch culture in 50ml shake flasks (n-7). The anti-glypican-3 antibody of pHyg/TauT-introduced cells was predominant over that of pHyg-introduced cells (P < 0.01).

FIG. 8 is a graph showing the survival rate of T10, a pHyg/TauT-introduced cell having a high proliferation potency in the course of expansion by static culture, in fed-batch culture in a 1L jar. The survival rate of T10 reached more than 80% at day 32 of culture. While the parental strain decreased by 80% on day 19.

FIG. 9 is a graph showing the amount of antibody produced in fed-batch culture in a 1L jar as pHyg/TauT-introduced cell T10 having high proliferation potency in the course of expansion by static culture. The anti-glypican-3 antibody production at day 35 of culture was 2.9 g/L.

FIG. 10 shows the results of flow cytometry analysis of TauT molecules expressed on the cell membrane of TauT-introduced T10 cells. A rabbit anti-rat taurine transporter antibody (A1phadiagnostics, u.s.) (antibody ±) was used as a primary antibody, and an donkey anti-rabbit IgG antibody was labeled with a phycoerythrin conjugate (Abcam, u.k.) as a secondary antibody.

FIG. 11 is a graph showing the intracellular ammonia content (concentration ratio) in 1L jar fed-batch culture. The pHyg/TauT-introduced strain exhibited significant ammonia inhibition relative to the parent strain.

FIG. 12 is a graph showing that incorporation of taurine into cells depends on the concentration of taurine in a medium. No difference was observed between the pHyg/TauT-introduced strain and the parent strain in the amount of taurine incorporated.

FIG. 13 is a graph showing glutamine consumption in the medium. The pHyg/TauT-introduced strain had a significantly higher average glutamine consumption per cell than the parent strain, and was independent of the taurine concentration in the medium.

FIG. 14 shows that the anti-glypican (proteo) glycan-3 antibody production amounts in the pHyg/TauT-introduced strains were comparable regardless of the taurine concentration in the medium at the start of the culture.

Best mode for carrying out the invention

Embodiments of the present invention will be described in more detail below.

The present invention provides a method for producing a polypeptide, which comprises culturing a cell which highly expresses a taurine transporter and into which a DNA encoding a desired polypeptide has been introduced, and producing the desired polypeptide.

In the method of the present invention, the cell may be a natural cell capable of producing the desired polypeptide, or a transformed cell into which a DNA encoding the desired polypeptide has been introduced, preferably a transformed cell into which a DNA encoding the desired polypeptide has been introduced.

In the method of the present invention, the desired polypeptide is not particularly limited, and may be any of antibodies (for example, anti-IL-6 receptor antibody, anti-glypican-3 antibody, anti-CD 3 antibody, anti-CD 20 antibody, anti-GPIIb/IIIa antibody, anti-TNF antibody, anti-CD 25 antibody, anti-EGFR antibody, anti-Her 2/neu antibody, anti-RSV antibody, anti-CD 33 antibody, anti-CD 52 antibody, anti-IgE antibody, anti-CD 11a antibody, anti-VEGF antibody, anti-VLA 4 antibody, etc.) or physiologically active proteins (granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin, interferon, interleukins such as IL-1 and IL-6, t-PA, urokinase, serum globulin, blood clotting factor, PTH, etc.), etc., with antibodies being particularly preferred. The antibody may be any of natural antibodies, low molecular weight antibodies such as Fab, scFv, and sc (Fv)2, chimeric antibodies, and humanized antibodies.

The present inventors have found that the incorporation of glutamine into cells can be promoted not only by specifically promoting taurine and β -alanine but also by using cells highly expressing a taurine transporter.

The taurine transporter is known as a membrane protein having a function of transporting taurine and β -alanine into cells, but it is not known that glutamine can be specifically transported into cells by highly expressing the taurine transporter in the cells. Since glutamine is known to be involved in antibody production in hybridomas (see: Yeon-Ho Jeong et al, Enzyme and microbial Technology (1995)17, 47-55), it is considered that the effect of enhancing the production of proteins such as antibodies by cells highly expressing a taurine transporter may be caused by the action of the taurine transporter in specifically transporting glutamine into cells.

The cell highly expressing the taurine transporter is not particularly limited as long as the expression level of the taurine transporter is increased as compared with that of a natural cell. The natural cells are not particularly limited, and examples thereof include CHO cells used as hosts in producing recombinant proteins.

Examples of the cells highly expressing a taurine transporter include cells artificially introduced with a taurine transporter gene. The cell into which the taurine transporter gene has been artificially introduced can be prepared by a method known in the art, for example, by integrating the taurine transporter gene into a vector and transforming the cell with the vector.

The taurine transporter gene that can express a cell at a high level is not particularly limited, and may be a taurine transporter derived from any organism. Specifically, for example, a taurine transporter derived from a living organism such as a rodent such as a human, a mouse, a rat, or a hamster, a taurine transporter derived from a human, a rodent, or the like of a host cell is preferable, and for example, when a cell highly expressing a taurine transporter is a chinese hamster ovary cell (CHO cell), a taurine transporter derived from a human or a hamster is preferable.

The taurine transporter gene highly expressed in the cell includes any of the following DNAs (a) to (e) encoding taurine transporter:

(a) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8,

(b) a DNA encoding a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or/and inserted in the amino acid sequence of SEQ ID NO. 2, 4, 6, or 8, and having a taurine transporter activity,

(c) DNA encoding a polypeptide having a taurine transporter activity and having a homology of 70% or more with the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8,

(d) DNA having the base sequence of SEQ ID NO. 1, 3, 5 or 7,

(e) a DNA which hybridizes to a DNA complementary to the DNA having the base sequence of SEQ ID NO. 1, 3, 5 or 7 under stringent conditions and encodes a polypeptide having a taurine transporter activity,

the cell highly expressing the taurine transporter may be any one of cells, preferably a CHO cell, particularly preferably a CHO dhf r-cell.

In order to produce a desired polypeptide, a gene encoding the desired polypeptide is introduced into a cell highly expressing a taurine transporter, and the cell is cultured in a medium.

When a desired polypeptide is produced using a cell into which a taurine transporter gene has been artificially introduced, the order of introduction of the taurine transporter gene and a gene encoding the desired polypeptide is not particularly limited, and a gene encoding the desired polypeptide may be introduced after the introduction of the taurine transporter gene, or a gene encoding the desired polypeptide may be introduced after the introduction of the taurine transporter gene. Alternatively, the taurine transporter gene and the gene encoding the desired polypeptide may be introduced simultaneously.

The taurine transporter gene and the gene encoding the desired polypeptide may be introduced simultaneously using a single vector, or may be introduced separately using a plurality of vectors.

For culturing the cells highly expressing the taurine transporter, a medium used for culturing ordinary cells (preferably animal cells) can be used. These typically include amino acids, vitamins, lipid factors, energy sources, osmotic pressure regulators, iron sources, and pH buffers. The content of these components is usually 0.05-1500mg/L amino acid, vitamin 0.001-10mg/L, lipid factor 0-200mg/L, energy 1-20g/L, osmotic pressure regulator 0.1-10000mg/L, iron source 0.1-500mg/L, pH buffer 1-10000mg/L, trace metal element 0.00001-200mg/L, surfactant 0-5000mg/L, proliferation auxiliary factor 0.05-10000 g/L and nucleoside 0.001-50mg/L, and is not limited to these, and can be further determined appropriately according to the kind of cultured cell, kind of desired polypeptide, etc.

In addition to the above components, for example, trace metal elements, surfactants, growth cofactors, nucleosides, and the like may be added.

Specifically, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-valine and the like are preferable, and L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-methionine and the like, Amino acids such as L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, pyridoxine hydrochloride, and vitamin B hydrochloride₆Riboflavin, thiamine hydrochloride, vitamin B₁₂Vitamins such as ascorbic acid; choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, and the like, preferably a lipid factor such as choline chloride; glucose, galactose, mannose, fructose, and the like, preferably an energy source such as glucose; sodium chloride, potassium nitrate, etc., preferably sodium chloride, etc.; ferric EDTA, ferric citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate nitrate, preferably ferric chloride, ferric EDTA, ferric citrate; sodium hydrogen carbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc., preferably a medium containing a pH buffer such as sodium hydrogen carbonate.

In addition to the above components, trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, sodium metasilicate, and the like, preferably copper sulfate, zinc sulfate, magnesium sulfate, and the like; surfactants such as Tween80, and a polymer of polypropylene glycol and ethylene oxide (polyether) F68(Pluronic F68); recombinant insulin, recombinant IGF-1, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-alpha, ethanolamine hydrochloride, sodium selenite, retinoic acid, putrescine hydrochloride and the like, preferably proliferation auxiliary factors such as sodium selenite, ethanolamine hydrochloride, recombinant IGF-1, putrescine hydrochloride and the like; nucleosides such as deoxyadenosine, deoxythymidine, deoxyguanosine, adenosine, thymidine, guanosine, uridine, etc. In addition, antibiotics such as streptomycin, penicillin G potassium and gentamicin, and pH indicators such as phenolate may also be contained in suitable examples of the above-mentioned medium.

The pH of the medium varies depending on the cells to be cultured, and is usually 6.8 to 7.6, and in many cases, preferably 7.0 to 7.4.

As the culture Medium, commercially available culture media for animal cell culture, for example, D-MEM (Dulbecco's Modified Eagle Medium), D-MEM/F-121: 1mix (Dulbecco' S Modified Eagle Medium: Nutrient mix F-12), RPMI1640, CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL301(JRH biosciences), CD-CHO (Invitrogen), ISCHO-V (Irvine Scientific, PF-ACF-CHO) (Sigma-Aldrich) and the like.

The medium may be a serum-free medium.

When the cell highly expressing the taurine transporter is a CHO cell, the culture of the CHO cell can be carried out by a method well known in the same industry. For example, CO may be generally in the gas phase₂The culture is carried out at a concentration of 0 to 40%, preferably 2 to 10%, in an atmosphere at 30 to 39 ℃, preferably about 37 ℃.

As shown in the examples below, in cells highly expressing a taurine transporter, the production of old waste such as lactic acid, which inhibits the growth of the cells, is suppressed. The results show that the effect of maintaining high survival rates, it is possible to culture the cells of the present invention for 3 months or more.

In addition, when a desired polypeptide such as an antibody is produced in the cultured cells, the cells are in a state of extremely high density (about 1X 10) in the late stage of the culture⁷Cell/ml), the influence of old waste such as lactic acid becomes very large. If a desired polypeptide is produced by the present invention, it is expected that not only a high survival rate is maintained but also the yield of the desired polypeptide is improved at the late stage of culture.

Suitable culture periods for producing the desired polypeptide using the cells of the invention are generally from 1 day to 3 months, preferably from 1 day to 2 months, more preferably from 1 day to 1 month.

As various culture apparatuses for culturing animal cells, for example, a fermentor type tank culture apparatus, an air generator type culture apparatus, a culture (glass) bottle type culture apparatus, a centrifuge bottle type culture apparatus, a microcarrier type culture apparatus, a fluidized bed type culture apparatus, a hollow fiber type culture apparatus, a roller bottle type culture apparatus, a packed tank type culture apparatus, and the like can be used for the culture.

The culture may be carried out by any method such as batch culture (bat culture), fed-batch culture (fed-batch culture), continuous culture (continuous culture), etc., preferably by fed-batch culture or continuous culture, more preferably by fed-batch culture.

In the case of culturing the cells of the present invention, taurine may be added to the medium in order to promote incorporation of taurine into the cells. The concentration of taurine to be added to the medium is not particularly limited, but is usually 0g/L to 100g/L, preferably 0g/L to 20g/L, and more preferably 0g/L to 10 g/L.

When the polypeptide produced by the method of the present invention has a biological activity usable as a medicine, a medicine can be produced by mixing the polypeptide with a pharmaceutically acceptable carrier or additive and then formulating the mixture.

Examples of the pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthene gum, gum arabic, cheese, agar, polyethylene glycol, diglycerin, glycerol, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, Human Serum Albumin (HSA), mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.

The actual additives may be selected from the above additives individually or in an appropriate combination according to the dosage form of the therapeutic agent of the present invention, and it is not limited thereto. For example, when the polypeptide is used as an injection preparation, the purified polypeptide may be dissolved in, for example, physiological saline, a buffer solution, a glucose solution, or the like, and a solution containing an adsorbent such as Tween80, Tween20, gelatin, human serum albumin, or the like may be added. Alternatively, the pharmaceutical composition may be a lyophilized sample for reconstitution into a dosage form to be dissolved before use, and sugar alcohols such as mannitol and glucose, or saccharides may be used as excipients for lyophilization.

The effective amount of the polypeptide to be administered may be appropriately selected depending on the kind of the polypeptide, the kind of the disease to be treated or prevented, the age of the patient, the severity of the disease, and the like. For example, when the polypeptide is an anti-glypican antibody, an effective administration amount of the anti-glypican antibody is selected in the range of from 0.001mg to 1000mg per 1kg body weight per one time. Or the administration amount of 0.01-100000 mg/body is selected for each patient. But is not limited to these amounts.

The method of administering the polypeptide may be oral administration or non-oral administration, and preferably is non-oral administration, specifically, such as injection (systemic or local administration such as intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection), nasal administration, pulmonary administration, or transdermal administration.

The present invention also provides any one of the following novel polypeptides (except polypeptides having amino acid sequences of SEQ ID Nos. 4, 6 and 8) of (A) to (E):

(A) polypeptide having amino acid sequence of SEQ ID No. 2

(B) A polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or/and inserted in the amino acid sequence of SEQ ID NO. 2, and having a taurine transporter activity,

(C) a polypeptide having a homology of 97% or more with the amino acid sequence of SEQ ID NO. 2 and having a taurine transporter activity,

(D) polypeptide encoded by DNA having base sequence of SEQ ID NO. 1

(E) A polypeptide encoded by a DNA which hybridizes to a DNA complementary to the DNA having the base sequence of SEQ ID NO. 1 under stringent conditions and encodes a polypeptide having taurine transporter activity

The novel polypeptide of the present invention is a hamster taurine transporter and a polypeptide functionally equivalent thereto.

In the present invention, functionally equivalent to a hamster taurine transporter means that the hamster taurine transporter has the same activity as the hamster taurine transporter, such as an activity of binding to taurine and an activity of transporting taurine into a cell. Such polypeptides include, for example, a mutant of hamster taurine transporter and the like.

As a method well known in the art for producing a polypeptide functionally equivalent to a certain polypeptide, a method of introducing a mutation into a polypeptide is known. For example, site-specific mutagenesis Methods (Hashimoto-Gotoh, T.et al. (1995) Gene152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol.100, 468-500, Kramer, W.et al. (1984) Nucleic Acids Res.12, 9441-9456, Kramer, W.and Fritz HJ (1987) Methods Enzymol.154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci. USA.82, 488-492, Kunkel (1988) Methods Enzymol.85, 2763-2766) and the like can be used in the same line to provide appropriate mutations in the amino Acids of taurine transporters and make hamster transporters functionally equivalent to those of taurine transporters. In addition, mutations of amino acids can also occur in nature. Therefore, a polypeptide having an amino acid sequence in which 1 or more amino acids are mutated in the amino acid sequence of the hamster taurine transporter of the present invention and functionally equivalent to the hamster taurine transporter is also included in the polypeptide of the present invention.

Specifically, the polypeptide functionally equivalent to the hamster taurine transporter of the present invention includes a polypeptide in which 1 or 2 or more, preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, further preferably 1 or more and 10 or less, and most preferably 1 or more and 5 or less amino acids are deleted from the amino acid sequence of the hamster taurine transporter, a polypeptide in which 1 or 2 or more, preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, further preferably 1 or more and 10 or less, and most preferably 1 or more and 5 or less amino acids are added to the amino acid sequence of the hamster taurine transporter, and a polypeptide in which 1 or 2 or more, preferably 1 or more and 30 or less, are substituted in the amino acid sequence of the hamster taurine transporter, and a polypeptide in which, More preferably 1 to 20, further preferably 1 to 10, most preferably 1 to 5 amino acids.

Although the amino acid residue to be mutated is not particularly limited, it is desirable to mutate the amino acid residue into another amino acid that retains the properties of the amino acid side chain. Examples of the properties of the amino acid side chain include a hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), a hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a side chain containing a hydroxyl group (S, T, Y), an amino acid having a side chain containing a sulfur atom (C, M), an amino acid having a side chain containing a carboxyl group and an amine group (D, N, E, Q), an amino acid having a side chain containing a base group (R, K, H), and an amino acid having an aromatic side chain (H, F, Y, W) (the parentheses indicate the one-letter abbreviations for any of the amino acids).

It is also known that a polypeptide having an amino acid sequence modified by deletion, addition and/or substitution of 1 or more amino acid residues of an amino acid sequence can maintain its original biological activity (Mark, D.F.et. al., Proc Natl Acad Sci USA (1984)81, 5662-.

The polypeptide to which one or more amino acid residues have been added to the hamster taurine transporter of the present invention is, for example, a fusion polypeptide containing the hamster taurine transporter. The fusion polypeptide is a product of fusion of the protein hamster taurine transporter of the invention with other polypeptides, and is included in the invention. As a method for producing a fusion protein, a method known in the same industry can be used, in which a gene encoding the hamster taurine transporter of the present invention and a gene encoding another polypeptide are ligated so as to be in frame, and then introduced into an expression vector to express the fusion protein. The other polypeptide to be added as a fusion with the polypeptide of the present invention is not particularly limited.

Examples of other polypeptides to be added as fusions to the polypeptide of the present invention include FLAG (Hopp, T.P.et al., Biotechnology (1988)6, 1204-1210), 6 XHis consisting of 6 His (histidine), 10 XHis, influenza lectin (HA), a fragment of human c-myc, a fragment of VSVSVGP, a fragment of p18HIV, T7-tag, HSV-tag, E-tag, a fragment of SV40T antigen, 1ck tag, a fragment of alpha-tubulin, B-tag, a fragment of ProteinC, GST (glutathione-S-transferase), HA (influenza lectin), an immunoglobulin constant region, beta-galactosidase, MBP (maltose binding polypeptide), and the like.

Fusion polypeptides can be prepared by fusing commercially available genes encoding these polypeptides with a gene encoding a polypeptide of the present invention and expressing the thus-prepared fusion gene.

In addition, the same lines as those for producing a polypeptide functionally equivalent to a certain polypeptide are well knownFor example, hybridization techniques (Sambrook, Jet al., Molecular Cloning 2) can be used^nded., 9.47-9.58, Cold Spring Harbor Lab. press, 1989). That is, in the case of the same lines, it is generally possible to isolate a DNA having a high homology to the DNA sequence encoding the hamster taurine transporter of the present invention or a part thereof, and isolate a polypeptide functionally equivalent to the hamster taurine transporter from the DNA. Therefore, a polypeptide encoded by a DNA that hybridizes to a DNA comprising a DNA encoding the hamster taurine transporter of the present invention or a part thereof is also included in the polypeptide of the present invention.

The conditions for hybridization for isolating a DNA functionally encoding a polypeptide equivalent to the hamster taurine transporter of the present invention can be appropriately selected. The hybridization conditions may be, for example, low stringency conditions. The low stringency conditions include 42 ℃ 2XSSC, 0.1% SDS, preferably 50 ℃ 2XSSC, 0.1% SOS. Or more preferably, for example, high stringency conditions, preferably 65 ℃, 2XSSC, 0.1% SDS. Under these conditions, lowering the temperature can not only yield DNA with high homology, but also only low homology. On the contrary, by increasing the temperature, only DNA having high homology can be obtained. However, as a factor affecting the stringency of hybridization, in addition to temperature, a plurality of factors such as salt concentration are considered, and the same stringency can be achieved by appropriately selecting these factors.

The DNA isolated by these hybridization techniques typically encodes a polypeptide having high amino acid sequence homology to the hamster taurine transporter of the invention. The polypeptide of the present invention is functionally equivalent to the hamster taurine transporter of the present invention, and includes a polypeptide having high homology to the hamster taurine transporter of the present invention. The term "high homology" generally means a homology of 97% or more, preferably 98% or more, more preferably 99% or more. To determine the homology of polypeptides, reference may be made to the algorithm described in the literature (Wilbur, W.J.and Lipman, D.J.Proc Natl Acad Sci USA (1983)80, 726-730).

The polypeptide of the present invention differs in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chain, form and the like depending on the cell producing the polypeptide described below, the host and the purification method. However, any polypeptide obtained is included in the present invention as long as it has the same function as the hamster taurine transporter of the present invention. For example, when the polypeptide of the present invention is expressed in a prokaryotic cell such as E.coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original polypeptide. And when expressed in eukaryotic cells, such as mammalian cells, the signal sequence at the N-terminus is removed. The polypeptides of the invention also include such polypeptides.

The polypeptide of the present invention may be prepared as a recombinant polypeptide, or a natural polypeptide, by methods well known in the art. In the case of a recombinant polypeptide, it can be prepared by incorporating a DNA encoding the polypeptide of the present invention into an appropriate expression vector, introducing the vector into an appropriate host cell, recovering the obtained transformant, obtaining an extract, and purifying the extract by chromatography such as ion exchange, reverse phase, or gel filtration, or by affinity chromatography in which an antibody against the polypeptide of the present invention is immobilized on a column, or by further combining a plurality of these columns.

In addition, in order to express a fusion polypeptide of the present invention and glutathione S-transferase or a recombinant polypeptide having a plurality of histidines added thereto in a host cell (e.g., an animal cell, E.coli, or the like), the expressed recombinant polypeptide may be purified using a glutathione column or a nickel column.

After purification of the fusion polypeptide, regions other than the target polypeptide in the fusion polypeptide may be cleaved and removed by thrombin or factor Xa as necessary.

In the case of a native polypeptide, purification and isolation can be carried out by a method known in the same lines, for example, affinity chromatography in which a tissue or cell extract expressing the polypeptide of the present invention is allowed to act on an antibody capable of binding to a hamster taurine transporter as described below. The antibody may be a polyclonal antibody or a monoclonal antibody.

The present invention also provides a taurine transporter-encoding DNA (excluding DNAs having base sequences of SEQ ID Nos. 3, 5 and 7) of any one of the following (a) to (e):

(a) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO. 2,

(b) a DNA encoding a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or/and inserted in the amino acid sequence of SEQ ID NO. 2 and having a taurine transporter activity,

(c) DNA encoding a polypeptide having a homology of 97% or more with the amino acid sequence of SEQ ID NO. 2 and having a taurine transporter activity,

(d) a DNA having the base sequence of SEQ ID NO. 1,

(e) DNA which hybridizes to DNA complementary to DNA having the base sequence of SEQ ID NO. 1 under stringent conditions and encodes a polypeptide having taurine transporter activity

The DNA of the present invention can be used for the production of the polypeptide of the present invention as described above in vivo or in vitro, and can also be used for the preparation of cells highly expressing a hamster taurine transporter. The DNA of the present invention may be in any form as long as it can encode the polypeptide of the present invention. That is, cDNA synthesized from mRNA, genomic DNA, chemically synthesized DNA, or the like may be used. But also includes DNA having an arbitrary nucleotide sequence derived from the degeneracy of the genetic code, as long as it encodes the polypeptide of the present invention.

The DNA of the present invention can be prepared by methods well known in the art. For example, the DNA fragment can be prepared by preparing a cDNA library from cells expressing the polypeptide of the present invention, probing a part of the sequence (for example, sequence 1) of the DNA of the present invention, and then hybridizing the probe. The cDNA library can be prepared by the method described in, for example, Sambrook, J.et al, Molecular Cloning, Cold Spring harborLab.Press, (1989), or a commercially available gene library can be used. On the other hand, RNA is prepared from cells expressing the polypeptide of the present invention, and an oligo DNA is synthesized based on the DNA sequence of the present invention (for example, SEQ ID NO: 1), and the oligo DNA is used as a primer for PCR reaction to amplify a cDNA encoding a taurine transporter.

The amino acid sequence of the polypeptide of the present invention can be obtained by determining the nucleotide sequence of the cDNA obtained and determining the translation region encoded by the cDNA. In addition, by screening a genomic DNA library using the obtained cDNA as a probe, genomic DNA can be isolated.

Specifically, the following procedure can be performed. First, mRNA is isolated from cells, tissues, etc. expressing the polypeptide of the present invention. Isolation of mRNA Total RNA was prepared by a well-known method, for example, guanidine ultracentrifugation (Chirgwin, J.M.et al., Biochemistry (1979)18, 5294-. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA purification kit (Pharmacia).

cDNA is synthesized from the resulting mRNA using reverse transcriptase. The synthesis of cDNA can be carried out using AMV reverse transcriptase first strand cDNA synthesis kit (Biochemical industry) or the like. In addition, cDNA synthesis and amplification can be carried out according to the 5 '-RACE method (Frohman, M.A.et. al., Proc.Natl.Acad.Sci.U.S.A. (1988)85, 8998-9002; Belyavsky, A.et. al., Nucleic Acids Res. (1989)17, 2919-2932) using a 5' -Ampli FINDE RRACE kit (manufactured by Clontech) and Polymerase Chain Reaction (PCR) using primers and the like.

From the obtained PCR product, a target DNA fragment was prepared and ligated to the vector DNA. Then, a recombinant vector is prepared therefrom, introduced into Escherichia coli or the like, and colonies are selected to prepare a desired recombinant vector. The nucleotide sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.

In the DNA of the present invention, a base sequence having higher expression efficiency can be designed in consideration of the codon usage frequency of the host used for expression (Grantham, R.et al., nucleic acids Res. (1981)9, r 43-74). In addition, the DNA of the present invention can be modified by a commercially available kit or a well-known method. As for the alteration, for example, by restriction enzyme digestion, synthesis of an oligonucleotide or insertion of an appropriate DNA fragment, addition of a linker, insertion of an initiation codon (ATG) and/or a termination codon (TAA, TGA or TAG), etc.

The DNA of the present invention is a DNA that hybridizes under stringent conditions to a DNA having the base sequence of SEQ ID NO. 1, and includes a DNA that encodes a polypeptide functionally equivalent to a taurine transporter.

As the stringent conditions, the same may be appropriately selected, for example, low stringent conditions. The low stringency conditions include 42 ℃, 2XSSC, 0.1% SDS, preferably 50 ℃, 2XSSC, 0.1% SDS. More preferably, high stringency conditions. The high stringency conditions are 65 ℃, 2XSSC, 0.1% SDS. Under these conditions, DNA having high homology can be obtained by increasing the temperature. The DNA to be hybridized is preferably derived from a natural DNA such as cDNA or chromosomal DNA.

The DNA isolated by these hybridization techniques has high homology in base sequence with the DNA encoding the hamster taurine transporter of the present invention. The DNA of the present invention also includes DNA encoding a polypeptide functionally equivalent to the hamster taurine transporter of the present invention and having high homology to the DNA encoding the hamster taurine transporter of the present invention. The term "high homology" generally means a homology of 96% or more, preferably 98% or more, more preferably 99% or more. The homology of the base sequences can be determined by the algorithm BLAST of Karlin and Altschul (Proc Natl Acad Sci U S A90: 5873-. Based on this algorithm, an algorithm called BLASTN and BLASTX was developed (Altschul et al.J.mol.biol.215: 403-. When the nucleotide sequence is analyzed by BLASTN according to BLAST, the data are, for example, score 100 and wordlength 12. Specific methods for these analysis methods are known (http:// www.ncbi.nlm.nih.gov.).

In addition, the present invention provides a vector into which the DNA of the present invention is inserted. The vector of the present invention retains the DNA of the present invention in a host cell and is used for expressing the polypeptide of the present invention (i.e., a hamster taurine transporter and a functionally equivalent polypeptide). In addition, the taurine transporter protein can be highly expressed in the host cell. By highly expressing the taurine transporter in a host cell, incorporation of taurine into the host cell can be promoted, and production of a desired polypeptide by the host cell can be increased.

When Escherichia coli is used as a host, it is preferable that the vector has "ori" for amplification in Escherichia coli and a selection gene of transformed Escherichia coli (for example, drug resistance genes that can be identified by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)) in order to amplify and prepare the vector in large quantities in Escherichia coli (for example, JM109, DH 5. alpha., HB101, XL1 Blue) and the like. Examples of the vector include M13 series vectors, pUC series vectors, PBR322, pBluscript, and pCR-script. Alternatively, when the objective is to subclone and cut cDNA, for example, pGEM-T, pDIRECT, pT7 and the like may be used in addition to the above-mentioned vector. Expression vectors are particularly useful when the vectors are used for the purpose of producing the polypeptides of the present invention. For example, when the expression vector is intended for expression in Escherichia coli, it is preferable to use Escherichia coli such as JM109, DH 5. alpha., HB101, XL1-Blue as a host in addition to the above-mentioned characteristics that the vector can be amplified in Escherichia coli, and to use a promoter capable of being expressed efficiently in Escherichia coli, such as lacZ promoter (Ward et al, Nature (1989)341, 544-doped 546; FASEB J. (1992)6, 2422-doped 2427), araB promoter (Better, Science (1988)240, 1041-doped 1043), T7 promoter, and the like. Examples of such vectors include pGEX-5X-1 (Pharmacia), "QIAexpresssystem" (Qiagen), pEGFP, and pET (in this case, BL21 expressing T7RNA polymerase is preferred as the host), in addition to the above vectors.

The vector may contain a signal sequence for secretion of the polypeptide. As a signal sequence for polypeptide secretion, the pe1B signal sequence can be used when the polypeptide is produced in the periplasm of E.coli (Lei, S.P.et al J.Bacteriol. (1987)169, 4379). The vector can be introduced into the host cell by, for example, the calcium chloride method or the electroporation method.

As a vector used for producing the polypeptide of the present invention, for example, in addition to Escherichia coli as a host, expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen corporation), pEGF-BOS (Nucleic Acids Res.1990, 18(17), p5322), pEF, pCDM8), expression vectors derived from insect cells (for example, "Bac-to-BAC Agrobacterium expression System" (manufactured by GIBCO BRL corporation) pBacPAK8), expression vectors derived from plants (for example, pMH1, pMH2), expression vectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (for example, pZIpneo), expression vectors derived from yeast (for example, "Pichia expression kit" (manufactured by Invitrogen corporation), pNV11, SP-Q01), expression vectors derived from Bacillus subtilis (for example, pPL, pPTH 50), and the like can be cited.

For the purpose of expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is preferable to include a promoter necessary for expression in the cells, for example, SV40 promoter (Mullingan et al, Nature (1979)227, 108), MMLV-LTR promoter, EF 1a promoter (Mizushima et al, Nucleic Acids Res. (1990)18, 5322), CMV promoter, etc., and more preferably a gene for selecting a gene for transforming cells (for example, a drug resistance gene that can be identified by providing a drug (neomycin, G418, etc.)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP 13.

In addition, for example, a method of introducing a vector (for example, pCHOI or the like) having a DHFR gene that complements a nucleic acid synthesis pathway into CHO cells deficient in the pathway and amplifying the gene by Methotrexate (MTX) is used for the purpose of stably expressing the gene and the gene copy number in the cells is amplified, and for the purpose of transient expression of the gene, a method of transforming COS cells having a gene expressing SV40T antigen on the chromosome with a vector (for example, pcD) having an SV40 replication origin is used for the purpose of transforming the gene. As the replication origin, polyoma virus or adenovirus can be used. Bovine Papilloma Virus (BPV), and the like. In addition, in order to amplify the copy number of the gene in the host cell line, the expression vector may contain, as selectable markers, an Aminoglycoside Phosphotransferase (APH) gene, a Thymidine Kinase (TK) gene, an escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, and the like.

In addition, the present invention provides a host cell into which the vector of the present invention has been introduced. The host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli, various animal cells, and the like can be used. The host cells of the invention may be used, for example, as production lines for the preparation and expression of the polypeptides of the invention. The host cell antigen highly-expressed taurine transporter of the present invention can promote incorporation of taurine, and increase production of a desired polypeptide. Production lines for polypeptide production include in vivo and in vitro production lines. As production lines in vivo, there are, for example, those using eukaryotic cells and those using prokaryotic cells.

When eukaryotic cells are used, for example, animal cells, plant cells, fungal cells can be used as the host. Mammalian cells, such as CHO (j.exp. med. (1995)108, 945), COS, 3T3, myeloma, bhk (baby hamsterkidney), HeLa, Vero, amphibian cells, such as xenopus laevis oocytes (valley, et al., Nature (1981)291.358-340), or insect cells, such as Sf9, Sf21, Tn5, are known as animal cells. As CHO cells, DHFR-CHO (Proc. Natl. Acad. Sci. USA (1980)77, 4216-4220) and CHO K-1(Proc. Natl. Acad. Sci. USA (1968)60, 1275) which lack the DHFR gene are particularly suitable. Among animal cells, CHO cells are particularly preferable for the purpose of large-scale expression. The vector can be introduced into the host cell by, for example, the calcium phosphate method, the DEAE dextran method, the method using cationic liposome DOTAP (manufactured by Boehringer Mannheim), electroporation, lipofection, or the like.

As a plant cell, a cell derived from tobacco (Nicotiana tabacum) is known as a polypeptide production line, and callus culture may be performed on the cell. Examples of known fungal cells include those of the genus Saccharomyces (Saccharomyces), such as Saccharomyces cerevisiae, and those of the genus Aspergillus (Aspergillus), such as Aspergillus niger (Aspergillus niger).

When prokaryotic cells are used, there are production lines using bacterial cells. As bacterial cells, Escherichia coli, such as JM109, DH 5. alpha., HB101, etc., and Bacillus subtilis are known.

The polypeptide encoded by the gene of interest can be obtained by transforming these cells with the gene of interest and culturing the transformed cells in vitro. The culture is carried out according to a well-known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as a culture medium for animal cells. In this case, a serum supplemented medium such as Fetal Calf Serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during the culture is preferably in the range of about 6 to 8. The culture is usually carried out at about 30 to 40 ℃ for about 15 to 200 hours, and medium exchange, aeration and agitation may be carried out as required.

As a system for producing a polypeptide in vivo, for example, a production system using animals and a production system using plants are used. The target gene is introduced into the animal or plant, and the polypeptide is produced in the animal or plant and recovered. The "host" in the present invention includes such animals and plants.

When animals are used, mammalian and insect production lines are used. As the mammal, goat, pig, sheep, mouse, cow (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993) can be used. In addition, when mammals are used, transgenic animals can be used.

For example, the gene of interest may be prepared as a fusion gene with a gene encoding a polypeptide inherently produced in milk such as goat beta casein. Then, the gene fragment containing the fusion gene is injected into an embryo of a goat, and the embryo is transplanted into a female goat. The polypeptide of interest can be obtained from the milk of a transgenic goat born by a goat harboring an embryo or its offspring. In order to increase the amount of milk containing the polypeptide produced from the transgenic goat, hormones may be suitably used for the transgenic goat (Ebert, K.M.et. al., Bio/Technology (1994)12, 699-702).

Further, as the insect, for example, silkworm can be used. When silkworms are used, the desired polypeptide can be obtained from the silkworms by infecting them with a baculovirus into which a gene encoding the desired polypeptide is inserted (Susumu, M.et al., Nature (1985)315, 592-594).

When plants are used, tobacco can be used. When tobacco is used, a gene encoding a polypeptide of interest is inserted into a plant expression vector, for example, pMON530, and the vector is inserted into a Bacillus such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). The bacterium is allowed to infect tobacco, such as Nicotiana tabacum, from which leaves the desired polypeptide can be obtained (Julian K. -C.Maet al., Eur.J.Immunol. (1994)24, 131-.

The polypeptide thus obtained can be isolated from the inside or outside of the host cell (e.g., culture medium) and purified to be a substantially pure and homogeneous polypeptide. The polypeptide may be isolated and purified by a method for isolating and purifying a polypeptide that is generally used for polypeptide purification, and is not limited at all. For example, the polypeptide can be separated and purified by appropriately selecting and combining the components from a chromatography column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization and the like.

As chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and gel filtration. Reversed phase chromatography, adsorption chromatography, etc. (stratgies for Protein Purification and chromatography: A Laboratory Course Manual. Ed DanielR. Marshak et al, Cold Spring Harbor Lab. press, 1996). These chromatographies may be liquid chromatographies such as HPLC, FPLC and the like. The invention also encompasses polypeptides that are highly purified using these purification methods.

In addition, before or after purification, a polypeptide may be optionally modified or a partial peptide may be removed by allowing an appropriate polypeptide-modifying enzyme to act on the polypeptide. Examples of the polypeptide-modifying enzyme include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glycosidase.

In addition, the present invention provides antibodies that bind to the polypeptides of the present invention. The form of the antibody of the present invention is not particularly limited, and may contain a monoclonal antibody in addition to a polyclonal antibody. The polypeptide of the present invention is used in immunizing rabbit, etc. to obtain serum containing polyclonal antibody and monoclonal antibody of all kinds.

The polypeptide used as the sensitizing antigen can be a complete polypeptide or a partial peptide fragment of the polypeptide. As partial peptide fragments of the polypeptide, there are, for example, an amino (N) -terminal fragment and a carboxyl (C) -terminal fragment of the polypeptide. An "antibody" as referred to herein refers to an antibody corresponding to a full length or fragment of a polypeptide.

The gene encoding the polypeptide of the present invention or a fragment thereof is inserted into a known expression vector system, the host cell mentioned in the present specification is transformed with the vector, and the target polypeptide or a fragment thereof can be obtained from the inside or outside of the host cell by a known method and used as a sensitizing antigen. In addition, cells expressing the polypeptide of the present invention, or a lysate thereof, or a chemically synthesized polypeptide of the present invention may be used as a sensitizing antigen.

The animal to be immunized with the sensitizing antigen is not particularly limited, and is preferably selected in consideration of suitability of the mother cell used for cell fusion, and animals of the order rodentia, leporia, and primates can be generally used.

Examples of the rodent include a mouse, a rat, and a hamster. Animals of the order Leporidae may be, for example, rabbits. Animals of the order primates include, for example, monkeys. The monkey may be a monkey of the order Tinospora (old world monkey), for example, a cynomolgus monkey (Macaca Fascicularis), a rhesus monkey, a baboon, or a chimpanzee.

For immunizing an animal with a sensitizing antigen, it can be carried out according to a known method. As a general method, a sensitizing antigen is injected into the abdominal cavity or subcutaneously of a mammal. Specifically, a solution obtained by diluting the sensitizing antigen with PBS (Phosphate Buffered Saline) or physiological Saline to an appropriate amount and suspending the diluted antigen in a suitable amount is mixed with a suitable amount of a general adjuvant, for example, freund's complete adjuvant, as necessary, and emulsified and administered to a mammal. Further, after injection, the sensitizing antigen mixed in an appropriate amount in Freund's incomplete adjuvant is administered several times every 4 to 21 days. In addition, an appropriate carrier can be used for immunization with a sensitizing antigen. Immunization as described above makes it possible to confirm whether or not the desired antibody level in serum has increased by a conventional method.

In order to obtain a polyclonal antibody against the polypeptide of the present invention, after confirming an increase in the level of a desired antibody in serum, the blood of a mammal immunized with an antigen can be taken out. Serum is separated from the blood by well-known methods. The polyclonal antibody may be used in the form of a serum containing the polyclonal antibody, or a fraction containing the polyclonal antibody may be further separated from the serum as necessary, and the fraction may be used. For example, by using an affinity chromatography column coupled with the polypeptide of the present invention, a fraction recognizing only the polypeptide of the present invention is obtained. The immunoglobulin G or M can be prepared by purifying the fraction with a protein A or protein G column.

In order to obtain a monoclonal antibody, after confirming that the serum level of a desired antibody in a mammal immunized with the antigen has increased, immune cells are removed from the mammal and cell fusion is performed. In this case, the immune cells used for cell fusion are preferably spleen cells. As another parent cell that can be fused with the above immune cell, a mammalian myeloma cell is preferable, and a myeloma cell that can obtain a characteristic for selecting a fused cell by a drug is more preferable.

Cell fusion of the above-mentioned immunocytes with myeloma cells is carried out, for example, according to the method of Milstein et al (Galfre, G.and Milstein, C., Methods Enzymol. (1981)73, 3-46).

Hybridomas obtained by cell fusion are selected by culturing in HAT (a culture medium containing hypoxanthine, aminopterin, and thymidine) which is a common selection medium. In order to cause the cells other than the target hybridoma (non-fused cells) to die by culturing in the HAT medium, a sufficient time is required, and the culture is usually continued for several days or several weeks. Then, a usual limiting dilution method is performed, and hybridomas producing the target antibody are selected and cloned.

The obtained hybridoma is then transplanted into the abdominal cavity of a mouse, ascites is recovered from the same mouse, and the obtained monoclonal antibody can be prepared by purification, for example, by ammonium sulfate precipitation, a protein a, a protein G column, DEAE ion exchange chromatography, an affinity chromatography column to which the polypeptide of the present invention is coupled, or the like. The antibody of the present invention can be used for purification and detection of the polypeptide of the present invention.

In addition, the MONOCLONAL antibody obtained as described above can be obtained as a recombinant antibody using a gene recombination technique (for example, refer to Borebaeck, C.A.K.and Larrick, J.W., THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United kingdom by MACMIMILLIAN PUBLISHERS LTD, 1990). The recombinant antibody can be produced by cloning a gene encoding the antibody from an immune cell such as a hybridoma or an antibody-producing sensitized lymphocyte, integrating the gene into an appropriate vector, and introducing the vector into a host. The present invention encompasses the recombinant antibody.

The antibody of the present invention may be a fragment of the antibody or a modified antibody as long as it binds to the polypeptide of the present invention. As antibody fragments, there may be mentioned, for example, Fab, F (ab') 2, Fv or single-chain variable region fragments Fv (scFv) in which the H chain and the L chain are joined by an appropriate linker (Huston, J.S.et al, Proc Natl.Acad.Sci.U.S.A. (1988)85, 5879-. Specifically, antibodies are treated with enzymes such as papain, pepsin to produce antibody fragments, or genes encoding these antibody fragments are constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, for example, Co, M.S. et al, J.Immunol. (1994)152, 2968-.

As the modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) may be used. These antibody modifications are also included in the "antibody" of the present invention. Such a modified antibody can be obtained by chemically modifying the obtained antibody. These methods are established in the art.

The antibody obtained as described above can be purified to a uniform degree. For the separation and purification of the antibody used in the present invention, separation and purification methods generally used for polypeptides can be used. For example, Antibodies can be separated and purified by appropriately selecting and combining a chromatography column such as affinity chromatography, filtration, ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold spring Harbor Lab.1988). But is not limited thereto. The concentration of the antibody obtained as described above can be measured by measuring the absorbance, Enzyme-linked immunosorbent assay (ELISA), or the like.

Examples of the column used for affinity chromatography include a protein A column and a protein G column. For example, as a column using protein A, Hyper D, POROS, Sepharose F.F (Pharmacia), etc. are exemplified.

As the chromatography other than affinity chromatography, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like (stratgies for protein purification and chromatography: A Laboratory Course Manual. Ed Daniel R. Marshak et al, Cold Spring Harbor Lab. press, 1996) are mentioned. These chromatographies can be carried out by liquid chromatography such as HPLC and FPLC.

In addition, as a method for measuring the antigen binding activity of the antibody of the present invention, a method of measuring absorbance, an Enzyme-linked immunosorbent assay (ELISA), EIA (Enzyme immunoassay), RIA (radioimmunoassay), or a fluorescent antibody method may be used. When ELISA is used, the polypeptide of the present invention is added to a plate on which the antibody of the present invention is immobilized, and then a sample containing the antibody of interest, for example, a culture supernatant of antibody-producing cells or purified antibody is added. The antigen binding activity can be evaluated by adding a secondary antibody that recognizes an antibody labeled with an enzyme, for example, alkaline phosphatase, incubating the plate, washing the plate, adding an enzyme substrate such as p-nitrophenylphosphate, and measuring the absorbance. The polypeptide may be a fragment of the polypeptide, for example, a fragment composed of the C-terminus or a fragment composed of the N-terminus. BIAcore (produced by Pharmacia) can be used for the evaluation of the activity of the antibody of the present invention.

Examples

The present invention will be specifically described below with reference to examples. These examples are only examples for illustrating the present invention and do not limit the scope of the present invention.

Example 1: cloning of a Gene encoding a taurine transporter from a CHO cell hamster

Total RNA was extracted from anti-IL-6 receptor antibody-producing cells (JP-A-8-99902) into which anti-IL-6 receptor antibody genes had been introduced into CHO DXB11 cells, and then cDNA dependent on poly A was synthesized. The hamster taurine transporter (TauT) gene was obtained by PCR using cDNA fragmented with three restriction enzymes SalI, XhoI, EcoRI as a template. PCR primers used were designed to contain 5 ', 3' sequences that preserve the gene sequence between known rat/mouse TauT. By determining the nucleotide sequence of the cloned gene, the coding hamster tauT was confirmed by homology with the TauT of known biological species (FIG. 1). The hamster tau amino acid sequence has high homology to mouse (96% identity), rat (96% identity), and human (93% identity), and is predicted to be a transporter with 12 transmembrane domains (fig. 2).

Example 2: increasing viable cell density, suppressing lactic acid production and increasing antibody production by introducing hamster taurine transporter

A Kozak sequence was added to the hamster TauT (hereinafter referred to as TauT) gene obtained by cloning in example 1, and a CMV promoter expression plasmid pHyg/TauT was constructed (fig. 3). The control plasmid pHyg from which the pHyg/TauT or TauT gene has been removed is introduced into a CHO cell that produces an anti-glypican (proteoglycan) 3 antibody as a parent strain by electroporation (see WO 2006/006693). Cells into which the expression plasmid was introduced were selected in the presence of hygromycin (400. mu.g/ml), and all stable and proliferating cell lines were expanded (pHyg/TauT: 8 strain, pHyg: 7 strain). In the preparation of TauT mRNA, 7 strains, which were confirmed to be preferentially expressed in the mother strain, were introduced into the cells as pHyg/TauT according to the TaqMan method. The average expression level of mRNA introduced into the cells (7 strains) was about 40 times that of the control (7 strains). The total number of 14 cells was 2X 10⁵Initial density of cell/mL batch (batch) culture and Fed-batch (Fed-batch) culture were performed by 50mL shake flasks, and viable cell density, lactic acid production amount, and anti-glypican (proteoglycan-3 antibody production amount at the 7 th day in the late stage of culture were compared. In batch culture, growth is inhibited by accumulation of growth-inhibiting substances such as lactic acid in the cell growth medium, while viable cell density (FIG. 4) and lactic acid production (FIG. 5) of pHyg/TauT-introduced cells are predominant over that of pHyg-introduced cells (t-test P)<0.05). As for the amount of anti-glypican-3 antibody produced, 4 strains out of 7 strains of pHyg/TauT-introduced cells were the highest value of pHyg-introduced cells or more (FIG. 6). Superiority of anti-glypican (proteoglycan) 3 antibody production amount of pHyg/TauT-introduced cell (t test P)<0.01, FIG. 7) As is clear from the fed-batch culture, among the above 4 strains, when pHyg/TauT-introduced cells (T10) having the highest proliferation potency and 1L jars of the mother strain were fed-batch cultured, the survival rate of T10 was maintained at 80% or more on the 32 th day of culture (FIG. 8), and lactic acid production was suppressed. It is composed ofAs a result, on day 35 of the culture, the amount of anti-glypican-3 antibody produced reached 2.9g/L (FIG. 9). The expression of the TauT molecule on the cell membrane by the TauT-introduced T10 cells was confirmed by flow cytometry analysis (fig. 10). These results indicate that the potential of antibody-producing cells was increased by artificially expressing hamster TauT, and antibody-producing cells with high productivity were obtained.

Example 3: suppression of ammonia production by hamster taurine transporter-introduced strain, incorporation of taurine, increase in glutamine consumption, and production of taurine-independent antibody

The stock strain and the pHyg/TauT transformant were initially expressed at 2X 10⁵1L jar fed-batch culture was carried out for cells/mL, and the cells containing 450X 10 cells were collected from the culture tank at appropriate times⁵A culture solution of cells. After the culture supernatant was separated and extracted by centrifugation, 1mL of chilled sterile water containing a protease inhibitor (Complete Mini, Roche Diagnostics, Proteasein ib cordtail tables) was added to the cell pellet, the cell pellet was placed on ice, and after a pulse operation for 5 seconds using an ultrasonic cell disruptor (MISONIX ASTRASON MODEL XL2020), treatment was repeated for 12 groups in total, thereby completely disrupting the cells. The treated solution was added to a centrifugal filtration unit to prepare a filtrate having a molecular weight of 5000 or less as a sample for intracellular amino acid measurement. The absorbance at 570nm of each sample was measured and compared with an improvement of ninhydrin reagent-L8500 kit (Wako pure chemical industries) and Hitachi full-automatic amino acid analyzer (L-8500) to determine the concentration of each amino acid in the sample. Since the concentrations of various amino acids and ammonia in the culture solution were directly measured, the concentrations were compared on the μ M scale, and on the one hand, the intracellular concentrations were measured by adding 1mL of chilled sterile water to the cell pellet, and then the cell pellet was subjected to ultrasonic cell disruption, and the measured values of various amino acids and ammonia were converted into values for each cell, and the converted values were compared. FIG. 11 shows the ammonia concentration ratio at every 450X 10 at the start of fed-batch culture in a 1L jar⁵The ammonia test value of the individual parent strain was defined as 1,the ratio was determined by comparing the measurement values at the start of culture, day 6, day 12 and day 18. Both taurine and glutamic acid of fig. 12 and 13 were measured by the above amino acid analysis.

As a result, it was considered that the pHyg/TauT-introduced strain contributed to the high production of the antibody by maintaining the intracellular ammonia concentration at a low level in the late stage of the culture (FIG. 11).

The intracellular taurine concentration ratio can be determined by a method substantially similar to the above-described ammonia (fig. 12). Except that 50mL of shake flasks were batch cultured every 200X 10 on day 4⁵The ammonia test value of each parent strain was defined as 1.

The results showed that taurine incorporation by the pHyg/TauT-introduced strain was dependent on the amount of taurine added, and the amount of incorporation was equivalent to that of the parent strain. However, as shown in FIG. 13, the glutamine consumption of the pHyg/TauT-introduced strain was very significant compared to the parent strain, and was not dependent on the initial taurine concentration. Glutamine has been reported to have the effect of improving cell proliferation and survival rates of hybridomas, as well as antibody production ability, and increasing antibody production (Enzyme and Microbial Technology 17: 47-55, 1995). Therefore, it is considered that the antibody production-enhancing effect of the pHyg/TauT-introduced strain is caused by incorporation of an amino acid (e.g., glutamine) other than taurine into the taurine transporter. The glutamine concentration was calculated by converting the measured value of amino acid analysis of the culture solution on the 4 th day of culture in FIG. 12 to 1X 10⁵The value of the cell.

The anti-glypican (proteo) glycan-3 antibody production amount was independent of the initial taurine concentration (0 to 500mM (62.575g/L)) in the 50mL shake flask fed-batch culture (FIG. 14), and no superiority difference was observed with respect to the effect of the initial taurine concentration on the antibody production amount of the mother strain.

The above results indicate that TauT has high antibody-producing ability even when taurine is not contained in the medium at the start of culture, and that incorporation of amino acids other than taurine and the like is likely to be promoted.

The present invention is applicable to all antibody-producing cells.

All publications, patents and patent applications cited in this specification are herein incorporated by reference.

Possibility of industrial utilization

The invention is useful for the production of polypeptides.

Sequence listing

< sequence 1>

Sequence No. 1 shows the nucleotide sequence of a gene encoding a hamster taurine transporter.

< sequence 2>

Sequence No. 2 shows an amino acid sequence of a hamster taurine transporter.

< sequence 3>

Sequence No. 3 shows the base sequence of the gene encoding rat taurine transporter.

< sequence 4>

The sequence No. 4 shows the amino acid sequence of the rat taurine transporter.

< sequence 5>

Sequence No. 5 shows the base sequence of a gene encoding a mouse taurine transporter.

< sequence 6>

The sequence No. 6 shows an amino acid sequence of a mouse taurine transporter.

< sequence 7>

SEQ ID NO. 7 shows a nucleotide sequence of a gene encoding human taurine transporter.

< sequence 8>

The sequence No. 8 shows an amino acid sequence of a human taurine transporter.

Claims (5)

1. A method for producing a polypeptide, which comprises culturing a cell which highly expresses a taurine transporter and into which a DNA encoding the desired polypeptide is introduced, and producing the desired polypeptide, wherein the DNA encoding the taurine transporter is any one of the following (a) to (b):

(a) DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8,

(b) a DNA comprising the base sequence of SEQ ID NO. 1, 3, 5 or 7.

2. The method according to claim 1, wherein the cell highly expressing a taurine transporter is a cell into which a DNA encoding the taurine transporter has been introduced.

3. The method of claim 2, wherein the cell is a Chinese hamster ovary cell.

4. The method of claim 3, wherein the desired polypeptide is an antibody.

5. The method according to any one of claims 1 to 4, which comprises culturing in a medium containing taurine at a concentration of 0 to 100 g/L.