Patent Grant
8697397
This application is a National Stage application of PCT/JP2008/064095, filed Aug. 6, 2008, which claims priority from Japanese application JP 2007-205158, filed Aug. 7, 2007.
1. Technical Field
The present invention relates to a method of producing a heterogenous protein, more specifically, a method of producing a polypeptide using a cell which strongly expresses alanine aminotransferase.
2. Background Art
When proteins useful as pharmaceuticals are produced with the recombinant DNA technique, use of animal cells enables complicated post-translational modification and folding which prokaryotic cells can not perform. Therefore, animal cells are frequently used as host cells for producing recombinant proteins.
Recently, a large number of biopharmaceuticals, such as antibodies and physiologically active proteins, have been developed. Techniques that permit efficient production of recombinant proteins by animal cells lead to cost reduction of biopharmaceuticals and promise their stable supply to patients.
Under these circumstances, a method of protein production with higher production efficiency is desired.
Alanine is one of the proteinogenic amino acids, and is a non-essential amino acid. In a living body, it is biosynthesized by transfer of an amino group from glutamate to pyruvate, and is degraded by a reverse reaction.
As an alanine degrading enzyme, alanine aminotransferase (EC 2.6.1.2.) (Non-patent document 1) has been known. This enzyme transfers an amino group from alanine to 2-oxoglutarate to synthesize glutamate. Alanine aminotransferase is also called glutamic-pyruvic transaminase, which is abbreviated as GPT (Non-patent document 2). GPT and GOP (aspartate aminotransferase) are enzymes found in the liver. Since GPT and GOP are released into the blood when hepatic cells are destroyed, the liver is diagnosed to have some kind of disorder when abnormally high levels of GPT and GOT are observed.
As shown above, alanine aminotransferase is used as a marker of hepatic function. However, it has not been known how host cells such as CHO cells behave if alanine aminotransferase is strongly expressed in them.
[Non-Patent Document 1]
Problem for Solution by the Invention
It is an object of the present invention to provide a method which is capable of producing a natural or recombinant protein in high yield.
Means to Solve the Problem
As a result of extensive and intensive researches toward the solution of the above problem, the present inventors have found that it is possible to increase the yield of a desired polypeptide by using a cell that strongly expresses alanine aminotransferase (hereinafter sometimes referred to as “ALT”). Thus, the present invention has been achieved. Moreover, the desired polypeptide could be produced in an even greater amount by using cells capable of co-expressing ALT and a taurine transporter. Since alanine is produced in large amount over time in cell culture, alanine accumulated in cells is secreted in the medium. If the reaction of biosynthesizing pyruvate and glutamate from alanine can be promoted by strongly expressing ALT, the products are utilized in metabolism during a TCA cycle and glucose production by glycogenesis. This will improve cell culture behavior, and thus high-yield production of the desired polypeptide is anticipated.
The present invention may be summarized as follows.
(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60;
(b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has alanine aminotransferase activity;
(c) a DNA encoding a polypeptide having 70% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 and yet having alanine aminotransferase activity;
(d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59;
(e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59 under stringent conditions and yet encodes a polypeptide having alanine aminotransferase activity.
According to the present invention, it has become possible to increase the yield of a desired polypeptide.
The present specification encompasses the contents disclosed in the specification and/or the drawings of Japanese Patent Application No. 2007-205158 based on which the present patent application claims priority.
C
Hereinbelow, embodiments of the present invention will be described in more detail.
The present invention provides a method of producing a polypeptide, comprising culturing a cell which strongly expresses ALT and has a transferred DNA encoding a desired polypeptide and thereby allowing the cell to produce the polypeptide.
In the method of the present invention, the cell may be either a natural cell capable of producing the desired polypeptide or a transformed cell into which a DNA encoding the desired polypeptide has been transferred. Preferably, a transformed cell into which a DNA encoding the desired polypeptide has been transferred is used.
In the method of the present invention, the desired polypeptide is not particularly limited. The polypeptide may be any polypeptide such as an antibody (e.g., anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb/IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2/neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, anti-VLA4 antibody, and the like) or a physiologically active protein (e.g., granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin, interferon, interleukin such as IL-1 or IL-6, t-PA, urokinase, serum albumin, blood coagulation factor, PTH, and the like). An antibody is particularly preferred, and may be any antibody such as a natural antibody, a low molecular sized antibody (e.g., Fab, scFv, sc(Fv)2), a chimeric antibody, a humanized antibody, etc.
By using strongly ALT expressing cells, the amount of a polypeptide produced by cells can be increased.
ALT is fundamentally known as an enzyme that produces glutamate by transferring an amino group from alanine to 2-oxoglutarate. The present inventors considered that if the reaction of biosynthesizing pyruvate and glutamate from alanine could be promoted by strongly expressing ALT in host cells such as CHO cells, the products might be utilized in metabolism during a TCA cycle and glucose production by glycogenesis, and this might improve cell culture behavior, leading to high-yield production of the desired polypeptide.
The strongly ALT expressing cells are not particularly limited as long as they are capable of ALT expression at higher levels than natural cells. Natural cells include, but are not particularly limited to, cells that are used as hosts in the production of recombinant proteins and may be exemplified by CHO cells.
A cell which strongly expresses ALT is not particularly limited as long as the cell has an increased expression level of ALT compared to a corresponding natural cell. The natural cell is not particularly limited. A cell which is used as a host in the production of a recombinant protein (e.g., CHO cells) may be used.
As a cell which strongly expresses ALT, a cell into which an ALT gene has been artificially transferred may be given. A cell into which an ALT gene has been artificially transferred can be prepared by methods known to those skilled in the art. For example, such a cell may be prepared by incorporating an ALT gene into a vector and transforming the vector into a cell. Furthermore, the concept of “cells into which an ALT gene has been artificially transferred” encompasses herein cells in which an endogenous ALT gene has been activated by gene activation technology (see, for example, International Publication WO94/12650) so that ALT is strongly expressed.
As ALT to be strongly expressed in a cell, ALT derived from any organism may be used. Specifically, ALTs derived from human, mouse, rat, dog, African clawed frog, fruit fly, nematode, Japanese rice, Cyanidioschyzon merolae, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, Cryptococcus neoformans, Dictyostelium discoideum, Trypanosoma brucei, Leishmania major, Entamoeba histolytica and Trypanosoma cruzi are known and can be used. Preferably, ALT derived from human, a rodent or the same species as the host cell may be used. For example, when the cell which is allowed to strongly express ALT is Chinese hamster ovary cells (CHO cells), ALT is preferably derived from human or hamster. For ALT in humans, mice, and yeast, variants (ALT1 and ALT2) exist. ALT2 has 80% or greater homology to ALT1 at the amino acid level. ALT1 was forcedly expressed in the Examples described later.
Further, as an ALT gene to be strongly expressed in a cell, any one of the following DNAs (a) to (e) encoding ALT may be used.
(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60;
(b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has alanine aminotransferase activity;
(c) a DNA encoding a polypeptide having 70% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 and yet having alanine aminotransferase activity;
(d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59;
(e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59 under stringent conditions and yet encodes a polypeptide having alanine aminotransferase activity.
The cell which strongly expresses ALT may be any cell, for example, eukaryotic cell such as animal, plant and yeast cells, prokaryotic cell such as E. coli and B. subtilis, etc. Preferably, animal cells such as CHO and COS cells are used, CHO cells are particularly preferred. In order to prepare a desired polypeptide, cells suitable for transfer of a gene encoding the desired polypeptide such as CHO-dhfr- cells are preferred.
Preferably, the cell of the present invention which strongly expresses ALT further expresses a taurine transporter strongly in order to prepare a desired polypeptide. By transferring a gene encoding the desired polypeptide into the cell and culturing the resultant cell in a medium, the desired polypeptide can be produced in a greater amount.
When a desired polypeptide is produced using a cell into which an ALT gene has been artificially transferred, the order of the transfer of an ALT gene and the transfer of a gene encoding a desired polypeptide is not particularly limited. A gene encoding a desired polypeptide may be transferred after the transfer of an ALT gene. Alternatively, an ALT gene may be transferred after the transfer of a gene encoding a desired polypeptide. It is also possible to transfer an ALT gene and a gene encoding a desired polypeptide simultaneously.
An ALT gene and a gene encoding a desired polypeptide may be transferred simultaneously in a single vector. Alternatively, they may be transferred separately using a plurality of vectors.
By using a cell which strongly expresses ALT and a taurine transporter, an intracellular ammonia concentration can decline.
It is known that taurine transporter is a membrane protein having the osmoregulatory function of taking up amino acids (such as taurine and β-alanine) into cells.
A cell which strongly expresses a taurine transporter is not particularly limited as long as the cell has an increased expression level of a taurine transporter compared to a corresponding natural cell. The natural cell is not particularly limited. A cell which is used as a host in the production of a recombinant protein (e.g., CHO cells) may be used.
As a cell which strongly expresses a taurine transporter, a cell into which a taurine transporter gene has been artificially transferred may be given. A cell into which a taurine transporter gene has been artificially transferred can be prepared by methods known to those skilled in the art. For example, such a cell may be prepared by incorporating a taurine transporter gene into a vector and transforming the vector into a cell.
As a taurine transporter to be strongly expressed in a cell, a taurine transporter derived from any organism may be used. Specifically, a taurine transporter derived from human or a rodent (such as mouse, rat or hamster) may be used. Preferably, a taurine transporter derived from human, a rodent or the same species as the host cell may be used. For example, when the cell which is allowed to strongly express a taurine transporter is Chinese hamster ovary cells (CHO cells), the taurine transporter is preferably derived from human or hamster.
Further, as a taurine transporter gene to be strongly expressed in a cell, any one of the following DNAs (a1) to (e1) encoding a taurine transporter may be used.
Production of a desired polypeptide may be performed by transferring a gene encoding the desired polypeptide into a cell which strongly expresses a taurine transporter gene and an ALT gene and culturing the resultant cell in a medium. Furthermore, a desired polypeptide can be prepared by using a cell in which an endogenous gene has been activated by gene activation technology (see, for example, International Publication WO94/12650) so that a desired polypeptide has been produced.
When a desired polypeptide is produced using a cell into which a taurine transporter gene and an ALT gene have been artificially transferred, the order of the transfer of a taurine transporter gene, the transfer of an ALT gene and the transfer of a gene encoding a desired polypeptide is not particularly limited. A gene encoding a desired polypeptide may be transferred after the transfer of a taurine transporter gene and an ALT gene. Alternatively, a taurine transporter gene and an ALT gene may be transferred after the transfer of a gene encoding a desired polypeptide. It is also possible to transfer a taurine transporter gene, an ALT gene and a gene encoding a desired polypeptide simultaneously.
A taurine transporter gene, an ALT gene and a gene encoding a desired polypeptide may be transferred simultaneously in a single vector. Alternatively, they may be transferred separately using a plurality of vectors.
For culturing the cell which strongly expresses ALT (and which may strongly express a taurine transporter), media used in conventional cell culture (preferably, animal cell culture) may be used. These media usually contain amino acids, vitamins, lipid factors, energy sources, osmotic regulators, iron sources and pH regulators. The contents of these components are usually as follows: amino acids 0.05-1500 mg/L, vitamins 0.001-10 mg/L, lipid factors 0-200 mg/L, energy sources 1-20 g/L, osmotic regulators 0.1-10000 mg/L, iron sources 0.1-500 mg/L, pH regulators 1-10000 mg/L, trace metal elements 0.00001-200 mg/L, surfactants 0-5000 mg/L, growth cofactors 0.05-10000 μg/L and nucleosides 0.001-50 mg/L. However, the contents are not limited to these ranges and may be appropriately selected depending on the type of the cell to be cultured, the type of the desired polypeptide, and so on.
In addition to these components, trace metal elements, surfactants, growth cofactors, nucleosides, and the like may be added.
Specific examples of such components include amino acids, such as 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, and L-valine, preferably, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine; vitamins, such as i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and ascorbic acid, preferably, biotin, folic acid, lipoic acid, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and ascorbic acid; lipid factors, such as choline chloride, choline tartrate, linoleic acid, oleic acid and cholesterol, preferably, choline chloride; energy sources, such as glucose, galactose, mannose, and fructose, preferably, glucose; osmotic regulators, such as sodium chloride, potassium chloride, and potassium nitrate, preferably, sodium chloride; iron sources, such as iron EDTA, ferric citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, and ferric nitrate, preferably, ferric chloride, iron EDTA, and ferric citrate; and pH regulators, such as sodium hydrogencarbonate, calcium chloride, sodium dihydrogen-phosphate, HEPES and MOPS, preferably, sodium hydrogencarbonate. Culture media containing any of these components may be given as examples.
Besides the above components, there may be added trace metal elements, such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride and sodium subsilicate, preferably, copper sulfate, zinc sulfate and magnesium sulfate; surfactants, such as Tween 80 and Pluronic F68; growth cofactors, such as recombinant insulin, recombinant IGF-1, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, sodium selenite, retinoic acid and putrescine dihydrochloride, preferably, sodium selenite, ethanolamine hydrochloride, recombinant IGF-1 and putrescine dihydrochloride; and nucleosides, such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine and uridine. In preferable examples of above media, antibiotics, such as streptomycin, penicillin-G potassium and gentamicin, and pH-indicators, such as Phenol Red, may be contained.
Further, α-ketoglutarate, serving as a substrate for ALT, can be added to the medium. The yield of the desired polypeptide (for example, an antibody) can be increased by addition of α-ketoglutarate. In this case, the amount of α-ketoglutarate to be added is normally in the range of 0.01 to 1000 mM, preferably 0.1 to 100 mM, and more preferably 1 to 10 mM.
The pH of the medium varies depending on the cell to be cultured. Generally, pH 6.8-7.6 is appropriate. In many cases, pH 7.0-7.4 is appropriate.
It is also possible to use a commercial medium for animal cell culture, e.g., D-MEM (Dulbecco's Modified Eagle Medium), D-MEM/F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium Nutrient Mixture F-12), RPMI1640, CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH Biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich) or the like.
Alternatively, the medium may be a serum-free medium such as CD-CHO (Invitrogen).
When the cell which strongly expresses ALT (and which may strongly express a taurine transporter) is CHO cells, CHO cells may be cultured by methods known to those skilled in the art. For example, CHO cells may be cultured usually in an atmosphere with a CO2 concentration in the gas phase of 0 to 40%, preferably 2 to 10%, at 30 to 39° C., preferably about 37° C.
Moreover, in the case where the desired polypeptide such as an antibody is produced by cell culture, cells become highly confluent at the late stage of the culture (approximately 1×107 cells/rill), and the effect of waste products such as lactate becomes extremely high. If the desired polypeptide is produced by strongly ALT expressing cells, a high survival ratio is maintained even at the late stage of the culture, and an improvement in the yield of the desired polypeptide can also be anticipated.
An appropriate culture period for producing a desired polypeptide using the cell which strongly expresses ALT is usually 1 day to 3 months, preferably 1 day to 2 months, more preferably 1 day to 1 month.
With respect to various culture devices for animal cell culture, a fermentor type tank culture device, an air lift type culture device, a culture flask type culture device, a spinner flask type culture device, a microcarrier type culture device, a fluidized bed type culture device, a hollow fiber type culture device, a roller bottle type culture device, a packed bed type culture device, or the like may be used.
Culture may be performed by any culture method such as batch culture, fed-batch culture or continuous culture. Preferably, fed-batch culture or continuous culture is used. Fed-batch culture is more preferred.
When the cell which strongly expresses ALT (and which may strongly express a taurine transporter) is cultured, taurine may be added to the medium in order to promote taurine uptake into cells. The concentration of taurine to be added to the medium is not specifically limited, but is normally in the range of 0 g/L to 100 g/L, preferably 0 g/L to 20 g/L, and more preferably 0 g/L to 10 g/L.
When the polypeptide produced according to the method of the present invention has a biological activity useful as a pharmaceutical, it is possible to produce a pharmaceutical by mixing this polypeptide with pharmaceutically acceptable carriers or additives and formulating into a preparation.
Specific examples of pharmaceutically acceptable carriers and additives include water, organic solvents that are pharmaceutically acceptable, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, carboxymethylcellulose sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, agar-agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and surfactants that are acceptable as pharmaceutical additives.
Actual additives may be selected from the above-mentioned additives singly or in combination according to the dosage form of the therapeutic of the present invention, but are not limited to those listed above. For example, when a polypeptide is used in an injectable formulation, the purified polypeptide may be dissolved in a solvent such as physiological saline, buffer or a glucose solution, and then an adsorption inhibitor such as Tween 80, Tween 20, gelatin or human serum albumin may be added to the solution. Alternatively, a freeze-dried agent may be used to prepare a dosage form which is dissolved and reconstituted prior to use. Examples of the excipient useful for freeze-drying include sugar alcohols and saccharides such as mannitol and glucose.
Effective doses of the polypeptide may be appropriately selected depending on the type of the polypeptide, the type of the disease to be treated or prevented, the age of the patient, the severity of the disease, etc. For example, when the polypeptide is anti-glypican antibody, the effective dose of anti-glypican antibody is selected within a range of 0.001 mg to 1000 mg per kg of body weight per administration. Alternatively, a dose of 0.01-100000 mg/body may be selected per patient. However, effective dose is not limited to these ranges.
The polypeptide may be administered either orally or parenterally, but parenteral administration is preferred. Specifically, injection (e.g., systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.), transnasal administration, transpulmonary administration, transdermal administration and the like may be enumerated.
In the present invention, as a gene encoding ALT, a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 may be used. Alternatively, a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has alanine aminotransferase activity may be used.
The polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has alanine aminotransferase activity is functionally equivalent to ALT derived from human, mouse, rat, dog, African clawed frog, fruit fly, nematode, Japanese rice, Cyanidioschyzon merolae, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, Cryptococcus neoformans, Dictyostelium discoideum, Trypanosoma brucei, Leishmania major, Entamoeba histolytica or Trypanosoma cruzi (hereinafter sometimes referred to as “ALT derived from human or the like”). Such a polypeptide encompasses, for example, mutants of ALT derived from human or the like. In Example described below, a mutant in which four out of 496 amino acids were replaced (R53S, Q72R, F286S and M332K) was used.
As methods well-known to those skilled in the art for preparing polypeptides functionally equivalent to a specific polypeptide, methods of introducing mutations into polypeptides may be given. For example, those skilled in the art could prepare polypeptides functionally equivalent to ALT derived from human or the like by appropriately introducing mutations into amino acids of ALT derived from human or the like by site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987) Methods. Enzymol. 154, 350-367; Kunkel, T A (1985) Proc Natl Acad Sci USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766). Mutations in amino acids may also occur in nature.
Specific examples of polypeptides functionally equivalent to the ALT derived from human or the like include, but are not limited to, a polypeptide having an amino acid sequence derived from the amino acid sequence (e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60) of the ALT derived from human or the like by deletion of one or more amino acids, preferably 1-30 amino acids, more preferably 1-10 amino acids; a polypeptide having an amino acid sequence derived from the amino acid sequence of the ALT derived from human or the like by addition of one or more amino acids, preferably 1-30 amino acids, more preferably 1-10 amino acids; and a polypeptide having an amino acid sequence derived from the amino acid sequence of the ALT derived from human or the like by substitution of one or more amino acids, preferably 1-30 amino acids, more preferably 1-10 amino acids, with other amino acids.
Amino acid residues to be mutated are not particularly limited. Preferably, amino acid residues are mutated to other amino acids in which the nature of the initial amino acid side chain is conserved. Specific examples of the nature of amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, H, K, S and T), amino acids with an aliphatic side chain (G A, V, L, I and P), amino acids with a hydroxyl group-containing side chain (S, T and Y), amino acids with a sulfur atom-containing side chain (C and M), amino acids with a carboxylic acid and amide-containing side chain (D, N, E and Q), amino acids with a base-containing side chain (R, K and H) and amino acids with an aromatic-containing side chain (H, F, Y and W) (In parentheses are one-letter codes for amino acids).
It has been reported that a polypeptide having an amino acid sequence derived from an original amino acid sequence by modification (such as deletion, addition and/or substitution of one or more amino acids) maintains the biological activity of the original polypeptide (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
As one example of the polypeptide in which one or more amino acid residues are added to the ALT derived from human or the like, a fusion polypeptide comprising the ALT derived from human or the like may be given. Such a fusion polypeptide is composed of the ALT derived from human or the like and other polypeptide fused thereto. Such a fusion polypeptide may be prepared by linking a gene encoding the ALT derived from human or the like in frame with a gene encoding the other polypeptide, transferring the resultant DNA into an expression vector and expressing the DNA in a host cell. Techniques known to those skilled in the art may be used. There is no limitation on the polypeptide to be fused to the ALT derived from human or the like.
Examples of polypeptides to be fused to the ALT derived from human or the like include, but are not limited to, FLAG (Hopp, T. P et al., BioTechnology (1988) 6, 1204-1210), 6×His comprising six histidine (His) residues, 10×His, influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, protein C fragment, glutathione-S-transferase (GST), influenza hemagglutinin (HA), immunoglobulin constant region, β-galactosidase and maltose-binding protein (MBP).
A commercially available gene encoding such polypeptide is fused to the gene encoding the ALT derived from human or the like. The fused gene thus prepared is expressed to prepare a fused polypeptide.
An alternative method known to those skilled in the art for preparing polypeptides functionally equivalent to a specific polypeptide is a method using the hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Those skilled in the art could routinely isolate a DNA highly homologous to the DNA sequence (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59) of the ALT derived from human or the like based on that DNA sequence or a part thereof, and isolate polypeptides functionally equivalent to the ALT derived from human or the like from that DNA.
Hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the ALT derived from human or the like can be appropriately selected by those skilled in the art. For example, low stringent hybridization conditions may be given. Low stringent hybridization conditions are, for example, 42° C., 2×SSC and 0.1% SDS, preferably 50° C., 2×SSC and 0.1% SDS. More preferably, high stringent conditions may be given. For example, high stringent conditions are 65° C., 2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is lowered, not only DNAs with high homology but also DNAs with only low homology are obtained. Conversely, it is expected that only those DNAs with high homology are obtained as the hybridization temperature is elevated. However, not only the temperature but also a plurality of factors (such as salt concentrations) affect the stringency of hybridization. Those skilled in the art could appropriately select these factors to realize similar stringency.
The polypeptide encoded by a DNA isolated by these hybridization techniques may have 70% or more homology and usually has high homology with the ALT derived from human or the like in the amino acid sequence. The term “high homology” refers to usually 97% or more homology, preferably 98% or more homology, more preferably 99% or more homology. For determination of the homology of polypeptides, the algorithm described in Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. USA (1983) 80, 726-730 may be followed.
The polypeptide may vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, morphology, etc. depending on the cell or host that produce the polypeptide or the purification method that will be described later. However, as long as the resultant polypeptide has functions equivalent to the functions of the ALT derived from human or the like, DNA encoding the polypeptide can be used in the present invention. For example, when the polypeptide of the present invention is expressed in a prokaryote (e.g., Escherichia coli), a methionine reside is added to the N-terminus of the initial amino acid sequence of the polypeptide. When the polypeptide is expressed in a eukaryote (e.g., a mammalian cell), the N-terminal signal sequence is removed. These polypeptides can be used in the present invention.
The polypeptide may be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art. A recombinant polypeptide may be prepared by incorporating a DNA encoding the polypeptide in an appropriate expression vector, introducing the vector into an appropriate host cell, collecting the resultant transformant, extracting a crude polypeptide, and then purifying the polypeptide by chromatography (such as ion exchange, reversed phase or gel filtration chromatography, or affinity chromatography in which an antibody to the polypeptide prepared by the method of the present invention is fixed in a column) or a combination of these chromatographic techniques.
When the polypeptide is expressed in a host cell (e.g., animal cell or E coli) as a fusion polypeptide with glutathione-S-transferase polypeptide or as a recombinant polypeptide with histidine residues added thereto, the expressed polypeptide may be purified with a glutathione column or a nickel column.
After purification of a fusion polypeptide, regions other than the polypeptide of interest may be cut off by thrombin or factor Xa and removed from the fusion polypeptide.
When the polypeptide is a natural polypeptide, the polypeptide may be isolated by purification methods known to those skilled in the art. For example, an extract from tissues or cells expressing the polypeptide functionally equivalent to the ALT derived from human or the like may be applied to an affinity column to which an antibody to the ALT derived from human or the like is bound. The antibody may be either a polyclonal antibody or a monoclonal antibody.
In the present invention, as DNA encoding ALT, a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59 may be used. Alternatively, a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59 under stringent conditions and yet encodes a polypeptide having alanine aminotransferase activity, may be used.
The DNA encoding ALT can be used in the in vivo or in vitro production of a desired polypeptide as described above. Further, the DNA encoding ALT may be used in the creation of a cell which strongly expresses ALT. The DNA encoding ALT may take any form as long as it is capable of encoding ALT. That is, the DNA may be, for example, a cDNA synthesized from mRNA, a genomic DNA or a chemically synthesized DNA. It should be noted that, as long as the DNA is capable of encoding ALT, the DNA may have any nucleotide sequence based on the degeneracy of genetic codes.
The DNA encoding ALT may be prepared by methods known to those skilled in the art. For example, the DNA may be prepared by preparing a cDNA library from a cell expressing ALT and performing hybridization using a part of the DNA sequence of ALT (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59) as a probe. The cDNA library may be prepared, for example, by the method described in Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989). Alternatively, a commercial cDNA library may be used. It is also possible to prepare the DNA encoding ALT by preparing RNA from a cell expressing ALT, synthesizing oligo DNA molecules based on the DNA sequence of ALT (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59), and performing PCR using the oligo DNA molecules as primers to thereby amplify a cDNA encoding ALT.
Further, by determining the nucleotide sequence of the resultant cDNA, it is possible to determine the translation region encoding ALT and to obtain the amino acid sequence of ALT. Further, by screening a genomic library using the resultant cDNA as a probe, it is possible to isolate a genomic DNA.
Specifically, the following procedures may be used. First, mRNA is isolated from cells, tissues or the like expressing ALT. For the isolation of mRNA, the total RNA is prepared by known methods, for example, the guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomczynski, P. and Sacchi, N., Anal. Biochem. (1987) 162, 156-159) or the like, and then mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia), etc. Alternatively, mRNA may be prepared directly using QuickPrep mRNA Purification Kit (Pharmacia).
From the resultant mRNA, cDNA is synthesized using a reverse transcriptase. Alternatively, cDNA may be synthesized using a kit such as AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (SEIKAGAKU CORPORATION). It is also possible to synthesize and amplify cDNA according to the 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using 5′-Ampli FINDER RACE Kit (Clontech) and polymerase chain reaction (PCR) with primers.
A DNA fragment of interest is prepared from the resultant PCR product and ligated to a vector DNA to thereby prepare a recombinant vector. The vector is introduced into a host (e.g., E. coli), followed by selection of resultant colonies to thereby obtain a desired recombinant vector. The nucleotide sequence of the DNA of interest may be confirmed by a known method such as the dideoxynucleotide chain termination method.
Further, a nucleotide sequence of higher expression efficiency can be designed for the DNA encoding ALT by considering the frequency of codon usage in the host to be used for expression (Grantham, R. et al., Nucleic Acids Research (1981)9, p. 43-74). Further, the DNA encoding ALT can be modified using commercially available kits or known methods. Examples of such modifications include, but are not limited to, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, and insertion of an initiation codon (ATG) and/or a termination codon (TAA, TGA or TAG).
The DNA encoding ALT also includes a DNA which hybridizes to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59 under stringent conditions and encodes a polypeptide functionally equivalent to ALT.
Stringent conditions can be appropriately selected by those skilled in the art, including, for example, low stringent conditions. Low stringent conditions refer to, for example, 42° C., 2×SSC and 0.1% SDS, preferably 50° C., 2×SSC and 0.1% SDS. More preferably, high stringent conditions may be selected. High stringent conditions refer to, for example, 65° C., 2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is elevated, DNAs with a higher homology can be obtained. The above-described DNA which hybridizes is preferably a DNA derived from nature, e.g., cDNA or chromosomal DNA.
These DNAs isolated by hybridization techniques usually have a high nucleotide sequence identity with a DNA encoding the ALT derived from human or the like. The DNA encoding ALT also includes a DNA which encodes a polypeptide functionally equivalent to the ALT derived from human or the like and has high identity with a DNA encoding the ALT derived from human or the like. The term “high identity” refers to usually 96% or more homology, preferably 98% or more homology, more preferably 99% or more identity. The identity of nucleotide sequences may be determined by algorithm BLAST (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Based on this algorithm, programs such as BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When nucleotide sequences are analyzed by BLASTN based on BLAST, parameters may be set as score=100 and wordlength=12, for example. Specific procedures for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).
The DNA encoding ALT may be inserted into a vector.
When the host cell to be used is E. coli, it is preferable that the vector has a replication origin (“ori”) so that the vector is largely amplified in E. coli (e.g., JM109, DH5α, HB101 and XL1-Blue) and prepared in large quantity, and also genes for selecting transformed E. coli (e.g., drug resistance genes that enable discrimination of transformant with some drugs such as ampicillin, tetracycline, kanamycin or chloramphenicol). Examples of preferable vectors include, but are not limited to, M13 vectors, pUC vectors, pBR322, pBluescript and pCR-Script. In addition to these vectors, pGEM-T, pDIRECT, pT7, etc. may be enumerated when the vector is used for the purpose of subcloning a cDNA and cutting off the subcloned cDNA. When the vector is used for the purpose of producing the polypeptide of the present invention, an expression vector is especially useful. When expression in E. coli is intended, the expression vector preferably has the above-described features so that the vector is amplified in E. coli, and it also preferably has a promoter which allows efficient expression in E. coli such as JM109, DH5α, HB101 or XL1-Blue, e.g., lacZ promoter (Ward et al, Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al, Science (1988) 240, 1041-1043) or T7 promoter. Specific examples of such vector include, in addition to those listed above, pGEX-5X-1 (Pharmacia), QIAexpress system (Qiagen), pEGFP, or pET (for its host, T7 RNA polymerase-expressing BL21 is preferred).
The vector may comprise signal sequences for polypeptide secretion. When the polypeptide is to be produced in the periplasm of E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used as a signal sequence for polypeptide secretion. Introduction of the vector into a host cell may be performed, for example, by the calcium chloride method or electroporation.
In cases where a host cell other than E. coli is used, vectors useful for producing a desired polypeptide include, but are not limited to, mammal-derived expression vectors [e.g., pcDNA3 from Invitrogen; pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p. 5322); pEF, pCDM8], insect cell-derived expression vectors (e.g., Bac-to-BAC baculovairus expression system from GIBCO BRL; pBacPAK8), plant-derived expression vectors (e.g., pMH1, pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (e.g., pZIpneo), yeast-derived expression vectors (e.g., Pichia Expression Kit from Invitrogen; pNV11; SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608, pKTH50).
When expression of the polypeptide in animal cells (such as CHO cells, COS cells, NIH3T3 cells, etc.) is intended, the vector preferably has a promoter necessary for expressing the polypeptide in those cells. Examples of such promoter include, but are not limited to, SV40 promoter (Mulligan et al, Nature (1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322) and CMV promoter. More preferably, the vector also has genes for selecting transformed cells (e.g., drug resistance genes that enable discrimination with drugs such as neomycin or G418). Examples of vectors having such properties include, but are not limited to, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
Further, when stable expression of a gene of interest and intracellular amplification of the copy number of the gene are indented, the following method may be used. Briefly, into CHO cells lacking a nucleic acid synthesis pathway, a vector having DHFR gene that complements the lack (e.g., pCHOI) is introduced, followed by amplification with methotrexate (MTX). On the other hand, when tentative expression of a gene of interest is intended, a method may be used in which COS cells carrying a gene expressing SV40T antigen on the chromosome is transformed with a vector having the replication origin of SV40 (e.g., pcD). As the replication origin, a replication origin derived from polyomavirus, adenovirus or bovine papillomavirus (BPV) may also be used. Further, the expression vector may contain selectable markers for amplifying the copy number of the gene in a host cell system. Examples of such selectable markers include, but are not limited to, aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene and dihydrofolate reductase (dhfr) gene.
The host cell into which the DNA encoding ALT (which may be incorporated in a vector) is transferred is not particularly limited. For example, E. coli or various animal cells may be used. If DNA encoding a desired polypeptide is transferred into a host cell into which DNA encoding ALT is transferred, this host cell can express ALT strongly, which leads to an increased production of the desired polypeptide. DNA encoding a taurine transporter (which may be incorporated into a vector) may be further transferred into the host cell into which DNA encoding ALT is transferred. By transferring DNA encoding a desired polypeptide and DNA encoding a taurine transporter into a host cell into which DNA encoding ALT is transferred, the yield of the desired polypeptide can be increased. For the production of the polypeptide, there are in vivo and in vitro production systems. Examples of in vitro production systems include systems using eukaryotes and systems using prokaryotes.
In the present invention, as DNA encoding a taurine transporter, a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 62, 64, 66 or 68 may be used. Alternatively, a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 62, 64, 66 or 68 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has taurine transporter activity may be used.
The polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 62, 64, 66 or 68 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has taurine transporter activity is functionally equivalent to a hamster, rat, mouse or human taurine transporter (hereinafter sometimes referred to as “taurine transporter derived from hamster or the like”). Such a polypeptide encompasses, for example, mutants of the taurine transporter derived from hamster or the like.
As methods well-known to those skilled in the art for preparing polypeptides functionally equivalent to a specific polypeptide, methods of introducing mutations into polypeptides may be given. For example, those skilled in the art could prepare polypeptides functionally equivalent to hamster taurine transporter by appropriately introducing mutations into amino acids of hamster taurine transporter by site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987) Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766). Mutations in amino acids may also occur in nature.
Specific examples of polypeptides functionally equivalent to the taurine transporter derived from hamster or the like include, but are not limited to, a polypeptide having an amino acid sequence derived from the amino acid sequence of the taurine transporter derived from hamster or the like by deletion of one or more amino acids, preferably 2-30 amino acids, more preferably 2-10 amino acids; a polypeptide having an amino acid sequence derived from the amino acid sequence of the taurine transporter derived from hamster or the like by addition of one or more amino acids, preferably 2-30 amino acids, more preferably 2-10 amino acids; and a polypeptide having an amino acid sequence derived from the amino acid sequence of the taurine transporter derived from hamster or the like by substitution of one or more amino acids, preferably 2-30 amino acids, more preferably 2-10 amino acids, with other amino acids.
Amino acid residues to be mutated are not particularly limited. Preferably, amino acid residues are mutated to other amino acids in which the nature of the initial amino acid side chain is conserved. Specific examples of the nature of amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, H, K, S and T), amino acids with an aliphatic side chain (G; A, V, L, I and P), amino acids with a hydroxyl group-containing side chain (S, T and Y), amino acids with a sulfur atom-containing side chain (C and M), amino acids with a carboxylic acid and amide-containing side chain (D, N, E and Q), amino acids with a base-containing side chain (R, K and H) and amino acids with an aromatic-containing side chain (H, F, Y and W) (In parentheses are one-letter codes for amino acids).
As one example of the polypeptide in which one or more amino acid residues are added to the taurine transporter derived from hamster or the like, a fusion polypeptide comprising the taurine transporter derived from hamster or the like may be given. Such a fusion polypeptide is composed of the taurine transporter derived from hamster or the like and other polypeptide fused thereto. Such a fusion polypeptide is included in the present invention. Such a fusion polypeptide may be prepared by linking a gene encoding the taurine transporter derived from hamster or the like in frame with a gene encoding the other polypeptide, transferring the resultant DNA into an expression vector and expressing the DNA in a host cell. Techniques known to those skilled in the art may be used. There is no limitation on the polypeptide to be fused to the taurine transporter derived from hamster or the like.
Examples of polypeptides to be fused to the taurine transporter derived from hamster or the like include, but are not limited to, FLAG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), 6×His comprising six histidine (His) residues, 10×His, influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, protein C fragment, glutathione-S-transferase (GST), influenza hemagglutinin (HA), immunoglobulin constant region, β-galactosidase and maltose-binding protein (MBP).
A commercially available gene encoding such polypeptide is fused to the gene encoding the taurine transporter derived from hamster or the like. The fused gene thus prepared is expressed to prepare a fused polypeptide.
An alternative method known to those skilled in the art for preparing polypeptides functionally equivalent to a specific polypeptide is a method using the hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Those skilled in the art could routinely isolate a DNA highly homologous to the DNA sequence of the taurine transporter derived from hamster or the like based on that DNA sequence or a part thereof, and isolate polypeptides functionally equivalent to the taurine transporter derived from hamster or the like from that DNA.
Hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the taurine transporter derived from hamster or the like can be appropriately selected by those skilled in the art. For example, low stringent hybridization conditions may be given. Low stringent hybridization conditions are, for example, 42° C., 2×SSC and 0.1% SDS, preferably 50° C., 2×SSC and 0.1% SDS. More preferably, high stringent conditions may be given. For example, high stringent conditions are 65° C., 2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is lowered, not only DNAs with high homology but also DNAs with only low homology are obtained. Conversely, it is expected that only those DNAs with high homology are obtained as the hybridization temperature is elevated. However, not only the temperature but also a plurality of factors (such as salt concentrations) affect the stringency of hybridization. Those skilled in the art could appropriately select these factors to realize similar stringency.
The polypeptide encoded by a DNA isolated by these hybridization techniques may have 70% or more homology and usually has high homology with the taurine transporter derived from hamster or the like in the amino acid sequence. The polypeptide also include those polypeptides which are functionally equivalent to the taurine transporter derived from hamster or the like and have high homology with the amino acid sequence of the taurine transporter derived from hamster or the like. The term “high homology” refers to usually 97% or more homology, preferably 98% or more homology, more preferably 99% or more homology. For determination of the homology of polypeptides, the algorithm described in Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. USA (1983) 80, 726-730 may be followed.
The polypeptide may vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, morphology, etc. depending on the cell or host that produce the polypeptide or the purification method that will be described later. However, as long as the resultant polypeptide has functions equivalent to the functions of the taurine transporter derived from hamster or the like, a DNA encoding the polypeptide can be used in the present invention. For example, when the polypeptide is expressed in a prokaryote (e.g., Escherichia coli), a methionine reside is added to the N-terminus of the initial amino acid sequence of the polypeptide. When the polypeptide is expressed in a eukaryote (e.g., a mammalian cell), the N-terminal signal sequence is removed. DNAs encoding such polypeptides can be used in the present invention.
The polypeptide may be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art. A recombinant polypeptide may be prepared by incorporating a DNA encoding the polypeptide in an appropriate expression vector, introducing the vector into an appropriate host cell, collecting the resultant transformant, extracting a crude polypeptide, and then purifying the polypeptide by chromatography (such as ion exchange, reversed phase or gel filtration chromatography, or affinity chromatography in which an antibody to the polypeptide is fixed in a column) or a combination of these chromatographic techniques.
When the polypeptide is expressed in a host cell (e.g., animal cell or E coli) as a fusion polypeptide with glutathione-S-transferase polypeptide or as a recombinant polypeptide with histidine residues added thereto, the expressed polypeptide may be purified with a glutathione column or a nickel column.
After purification of a fusion polypeptide, regions other than the polypeptide of interest may be cut off by thrombin or factor Xa and removed from the fusion polypeptide.
When the polypeptide is a natural polypeptide, the polypeptide may be isolated by purification methods known to those skilled in the art. For example, an extract from tissues or cells expressing the polypeptide may be applied to an affinity column to which an antibody to the taurine transporter derived from hamster or the like described later is bound. The antibody may be either a polyclonal antibody or a monoclonal antibody.
In the present invention, as DNA encoding a taurine transporter, a DNA having the nucleotide sequence as shown in SEQ ID NO: 61, 63, 65 or 67 may be used. Alternatively, a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 61, 63, 65 or 67 under stringent conditions and yet encodes a polypeptide having taurine transporter activity may be used.
The DNA encoding a taurine transporter can be used in the in vivo or in vitro production of a desired polypeptide as described above. Further, the DNA encoding a taurine transporter may be used in the creation of a cell which strongly expresses a taurine transporter. The DNA encoding a taurine transporter may take any form as long as it is capable of encoding a taurine transporter. That is, the DNA may be, for example, a cDNA synthesized from mRNA, a genomic DNA or a chemically synthesized DNA. It should be noted that, as long as the DNA is capable of encoding a taurine transporter, the DNA may have any nucleotide sequence based on the degeneracy of genetic codes.
The DNA encoding a taurine transporter may be prepared by methods known to those skilled in the art. For example, the DNA may be prepared by preparing a cDNA library from a cell expressing a taurine transporter and performing hybridization using a part of the DNA sequence encoding a taurine transporter (e.g., SEQ ID NO: 61, 63, 65 or 67) as a probe. The cDNA library may be prepared, for example, by the method described in Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989). Alternatively, a commercial cDNA library may be used. It is also possible to prepare the DNA encoding a taurine transporter by preparing RNA from a cell expressing the taurine transporter, synthesizing oligo DNA molecules based on the DNA sequence of the taurine transporter (e.g., SEQ ID NO: 61, 63, 65 or 67), and performing PCR using the oligo DNA molecules as primers to thereby amplify a cDNA encoding the taurine transporter.
Further, by determining the nucleotide sequence of the resultant cDNA, it is possible to determine the translation region encoding a taurine transporter and to obtain the amino acid sequence of the taurine transporter. Further, by screening a genomic library using the resultant cDNA as a probe, it is possible to isolate a genomic DNA.
Specifically, the following procedures may be used. First, mRNA is isolated from cells, tissues or the like expressing a taurine transporter. For the isolation of mRNA, the total RNA is prepared by known methods, for example, the guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomczynski, P. and Sacchi, N., Anal. Biochem. (1987) 162, 156-159) or the like, and then mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia), etc. Alternatively, mRNA may be prepared directly using QuickPrep mRNA Purification Kit (Pharmacia).
From the resultant mRNA, cDNA is synthesized using a reverse transcriptase. Alternatively, cDNA may be synthesized using a kit such as AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (SEIKAGAKU CORPORATION). It is also possible to synthesize and amplify cDNA according to the 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using 5′-Ampli FINDER RACE Kit (Clontech) and polymerase chain reaction (PCR) with primers.
A DNA fragment of interest is prepared from the resultant PCR product and ligated to a vector DNA to thereby prepare a recombinant vector. The vector is introduced into a host (e.g., E. coli), followed by selection of resultant colonies to thereby obtain a desired recombinant vector. The nucleotide sequence of the DNA of interest may be confirmed by a known method such as the dideoxynucleotide chain termination method.
Further, a nucleotide sequence of a higher expression efficiency can be designed for the DNA encoding a taurine transporter by considering the frequency of codon usage in the host to be used for expression (Grantham, R. et al., Nucleic Acids Research (1981)9, p. 43-74). Further, the DNA encoding a taurine transporter can be modified using commercially available kits or known methods. Examples of such modifications include, but are not limited to, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, and insertion of an initiation codon (ATG) and/or a termination codon (TAA, TGA or TAG).
The DNA encoding a taurine transporter also includes a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 61, 63, 65 or 67 under stringent conditions and encodes a polypeptide functionally equivalent to a taurine transporter.
Stringent conditions can be appropriately selected by those skilled in the art, including, for example, low stringent conditions. Low stringent conditions refer to, for example, 42° C., 2×SSC and 0.1% SDS, preferably 50° C., 2×SSC and 0.1% SDS. More preferably, high stringent conditions may be selected. High stringent conditions refer to, for example, 65° C., 2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is elevated, DNAs with a higher homology can be obtained. The above-described DNA which hybridizes is preferably a DNA derived from nature, e.g., cDNA or chromosomal DNA.
These DNAs isolated by hybridization techniques usually have a high nucleotide sequence identity with a DNA encoding the taurine transporter derived from hamster, etc. The DNA also includes a DNA which encodes a polypeptide functionally equivalent to the taurine transporter derived from hamster, etc. and has high identity with a DNA encoding the taurine transporter derived from hamster, etc. The term “high identity” refers to usually 96% or more homology, preferably 98% or more homology, more preferably 99% or more identity. The identity of nucleotide sequences may be determined by algorithm BLAST (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Based on this algorithm, programs such as BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When nucleotide sequences are analyzed by BLASTN based on BLAST, parameters may be set as expect value=100 and word size=12, for example. Specific procedures for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).
The DNA encoding a taurine transporter may be inserted into a vector.
When the host cell to be used is E. coli, it is preferable that the vector has a replication origin (“ori”) so that the vector is largely amplified in E. coli (e.g., JM109, DH5α, HB101 and XL1-Blue) and prepared in large quantity, and also genes for selecting transformed E. coli (e.g., drug resistance genes that enable discrimination of transformant with some drugs such as ampicillin, tetracycline, kanamycin or chloramphenicol). Examples of preferable vectors include, but are not limited to, M13 vectors, pUC vectors, pBR322, pBluescript and pCR-Script. In addition to these vectors, pGEM-T, pDIRECT, pT7, etc. may be enumerated when the vector is used for the purpose of subcloning a cDNA and cutting off the subcloned cDNA. When the vector is used for the purpose of producing the polypeptide of the present invention, an expression vector is especially useful. When expression in E. coli is intended, the expression vector preferably has the above-described features so that the vector is amplified in E. coli, and it also preferably has a promoter which allows efficient expression in E. coli such as JM109, DH5α, HB101 or XL1-Blue, e.g., lacZ promoter (Ward et al, Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al, Science (1988) 240, 1041-1043) or T7 promoter. Specific examples of such vector include, in addition to those listed above, pGEX-5X-1 (Pharmacia), QIAexpress system (Qiagen), pEGFP, or pET (for its host, T7 RNA polymerase-expressing BL21 is preferred).
The vector may comprise signal sequences for polypeptide secretion. When the polypeptide is to be produced in the periplasm of E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used as a signal sequence for polypeptide secretion. Introduction of the vector into a host cell may be performed, for example, by the calcium chloride method or electroporation.
In cases where a host cell other than E. coli is used, vectors useful for producing a desired polypeptide include, but are not limited to, mammal-derived expression vectors [e.g., pcDNA3 from Invitrogen; pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p. 5322); pEF, pCDM8], insect cell-derived expression vectors (e.g., Bac-to-BAC baculovairus expression system from GIBCO BRL; pBacPAK8), plant-derived expression vectors (e.g., pMH1, pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (e.g., pZIpneo), yeast-derived expression vectors (e.g., Pichia Expression Kit from Invitrogen; pNV11; SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608, pKTH50).
When expression of the polypeptide in animal cells (such as CHO cells, COS cells, NIH3T3 cells, etc.) is intended, the vector preferably has a promoter necessary for expressing the polypeptide in those cells. Examples of such promoter include, but are not limited to, SV40 promoter (Mulligan et al, Nature (1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322) and CMV promoter. More preferably, the vector also has genes for selecting transformed cells (e.g., drug resistance genes that enable discrimination with drugs such as neomycin or G418). Examples of vectors having such properties include, but are not limited to, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
Further, when stable expression of a gene of interest and intracellular amplification of the copy number of the gene are indented, the following method may be used. Briefly, into CHO cells lacking a nucleic acid synthesis pathway, a vector having DHFR gene that complements the lack (e.g., pCHOI) is introduced, followed by amplification with methotrexate (MTX). On the other hand, when tentative expression of a gene of interest is intended, a method may be used in which COS cells carrying a gene expressing SV40T antigen on the chromosome is transformed with a vector having the replication origin of SV40 (e.g., pcD). As the replication origin, a replication origin derived from polyomavirus, adenovirus or bovine papillomavirus (BPV) may also be used. Further, the expression vector may contain selectable markers for amplifying the copy number of the gene in a host cell system. Examples of such selectable markers include, but are not limited to, aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene and dihydrofolate reductase (dhfr) gene.
The present invention provides a cell which has a transferred DNA encoding ALT and a transferred DNA encoding a taurine transporter, both or either of which may be incorporated into a vector.
When eukaryotes are used, animal cells, plant cells, fungal cells, etc. may be used as the host. Specific examples of animal cells include mammalian cells, such as CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, 3T3 cells, myeloma cells, BHK (baby hamster kidney) cells, HeLa cells and Vero cells; amphibian cells, such as oocytes of Xenopus laevis (Valle, et al., Nature (1981) 291, 358-340); or insect cells, such as sf9, sf21 and Tn5 cells. Amoung CHO cells, dhfr-CHO lacking DHFR gene (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4420) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) are used with particular advantage. When high expression is intended in an animal cell, CHO cells are especially preferred. Introduction of the DNA which may be incorporated into a vector into the host cell may be performed by such methods as the calcium phosphate method, the DEAE dextran method, a method using a cationic ribosome DOTAP (Boehringer-Mannheim), electroporation, lipofection, etc.
As plant cells for polypeptide production, a Nicotiana tabacum-derived cell is known as a polypeptide production system and this may be subjected to callus culture. As fungal cells for polypeptide production, specific examples include yeast belonging to the genus Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi belonging to the genus Aspergillus, e.g., Aspergillus niger.
When prokaryotes are used, production systems using bacterial cells are known. Specific examples of such bacterial cells include E. coli (such as JM109, DH5α, HB101) and Bacillus subtilis.
The polypeptide encoded by a gene of interest may be obtained by transforming these cells with the gene of interest and culturing the transformed cells in vitro. The culture may be performed by known methods. For example, as a culture broth for animal cells, a medium such as DMEM, MEM, RPMI1640 or IMDM may be used. A serum supplement such as fetal calf serum (FCS) may be used jointly. Alternatively, serum-free culture may be performed. The pH during culture is preferably about 6 to 8. The culture is usually performed at about 30-40° C. for about 15-200 hours. If necessary, replacement of the medium, aeration and agitation are carried out.
On the other hand, in vivo production systems include those using animals or plants. A gene of interest is transferred into these animals or plants to produce the polypeptide in the animal bodies or plant bodies. Then, the polypeptide is collected. The term “host” as used herein includes such animals or plants.
When animals are used, available production systems include those using mammals or insects. Goat, pig, sheep, mouse and cattle may be used as mammals (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When mammals are used, transgenic animals may be used.
First, a gene of interest is fused to a gene encoding a polypeptide produced inherently in milk (such as goat (3-casein) to thereby prepare a fusion gene. A DNA fragment containing this fusion gene is injected into a goat embryo, which is then implanted in the uterus of a female goat. The polypeptide of interest can be obtained from the milk produced by transgenic goats born from the goat which accepted the embryo or the offspring of the transgenic goats. In order to increase the yield of milk containing the polypeptide produced by the transgenic goats, hormones may be appropriately administered to the transgenic goats (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
Examples of insects which may be used include silkworm. In this case, silkworm is infected with baculovirus carrying a transferred gene encoding the polypeptide of interest. The polypeptide of interest can be obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594).
Furthermore, when plants are used, tobacco can typically be used. When tobacco is used, a gene encoding the polypeptide of interest is inserted into a plant expression vector (e.g., pMON 530), which is then transferred into a bacterium such as Agrobacterium tumefaciens. A tobacco plant (e.g., Nicotiana tabacum) is infected with the resultant bacterium. The polypeptide of interest can be obtained from leaves of this plant (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
The polypeptide thus obtained can be isolated from the inside of the host cell or from its outside (e.g., medium), and purified to a substantially pure and homogeneous polypeptide. Isolation and purification of polypeptides can be performed using conventional isolation and purification methods for polypeptides, and are not limited in any way. For example, polypeptides can be isolated and purified by appropriate selection and combination of various tools and techniques, such as chromatography columns, filters, ultrafiltration, salting-out, precipitation with solvent, extraction with solvent, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.
Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographic techniques can be carried out using liquid phase chromatography, for example, HPLC, FPLC, etc. The present invention also includes those polypeptides highly purified using these purification methods.
Before or after the purification, it is also possible to give optional modifications to the polypeptide or remove a partial peptide therefrom by reacting the polypeptide with an appropriate polypeptide modification enzyme. Examples of such enzyme include, but are not limited to, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase and glucosidase.
In the present invention, the concept of “cells into which DNA has been transferred” encompasses not only cells into which exogenous DNA has been incorporated by genetic recombination technology; but also cells in which endogenous DNA has been activated by gene activation technology (see, for example, International Publication WO94/12650) so that expression of a protein corresponding to the endogenous DNA or transcription of the DNA has been initiated or increased.
Hereinbelow, the present invention will be described in more detail with reference to the following Examples. It should be noted that these Examples are provided only for illustrating the present invention and not for limiting the scope of the present invention.
Using a commercial Human Liver QUICK-Clone cDNA (Clontech Laboratories, Inc.) as a template, alanine aminotransferase (ALT1) gene derived from a human liver was obtained by a PCR method. The gene thus cloned was sequenced and confirmed to encode ALT 1 based on its homology with published human ALT 1. The ALT 1 gene thus obtained had mutations at five sites in the sequence of 1488 bases (c157a, a215g, c765t, t857c, t995a) and coded for 496 amino acids including four different amino acids (R53S, Q72R, F286S, M332K), but this was used as a PCR clone of the human liver derived ALT1 for cell modulation.
By adding a Kozak sequence to the human ALT1 obtained by cloning in Example 1 (which is hereinafter called ALT1), pPur-ALT1 (
Then, pPur-ALT1 or pPur was co-transferred into T10 which was a pHyg-TauT-transferred cell used as a parent strain (see Referential Example 2 described later). TauT/ALT1 co-expressing cells that exhibited high proliferation and expressed human ALT1 at high level (six strains) and TauT/pPur co-expressing cells that exhibited high proliferation (eight strains) were selected and subjected to fed-batch culture in 50 mL shaker flasks (an initial cell density of 10×105 cells/mL). The anti-glypican-3 antibody yield of TauT/ALT1 co-expressing cells, which were ALT expressing cells, on day 4 of the shaker culture was significantly higher than that of TauT/pPur cells (t-test: p<0.01,
TA41, which was a TauT/ALT1 co-expressing strain that produced the largest amount of an antibody (881 mg/L/4 days) and expressed ALT1 mRNA at the highest level in the study using the shaker fed-batch culture, was subjected to fed-batch culture in a 1 L jar (an initial cell density of 10×105 cells/mL). The antibody yields were as high as 1.3 g/L on day 7 of the culture, 3.0 g/L on day 10 of the culture, 3.5 g/L on day 12 of the culture, 4.6 g/L on day 17 of the culture, and 5.3 g/L on day 21 of the culture (
The anti-glypican-3 antibody yield of TA41, a TauT/ALT1 co-expressing strain, on day 14 of the fed-batch culture in a 50 ml shaker flask was increased by addition of α-ketoglutarate which, like alanine, served as a substrate for ALT (
The above results suggest that cells capable of high-yield antibody production at the late stage of culture can be obtained by artificially expressing ALT 1.
The present invention is applicable to any antibody-producing cell.
Total RNA was extracted from anti-IL-6 receptor antibody-producing cells (A CHO DXB11 cell line into which an anti-IL-6 receptor antibody gene had been transferred) (Japanese Unexamined Patent Publication No. Hei 8-99902), and then cDNA was synthesized therefrom in a poly(A) dependent manner. Hamster taurine transporter (TauT) gene was obtained by PCR using as a template the cDNA fragmented with three restriction enzymes, SalI, XhoI and EcoRI. As PCR primers, those containing the 5′-end and the 3′-end sequence conserved between rat and mouse TauTs were designed. The nucleotide sequence of the cloned gene was determined. From its homology with other TauT genes of known species, the cloned gene was confirmed to encode hamster TauT (
CMV promoter expression plasmid pHyg/TauT (
The parent strain and pHyg/TauT transferred strain were fed-batch cultured in 1 L jar with an initial cell density of 2×105 cells/ml. A part of the culture broth containing 450×105 cells was taken from the jar at appropriate time points. After the culture supernatant was separated by centrifugation, 1 ml of cooled sterile water containing a protease inhibitor (Complete Mini; Roche Diagnostics; Protease inhibitor cocktail tablets) was added to the cell pellet. Then, the cells were completely disrupted on ice in a sonicator (MISONIX ASTRASON MODEL XL2020) with a set of 5 seconds pulse-on and 5 seconds pulse-off being repeated 12 times. The total volume of the thus treated cells was applied to a centrifugal filter unit to thereby prepare a filtrate with a molecular weight of 5000 or less. This filtrate was used as a sample for determining intracellular amino acids. Each sample was subjected to detection and comparison of absorbance at 570 nm using a ninhydrin reagent L-8500 set (Wako Pure Chemical Industries) and an improved model of Hitachi fully automated amino acid analyzer (L-8500). Thus, various amino acid concentrations in samples were determined. Since the concentrations of amino acids and ammonia in culture broth were directly measured values, concentration comparisons in the order of μM were performed. On the other hand, since intracellular concentrations were obtained after addition of 1 ml of cooled sterile water to the cell pellet and sonication thereof, the measured concentrations of various amino acids and ammonia were converted into values per cell, followed by comparison of the converted values. To determine the ammonia concentration ratios shown in
As a result, the intracellular ammonia in pHyg/TauT transferred strain was maintained at a low concentration at the late stage of culture; it is believed that this contributes to high antibody yield (
Intracellular taurine concentration ratios were determined in the same manner as described above for ammonia concentrations (
As a result, it was found that pHyg/TauT transferred strain had taken up taurine in a manner dependent on the amount of taurine added and that its uptake was almost equal to that by the parent strain. However, as shown in
Actually, anti-glypican-3 antibody yield was not dependent on the initial taurine concentration (0-500 mM (62.575 g/L)) at the start of 50 ml shaker fed-batch culture (
The results described so far suggest that TauT strongly expressing strains have high antibody production ability even if the medium does not contain taurine at the start of culture and that there is a possibility that such strains also promote uptake of amino acids other than taurine.
The present invention is applicable to any antibody-producing cell.
All publications, patent and patent applications cited herein are incorporated herein by reference in their entirety.
Industrial Applicability
The present invention is applicable to production of polypeptides.
Sequence Listing Free Text
<SEQ ID NO: 1>
SEQ ID NO: 1 shows the nucleotide sequence of a gene encoding human alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 2875).
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the amino acid sequence of human alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 2875)
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the nucleotide sequence of a gene encoding human alanine aminotransferase mutant (ALT2) (KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706).
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the amino acid sequence of human alanine aminotransferase mutant (ALT2) (KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706).
<SEQ ID NO: 5>
SEQ ID NO: 5 shows the nucleotide sequence of a gene encoding mouse alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 76282).
<SEQ ID NO: 6>
SEQ ID NO: 6 shows the amino acid sequence of mouse alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 76282).
<SEQ ID NO: 7>
SEQ ID NO: 7 shows the nucleotide sequence of a gene encoding mouse alanine aminotransferase mutant (ALT2) (KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 108682).
<SEQ ID NO: 8>
SEQ ID NO: 8 shows the amino acid sequence of mouse alanine aminotransferase mutant (ALT2) (KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 108682).
<SEQ ID NO: 9>
SEQ ID NO: 9 shows the nucleotide sequence of a gene encoding rat alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670).
<SEQ ID NO: 10>
SEQ ID NO: 10 shows the amino acid sequence of rat alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670).
<SEQ ID NO: 11>
SEQ ID NO: 11 shows the nucleotide sequence of a gene encoding dog alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 609510).
<SEQ ID NO: 12>
SEQ ID NO: 12 shows the amino acid sequence of dog alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 609510).
<SEQ ID NO: 13>
SEQ ID NO: 13 shows the nucleotide sequence of a gene encoding African clawed frog alanine aminotransferase (ALT 1) (KEGG/ENZYME: 2.6.1.2/Xenopus laevis (African clawed frog): 444533).
<SEQ ID NO: 14>
SEQ ID NO: 14 shows the amino acid sequence of African clawed frog alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Xenopus laevis (African clawed frog): 444533).
<SEQ ID NO: 15>
SEQ ID NO: 15 shows the nucleotide sequence of a gene encoding fruit fly alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Drosophila melanogaster (fruit fly): Dmel_CG1640).
<SEQ ID NO: 16>
SEQ ID NO: 16 shows the amino acid sequence of fruit fly alanine aminotransferase (ALT 1) (KEGG/ENZYME: 2.6.1.2/Drosophila melanogaster (fruit fly): Dmel_CG1640).
<SEQ ID NO: 17>
SEQ ID NO: 17 shows the nucleotide sequence of a gene encoding nematode alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Caenorhabditis elegans (nematode): C32F10.8).
<SEQ ID NO: 18>
SEQ ID NO: 18 shows the amino acid sequence of nematode alanine aminotransferase (ALT 1) (KEGG/ENZYME: 2.6.1.2/Caenorhabditis elegans (nematode): C32F10.8).
<SEQ ID NO: 19>
SEQ ID NO: 19 shows the nucleotide sequence of a gene encoding one of two kinds of Japanese rice alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): 4342210).
<SEQ ID NO: 20>
SEQ ID NO: 20 shows the amino acid sequence of one of two kinds of Japanese rice alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): 4342210).
<SEQ ID NO: 21>
SEQ ID NO: 21 shows the nucleotide sequence of a gene encoding one of two kinds of Japanese rice alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): 4348524).
<SEQ ID NO: 22>
SEQ ID NO: 22 shows the amino acid sequence of one of two kinds of Japanese rice alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): 4348524).
<SEQ ID NO: 23>
SEQ ID NO: 23 shows the nucleotide sequence of a gene encoding Cyanidioschyzon merolae alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon merolae: CMM066C)
<SEQ ID NO: 24>
SEQ ID NO: 24 shows the amino acid sequence of Cyanidioschyzon merolae alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon merolae: CMM066C).
<SEQ ID NO: 25>
SEQ ID NO: 25 shows the nucleotide sequence of a gene encoding Saccharomyces cerevisiae alanine aminotransferase (ALT1) (KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089C).
<SEQ ID NO: 26>
SEQ ID NO: 26 shows the amino acid sequence of Saccharomyces cerevisiae alanine aminotransferase (ALT 1) (KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089C).
<SEQ ID NO: 27>
SEQ ID NO: 27 shows the nucleotide sequence of a gene encoding Saccharomyces cerevisiae alanine aminotransferase mutant (ALT2) (KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: YDR111C).
<SEQ ID NO: 28>
SEQ ID NO: 28 shows the amino acid sequence of Saccharomyces cerevisiae alanine aminotransferase mutant (ALT2) (KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: YDR111C).
<SEQ ID NO: 29>
SEQ ID NO: 29 shows the nucleotide sequence of a gene encoding Ashbya gossypii alanine aminotransferase KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Eremothecium gossypii): AGOS_AGRO85W).
<SEQ ID NO: 30>
SEQ ID NO: 30 shows the amino acid sequence of Ashbya gossypii alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Eremothecium gossypii): AGOS_AGRO85W).
<SEQ ID NO: 31>
SEQ ID NO: 31 shows the nucleotide sequence of a gene encoding Candida albicans alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Candida albicans: CaO19—346).
<SEQ ID NO: 32>
SEQ ID NO: 32 shows the amino acid sequence of Candida albicans alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Candida albicans: CaO19—346).
<SEQ ID NO: 33>
SEQ ID NO: 33 shows the nucleotide sequence of a gene encoding Schizosaccharomyces pombe alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08).
<SEQ ID NO: 34>
SEQ ID NO: 34 shows the amino acid sequence of Schizosaccharomyces pombe alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08).
<SEQ ID NO: 35>
SEQ ID NO: 35 shows the nucleotide sequence of a gene encoding Aspergillus nidulans alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2).
<SEQ ID NO: 36>
SEQ ID NO: 36 shows the amino acid sequence of Aspergillus nidulans alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2).
<SEQ ID NO: 37>
SEQ ID NO: 37 shows the nucleotide sequence of a gene encoding Aspergillus fumigatus alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA—6G07770).
<SEQ ID NO: 38>
SEQ ID NO: 38 shows the amino acid sequence of Aspergillus fumigatus alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA—6G07770).
<SEQ ID NO: 39>
SEQ ID NO: 39 shows the nucleotide sequence of a gene encoding Aspergillus oryzae alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: AO090003000164).
<SEQ ID NO: 40>
SEQ ID NO: 40 shows the amino acid sequence of Aspergillus oryzae alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: AO090003000164).
<SEQ ID NO: 41>
SEQ ID NO: 41 shows the nucleotide sequence of a gene encoding Cryptococcus neoformans alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Cryptococcus neoformans JEC21: CNG01490).
<SEQ ID NO: 42>
SEQ ID NO: 42 shows the amino acid sequence of Cryptococcus neoformans alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Cryptococcus neoformans JEC21: CNG01490).
<SEQ ID NO: 43>
SEQ ID NO: 43 shows the nucleotide sequence of a gene encoding Dictyostelium discoideum alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Dictyostelium discoideum: DDB—0232139).
<SEQ ID NO: 44>
SEQ ID NO: 44 shows the amino acid sequence of Dictyostelium discoideum alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Dictyostelium discoideum: DDB—0232139).
<SEQ ID NO: 45>
SEQ ID NO: 45 shows the nucleotide sequence of a gene encoding Trypanosoma brucei alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950).
<SEQ ID NO: 46>
SEQ ID NO: 46 shows the amino acid sequence of Trypanosoma brucei alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950)<
SEQ ID NO: 47>
SEQ ID NO: 47 shows the nucleotide sequence of a gene encoding Leishmania major alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Leishmania major: LmjF12.0630).
<SEQ ID NO: 48>
SEQ ID NO: 48 shows the amino acid sequence of Leishmania major alanine aminotransferase (KEGG/ENZYME: 2.6.1.2/Leishmania major: LmjF12.0630).
<SEQ ID NO: 49>
SEQ ID NO: 49 shows the nucleotide sequence of a gene encoding one of two kinds of Entamoeba histolytica alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica: 233.t00009).
<SEQ ID NO: 50>
SEQ ID NO: 50 shows the amino acid sequence of one of two kinds of Entamoeba histolytica alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica: 233.t00009).
<SEQ ID NO: 51>
SEQ ID NO: 51 shows the nucleotide sequence of a gene encoding one of two kinds of Entamoeba histolytica alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica: 24.t00016).
<SEQ ID NO: 52>
SEQ ID NO: 52 shows the amino acid sequence of one of two kinds of Entamoeba histolytica alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica: 24.t00016).
<SEQ ID NO: 53>
SEQ ID NO: 53 shows the nucleotide sequence of a gene encoding one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 506529.420).
<SEQ ID NO: 54>
SEQ ID NO: 54 shows the amino acid sequence of one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 506529.420).
<SEQ ID NO: 55>
SEQ ID NO: 55 shows the nucleotide sequence of a gene encoding one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 506529.430).
<SEQ ID NO: 56>
SEQ ID NO: 56 shows the amino acid sequence of one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 506529.430).
<SEQ ID NO: 57>
SEQ ID NO: 57 shows the nucleotide sequence of a gene encoding one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 510889.120)
<SEQ ID NO: 58>
SEQ ID NO: 58 shows the amino acid sequence of one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 510889.120).
<SEQ ID NO: 59>
SEQ ID NO: 59 shows the nucleotide sequence of a gene encoding one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 510889.140).
<SEQ ID NO: 60>
SEQ ID NO: 60 shows the amino acid sequence of one of four kinds of Trypanosoma cruzi alanine aminotransferases (KEGG/ENZYME: 2.6.1.2/Trypanosoma cruzi: 510889.140).
<SEQ ID NO: 61>
SEQ ID NO: 61 shows the nucleotide sequence of a gene encoding hamster taurine transporter.
<SEQ ID NO: 62>
SEQ ID NO: 62 shows the amino acid sequence of hamster taurine transporter.
<SEQ ID NO: 63>
SEQ ID NO: 63 shows the nucleotide sequence of a gene encoding rat taurine transporter (GenBank NM—017206).
<SEQ ID NO: 64>
SEQ ID NO: 64 shows the amino acid sequence of rat taurine transporter (GenBank NM 017206).
<SEQ ID NO: 65>
SEQ ID NO: 65 shows the nucleotide sequence of a gene encoding mouse taurine transporter (GenBank NM—009320).
<SEQ ID NO: 66>
SEQ ID NO: 66 shows the amino acid sequence of mouse taurine transporter (GenBank NM 009320).
<SEQ ID NO: 67>
SEQ ID NO: 67 shows the nucleotide sequence of a gene encoding human taurine transporter (GenBank NM—003043).
<SEQ ID NO: 68>
SEQ ID NO: 68 shows the amino acid sequence of human taurine transporter (GenBank NM—003043).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-205158 | Aug 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/064095 | 8/6/2008 | WO | 00 | 2/4/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/020144 | 2/12/2009 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5658786 | Smith et al. | Aug 1997 | A |
| 6184007 | Dusch et al. | Feb 2001 | B1 |
| 6225115 | Smith et al. | May 2001 | B1 |
| 6251613 | Kishimoto et al. | Jun 2001 | B1 |
| 6316238 | Nakamura et al. | Nov 2001 | B1 |
| 6812339 | Venter et al. | Nov 2004 | B1 |
| 7413536 | Dower et al. | Aug 2008 | B1 |
| 7919086 | Nakano et al. | Apr 2011 | B2 |
| 20030165495 | Carulli et al. | Sep 2003 | A1 |
| 20050221466 | Liao et al. | Oct 2005 | A1 |
| 20050265983 | Melamed et al. | Dec 2005 | A1 |
| 20060014937 | Kang et al. | Jan 2006 | A1 |
| 20070162995 | Good et al. | Jul 2007 | A1 |
| 20070166362 | Sakuma et al. | Jul 2007 | A1 |
| 20070190599 | Nakano et al. | Aug 2007 | A1 |
| 20090191591 | Tabuchi et al. | Jul 2009 | A1 |
| 20090221442 | Dower et al. | Sep 2009 | A1 |
| 20100233759 | Tabuchi et al. | Sep 2010 | A1 |
| 20100248359 | Nakano et al. | Sep 2010 | A1 |
| 20110003334 | Tabuchi et al. | Jan 2011 | A1 |
| 20110014654 | Tabuchi et al. | Jan 2011 | A1 |
| 20120045795 | Tabuchi et al. | Feb 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| 1612689 | May 2005 | CN |
| 1838969 | Sep 2006 | CN |
| 1 212 619 | May 2007 | EP |
| 2 213 746 | Aug 2010 | EP |
| 08-191693 | Jul 1996 | JP |
| 10-075787 | Mar 1998 | JP |
| 10-191984 | Jul 1998 | JP |
| 2000-228990 | Aug 2000 | JP |
| 2005-525100 | Aug 2005 | JP |
| 2006-506086 | Feb 2006 | JP |
| WO-9204381 | Mar 1992 | WO |
| WO-9727485 | Jul 1997 | WO |
| WO-0120331 | Mar 2001 | WO |
| WO 02092768 | Nov 2002 | WO |
| WO-03039485 | May 2003 | WO |
| WO-2005076015 | Aug 2005 | WO |
| WO-2006006693 | Jan 2006 | WO |
| WO-2006119115 | Nov 2006 | WO |
| WO-2007056507 | May 2007 | WO |
| WO-2007119774 | Oct 2007 | WO |
| WO-2008114673 | Sep 2008 | WO |
| WO-2009020144 | Feb 2009 | WO |
| WO-2009051109 | Apr 2009 | WO |
| WO-2009054433 | Apr 2009 | WO |
| Entry |
|---|
| Kennell, “Principles and Practices of Nucleic Acid Hybridization”, Progr. Nucleic Acid Res. Mol. Biol. 11: 259-270, 1971. |
| Ngo, in “The Protein Folding Problem and Tertiary Structure Prediction”, Merz et al. (eds.), Birkhauser Boston: Boston, MA, pp. 433 and 492-495, 1994. |
| Rudinger, in “Peptide Hormones”, Parsons (ed.), University Park Press: Baltimore, MD, pp. 1-7, 1976. |
| Ito, T. et al., “Expression of taurine transporter is regulated through the TonE/TonEBP pathway and contributes to cytoprotein in HepG2 cells”, 2004, Biochem. J. vol. 382: pp. 177-182. |
| Kim N. et al., “Response of Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression”, 2002, J. Biotech., vol. 95: pp. 237-248. |
| Ramamoorthy, S. et al. “Functional characterization and chromosomal location of a cloned taurine transporter from human placecnta”, 1994, Biochem. J., vol. 30: pp. 893-900. |
| Jhiang et al., “Cloning of the human taurine transporter and characterization of taurine uptake in thyroid cells,” FEBS, 318(2):139-144. |
| Supplementary European Search Report dated Aug. 3, 2010, in corresponding EP 08792251.4, 10 pages. |
| Bell et al., “Genetic Engineering of Hybridoma Glutamine Metabolism,” Enzyme and Microbial Technology, 1995, 17(2):98-106. |
| Butler, Michael, “Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals,” Appl. Microbiol. Biotechnol., Aug. 2005, 68(3):283-291. |
| Christie et al., “The Adaptation of BHK Cells to a Non-Ammoniagenic Glutamate-Based Culture Medium,” Biotechnology and Bioengineering, Aug. 5, 1999, 64(3):298-309. |
| de la Cruz Edmonds et al., “Development of Transfection and High-Producer Screening Protocols for the CHOK1SV Cell System,” Molecular Biology, Oct. 1, 2006, 34(2):179-190. |
| Good et al., “Engineering nitrogen use efficiency with alanine aminotransferase,” Canadian Journal of Botany, Mar. 1, 2007, 85(3):252-262. |
| Kalwy et al., “Toward More Efficient Protein Expression,” Molecular Biology, Oct. 2006, 34(2):151-156. |
| Voss et al., “Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts,” Eur. J. Biochem., Dec. 2004, 271(23-24):4646-4658. |
| Yang et al., “cDNA Cloning, Genomic Structure, Chromosomal Mapping, and Functional Expression of a Novel Human Alanine Aminotransferase,” Genomics, Mar. 1, 2002, 79(3):445-450. |
| Zhang et al., “Metabolic characteristics of recombinant chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine,” Cytotechnology, 2006, 51(1):21-28. |
| U.S. Appl. No. 13/368,945, filed Feb. 8, 2012, Tabuchi et al. |
| Alper, Seth L., “Molecular physiology of SLC4 anion exchangers,” Exp. Physiol., 2006, 91:153-161. |
| Arden et al., “Life and death in mammalian cell culture: strategies for apoptosis inhibition,” Trends in Biotechnology, Apr. 2004, 22(4):174-180. |
| Butler, Michael, “Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals,” Appl. Microbiol. Biotechnol., 2005, 68:283-291. |
| Chambard et al., “Sugar transport by mammalian members of the SLC26 superfamily of anion-bicarbonate exchangers,” J. Physiol., 2003, 550:667-677. |
| Christensen et al., “High expression of the taurine transporter TauT in primary cilic of NIH3T3 fibroblasts,” Cell Biology International, 2005, 29:347-351. |
| Database DDBJ/EMBL/GenBank [online], Accession No. NM—000342, uploaded Sep. 25, 2007, Keskanokwong et al., Definition: Homo sapiens solute carrier family 4, anion exchanger, member 1 (erythrocyte membrane protein band 3, Diego blood group) (SLC4A1), mRNA, retrieved Nov. 11, 2008, 12 pages. |
| Database EMBL [Online] Jul. 23, 1992, XP002593029, retrieved from EBI accession No. EMBL:M95495, 3 pages. |
| Database Uniprot [Online] Jan. 10, 2006, XP002593032, retrieved from EBI accession No. UNIPROT:Q2VRP7, 1 page. |
| Database UniProt [Online] Jul. 1, 1993, XP002593028, retrieved from EBI accession No. UNIPROT:Q00589, 2 pages. |
| Database UniProt [Online] Jun. 1, 2001, “RecName: Full=Cysteine sulfinic acid decarboxylase; EC=4.1.1.29; AltName: Full=Cysteine-sulfinate decarboxylase; AltName: Full=Sulfinoalanine decarboxylase;” XP002597738 retrieved from EBI accession No. UNIPROT:Q9DBE0 Database accession No. Q9DBE0, 2 pages. |
| Database Uniprot [Online] Mar. 15, 2005, XP002593030, retrieved from EBI accession No. UNIPROT:Q5F431, 1 page. |
| Database Uniprot [Online] Oct. 1, 2000, XP002593031, retrieved from EBI accession No. UNIPROT:Q9MZ34, 2 pages. |
| de la Rosa et al., “Evidence for a Rate-Limiting Role of Cysteinesulfinate Decarboxylase Activity in Taurine Biosynthesis in Vivo,” Comp. Biochem. Physiol., 1985, 81B(3):565-571. |
| De La Rosa et al., “Evidence for a Rate-Limiting Role of Cysteinesulfinate Decarboxylase Activity in Taurine Biosynthesis In Vivo,” Comp. Biochem. Physiol. B, 1985, 81(3):565-571. |
| Dusch et al., “Expression of the Corynebacterium glutamicum panD Gene Encoding L-Aspartate-alpha-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli,” Applied and Environmental Microbiology, Apr. 1999, 65(4):1530-1539. |
| Final Office Action dated Dec. 17, 2010 in U.S. Appl. No. 12/226,195. |
| Final Office Action dated Mar. 2, 2012 in U.S. Appl. No. 12/734,283. |
| Final Office Action dated Aug. 23, 2011 in U.S. Appl. No. 12/733,815. |
| Fu et al., “Direct interaction and cooperative role of tumor suppressor p16 with band 3 (AE1),” FEBS Letters, 2005, 579(10):2105-2110. |
| Ganapathy et al., “Expression and Regulation of the Taurine Transporter in Cultured Cell Lines of Human Origin,” Advances in Experimental Medicine and Biology, 1994, 359:51-57, XP009123192. |
| GenBank Accession No. AEQ38544, Oct. 2011, 2 pages. |
| GenBank Accession No. EGW01898, Aug. 2011, 2 pages. |
| Griffith, Owen W., “Crysteinesulfinate Metabolism, Altered Partitioning Between Transamination and Decarboxylation Following Administration of β-Methyleneaspartate,” J. Biol. Chem., Feb. 10, 1983, 258(3):1591-1598. |
| Hammer et al., “β-Alanine but not taurine can function as an organic osmolyte in preimplantation mouse embryos cultured from fertilized eggs,” Molecular Reproduction and Development, Oct. 2003, 66(2):153-161. |
| Han et al., “Is TauT an Anti-Apoptotic Gene?” Taurine 6, Oja et al. Eds., 2006, 59-67. |
| Hwang et al., “Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells,” Protein Expression and Purification, 2005, 39:175-183. |
| Ifandi et at., “Regulation of Cell Proliferation and Apoptosis in CHO-K1 Cells by the Coexpression of c-Myc and Bcl-2,” Biotechnol. Prog., 2005, 21:671-677. |
| Kim et al., “Characterization of Chimeric Antibody Producing CHO Cells in the Course of Dihydrofolate Reductase-Mediated Gene Amplification and Their Stability in the Absence of Selective Pressure,” Biotechnology and Bioengineering, Apr. 5, 1998, 58(1):73-84. |
| Kondo et al., “Modulation of apoptosis by endogenous Bcl-xL expression in MKN-45 human gastric cancer cells,” Oncogene, 1998, 17:2585-2591. |
| Lee et al., “Development of Apoptosis-Resistant Dihydrofolate Reductase-Deficient Chinese Hamster Ovary Cell Line,” Biotechnol. Bioengineer., 2003, 82:872-876. |
| Liu et al., “Cloning and expression of a cDNA encoding the transporter of taurine and β-alanine in mouse brain,” Proc. Natl. Acad. Sci. USA, Dec. 1992, 89(24):12145-12149. |
| Lux et al., “Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1),” Proc. Natl. Acad. Sci. USA, Dec. 1989, 86:9089-9093. |
| Miyasaka et al., “Characterization of Human Taurine Transporter Expressed in Insect Cells Using a Recombinant Baculovirus,” Protein Expression and Purification, 2001, 23(3):389-397. |
| Morgan et al., “Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters,” Am. J. Physiol. Cell Physiol., Aug. 2007, 293(2):C738-C748. |
| Office Action dated Jan. 6, 2011 in U.S. Appl. No. 12/733,815. |
| Office Action dated May 18, 2010 in U.S. Appl. No. 12/226,195. |
| Office Action dated Aug. 3, 2011 in U.S. Appl. No. 12/734,283. |
| Office Action dated Aug. 9, 2011 in U.S. Appl. No. 12/450,161. |
| Porter et al., “Non-steady-state kinetics of brain glutamate decarboxylase resulting from interconversion of the apo- and holoenzyme,” Biochimica et Biophysica Acta, 1988, 874:235-244. |
| Reymond et al., “Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD),” Biochimica et Biophysica Acta, 1996, 1307:152-156. |
| Shen et al., “Expression of Anion Exchanger 1 Sequestrates p16 in the Cytoplasm in Gastric and Colonic Adenocarcinoma,” Neoplasia, Oct. 2007, 9(10):812-819. |
| Smith et al., “Cloning and Expression of a High Affinity Taurine Transporter from Rat Brain,” Mol. Pharmacol., 1992, 42(4):563-569. |
| Tabuchi et al., “Overexpression of Taurine Transporter in Chinese Hamster Ovary cells Can Enhance Cell Viability and Product Yield, While Promoting Glutamine Consumption,” Biotechnology and Bioengineering, 2010, 107(6):998-1003. |
| Tang et al., “Protein Phosphorylation and Taurine Biosynthesis In Vivo and In Vitro,” Journal of Neuroscience, Sep. 15, 1997, 17(18):6947-6951. |
| Tappaz et al., “Characterization of the cDNA Coding for Rat Brain Cysteine Sulfinate Decarboxylase: Brain and Liver Enzymes are Identical Proteins Encoded by Two Distince mRNAs,” J. Neurochem., 1999, 73(3):903-912. |
| Tinland et al., “Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus,” Proc. Natl. Acad. Sci. USA, Aug. 1994, 91:8000-8004. |
| Trill et al., “Production of monoclonal antibodies in COS and CHO cells,” Current Opinion in Biotechnology, 1995, 6:553-560. |
| Uchida et al., “Molecular cloning of the cDNA for an MDCK cell Na+- and Cl−-dependent taurine transporter that is regulated by hypertonicity,” Proc. Natl. Acad. Sci. USA, Sep. 1992, 89:8230-8234. |
| Voss et al., “Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts,” Eur. J. Biochem., 2004, 271:4646-4658. |
| Wirth et al., “Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure,” Gene, 1988, 73:419-426. |
| Wu et al., “Overexpression of Anion Exchanger 2 in Human Hepatocellular Carcinoma,” Chinese Journal of Physiology, 2006, 49(4):192-198. |
| Yang et al., “Human Hepatitis B Viral e Antigen Interacts with Cellular Interleukin-1 Receptor Accessory Protein and Triggers Interleukin-1 Response,” Journal of Biological Chemistry, Nov. 10, 2006, 281(45):34525-34536. |
| Office Action dated Sep. 21, 2012 in U.S. Appl. No. 13/368,945. |
| Shibayama et al., “Effect of Methotrexate Treatment on Expression Levels of Organic Anion Transporter Polypeptide 2,P-Glycoprotein and Bile Salt Export Pump in Rats,” Biol. Pharm. Bull., Mar. 2009, 32(3):493-496. |
| Office Action dated Feb. 27, 2013 in U.S. Appl. No. 13/138,909. |
| Final Office Action dated May 24, 2013 in U.S. Appl. No. 13/368,945. |
| Tanner et al., “The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence,” Biochem. J., 1988, 256:703-712. |
| Beckmann et al., “Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide,” Blood, Apr. 15, 2001, 97(8):2496-2505. |
| Han et al., “Regulation of TauT by cisplatin in LLC-PK1 renal cells,” Pediatr. Nephrol., 2005, 20:1067-1072. |
| Ishiguro et al., “CO2 permeability and bicarbonate transport in microperfused interlobular ducts isolated from guinea-pig pancreas,” Journal of Physiology, 2000, 528.2:305-315. |
| Mount et al., “The SLC26 gene family of multifunctional anion exchangers,” Pflugers Arch.—Eur. J. Physiol., 2004, 447:710-721. |
| Pushkin et al., “SLC4 base (HCO-3, CO-23) transporters: classification, function, structure, genetic diseases, and knockout models,” Am. J. Physiol. Renal Physiol., 2006, 290:F580-F599. |
| International Search Report mailed Sep. 2, 2008, in PCT/JP2008/064095, 5 pages. |
| Jhiang et al., “Cloning of the human taurine transporter and characterization of taurine uptake in thyroid cells,” FEBS, 318(2):139-144, 1993. |
| Number | Date | Country | |
|---|---|---|---|
| 20100167346 A1 | Jul 2010 | US |
