JPH05252956A - Production of recombinant cell capable of highly manifesting polypeptide - Google Patents

Abstract

PURPOSE:To provide the subject new recombinant cell capable of highly manifesting the objective polypeptide. CONSTITUTION:A vector having a nucleotide sequence coding a polypeptide, another nucleotide sequence amplifying it and imparting resistance properties against a selected chemical to a mammal cell and one more nucleotide sequence controlling manifestation of both the nucleotide sequences is introduced into a mammal cell. From the resultant recombinant cells, recombinant cells exhibiting resistance against the selected chemical are separated each in the form of a unicell-derived recombinant cell. Candidate recombinant cells capable of highly manifesting a polypeptide are selected by quantitative analysis of the polypeptide-manifestation properties. The selected candidate cells are then cultured several times each in a culture medium containing a gradually increased concentration of the selected chemical, thus selecting the objective recombinant cell capable of highly manifesting a polypeptide. For example, hTGF-beta1- producing cell strain 29E6-2 (FERM BP-3218) highly manifesting hTGF-beta1 is selected out.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the high expression of a target polypeptide from a recombinant cell group obtained by introducing a vector having a nucleotide sequence encoding a polypeptide useful as a medicine into mammalian cells. To select a recombinant cell line to be used, and utilizing this method, human transforming growth factor β1 (human transfo
rming growth factor β1; hereinafter, abbreviated as “hTGF-β1” hereinafter, a recombinant cell line highly expressing transforming growth factor β (hereinafter abbreviated as “TGF-β”) is selected, and a selected group is selected. The present invention relates to a method for producing TGF-β using a recombinant cell line.

[0002]

2. Description of the Related Art In recent years, various kinds of useful polypeptides have been introduced into a bacterium or the like by introducing a nucleotide sequence encoding the polypeptide into the bacterium by various transformation methods, and the transformed bacterium or the like is used in or in the bacterium. It is produced by expressing and producing it in the supernatant and purifying it. In particular, Escherichia coli is a bacterium that has been most studied biochemically, and has been successfully used for mass production of insulin and growth hormone. However, many useful polypeptides have been modified by glycosylation, amidation, phosphorylation, etc., and these modifications are associated with expression of their biological activities, lifespan, and binding with plasma proteins for transport. It is often required for enhancement and is involved in physical properties, stability, and solubility in vivo. However, since bacteria lack many functions of modifying polypeptides, attempts to recombinantly produce polypeptides using these as hosts often fail to yield polypeptides with the same modifications as the natural type. .. On the other hand, these polypeptides produced by bacteria often do not show the activity as they are even if the activity does not depend on the presence or absence of modification. This is because the three-dimensional structure of the polypeptide is different from that of the natural product, and reconstitution of the polypeptide is necessary to restore the activity. However, it is difficult to reconstitute a polypeptide having a large number of SS bonds or a plurality of subunit structures, and bacteria such as Escherichia coli are unsuitable as a host for the purpose of producing a polypeptide having such a complicated three-dimensional structure. is there. Furthermore, there is concern that a polypeptide produced using Escherichia coli as a host may be contaminated with a pyrogen (pyrogen) derived from Escherichia coli, and its removal is a problem in the production of pharmaceuticals.

For these reasons, the method using a microorganism as a host is very important in the production of a mammal-derived polypeptide which has been modified by glycosylation or has a complicated higher-order structure (hereinafter referred to as a target polypeptide). However, it is advantageous to use a cell originally having the ability to produce the polypeptide or a mammalian cell close thereto as a host.

However, when a desired polypeptide is produced by using a mammalian cell as a host, a plasmid vector as used in bacteria cannot be used.
The following two methods are performed. One is a method in which a recombinant virus or the like is used as a vector to temporarily increase the copy number of the nucleotide sequence encoding the target polypeptide in the host cell, and a large amount of the target polypeptide is expressed in a short period of time. Is a method for stably expressing a target polypeptide by inserting a nucleotide sequence encoding the target polypeptide into chromosomal DNA of a host cell.

As the former method, there is a method using a recombinant virus such as SV40 or adenovirus. After infecting animal cells, these viral vectors undergo remarkable amplification to obtain a high copy number and transiently produce a large amount of the desired polypeptide, resulting in death of the host cell.
Also, a vector containing the replication origin of SV40 (eg P91
(023B, CDM8, etc.) amplifies in a large amount when introduced into monkey COS cells constitutively expressing the SV40 T antigen, which also kills the cells. Although the method of temporarily increasing the copy number of the vector and expressing the target polypeptide in this manner is advantageous in conducting research on the polypeptide expressed from the introduced vector, the target polypeptide can be used as a drug substance or the like. Is a disadvantage as a means for continuous mass production. Further, in the method using a bovine papillomavirus (BPV) or BK virus vector, these vectors exist without being integrated into the chromosome in the introduced cells, and the cells are not killed, so that a relatively stable transformed cell can be obtained. .. However, these vectors are generally unstable, and although the copy number is retained, transcription is not performed,
Since some of the replicating vectors are deficient, it is a disadvantage as a means for stably obtaining the target polypeptide.

For these reasons, the latter method of using an integrative vector in which the nucleotide sequence encoding the target polypeptide is retained on the chromosome is preferable for obtaining the target polypeptide stably. For example, a retrovirus has an RNA genome, but during its replication, the genome is reverse transcribed into DNA, and it is known that this proviral DNA proliferates without killing host cells and is retained on the chromosome in the process. There is. Utilizing this fact, D containing a region required for replication of retroviral proviral DNA
An expression vector for producing the desired polypeptide can be obtained by using NA as a vector and substituting a part thereof with a nucleotide sequence encoding the desired polypeptide.

However, the expression level is extremely low even if the nucleotide sequence encoding the desired polypeptide is retained in the chromosome. For example, in order to express hTGF-β1 which is one of the target polypeptides, hTGF-β1
When cDNA was introduced into 293 cells derived from human embryonic kidney transformed with adenovirus, hTGF
Most of the expression levels of -β1 were 1 ng / ml or less. on the other hand,
It is known that certain genes such as dihydrofolate reductase (hereinafter referred to as DHFR) have their copy numbers amplified on chromosomal DNA under certain conditions. Therefore, in order to increase the expression level of the target polypeptide, a gene amplifiable gene (hereinafter referred to as an amplified gene) and a nucleotide sequence encoding the target polypeptide are allowed to coexist on one vector, and a vector capable of inserting these into a host DNA is constructed. It is preferable to use. In fact, DHFR and adenosine deaminase [Kantman RJ: Pro
c. Natl. Acad. Sci. USA, 83 31
36 (1986)] or glutamine synthetase [Hay
word BE: Nucleic Acids Re
s. 14 999 (1986)] and the like are used as amplification genes to enable the production of interleukin 2 (IL-2) and the like.

On the other hand, TGF-β is a homodimeric polypeptide having a molecular weight of 25,000, and is a factor having a physiologically extremely important effect of controlling various cell growth and differentiation. From this, the application development of TGF-β is expected as a therapeutic drug for wound healing, bone fracture, inflammation, autoimmune disease, cancer and the like. TGF-β is known to exist in the kidney, placenta and platelets of normal animals, but the amount thereof is extremely small, and it is impossible to obtain TGF-β stably and in large amounts. there were. This T
Among GF-β, a cDNA encoding a precursor of hTGF-β1 was cloned from a human placenta by Derinkk et al. In 1985 [Nature,
316 701 (1985)]. According to this announcement, hT
The GF-β1 monomer is a polypeptide consisting of 112 amino acids and is a relatively small polypeptide having 9 cysteine residues. By forming a complex three-dimensional structure by cross-linking these cysteine residues, hTG
F-β1 exerts its biological activity.

[0009] This TGF- represented by hTGF-β1
β has been attempted to be produced by a gene recombination technique, and as described above, a vector containing a nucleotide sequence encoding a target polypeptide has been inserted into a host chromosome, and gene amplification using an amplified gene such as DHFR has been performed so far. By using the method, production by a gene recombination method using a mammalian cell as a host has been attempted. Japanese Patent Laid-Open No. 1-25
No. 7497, monkey TGF-β
28 μg / 10 ⁷ ce of biologically active mature TGF-β1 using the cDNA encoding the precursor of 1.
It is produced at a rate of 11s / 24 hr. Then, Gopal et al. Used 293 cells and hTGF
Attempts are being made to produce hTGF-β1 using a vector having a nucleotide sequence encoding -β1 and an amplification gene. However, the expression level of hTGF-β1 was 10 μg.
/ 10 ⁷ only cells / 24 hr or, production of these reported TGF-beta were those either insufficient as preparation for practical use.

[0010]

As described above, when obtaining an expression cell transformed with a vector having a nucleotide sequence encoding a target polypeptide using a mammalian cell, the transduced cell obtained is There is no particular problem when a large amount of target polypeptide can be produced, but how to increase the expression level is important when the expression level is low such as TGF-β and production at a practical level is difficult. It is a problem.

Several methods are conceivable as means for increasing the expression level of the target polypeptide. That is, one major factor that regulates the expression level of the target polypeptide is an expression control factor such as a promoter or an enhancer, but many studies have already been conducted on these factors, and the expression control factors currently used are All are excellent, and we cannot expect much improvement. The expression level of the target polypeptide may vary greatly depending on the type of host cell used, the medium, or the combination of culture methods.These combinations depend on the individual properties of the target polypeptide or the host cell used. It should be decided individually according to the above, and since trial and error must be performed, it cannot be established as a general methodology. On the other hand, in the gene amplification method, in order to amplify the amplified gene, the concentration of the selective drug is sequentially increased, the transduced cells are cultured, and cells having higher drug resistance are selected to obtain a highly expressing strain of the target polypeptide. However, at that time,
The higher the drug resistance of the cell, the higher the expression level of the target polypeptide does not necessarily mean, but there is always a limit.

[0012] As described above, the means for selecting recombinant cells capable of highly expressing the desired polypeptide over recombinant mammalian cells has not yet reached a sufficiently satisfactory level. Further, it is the current situation that a low-expressing polypeptide such as TGF-β cannot be industrially produced due to lack of the selection means.

Therefore, an object of the present invention is to provide a method for selecting a recombinant cell line which is genetically stable and homogeneous and which highly expresses a polypeptide, using a mammalian cell as a host. Still another object of the present invention is to use TGF-β, particularly h by recombinant DNA technique using mammalian cells as hosts.
It is to provide a method for mass-producing TGF-β1.

[0014]

Therefore, as a result of various studies on the conventional means for selecting a recombinant cell line, the present inventors have found that their methodologies have the following major defects. .. That is, when the vector containing the nucleotide sequence encoding the target polypeptide is first inserted into the host chromosomal DNA, the insertion position is any one point on the huge chromosomal DNA, and the resulting transduction Despite the fact that the cells are different from each other, the transduced cells are not regarded as individual but only as a group. This is in contrast to the fact that all vectors introduced into transformed cells, such as plasmid vectors in bacteria, behave uniformly as foreign genes. One of the problems that arises from the treatment of recombinant cells having individual characteristics as described above as a group is that recombinant cells with a high expression ability, which may have been present at the beginning of the transformation, are selected by the gene amplification method. There is a possibility that it will be culled and disappear.

The causes of selection in the selection process are as follows:
The following items 1) to 4) can be considered. 1) When a certain transduced cell produces a large amount of a gene product such as an enzyme that confers a desired polypeptide and drug resistance, the energy of cell activity to be used for the growth of the cell has priority over the production of these gene products. , The growth rate of the cells is slower than that of other transduced cells. As a result, the difference in growth rate between adjacent recombinant cells
Each time cell division is repeated, the cells are expanded sequentially, and finally cells with high expression ability and poor proliferation ability may be eliminated. For example, animal cells are colonies (about 10 mm in diameter)
Calculation requires about 10 cell divisions to form ³ cells). When the first transduced cells are adjacent to each other and the growth rate of the other recombinant cell line is about 20% slower than that of the other recombinant cell line, these cells form colonies of 1 mm. When formed, about 90% of the cells in this colony are the former cells and the latter cells are less than about 10%. When such a mixed cell line is repeatedly cultured, it is considered that the latter cells are selected and eventually disappear. Furthermore, when the animal cells are adherent cells, if they are adjacent to each other and have different growth rates as described above, the former cells surround the latter cells, and the latter cells are prevented from growing. It is considered that the number of cells is smaller than that in the above calculation.

2) The gene amplification method is based on the assumption that the amplified gene and the nucleotide sequence encoding the target polypeptide are amplified in the same manner, but whether this assumption is correct or not is unclear. In other words, there is a good possibility that some of the cell populations selectively amplify only the amplified gene, and in the presence of the selective drug, such cells are more likely to produce the desired polypeptide than other cells. Since there is no energy loss, it can grow favorably. Therefore, recombinant cells with low ability to produce the desired polypeptide will survive selectively.

3) When the nucleotide sequence encoding the target polypeptide is structurally unstable, or when producing the target polypeptide is unfavorable to the survival of the host cell, the target polypeptide is amplified during amplification of the amplified gene. It is possible that the nucleotide sequence that encodes L. From this, as in the case of 2), recombinant cells having a low production ability of the target polypeptide selectively survive.

4) When the vector is inserted at a position on the host chromosomal DNA which is preferable for the expression of the amplified gene but is not preferable for the expression of the nucleotide sequence encoding the target polypeptide, this recombinant cell line shows high drug resistance. However, since the ability to produce the desired polypeptide is low, undesired cells will advantageously grow. On the contrary, when it is not preferable for the expression of the amplified gene, but when it is inserted at a position preferable for the expression of the nucleotide sequence encoding the target polypeptide, the productivity of the target polypeptide is high, but the drug resistance becomes low. The cells are easily culled by increasing the concentration of the selective drug.

When the cells that are originally hetero and should be regarded as individual cells are treated as a group and selection by the gene amplification method is performed, the best target polypeptide in the group is produced for the reason described above. Since the cells are selected, it may be difficult to obtain cells having a sufficiently high expression ability as a result. Therefore, the present inventors have completed the present invention as a result of conducting research in order to solve the above-mentioned problems from the viewpoint of treating transduced mammalian cells not as a group but as an individual.

That is, the present invention provides a nucleotide sequence (1) encoding a polypeptide, a nucleotide sequence (2) which amplifies the nucleotide sequence (1) and imparts resistance to a selected drug to a mammalian cell, Also, a vector having a nucleotide sequence (3) that regulates the expression of these nucleotide sequences (1) and (2) was introduced into mammalian cells, and a polypeptide was highly expressed using a selective drug from the resulting recombinant cell group. In the case of selecting a sex recombinant cell, (a) a recombinant cell having drug resistance to the selected drug is separated from the recombinant cell group as a recombinant cell derived from each single cell, and (b) the single recombinant cell. Quantitatively confirming the expression of the polypeptide in the cell-derived recombinant cells,
After selecting as a candidate recombinant cell with high expression of the polypeptide, (c) the candidate recombinant cell with high expression is cultured in a medium in which the concentration of the selective drug is increased stepwise to obtain a polypeptide with high expression of the polypeptide. The present invention provides a method for producing a recombinant cell highly expressing a polypeptide, which comprises selecting a replacement cell.

Further, the present invention is characterized in that the TGF-β high-expressing recombinant cells obtained by the above selection method are cultured, and TGF-β is collected from the culture solution.
The present invention provides a method for manufacturing the same.

The polypeptide highly expressed by the method of the present invention is not particularly limited as long as it is a polypeptide which produces an active form when mammalian cells are used as a host. When a microorganism such as Escherichia coli is used as a host, an active form is not produced, and a polypeptide that requires modification such as sugar chain addition, amidation, phosphorylation or the like for activity expression, or a polypeptide having a complex higher-order structure Peptides are preferred. Specific examples thereof include tissue plasminogen activator, erythropoietin, interleukins, inhibin, growth factors such as TGF-β, activin, prorelaxin, superoxide dismutase, virus antigen and the like. The nucleotide sequences (1) encoding these polypeptides are not particularly limited as long as they are the nucleotide sequences encoding the active amino acid sequences of these polypeptides.

The nucleotide sequence (2), which amplifies the nucleotide sequence (1) and imparts resistance to a selective drug to a mammalian cell, is used when the target polypeptide is produced by using the mammalian cell as a host. There is no limitation as long as it can increase the copy number of the nucleotide sequence encoding the target polypeptide introduced into the cell adjacent thereto. For example, it is known that the nucleotide sequence encoding DHFR is amplified by methotrexate (hereinafter referred to as MTX) which is an analogue of folic acid which is its inhibitor. In addition, the nucleotide sequence encoding adenosine deaminase is deoxycoformycin, and the nucleotide sequence encoding aspartate transcarbamylase is N-.
Due to (phosphocetyl) -L-aspartate, the nucleotide sequence encoding glutamine synthetase is methionine sulfoximine, and the nucleotide sequence encoding metallothionein-I is a multi-drug resistant compound due to the action of heavy metals such as Ca ^2+. Nucleotide sequences encoding factors are known to be amplified in cells by the action of adreamycin, and therefore it is preferable to use these nucleotide sequences.

The nucleotide sequences (1) and (2)
The nucleotide sequence (3) that regulates the expression of E. coli preferably has a control region, a promoter, an enhancer, a translation initiation signal, a transcription termination site, a non-translation site, and the like. It is desirable that these sequences (3) are incorporated into the vector together with the nucleotide sequences (1) and (2) in an appropriate order.

The control region is a unique sequence of the 5'or 3'non-coding region of the eukaryotic gene which is involved in the control of either transcription or translation. In fact, most eukaryotic genes have a characteristic AT-rich sequence about 25-30 bases upstream from the transcription start site. And at the 3'end of most eukaryotic genes, AATAAA
There is a sequence, which is a signal to add a poly A chain to the 3'end of the transcribed mRNA.

The promoter for controlling transcription from the vector in mammalian cells is derived from viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, cytomegalovirus and the like. , Or a heterologous mammalian source such as the beta-actin promoter. The early and late promoters of the SV40 virus also contain the replication initiation region of the SV40 virus. Of course, in the present invention, a promoter obtained from the host cell or its related species can also be used. The promoter is linked to a nucleotide sequence to be expressed, for example, a nucleotide sequence that confers resistance to a selective drug on a host cell, and the direction of the promoter linkage and the distance from the nucleotide sequence can be appropriately determined.

Enhancers are inserted into the vector as needed. Enhancers are usually about 10-300 bp c
A nucleotide sequence that acts is and is an element that acts on a promoter and increases its transcription. Transcription of the nucleotide sequence encoding the polypeptide of interest by eukaryotic cells is enhanced by inserting an enhancer sequence into the vector. Enhancers are relatively orientation- and position-independent, and are 5'to the transcription unit [Reymins et al. Proc. Natl. Acad. Sci. USA 7
8 993 (1981)] and 3'side [Rusky Mo
l. Cell. Biol. 3 1108 (198
3)], or in the intron [Banergy Cell
133 729 (1983)] or in the structural gene [Osborn Mol. Cell. Biol. 4 12
93 (1984)]. Many enhancers from mammalian genes are known today (globin, elastase, albumin, alpha-fetoprotein and insulin). However, in general, an eukaryotic virus-derived enhancer is used. For example, an enhancer existing at the late site of the replication initiation region of SV40, an enhancer of the early promoter of cytomegalovirus, an enhancer existing at the late site of the replication initiation region of polyomavirus, and an adenovirus enhancer can be mentioned.

A translation initiation signal is also required for translation of the inserted nucleotide sequence encoding the polypeptide of interest. These signals include the ATG initiation codon and adjacent sequences. No additional translation initiation signal is required if the polypeptide of interest is introduced into the vector with its own initiation codon. Furthermore,
The vector has a transcription termination site and a non-translation site.

Construction of a vector having these sequences (1), (2) and (3) is carried out according to a conventional method. In addition, a vector that can be replicated in E. coli is convenient when preparing the vector.

Further, modification (eg, glycosylation) and processing of the target polypeptide are important for the function of the target polypeptide. For post-translational processing and modification of the polypeptide of interest, each host cell has a characteristic and specific mechanism, and it is necessary to select an appropriate cell line or host system.

The mammalian cell used in the present invention is not limited as long as it is a cell into which a vector can be introduced as a host. However, an animal cell of the same type of origin as the target polypeptide is advantageous in maintaining the higher-order structure of the target polypeptide, and in the case of a human-derived polypeptide, human-derived cells give better results. Easy to get.

The host cells preferably use cell lines which are usually immortalized, ie stabilized. Suitable host cells include monkey kidney CV transformed with SV40
I strain, human embryonic kidney cell line, baby hamster kidney cell, Chinese hamster ovary cell, monkey kidney cell, African green monkey kidney cell, human jaw cancer cell, dog kidney cell, buffalo rat hepatocyte, human lung cell, human liver Cells, mouse breast cancer cells, rat liver cancer cells, TR-1 cells and the like.

As the host-vector system, many mammalian host / expression vector systems which are already well known to those skilled in the art (ie, replication, transcription and translation of a DNA encoding a polypeptide of interest in an appropriate host cell) can be used. Vectors having a group of nucleotide sequences that regulate expression can be used to direct. The system is not limited to the above-described mammalian cells and vectors, but the system may be a viral expression vector / mammalian host cell system, or a non-viral promoter expression system vector / mammalian derived from the genome of mammalian cells. There are animal host cells (eg, mouse metallothionein promoter) and the like.

In the present invention, the method for introducing the vector into mammalian cells is not particularly limited, but examples thereof include the calcium-phosphate method, electroporation method, microinjection method, ribosome fusion method, protoplast fusion method and the like. After introducing the vector by these methods, it can be considered that the vector has been introduced into mammalian cells if the functions derived from various vectors are expressed.

In order to select a polypeptide-highly expressing recombinant cell from the recombinant cell group obtained as described above using a selective agent, the following procedures (a), (b) and (c) are carried out. Do. (A) A recombinant cell having drug resistance to the selected drug is isolated from the recombinant cell group as a recombinant cell derived from each single cell. (B) The expression of the polypeptide is quantitatively confirmed for the recombinant cells derived from the single cell, and candidate recombinant cells with high polypeptide expression are selected. (C) Recombinant cells highly expressing a polypeptide are selected by culturing the candidate recombinant cells having a high expression level in a medium in which the concentration of the selective drug is gradually increased.

Prior to carrying out the operation (a), as a preliminary test, the lethal concentration of the host mammalian cells with respect to the selective drug is determined, and then the vector-treated mammalian cells are introduced on the basis of the lethal concentration of the selective drug. That is, the transduced cells are selected, and the efficiency with which the vector is introduced, that is, the transduction rate is determined. Here, the lethal concentration of the drug in the mammalian cell that is the host is determined by a conventionally known method. In addition, the “lethal concentration of drug” in this case means the concentration at which the host cell is killed by the drug related to drug resistance that is expressed based on the gene that is the target of gene amplification described in step (c). When the target polypeptide is hTGF-β1, for a human-derived cell that is a host, for example, a nucleotide sequence encoding DHFR is used as a nucleotide sequence having a gene-amplifying action, and a DHFR competitive inhibitor MTX is used as a selective agent. Using the MTX of the cells
The lethal concentration by The transduction rate is determined by the ratio of the number of cells having drug resistance expressed by the introduction of the vector to the number of cells subjected to the vector introduction treatment. This is also obtained by a conventionally known method.

The operation (a) is a treatment of introducing a vector into host mammalian cells, and is calculated from the transduction rate,
It is carried out by inoculating the culture medium containing the selective drug with cells diluted to a density sufficient to separate individual single cell-derived colonies generated in the incubator, and culturing until the colonies have a size that can be separated. .. When using a normal incubator, adjacent colonies are often mixed with each other, and therefore it is preferable to use one in which the bottom of the incubator is partitioned by many wall surfaces. For example, a hybridoma dish having 700 cells can be used, but an animal cell culture plate having a large number of cells can also be used. that time,
A cell suspension may be prepared and cultured at a cell density at which one single cell-derived colony appears in one cell. In the culture, in order to recover the damage caused by the transduction treatment, it is preferable that the culture is performed in a medium to which the selective drug is not added, and then the medium containing the selective drug is replaced and cultured. The culture conditions may be such that host cells can grow. For example, it is carried out by culturing using RPMI1640 medium supplemented with bovine serum, Eagle medium supplemented with bovine serum, serum-free medium and the like.

Next, the operation (b) is carried out by quantifying the production amount of the target polypeptide in the single cell-derived recombinant cells obtained in (a). When the target polypeptide is hTGF-β1, it is preferable to determine the growth inhibitory activity of mink lung epithelial cells MvlLu (hereinafter referred to as CCL-64 cells) by simply measuring. The development of this simple measurement method has made it possible to process a large number of recombinant cells in a short time. Traditionally, neutral red is taken up in proportion to the number of undamaged cells,
It is known that the incorporated neutral red is extracted with an ethanol solution containing monosodium phosphate, and the amount of neutral red, that is, the number of living cells can be determined by measuring the absorbance of the extract at 540 nm. [Babich: J. Pharm. Sci. 795
92 (1990)]. Therefore, by utilizing this feature, CCL
To the -64 cell suspension, a culture supernatant of recombinant cells or a solution containing platelet-derived hTGF-β1 as a control was added, and after culture, stained with a neutral red solution, and the absorbance was measured with a spectrophotometer. To do. Then, the number of living cells of CCL-64 is determined, and compared with the number of living cells of CCL-64 when a known amount of platelet-derived hTGF-β1 is added, the amount of hTGF-β1 in the culture supernatant of the recombinant cells is determined. .. Then, a cell group that produces a large amount of polypeptide is used as a candidate recombinant cell that highly expresses the polypeptide.

The operation (c) is a step of subjecting the highly expressing candidate recombinant cells obtained in the operation (b) to a gene amplification treatment to select a target highly expressing recombinant cell. The gene amplification treatment may be carried out by culturing in a medium containing a selective drug at a concentration higher than the drug concentration used in the operation (a), and selecting cells having high expression of the target polypeptide from the surviving cells. That is, while increasing the concentration of the selective drug in the medium kills many cells, some cells survive by increasing the copy number of the nucleotide sequence involved in drug resistance. At that time, the copy number of the nucleotide sequence adjacent to the nucleotide sequence involved in drug resistance is also increased. This means that the copy number of the nucleotide sequence encoding the target polypeptide adjacent to the nucleotide sequence involved in the drug resistance is amplified, and as a result, the productivity of the target polypeptide is increased. At this time as well, an increase in expression ability can be confirmed by quantifying the expression level of the target polypeptide in the same manner as in the operation (b).

When the target polypeptide is hTGF-β1, the number of recombinant cells subjected to this operation (c) is 9 cells (third screening). The 9 cells showed resistance to each concentration of the selective drug, and the proliferating cells were measured for the expression level of hTGF-β1. As a result, the MTX resistance of recombinant cells derived from each single cell and h
Looking at the expression level of TGF-β1, it is clear that not only there is a large difference among the cells, but also the expression level of the target polypeptide is different among cells showing the same level of drug resistance. Then, 29Al, which is a transduced strain selected by a conventional selection method using MTX and used as a positive control in Examples, has an MTX tolerance of 5 μM, and the expression level of hTGF-β1 is about 1 mg / l. However, when the method for isolating highly expressed strains of the present invention was used, there were 3 strains that showed an expression ability higher than 29Al with the same 5 μM resistance. Further, among the strains exhibiting the same expression ability, the strains having acquired higher drug resistance could be isolated. Furthermore, one of the cell lines resistant to MTX of 150 μM, which is the highest drug resistance, acquired 6 times the ability to express the target polypeptide as compared with the positive control.

By culturing the highly expressing recombinant cells obtained by the present invention and collecting the target polypeptide from the culture solution, the target polypeptide can be efficiently produced in a large amount.

As a method for culturing the recombinant cell line obtained by the present invention, a known method may be used. Any medium can be used as long as the recombinant cells can grow. For example, a culture can be obtained by culturing using RPMI1640 medium supplemented with bovine serum, Eagle medium supplemented with bovine serum, serum-free medium or the like. By subjecting the obtained culture to operations such as centrifugation and membrane filtration according to a conventional method, a culture supernatant containing a target polypeptide, for example, hTGF-β1 can be separated and obtained. Here, in the case of hTGF-β1, since the hTGF-β1 precursor is a secretory protein having a signal sequence, it is necessary to separate and obtain the culture supernatant. Therefore, hTGF-β1 produced by recombinant cells is also a medium. It is based on the idea of being secreted into.

HTGF from the culture supernatant thus obtained
The production of a crude sample containing TGF-β represented by -β1
It can be carried out by appropriately combining various conventionally known operations. The operation includes, for example, treatment with a protein precipitation agent such as salting out with ammonium sulfate, centrifugation, dialysis, ultrafiltration,
An example is concentration.

Also, the purification operation of the crude product is carried out by various known methods such as gel filtration, liquid chromatography, electrophoresis, affinity chromatography, chromatofocusing, reverse phase high performance liquid chromatography, ion exchange column chromatography, and the like. Can be performed by a combination of the above. More specifically, for example, TS
Cation exchange column chromatography using K gel SP-Toyopearl 650M (manufactured by Tosoh Corp.), HA
-1000 (manufactured by Tosoh Corporation) and the like, hydroxyapatite chromatography, Vydac protein C
4 columns (The Separations Group
The product can be purified by reverse-phase high performance liquid chromatography or the like using a product manufactured by Company p. Also SDS-PAGE
Is the method of Laemli [Laemmli, U .; K. , Nat
ure 227 680 (1970)] and the like.

As mentioned above, TGF-β is 10%.
It can be measured by using mink lung epithelial cells maintained in Dulbecco's modified Eagle's medium containing (v / v) fetal bovine serum. More specifically, 0.
Release of the cells from tissue culture plates by addition of 05% (w / v) trypsin and 0.1% (w / v) EDTA phosphate buffered saline (PBS) without calcium and magnesium. And inoculate 0.5 ml of the resulting cells (approximately 5 × 10 ⁴ cells / ml) into 24-well tissue culture plates. At this point, the platelet-derived standard h
Add TGF-β1 and unknown sample to be assayed. 24% in a 5% (v / v) CO ₂ atmosphere at 37 ° C.
After culturing for an hour, 50 μl of 0.01 mCi / ml ³ H-thymidine, specific activity = 6.7 Ci / mM) is added to each well. After about 16 hours, cold 10% (w /
v) Wash each well twice with trichloroacetic acid. After removing trichloroacetic acid, 1.0 N sodium hydroxide 0.5
Add ml to lyse the cells. Take 450 μl of lysed cells and measure with a liquid scintillation counter. Further, as described above, the standard hTGF-β1 derived from platelets and an unknown sample to be assayed were added to the mink lung epithelial cell suspension, and at the same time, a 0.2% neutral red solution was added, and the mixture was incubated at 37 ° C. Incubate for 24 hours in a 5% (v / v) CO ₂ atmosphere. And after washing the cells, 7.8%
The viability of mink lung epithelial cells was determined by measuring the absorbance at 540 nm of a solution extracted with a 50% ethanol solution containing monosodium phosphate, and from this value, hTGF- in the sample was determined.
The β1 amount can be quantitatively measured. This quantification method is based on the fact that the proliferation of mink lung epithelial cells is inhibited in the presence of TGF-β1.

[0046]

According to the present invention, all the problems of selection that occur during the process of selecting highly expressing recombinant cells from the above-mentioned recombinant cells are solved. That is, regarding item 1) regarding the growth rate of cells, at the time of subjecting the transduced cells to a selection method utilizing drug resistance, the transduced cells are grown under the condition that single-cell-derived colonies grow. It was solved by letting. This is much more time-consuming than the conventional method of selecting a transduced cell only by the expression of a selection marker such as drug resistance, but it is a selection method in the form of taking into consideration the characteristics peculiar to the cell. Is extremely large. From this, a very useful idea for establishing a highly-expressing recombinant cell line of a useful substance that has not been obtained hitherto is shown. That is, it shows an excellent selection method capable of coping with various problems derived from cell-specific properties other than the difference in proliferation rate described in item 1). Regarding item 2) in the isolation of highly expressing strains, the transduced cells were subjected to a selection method utilizing drug resistance, and then subjected to selection based on the expression amount of the target polypeptide for each single cell-derived colony. Thus, not only selection of drug resistance but also expression level of target polypeptide could be confirmed in parallel. This can be done for the first time in the present invention by establishing a method for easily measuring the expression level of the target polypeptide, hTGF-β1. This can be achieved for the first time by eliminating the genetic difference between the recombinant cells derived from each single cell and adjusting the conditions for obtaining the most preferable recombinant cells. This allows selection of a highly expressing recombinant cell line without being affected by changes other than the expression level and drug resistance in the amplification of the nucleotide sequence encoding the amplified gene and the target polypeptide, such as changes in growth rate. It was the first condition that could be achieved. Also, regarding the problem of the loss of the nucleotide sequence encoding the target polypeptide in item 3) in the isolation of the high expression strain, the target polypeptide is not expressed in the one in which the gene has been dropped, so that it can be easily eliminated by screening. Has been resolved. By these, it became possible to select cells having both increased drug resistance and high expression, and it was possible to solve item 4). This shows that drug resistance is also comparable to recombinant cells obtained by the conventional selection method using the same host and the same vector, that is, the selection method in which transduced cells are grouped. 30-fold in hTGF-β1
It is also clear from the expression level of 6 times.

[0047]

EXAMPLES Examples will be described below to specifically explain the present invention. However, the present invention is not limited to the examples.

Example 1 Selection of strains highly expressing TGF-β1 which is one of the target polypeptides a) hTGF-β1 expression plasmid and cells hTGF-β1 expression plasmid CMV-TGF-β-S
V2-DHFR was donated by Dr. Gopal of Otsuka America Pharmaceutical, Inc. [T. V. Gopal et al. In Vitro Cell.
Dev. Biol. 25 1147-1154 (198)
9)]. CMV-TGF-β-SV2-DHFR is a plasmid of about 7.25 Kb, hTG cloned from human erythroid leukemia cell line (HEL) mRNA.
It has F-β1 cDNA (1.3 Kb), upstream of which is linked to the cytomegalovirus early promoter, and downstream of which is linked SV40t splice and poly A addition sequence derived from plasmid pMSG (Pharmacia). Has been done. Furthermore, the plasmid is a wild type human DH
It also contains the FR gene, with the SV40 early promoter upstream and the SV40t splice and poly A addition sequence downstream. In addition, as a host cell, a human fetal kidney-derived cell transformed with adenovirus (accession number 293 American Type Culture Collection: hereinafter referred to as 293 cell)
Was used. It has been confirmed that 293 cells are quickly killed when cultured in the presence of 200 nM or more of methotrexate (MTX).

B) Comparative Example As a positive control of the present invention, CMV-TG was added to 293 cells.
F-β-SV2-DHFR was introduced, and 29A1 cells obtained by the selection method using MTX were used. 29A1 cells are a cell line donated by Dr. Gopar and were obtained by the method described below. First, 1 × 10 ⁶ 293
The cells were inoculated into 10 ml of RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 300 μg / l of L-glutamine and 10% (v / v) of fetal bovine serum (FBS: Biocell), and 5% (v / v). After culturing at 37 ° C. for 3 days in a CO ₂ atmosphere, the medium was changed to RPMI1640 medium and further cultivated for 24 hours under the same conditions to form a cell monolayer in an incubator (φ100 mm petri dish). In the following, RPM
RPMI164 with I1640 or FBS added
When we call 0 medium, these are all 300 μg / l of L
-It contains glutamine. Example 1. a) Plasmid CMV-TGF-β-SV2-DHFR 40μ
g was dissolved in 1 ml of water, and 0.12 ml of 1M calcium chloride was added thereto with good stirring to prepare a plasmid solution. In addition, 2 ml of HBS buffer solution (280 mM sodium chloride 50 mM HEPES pH 7.07) 1 ml and 1
00 × PO ₄ solution (70mM Na ₂ HPO ₄ and 70m
The plasmid solution was slowly added dropwise to a mixed solution of 25 μl of M NaH ₂ PO ₄ ) while blowing air with a pipette, and the obtained suspension was left on ice for 30 minutes and then gently stirred. The suspension was then gently cast onto the cell monolayer. After leaving at room temperature for about 30 minutes, RP
10 ml of MI1640 medium was added to the incubator. Then 5%
After culturing at 37 ° C. for 4 hours in a (v / v) CO ₂ atmosphere, the medium was removed from the incubator, 10 ml of RPMI1640 medium containing 25% (v / v) glycerol was added, and the mixture was allowed to stand at room temperature for 100 seconds. The cells are then treated with 20 ml of RPMI1
After washing twice with 640 medium, RPMI1640 medium 1
0 ml was added and the cells were cultured under a 5% (v / v) CO ₂ atmosphere at 37 ° C. for 48 hours. Furthermore, the medium in the above incubator is replaced with 20
Replace with selective medium containing 0 nM MTX, 5% (v / v)
Culturing was continued for 14 days at 37 ° C. in a CO ₂ atmosphere. After culturing, a large number of colonies thought to be derived from the transduced cells were formed in the incubator. Next, the transduced cells (hTGF-β1 expressing strain) were selected by further increasing the MTX concentration in the medium stepwise. First, 0.02% (v /
v) 0.3 ml of PBS supplemented with EDTA and 0.1% (v / v) trypsin (hereinafter referred to as a trypsin-EDTA treatment solution) was added, and treated at 25 ° C. for 2 minutes to release it.
Approximately 1 × 10 ⁵ cells of the cells were cultured in a selective medium containing 300 nM MTX under a 5% (v / v) CO ₂ atmosphere at 37 ° C. for 14 hours.
Cultured for a day. After the cells were released from the colonies generated in the incubator again with a trypsin-EDTA treatment solution as described above, the cells were cultured in a selective medium containing 600 nM MTX until colonies were formed. These series of operations are sequentially MT
By repeating using a selective medium with an increased X concentration,
Finally, as a cell line resistant to 5 μM MTX, 29
A1 strain was obtained. The above selection medium is MT of various concentrations
RPMI 16 containing X and dialyzed FBS 10% (v / v)
It is called 40 medium. The dialyzed FBS was prepared by dialyzing FBS against PBS and then against Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 4 ° C. for 24 hours.

C) Bioassay Method for hTGF-β1 As a bioassay method for hTGF-β1, hTGF-β1 was used.
Mink lung epithelial cell Mv1L whose growth is suppressed by β1
u (Accession number CCL-64, American Type Culture Collection: hereinafter CCL-64 cells)
The method of using the growth inhibitory activity of the sample for the above as an index was established as follows. CCL-64 cells are non-mobilized FBS10
The cells were cultured in 10 ml of Eagle's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% (v / v) in a 5% (v / v) CO ₂ atmosphere at 37 ° C. for 3 days. Then, the cells were treated with 0.4 ml of trypsin-EDTA treatment solution at 37 ° C. for 4 minutes, and then the cell suspension prepared in Eagle MEM medium at 4 × 10 ⁴ cells / ml was added to a 24-well plate in 0.5 ml portions. Was added.
After culturing at 37 ° C. for 2 hours in a 5% (v / v) CO ₂ atmosphere, a sample solution or a known amount of platelet-derived hTGF-β1
0.1 ml of a solution containing ## STR3 ## was added to each well and gently mixed with a pipette, and then 37% in a 5% (v / v) CO ₂ atmosphere.
Culturing was carried out at 0 ° C for 3 days. Next, 0.25 ml of Neutral Red reagent solution [0.05% (w / v) Neutral Red (manufactured by Kanto Kagaku), 4.5 g / l Eagle MEM medium]
Was added to each well and incubated at 37 ° C. for 2 hours to obtain CCL-64
The cells were stained. And PBS (+) solution (PB containing 1 mM calcium chloride and 0.5 mM magnesium chloride)
S) twice, then 7.8% (w / v) phosphoric acid 1
0.5 ml of 50% (v / v) ethanol solution containing sodium (hereinafter referred to as sodium phosphate ethanol solution)
Neutral red that had been added and taken up in the cells was extracted, and the result was measured with a spectrophotometer (manufactured by Titertek) at 540.
The absorbance at nm was measured. FIG. 1 shows the growth inhibition curve of hTGF-β1 for CCL-64 cells using neutral red. And the platelet-derived hTGF-β of FIG.
The hTGF-β1 expression level of each transformant was calculated by comparison with the CCL-64 cell growth prevention standard curve of 1.
FIG. 1 is a growth inhibition curve of hTGF-β1 for CCL-64 cells using Neutral Red. In the figure, the vertical axis is the absorbance at 540 nm and the horizontal axis is hTGF-β.
1 shows the concentration (ng / ml), and the curves in the figure are absorbance and hT.
The correlation curve of GF-β1 is shown. By the above bioassay method, hTGF-β was obtained from the 29A1 strain of Comparative Example.
The expression level of 1 activity was examined. That is, 1 x 10 ⁷ 29A
1 cell was inoculated into a petri dish (φ10 cm) containing 10 ml of RPMI1640 medium, cultured at 37 ° C. for 24 hours in a 5% (v / v) CO ₂ atmosphere, and 1 ml of a 1 M acetic acid solution was added to 1 ml of the obtained culture supernatant. After adding 2 hours at 4 ℃,
The mixture was neutralized by adding 0.2 ml of 1N sodium hydroxide solution.
As a result of the above bioassay using this as a measurement sample, the expression level of hTGF-β1 activity in the culture supernatant of the 29A1 strain was 1.0 mg / l.

D) hTG by single cell isolation and growth method
Selection of F-β1 highly expressing strain 1 × 10 ⁶ 293 cells with FBS 1% (v / v) added R
After culturing in 10 ml of PMI1640 medium in a 5% (v / v) CO ₂ atmosphere at 37 ° C. for 3 days, the medium was RPMI16.
The medium was changed to 40 ml of 40 medium and further cultured for 24 hours to form a cell monolayer in the incubator. Example 1 a) plasmid CMV-TGF-β-SV2-DHF
After dissolving 40 μg of R in 1 ml of water, 0.12 ml of 1M calcium chloride was added while stirring well to prepare a plasmid solution. And 2 × HBS buffer (280 mM sodium chloride and 50 mM HEPES; pH 7.0)
7] 1 ml and 100x phosphoric acid solution (solution containing 70 mM disodium phosphate and 70 mM monosodium phosphate)
The plasmid solution was slowly added dropwise to 25 μl of the mixed solution while blowing air with a pipette, and the obtained suspension was allowed to stand on ice for 30 minutes and then gently stirred. The suspension was then gently cast onto the cell monolayer. About 30
After leaving at room temperature for 10 minutes, further 10 ml of RPMI1640 medium
Was added to the incubator. Then, after culturing at 37 ° C. for 4 hours in a 5% (v / v) CO ₂ atmosphere, the medium was removed from the incubator, and RPMI164 containing 25% (v / v) glycerol was added.
10 ml of 0 medium was added, and the mixture was left standing at room temperature for 100 seconds. Then, the cells were washed twice with 20 ml of RPMI1640 medium and then 10 ml of RPMI1640 medium was added to give 5% (v / v) C.
The cells were cultured under an O ₂ atmosphere at 37 ° C. for 48 hours. Then, the medium was removed from the incubator, and cells released by treatment with 0.3 ml of trypsin-EDTA treatment solution for 3 minutes at 37 ° C. were suspended in 10 ml of RPMI1640 medium. It was confirmed by preliminary studies that about 500 colonies were obtained by culturing the cell suspension obtained by the above transduction treatment in a selection medium supplemented with 250 nM MTX. Therefore, in order to ensure the isolation of individual colonies derived from a single cell, the suspension was transferred to a hybridoma dish (φ10 cm Greiner) and incubated at 37 ° C. for 24 hours in a 5% (v / v) CO ₂ atmosphere. Cultured. The hybridoma dish used here has 700 cells formed on the bottom of the dish by a lattice-like shallow cut, and the medium can move freely inside the dish, but the cells attached to the bottom of the dish move over the cut. There is nothing to do. Then, the medium was removed from the incubator, and 10 ml of selective medium containing 250 nM MTX was added.
12% at 37 ° C. under a 5% (v / v) CO ₂ atmosphere.
Cultured for a day. As a result, since single cell-derived colonies were formed in most cells of the hybridoma dish, each colony was individually inoculated into each well of a 96-well plate (manufactured by Nunc) containing 0.2 ml of RPMI1640 medium, 4 at 37 ° C under 5% (v / v) CO ₂ atmosphere
Cultured for a day. As a result, 11712 single cell-derived transduced cell lines were isolated. Selection of hTGF-β1 high-producing strains was performed as a primary screen for each of the obtained transduced strains. Example 1. The expression level of hTGF-β1 was measured by the bioassay method described in c), and the 29A1 cell line was evaluated as a positive control. As a result, 988 hTGF-β1 producing transduced strains showing the same or higher hTGF-β1 expression level as the 29A1 cell line were obtained.

Secondary screening: Cells of 988 candidate strains obtained by the primary screening were treated with trypsin-ED.
RPMI1640 for each cell line treated with TA treatment solution
A cell suspension was prepared using the medium. 0.5 ml of cell suspension (10 ⁴ cells per well) was added to a 24-well plate (manufactured by Costor) and 5% (v / v) CO was added.
_It was cultured at 37 ° C. for 24 hours in ₂ atmospheres. Remove the medium,
Replace with 2 ml of selective medium containing 400 nM MTX, 5%
(V / v) After culturing at 37 ° C. for 18 days in a CO ₂ atmosphere,
The medium was removed, 0.1 ml of a trypsin-EDTA treatment solution was added, and the mixture was treated at 37 ° C. for 2 minutes to prepare a cell suspension. 2 ml of RPMI1640 medium was inoculated into a 24-well plate to give 1 × 10 ⁴ cells per well, and 5%
(V / v) CO ₂ atmosphere, it culture | cultivated at 37 degreeC for 24 hours. And hTGF in the culture supernatant separated from each well
-The expression level of β1 activity was CCL-established in Example c)
It was measured by an assay method using 64 cells. FIG. 2 shows the first embodiment. 54 measured according to the bioassay method described in c.
HTGF-β1 of each cell line based on the absorbance at 0 nm
It is the result of displaying the activity by frequency distribution, and the vertical axis shows the number of cell lines. Further, in FIG. 2, the thin arrows indicate the activity of 0.1 and 1 ng / ml platelet-derived hTGF-β1, and the thick arrow indicates the activity of the culture supernatant of 29A1 strain. The following third screening was carried out for 9 cell lines showing an expression level of hTGF-β1 activity equivalent to or higher than that of the positive control 29A1 cells.

Third screening: Each cell obtained by the second screening was treated with a trypsin-EDTA treatment solution, and a cell suspension was prepared for each cell using RPMI1640 medium. Using the cell suspension, 1 ×
10 ⁵ cells / dish to become like tissue culture dish (φ
5% (v / v) CO _{2 in} 100 mm Falcon)
After culturing at 37 ° C. for 24 hours in the atmosphere, the cells were attached to the dish. Next, selection medium 1 containing 500 nM MTX
The cells were replaced with 0 ml and cultured at 37 ° C. for 15 days in a 5% (v / v) CO ₂ atmosphere. And for the cells in each dish,
Add 0.3 ml of trypsin-EDTA treatment solution, and add 37
A cell suspension was prepared by treatment at 2 ° C for 2 minutes. The cell suspension was inoculated into each of two cell culture petri dishes at 1 × 10 ⁷ cells and cultured. One plate was added with 10 ml of RPMI1640 medium and cultured in a 5% (v / v) CO ₂ atmosphere for 24 hours, and then the culture supernatant was separated. The expression level of hTGF-β1 activity was measured by the bioassay method described in c). 1 μM MTX for the remaining one
Was added to the medium, and the cells were cultured under the same conditions until colony formation was confirmed. The concentration of MTX in the selective medium was 1 μM, 5 μM, 50 μM and 15 for 9 cell lines.
A strain having a high degree of MTX resistance was selected by sequentially increasing the concentration to 0 μM and repeating the above operation, and as a result, a strain showing resistance to 150 μM MTX was finally obtained.
This is the 29E6-2 strain [Mikoken Kenjoyori No. 3218 (FE
RM BP-3218) deposited with Institute of Microbial Technology, Institute of Industrial Science and Technology]. As shown in Table 1, 29E6-
The expression level of hTGF-β1 in the culture supernatants of the two strains was 6.
The value was 04 mg / l (64 μg / 10 ⁷ cells / 24 h), which was the highest among all candidate strains. And 150 μM M
Growth in the presence of TX was similar to that of parental strain 293 in the absence of MTX. That is, the 29E6-2 strain is 10% RPMI1640 medium containing 150 μM MTX.
ml or 293 strains are RPMI 1640 medium 10
For each ml, 1.0 × 10 ⁵ (cells / ml) was prepared and 5% (v / v
v) The cells were cultured in an atmosphere at 37 ° C for 4 days. The resulting adherent cells were treated with EDTA-trypsin, and the number of cells was measured with a hemocytometer (Kayagaki Irikaka Kogyo Co., Ltd.). As a result 2
9E6-2 strain has 17.0 × 10 ⁵ cells / ml, 29
The 3 strains were almost equal to 15.5 × 10 ⁵ cells / ml.

[0054]

[Table 1]

Example 2 Purification and Isolation of Recombinant hTGF-β1 a) Acid Ethanol Crude Extraction The 29E6-2 cells obtained in Example 1 were treated with RPMI164.
Inoculate 5 × 10 ⁵ cells per 10 ml of 0 medium, 5% (v / v)
It was cultured at 37 ° C. for 4 days in a CO ₂ atmosphere. The culture solution 1
1 was centrifuged with RPR10-2 rotor (manufactured by Hitachi Koki Co., Ltd.) for 30 minutes at 9500 rpm, and
4 l of a 70% (v / v) ethanol solution containing 5N hydrochloric acid was added. The resulting suspension (pH 3.5) was thoroughly stirred and then left overnight at 4 ° C. Then, 1N sodium hydroxide solution was added to adjust the pH to 5.5, ethanol was distilled off under reduced pressure, and the precipitate was removed by centrifugation at 9500 rpm at 4 ° C. for 45 minutes. Crude extract obtained (2.5 l)
The protein content in the
Lowry method (Lowry OH et al. (1951)) using io-Rad Laboratories.
J. Biol. Chem. As a result of measurement in Example 193 256), the amount was 687 mg, and the amount of hTGF-β1 activity was 1. As a result of measurement by the method c), it was 5.5 mg.

B) Cation exchange separation The resulting crude extract was then loaded onto a SP-Toyopearl column (φ45 mm × 50 mm) pre-equilibrated with 25 mM sodium acetate buffer (pH 5.5), and 0 → 3.0M
Eluting with 25 mM sodium acetate buffer containing a linear gradient of sodium chloride for 140 minutes at a flow rate of 4 ml / min,
Fractions were collected every 2 minutes. The result of SP-Toyopearl column chromatography is shown in FIG. In FIG. 3, the left vertical axis is the absorbance at 280 nm, and the right vertical axis is hTGF-.
The β1 activity and the sodium chloride concentration (M) in the solution are plotted on the horizontal axis. The solid line is the absorbance at 280 nm,
White circles indicate hTGF-β1 activity, and broken lines indicate sodium chloride concentration. From this FIG. 3, fractions 31 to 39 (72
ml) showed hTGF-β1 activity, these fractions were pooled. The total amount of protein in the pooled sample was about 4.2 mg, and the amount of hTGF-β1 activity was 4.1 mg.

C) Reversed phase HPLC The pH of the sample obtained in b) was adjusted to 2.5 with 6N hydrochloric acid, and then the sample was applied to a Vydac Protein C4 column (4.6 mm × 2).
50mm) (The Separations Group
Applied by HPLC (manufactured by Waters), and two kinds of eluents, that is, 0.1% (v / v) trifluoroacetic acid (hereinafter referred to as “TFA”) aqueous solution [A]. And 90% (v / v) containing 0.1% (v / v) TFA
/ V) using [B] which is an aqueous acetonitrile solution.
Elution was at a rate of 0 ml / min. The elution condition is 100 → 70% [A] / 0 → 30% [B] for the first 5 minutes, and 70 → 55% [A] / 30 → 45% [B] for 55 minutes.
In addition, 55 → 0% [A] / 45 → 100 for 5 minutes
% [B] was used as the linear gradient. Fractions were collected every minute and the absorbance at 280 nm was measured. In addition, the amount of hTGF-β1 activity in each fraction was determined as in Example 1. It was measured by the method of c). The result is shown in FIG. In FIG. 4, the left vertical axis shows the absorbance at 280 nm, the right vertical axis shows the hTGF-β1 activity amount, and the concentration of acetonitrile (%) in the eluate, and the horizontal axis shows the fraction number. The solid line is the absorbance at 280 nm, the straight line with white circles is hTG
The amount of F-β1 activity, the broken line indicates the concentration of acetonitrile. From this figure, fractions 22 to 26 show hTGF-β1 activity, and these fractions were pooled. The amount of hTGF-β1 activity in the pooled sample was 2 mg. Hereinafter, this sample is referred to as purified recombinant hTGF-β1.
Table 2 shows the outline of the purification process.

[0058]

[Table 2]

Example 3 Identification of Physical Properties of Purified Recombinant hTGF-β1 a) SDS-Polyacrylamide Gel Analysis 5.0% of 5.0% of purified recombinant hTGF-β1 and platelet-derived hTGF-β1 obtained in Example 2 ( v / v) 2
-Mercaptoethanol (600 μl) was added, and the mixture was reacted at 100 ° C. for 5 minutes for reduction. Then, each reduced sample and each non-reduced sample were respectively added to Laemml (Laemml).
i, U. K. ) Et al. (Natura 227 680)
Electrophoresis was carried out using SDS-polyacrylamide gel (18%, w / v) prepared by ~ 685 (1970)). Further, the gel was applied to Coomassie brilliant blue R-250 (Coomasie brilliant).
tblue R-250) and Weber (Web
er. K. ) Et al. (J. Biol. Chem., 244 ).
4406 to 4412 (1969)).
The result is shown in FIG. In the figure, samples are molecular weight markers from the left, reduced platelet-derived hTGF-β1, reduced recombinant hTGF-β1, and non-reduced platelet-derived hTGF-β.
1, non-reducing recombinant hTGF-β1, and the numbers indicate the molecular weight determined from the molecular weight marker. By recombinant SDS-polyacrylamide gel electrophoresis as described above, recombinant hTGF
-Β1 showed the same behavior as that of platelet-derived hTGF-β1 under reducing or non-reducing conditions, and was a single band. From this, hTGF-obtained by the present invention
It was confirmed that β1 was homogeneous and had a purity of 95% or more. Furthermore, the recombinant hTGF-β1 of the present invention is a platelet-derived h
Similar to TGF-β1, it was confirmed to be a 25 K dalton dimer under non-reducing conditions and a 12.5 K dalton monomer under reducing conditions.

B) Comparison of reverse phase HPLC elution patterns 5 μg each of the purified recombinant hTGF-β1 and the platelet-derived hTGF-β1 obtained in Example 2 was added to 75% of [A] described in Example 2-c) and [B]. Was applied to a Vydac Protein C4 column that had been pre-equilibrated with a 25% mixture. The elution condition is 75 → 50% [A] for the first 30 minutes.
/ 25 → 50% [B], 50 → 0% for the next 5 minutes
The linear gradient was [A] / 50 → 100% [B]. The result is shown in FIG. In the figure, the left vertical axis is 2
Absorbance at 80 nm, right vertical axis shows acetonitrile concentration (%)
And the horizontal axis indicates the elution time (minutes), the continuous line indicates the absorbance, and the broken line indicates the acetonitrile concentration. The upper part is purified recombinant hTGF-β1, and the lower part is platelet-derived hTGF-β1.
The behavior of β1 was shown. Comparing these, purified recombinant h
The elution times of TGF-β1 and platelet-derived hTGF-β1 were completely the same.

C) Amino acid analysis 6N HC1 (manufactured by Wako Pure Chemical Industries) containing 1% phenol (manufactured by Wako Pure Chemical Industries) containing 20 μg of each of the purified recombinant hTGF-β1 and platelet-derived hTGF-β1 obtained in Example 2. 200
Teflon sealed glass hydrolysis vial containing μl (W
The product was hydrolyzed at 130 ° C. for 24 hours in the presence of N ₂ . The results of analysis of the obtained hydrolyzate with a 6300E type amino acid analyzer (manufactured by Beckman) are shown in Table 3. The theoretical amino acid composition of purified recombinant hTGF-β1 (Derynck. R. et al. (1985) Nature).
316 701) and the platelet-derived hTGF-β1 amino acid composition.

[0062]

[Table 3]

D) N-Terminal Amino Acid Sequence Analysis 20 μg of the purified recombinant hTGF-β1 obtained in Example 2 was subjected to reductive carboxamide methylation and then model 470.
Amino acid sequence analysis was performed using an A amino acid sequencer (manufactured by Applied Biosystems). The result is shown in SEQ ID NO: 1. The amino acid sequence of purified recombinant hTGF-β1 was determined from the N terminus to the 61st position, and this sequence was the amino acid sequence of hTGF-β1 shown in SEQ ID NO: 2 (the 1-112th position) (Derynck. R. et al.
985) It completely matched the sequence below the N terminus of Nature 316 701). From the results of the above SDS-polyacrylamide gel analysis, reverse phase HPLC elution pattern comparison, amino acid analysis and N-terminal amino acid sequence analysis, it was confirmed that purified recombinant hTGF-β1 has the same physical properties as platelet-derived hTGF-β1. Was done.

Example 4 Identification of Biological Activity of Purified Recombinant hTGF-β1 a) CCL-64 Cell Growth Inhibitory Activity Measurement Using a flat bottom 24-well tissue culture plate (Costar), 4 × 10 ³ CCLs per well were used. -64 cells and 500 ml of Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% immobilized FBS were added. After culturing at 37 ° C for 2 hours,
The purified recombinant hTGF-β1 obtained in Example 2 was added to Eagle M
It was diluted with EM medium and added to each well. 5% (v
/ V) Incubated at 37 ° C. for 72 hours in a CO ₂ atmosphere. Next, 250 μl of Neutral Red reagent solution was added to each well and incubated at 37 ° C. for 2 hours. And the cells are PBS
After washing twice with (+) solution, 500 μl of sodium phosphate ethanol solution was added, and the absorbance at 540 nm was measured. The result is shown in FIG. 7. In FIG. 7, the vertical axis is 540
Absorbance in nm, horizontal axis is hTGF-β1 concentration (ng / ml)
And a continuous line in which white circles continue is platelet-derived hTGF-
β1 and continuous line with black circles are purified recombinant hTG
F-β1 is shown. From this figure, purified recombinant hTGF-β1
And platelet-derived hTGF-β1 were confirmed to have the same dose-dependent growth inhibitory activity.

B) Proliferation analysis in soft agar 6 Dulbecco's modified Eagle medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 0.6% (v / v) agar and 6% bovine serum was used in 6 multiwell plates (manufactured by Corning Incorporated). 2 ml / well was dispensed into each well and allowed to stand to solidify. Meanwhile, 5% (v / v) bovine serum, hEGF2ng and 0.05-10 mg hTG
To 1 ml of 0.3% agar containing F-β1, 3 × 10 ³ normal rat fibroblasts (1NRK-49F cells, distributed by RIKEN) were added, and this was added on the above solidified medium. , Solidified at room temperature. 5% (v / v) of the plate
After culturing in a CO ₂ atmosphere at 37 ° C. for 7 days, the diameter was 60 μm.
The number of colonies of m or more was measured. The result is shown in FIG. In FIG. 8, the vertical axis represents the number of colonies with a diameter of 60 μm or more (× 10 ³ ) and the horizontal axis represents the hTGF-β1 concentration (ng /
ml), and a continuous line with continuous white circles is platelet-derived hTG
The continuous line connecting F-β1 and black circles is purified recombinant hTG.
F-β1 is shown. Purified recombinant hTGF-β1 is 1 ng /
It showed the maximum dose-dependent colony formation promoting activity in ml, and its pattern was the same as that of platelet-derived hTGF-β1. Based on the results of the CCL-64 cell growth inhibitory activity and the proliferation ability analysis in NRK cell soft agar, purified recombinant hTG
It was demonstrated that F-β1 has the same biological activity as platelet-derived hTGF-β1.

[0066]

INDUSTRIAL APPLICABILITY According to the present invention, a method for producing high-producing cells of a useful polypeptide has been established, and a novel hTGF-β1-producing cell line 29E6-2 or the like has been obtained by the method, and the cells are more homogeneous. Since hTGF-β1 can be mass-produced, it is possible to provide a substance useful as a therapeutic agent for wound healing, bone fracture, inflammation, autoimmune disease, cancer, etc., or in the field of diagnostic agent for diseases related to hTGF-β1. ..

[0067]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 61 Sequence type: Amino acid Sequence type: Peptide Sequence 1 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg 20 Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu Pro 40 Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu 60 Asp Thr Gln Tyr Ser Lys Val

SEQ ID NO: 2 Sequence length: 112 Sequence type: Amino acid Sequence type: Peptide sequence 1 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg 20 Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu Pro 40 Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu 60 Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala 80 Ser Ala Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr 100 Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser 112 Cys Lys Cys Ser

[Brief description of drawings]

FIG. 1 is a drawing showing a growth inhibition curve of hTGF-β1 against CCL-64 cells using Neutral Red.

FIG. 2 Example 1. FIG. 3 is a drawing showing the frequency distribution of hTGF-β1 activity of each cell line based on the absorbance at 540 nm measured according to the bioassay method described in c).

FIG. 3 is a drawing showing the results of SP-Toyopearl column chromatography of a crude hTGF-β1 extract.

FIG. 4 is a drawing showing the results of reverse phase-HPLC of hTGF-β1 extract.

FIG. 5: Purified recombinant hTGF-β1 and platelet-derived hTG
It is a figure which shows the result of SDS-polyacrylamide electrophoresis of F- (beta) 1.

FIG. 6: Purified recombinant hTGF-β1 and platelet-derived hTG
It is a figure which shows the result of the reverse phase HPLC of F- (beta) 1.

FIG. 7: Recombinant hTGF-β1 and platelet-derived hTGF-
It is a figure which shows the growth inhibitory activity of (beta) 1 with respect to CCL-64 cell.

FIG. 8: Recombinant hTGF-β1 and platelet-derived hTGF-
It is a figure which shows the NRK cell proliferation ability in soft agar of (beta) 1.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // A61K 37/02 ABC ABE ABJ ADS ADU 8314-4C (C12N 5/10 C12R 1:91) ( C12P 21/02 C12R 1:91)

Claims (8)

[Claims] 1. A nucleotide sequence (1) encoding a polypeptide, a nucleotide sequence (2) which amplifies the nucleotide sequence (1) and imparts resistance to a selected drug to a mammalian cell, and these nucleotide sequences. A vector having a nucleotide sequence (3) that regulates the expression of (1) and (2) is introduced into a mammalian cell, and a recombinant cell group that highly expresses a polypeptide using a selective drug from the resulting recombinant cell group. In the case of selecting, (a) a recombinant cell having drug resistance to the selected drug from the recombinant cell group is separated as a recombinant cell derived from each single cell, and (b) a group derived from the single cell. After confirming quantitatively the polypeptide expressibility of the replacement cells and selecting them as the polypeptide highly expressing candidate recombinant cells, (c) the high expressing candidate recombinant cells are selected. A method for producing a recombinant cell highly expressing a polypeptide, comprising culturing in a medium in which the concentration of a selective drug is gradually increased to select the recombinant cell highly expressing the polypeptide. 2. A polypeptide-high-expressing recombinant cell obtained by the method according to claim 1. 3. The method for producing a recombinant cell highly expressing a polypeptide according to claim 1, wherein the polypeptide is transforming growth factor β. 4. The method for producing a recombinant cell highly expressing a polypeptide according to claim 1 or 3, wherein the polypeptide is human transforming growth factor β1. 5. A recombinant cell highly expressing a polypeptide according to claim 2, wherein the polypeptide is transforming growth factor β. 6. The recombinant cell highly expressing the polypeptide according to claim 2, wherein the polypeptide is human transforming growth factor β1. 7. A method for producing human transforming growth factor β1 which comprises culturing the recombinant cell highly expressing the polypeptide according to claim 6 and collecting human transforming growth factor β1 from the culture solution. 8. It is stable in the presence of methotrexate at 50 μg / ml or more, and its culture supernatant is 680 fg / cell / 2.
A transformant growth factor β high-expressing recombinant cell having a CCL-64 cell growth inhibitory activity for 4 hours or more.