JPH03180192A - Production of human pro-tgf-beta1 by genetic recombination - Google Patents

Abstract

PURPOSE:To efficiently obtain a human pro-TGF-beta1 having antitumor action, etc., with hardly any side effects by preparing a DNA chain capable of coding a human prepro-TGF-beta1, interpreting the prepared DNA chain into an expression vector, transforming a host and culturing the resultant transformant. CONSTITUTION:A cDNA is synthesized from an mRNA isolated from a human placenta and a DNA chain containing a base sequence capable of coding a human prepro-TGF-beta1 is then prepared using a DNA cloning technique, etc. The resultant DNA chain is subsequently integrated into an expression vector and the obtained expression vector is used to transform a host cell (e.g. CHO cell). The resultant transformant is then cultured to produce a human pro- TGF-beta1 in the culture. The produced human pro-TGF-beta1 is separated and purified by ultrafiltration, ion exchange chromatography, etc., to afford the objective human pro-TGF-beta1.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a technology for producing human proTGF-β1, which has physiological effects such as bone formation promoting action and antitumor action, by genetic recombination. Specifically, human preproTGF-
The present invention relates to a method for producing human proTGF-β1 using a DNA strand containing a base sequence encoding β1, an expression vector and a transformant used in this production, and human proTGF-β1 produced by the transformant.

<Prior Art> TGF-β1 is a mature protein that exhibits physiological effects, but when it is biosynthesized in vivo, it is first produced as a pro-type to which a precursor portion is added. It is thought that it is then cleaved into the mature form by acidic conditions and proteases in the body. Conventionally, human proTGF-
β1 had been isolated from human platelets. The yield is about 40 μg from 300 g of platelets (0,00001
3%), making it an impractical production method for making it into a drug, both in terms of its source and yield.
Journal of Biological Chemistry, 2
63 Near 64B-7854, 1988, Miyazon
OK.

et al: Latent hlgh 5o
regular velght complexo
f transforming growth f'a
ctor beta 1. J, Blol.

Ches+, 288 Near 64B-7654, 1988,
), and now that the gene for human proTGF-β1 has been cloned [Plink et al., Nature,
318 Near 01-705.1985. Deryne
k et al: Nature, 31B: 7G
1-705,. 1985. , Prink et al., Nucleic Acid Research, 15:318g-3189,
1987, Deryck etat: Nucle
le, Ac1d, Res, 15:31gg-31
89, 1987,), it has been realized to produce human mature TGF-β1 using genetic recombination technology.

That is, human mature TGF-β1 is expressed and produced in CHO cells transformed with DNA encoding a protein to which a precursor portion has been added (Japanese Patent Application Laid-open No. 219395-1983). Nucleic acids and their uses). The differences between the mature type and the pro type are as shown in FIG. 7, and pro TGF-β1 is the mature TGF-β1 with a precursor protein added. It is thought that when secreted from within cells, it exists as a pro-form, and this is transported locally within the body and becomes the mature form.

The details of this mature type recombinant production method disclosed in the prior patent are as follows.

That is, the DNA for TGF-β1 was introduced into CHO cells in a form containing part of the pro-type precursor part and part of the herpes simplex virus membrane protein (HSV-1gD), and the precursor part was 359 The chimeric peptide is produced as a chimeric peptide consisting of 5 amino acids (B'-C portions in Figure 8).

Thereafter, the C portion is artificially cut in an acidic environment to produce a mature portion. However, the fusion pro-TGF-β1 (8th
(see figure) expression level is 0.0005μg per mL of medium.
The amount is extremely small and is considered inappropriate for industrial production. In addition, mature TGF-β1 itself suppresses the immune system (i.e., it inhibits macrophages, which play an important role in immune regulation, such as feeding on bacteria that have invaded the body from the outside and communicating the strength of antigens to T cells. It has the side effect of suppressing the function [Tsunawaki et al.
"Inactivation of macrophages by TGF-β1" Nature 314:28G-282, 1988゜(Tsuna
vakl S, et at: Deactlvatl
on of' macro-phagcs by
TGP-Beta, Nature 3342280-282゜1988). That is, when the mature type is administered into a living body by intravenous injection or the like, it acts on macrophages in the blood or in normal organs and suppresses their functions.

Monkey proTGF-β1 is known as a prior art related to the production of non-human TGF-β1, but compared to human proTGF-β1, there are three amino acids at the amino acid level (in monkeys, there are three (B ) - (C> 48th Ala
al91st Asp.

and 228th Arg are respectively Thr.

Asn and L y s l (substituted) are different, and as mentioned above, when administered to humans, there is a concern that it may show antigenicity, and is not in a satisfactory state as a drug. Yi et al., “Genetically modified T
“Molecular structural changes in the processing of GF-β1 from prepro to mature forms”, Molecular Cellular Biology, 8:4.182-4188.1
988. (Gentry LE, et al:Mo1
ecular events In the proc
essing or recombinant type
e 1 pre-pro-transrorltngg
rowth I'actor beta 10 Lhe
mature polypeptide.

Mo1. Ce11. Biol, 8:41B2-41B8
．． 1988. ) ; Planner N.M. et al., “
"Recombinant TGF-β1 precursor produced in Chinese hamster oocytes (CHO) is glycosylated and phosphorylated", Molecular Cellular Biology, 8:2229-2282.198
8. (Brunner AM.

et al: Recombinant type l
transl'orilng growth1'act
or beta precursor produce
d In Chinese hamster overary
cells is g+ycosy+atedand
phosphorylated, Mo1. Ce11.
Blol, 8:2229-2232゜1988, );
Zou Shah ``The importance of glycosylation and acid proteases for processing and secretion of rTGF-β1'', Molecular Endocrinology,
13:1090-1098゜1989, (Xue Sh
a, et al: Transf'orming g
rowstractor beta 1: Import
ance or Glycosylatlonand
Ac1dic Proteases for Proc
essing andSecretion, Mo1.
Endo, 13:1090-1098.1989)
).

TGF-β1 in other animals is still at the research stage, and only a part of the amino acid sequence is known [(Seidin Nis. M et al., “Chondrogenic factor B is structurally and functionally "A unique protein associated with
2G2:194B-1949゜1987, (Seyed
in SM, et al:cartllage-1n
ducing factor B Is a unique
e protein 5 structurally and
f'unct1onally related to
TGF-beta, J, Blol.

Chem, 282:194B-1949, 1987,)
; Chaifets et al., rTGF-β system: complex pattern of cross-reacting ligands and receptors," Cell, 48:409-415°1987, Chcifct
s S, at al: Thc TGF-beta s
system.

a cos+plcx pattern of cro
ss-reacttve Ilgandsand rc
ccptors, ccll 48: 409-415
．． 1987) ).

[Summary of the invention]

<Problems to be Solved by the Invention> As described above, since mature TGF-β1 has side effects due to immunosuppression, pro-type TGF-β1 is considered to be less toxic. That is, when the mature type is administered into a living body by intravenous injection, it acts on macrophages in the blood or in normal organs and suppresses their functions, but even if the pro type is administered in the same way, the active site is in the precursor region (the seventh (B) to (C) in the figure), it is thought that the pro-type does not suppress macrophage function. However, in bone formation and tumor growth, the environment around cells becomes acidic due to acid release or lactic acid accumulation (Tatsuo Suda et al., "Osteoscience", published by Ishiyaku Publishing Co., Ltd., 1988 edition). , in p185r bone resorption (osteoclasts), local pH decreases...'',
Kazuo Shizume et al., “Bone Metabolism Regulator”, published by Hijikamisha Co., Ltd.
1987 edition, β35 "Bone mineral dissolution is said to be carried out by acid secreted from bamboo shoot cells. There are reports that the acid is lactic acid, pyruvic acid, or citric acid, but in recent years it has been reported that it is hydrochloric acid, similar to stomach acid. ), this low pH
The effect cleaves the pro-type C site. As a result, TGF can be used in normal tissues in its professional form without any side effects.
-It becomes possible for β1 to be used effectively and specifically in the body where it is most desired to act. Such an effect is expected only with human pro-TGF-β1, and it is expected that TGF-β1 with a heterologous protein added to the pro portion or non-human monkey pro-TGF-β1 produced using conventional techniques. Since there is a risk that antigenicity may appear when administered in the body, there is little possibility that it will be used as a medicine.

Based on the above, efficient production of human pro-type TGF-β1 is desired.

The present invention solves the above-mentioned problems and
Human pro-TGF that is expected to be safer than β1 and exhibits osteogenic and antitumor effects in vivo
- The purpose of this invention is to provide a method for high production through genetic recombination of β1.

Means for Solving the Problems The present inventors have developed a method for producing human pro-TGF-β1 that is expected to have fewer side effects as described above, and using this method, human pro-TGF-β1 can be produced at a high rate in cells. It was confirmed that it was expressed well.

In other words, the present inventors found that when human pro-TGF-β1 is expressed in the same natural sequence as opposed to the fusion type, the production amount increases approximately 1000 times compared to the fusion type, and based on this knowledge, The present invention was finally completed.

The present invention provides a means for efficiently producing pro-TGF-β1 in cells. That is, the method for producing human pro-TGF-β1 according to the present invention
Prepare a DNA strand containing a base sequence encoding F-β1, create an expression vector containing this DNA strand in a state in which its genetic information can be expressed, transform a host cell with this expression vector, and transform the resulting transformation. The method is characterized in that human proTGF-β1 is produced in the culture by subjecting it to a step consisting of culturing the body.

The invention also relates to human proTGF-β1. That is, human proTGF-β1 according to the present invention is produced using a transformant transformed with DNA containing this gene, and is novel as produced using genetic recombination technology.

Furthermore, the present invention relates to recombinant DNA and transformants. That is, the recombinant DNA according to the present invention is one in which a DNA strand containing a base sequence encoding human preproTGF-β1 is integrated into an expression vector. The transformant according to the present invention is one in which a host cell is transformed with the above recombinant DNA.

<Effects of the Invention> According to the present invention, human pro-TGF-β1 containing a complete TGF-β1 precursor can be produced at a high rate in host cells. As shown in the experimental examples below, the production amount is approximately 1,000 times greater than that of the conventional technology.

Further, pro-TGF-β1 is not limited to use simply as an intermediate for producing TGF-β1, but can also be used as a dosage form for pharmaceuticals such as tumor suppressants. Originally TGF-β1
The main action of Mature TGF-β1 is a polypeptide composed of 112 amino acids, but when administered directly into the body, Mature TGF-β1 exhibits toxicity due to suppression of macrophage function, making it unsuitable as a pharmaceutical form. Therefore, the concept of "prodrug" was proposed, but this method does not require mature TGF-β1.
It is difficult to select a modifying group to eliminate the drug efficacy of TGF-β1, and there are problems with antigenicity, so it is difficult to immediately apply this method to TGF-β1. In contrast, human pro-TGF that can be produced by the present invention
-β1 has its complete pro portion, so
At the time of administration into the body, it shows no physiological activity and therefore no biotoxicity. On the other hand, when it reaches a local site during internal circulation, the precursor portion is removed (processed) and takes the form of mature TGF-β1, which has physiological effects. It can be expected to be effective as a so-called ``natural prodrug.''

Since TGF-β1 is a substance that exhibits its physiological activity in the mature form, conventional technology
Emphasis was placed only on the expression of the precursor, and the importance of the precursor portion was not recognized. Behind this idea is the recognition that the precursor is not involved in the regulation of expression within the cell. One example is that it has become a well-established theory. In view of the above, it can be said that the expression-enhancing effect of the complete precursor portion of human proTGF-β1 according to the present invention is unexpected.

[Detailed description of the invention]

(I) Method for producing human pro-TGF-β1 The method for producing human pro-TGF-β1 according to the present invention uses a DNA containing a base sequence encoding human pre-proTGF-β1.
From preparing a DNA strand, creating an expression vector (i.e., recombinant DNA) containing this DNA strand in a state in which its genetic information can be expressed, transforming a host cell with this recombinant DNA, and culturing the resulting transformant. The method is characterized in that human proTGF-β1 is produced in a culture by the steps of:

A DNA strand containing a base sequence encoding human preproTGF-β1 The base sequence encoding human preproTGF-β1 is a polypeptide having a signal peptide on the N-terminal side of human proTGF-β1 that is removed after secretion from the transformant. (
That is, a polypeptide (i.e., human preproTGF-β1) that encodes human preproTGF-β1) and whose amino acid sequence is substantially from A to D among the amino acid sequences shown in FIG. This is the code. Here, "the amino acid sequence is substantially the first
Amino acid sequences A to D shown in the figure" means that this peptide is human preproTGF-
This indicates that some of the amino acids may be deleted, substituted, added, etc. as long as they have the function of β1.

A typical human preproTGF-β1 polypeptide in the present invention has the amino acid sequence A to D of the amino acid sequence shown in FIG. 1, and is a known polypeptide consisting of 390 amino acid sequences. In FIG. 1, the amino acid sequence A-B is the signal peptide part (29 amino acids), B-D is the human proTGF-β1 part (361 amino acids), and C-
D is TGF-β1 part (mature part) (amino acid 11
2 pieces).

Therefore, the nucleotide sequence encoding human preproTGF-β1 in the present invention corresponds to the nucleotide sequence A to D in FIG. 1, degenerate isomers thereof, and changes in the amino acid sequence of the polypeptide as described above. It has a base sequence or its degenerate isomer. Here, the term "degenerate isomer" refers to those having base sequences that can code for the same polypeptide that differ only in degenerate codons.

In the present invention, the DNA strand containing the above base sequence is condensed into an appropriate expression vector and introduced into a host cell for efficient expression. A DNA strand of an appropriate length, for example, a DNA strand as an untranslated region having one or more stop codons at the 3' end may be bound to the end. A typical example of the base sequence of such an NA strand is that shown in FIG. 1, and this base sequence is known.

One way to obtain a DNA strand containing a base sequence encoding human preproTGF-β1 is to chemically synthesize at least a portion of the strand length according to a nucleic acid synthesis method.

Considering the number of bonded amino acids, it is better to synthesize cDNA from a chromosomal gene library or from mRNA isolated from a tissue, such as human placenta, than this chemical synthesis method, and use this gene library to synthesize cDNA in the field of genetic engineering. It is preferable to obtain this by a commonly used method, for example, a hybridization method using an appropriate probe (see Experimental Examples below).

Recombinant DNA> The recombinant DNA according to the present invention is obtained by incorporating the above DNA chain into an expression vector and ligating it.

This expression vector is a carrier D that plays the role of transferring the above DNA strand into host cells for transformation.
It is a circular or linear NA that grows autonomously within the host cell or integrates into the host chromosome.

Such expression vectors include plasmids, phages,
Cosmids and the like are included, but plasmids are more preferred.

Expression vectors include pBKTK-1 (BK virus vector), EBV (EB virus vector), H
Various known vectors can be used, such as SV (herpes simplex virus vector).

On the other hand, in order to express the human preproTGF-β1 gene in host cells, it is necessary to transcribe the DNA into mRNA. For this purpose, a promoter gene, which is a signal for transcription, may be inserted upstream of the 5' side of the DNA strand. There are various known promoters such as the 5v40 early or late promoter, the Rous sarcoma virus promoter, the human T-cell leukemia type promoter, the adenovirus major rate promoter, and the metallothionein mA promoter, and any of them can be used.

In addition, the terminator for terminating the transcription was added to the above D.
Preferably, it is incorporated downstream of the 3' side of the NA strand. Plasmid stability can be increased by inserting a terminator. TRP as a terminator
T terminator (tryptophan operon), λtR
2 terminator, λtR1 terminator, etc. can be used.

Furthermore, in the step of translating mRNA into protein, a sequence necessary for the liposome, which is the site of protein synthesis, to bind to the tip of the translation initiation site must be added in front of ATG, which is the initiation signal for protein synthesis.

Transformant> The transformant according to the present invention is obtained by transforming a host cell with the above recombinant DNA.

As the host cell, various types of cells can be used as long as a suitable vector exists, but animal cells such as N51 cells, VERO cells, HeLa cells, etc. are preferred examples, and CHO cells are particularly preferred.

Transformation of host cells with recombinant DNA can be carried out by any convenient method commonly used in the field of genetic engineering or biotechnology.

Appropriate reviews or reviews on boat fishing matters, such as T, Manlatls, E, P, Pr1t.
sch, J.

5aIlbrook: rMolecular CI
onIng A LaboratoryManualJ
, Co1d Spring Harbor Lab
oratory.

(19g2), can be referred to.

The transformant has a new trait (i.e., human preproTGF-β) due to the genetic information introduced by the DNA strand.
1 and human proTGF-β1) and the vector used, and the genotype, except for the missing corresponding gene due to the lack of some genetic information from the vector used during gene recombination, which may have occurred in some cases. or are identical in phenotype or cytological properties to the host cells used.

Production of human pro-TGF-β1> When the transformant clone obtained as described above is cultured, human pre-pro TGF-β1 is produced in the cells of the culture.
β1 is produced, the signal peptide portion is cleaved in the periplasm, and human proTGF-β1 accumulates in the periplasm and outside the cells, that is, in the culture medium.

The culture or growth conditions for the transformant are essentially the same as those for the host cell used. Further, the produced protein can be recovered from the culture, that is, the cells and/or the culture solution, according to any suitable method (for example, the method described in Experimental Example 6 below).

When CHO cells are used as host cells, the production amount is approximately 1,000 times greater than that of the conventional technology, as shown in the experimental examples below.

In addition, if necessary, human pro-TGF-β1 can be prepared using known methods (for example, Mjyazono, eL at: L
agent hlgh 5olecular weig
ht complex of'transl'ormi
ng growth f'actor beta 1.
, J. Blot.

Chew, , 263: 7G48-7854.19
Mature TGF-β1 can also be obtained by the method of H.

(n) Human proTGF-β1 Human proTGF-β1 according to the present invention is a polypeptide produced by a transformant transformed with a DNA chain containing the human proTGF-β1 gene described above.

The amino acid sequence of this human pro-TGF-β1 polypeptide includes, in addition to the typical amino acid sequence B-D shown in FIG. etc. may be included.

(m) Uses of human pro-TGF-β1/bone formation promoting agent or antitumor agent TGF-β1 acts as a strong growth inhibitory factor for many cells, but it has a differentiation-inducing effect on certain types of cells. show. Among these, it is known to increase and activate the proliferation and metabolism of cells involved in bone formation, and to promote bone formation in collaboration with bone morphogenetic factors. Specifically, osteoblasts (
It has the effect of activating osteoblasts and also stimulating osteoclasts to activate bone metabolism.

When mature TGF-β1 is administered directly into living organisms, it exhibits toxicity due to suppression of macrophage function [Tsunawaki et al., "Inactivation of macrophages by rTGF-β", Nature 334: 200-262% 1988
(Tsunavakl s, at al:Deact
lvatlonof macrophages by
TGF-Beta, Nature 334: 280-
282.1988) ), although it is unsuitable as a pharmaceutical dosage form, human proTGF-β1 is expected to reach local sites by administering it to the body in its original form, so it may have osteogenesis promoting or antitumor effects. In order to show that
It can be used in its pro form as an osteogenesis promoting agent or an antitumor agent, that is, as a "prodrug."

Prodrug technology was conceived as a useful method for drug formulation, as it has no effect when administered in the body and only exhibits drug efficacy once it reaches a localized site. This method is 19
This is a concept proposed by Alberi in 1958, in which a target substance loses its medicinal efficacy through special chemical modification, but when administered into the body, it is metabolized and the modifying group is separated and the medicinal efficacy is expressed. However, it has the drawbacks of difficulty in selecting a modifying group and problems with antigenicity.
It was difficult to apply this immediately for TGF-β1. Furthermore, since precursors are destined to be removed through processing, the conventional idea was to temporarily produce them as fusions with other proteins within cells.

In contrast, when administered in the form of proTGF-β1 according to the present invention, since it has its complete pro portion,
At the time of in vivo administration, it exhibits no physiological activity and therefore no biotoxicity. On the other hand, once it reaches a local site during body circulation, the precursor portion is removed by processing and takes the form of mature TGF-β1, which exhibits no physiological activity. To show,
It can be expected to be effective as a so-called "natural prodrug."

When used as an osteogenesis promoting agent or an antitumor agent, human proTGF-β1 can be administered by any convenient administration route and in a dosage form determined by the administration route employed. The drug is usually used as it is or diluted with a pharmaceutically acceptable carrier or diluent.

When this compound is actually administered as an osteogenesis promoting agent or an antitumor agent, one typical method is to dissolve the compound in distilled water for injection or physiological saline and inject it. Specifically, in the case of animals, injection methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration, and in humans, intravascular injection or injection into a vein or artery. There are methods such as local administration.

The dosage of this compound is determined in consideration of the results of animal tests and various situations so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage will vary depending on the method of administration, the circumstances of the patient or treated animal, such as age, weight, sex, sensitivity, diet, time of administration, concomitant drugs, and the degree of the patient or his or her disease. Needless to say, the appropriate dose and frequency of administration under certain conditions must be determined by a specialist's appropriate dose determination test based on the above guidelines.

Specifically, it is about 5 μg to 100 μg per day for adults.

Experimental Examples The following are experimental examples of the present invention, but the present invention is not limited thereto.

Experimental Example 1: Isolation of human preproTGF-β1 cDNA cD for isolation of human preproTGF-β1 cDNA
The NA library was prepared from mRNA isolated from human placenta using the method of Gubler & HofTman et al.
Gene, 25; 2B3
．． After cDNA synthesis according to (1983)), λ
gtlo vector [Fuyun T.V. et al., DN
A practical approach in cloning technology, IRL
Publishing, Oxford, P49. 1985 (IIu
ynh, T, V, etal, In DNA Clo
nlng Techniques, A Pract
lcalApproach(IRL Press, 0
xford, D, GIover ed) P49゜19
85). As a result of screening a library of about 300,000 clones using the synthetic probe shown in FIG. 2, about 50 positive clones were obtained.

1 from the clone hybridized with this synthetic probe.
Select a clone, name it TI, and select this T1 clone.
Further analysis was carried out. cDNA of clone T1
is excised by EcoR1 digestion and then inserted into the M13 vector [Janisch, Perron◆See et al., Gene vol. 33, p.
2O3-119, 1985 (Yannlsh-Per
ron C,et a! , Gene, 33.103
-119°1985), and the nucleotide sequence was determined by the dideoxy method.

This is shown in FIG. This clone T1 contains the entire coating sequence of human preproTGF-β1 and is retransferred into the pUc19 vector [Janisch, Peron et al.
Yanlsh-Perron C, et al, Gene 33, P2O3-119, 1985.
Gene.

pUC19-TGF was obtained by subcloning into the EcoR1 site of 33.103-119.191i5).

Experimental Example 2: Creation of vector for human proTGF-β1 expression Human preproTGF-β1 cloned in Experimental Example 1 above
A plasmid for expressing β1 cDNA in CHO cells was constructed. This plasmid pECβ contains the SV40 early and late promoters, the avian sarcoma virus promoter, the human cytomegavirus promoter, the cDNA encoding human preproTGF-β1, the SV40 late polyadenylation sequence, and the mouse dihydrofolate reductase cD.
Contains NA and some sequences of pBR322 (E. coli ampicillin resistance marker and replication origin).

A diagram of the construction process of the plasmid pECβ for expressing human preproTGF-β1 is shown in FIG. The construction method will be explained in detail below.

1) cD encoding human preproTGF-β1
Cloning vector pUc19T of NA1800bp
Obtained from GF by EcoR1 digestion. This fragment is DNA
The ends were made blunt with polymerase 1 (Flenow fragment).

2) Plasmid expression vector CDM8 (Seed Be Nature, Volume 329, P840-842.198
7 (Seed, B,, NaLure, 329.8
40-842.1987) from 5aC2, suppressor tRNA (SupF) gene by BamH1 digestion,
A 2324 bp fragment containing the avian sarcoma virus LTR, cytomegalovirus promoter, T7 promoter and SV40 early polyadenylation sequence was obtained. This fragment was blunt-ended with DNA polymerase 1 (Frenow Fragment).

3) Plasmid expression vector pSV2dhfr [Subramani Nis et al., Molecular Cellular Biology, Vol. 1, P2S5-864.1981 (Subra
lani, S., et at, Mo1. Ce1
1. BIOl, l1854-864.1981)]
was digested with Pvu2 and then dephosphorylated with alkaline phosphatase. This and the 2324 bp fragment of 2) were ligated using T4 ligase.

4) Plasmid expression vector pSVL (Pharmacia) was digested with Sma1 and then dephosphorylated with alkaline phosphatase.This and 1)
The 1800 bp fragment was ligated with T4 ligase.

5) Xbal from the plasmid of 3).

By EcoR1 digestion, T7 promoter, cytomegalovirus promoter, avian sarcoma virus LTRSS
A 3700 bp fragment of upFSSV40 early and late promoters, origin of replication, mouse dihydrofolate reductase cDNA, and SV40 early polyarylation sequence was obtained.

6) Xbal from the plasmid of 4).

Human preproTGF-β1 cDNA by EcoR1 digestion
A, A 4500 bp fragment containing the SV40 late polyadenylation sequence, E. coli ampicillin resistance marker and origin of replication was obtained.

7) pECβ was obtained by ligating the fragments of 5) and 6) with T4 ligase.

Experimental Example 3: Expression of human proTGF-β1 in CHO cells
Volume 77, P421B-4220, 1980, (C
hasin, L. A., & Urlaub, G.
P#AS; 77, 4218-4220 (1980)
)> was seeded in 60 mm dates, and 24 hours later, the above plasmid pECβ (10 mg) was transfected by the calcium phosphate method. Cells were grown in non-selective medium (DMEM + 10% FCS + HT) for 2 days and then re-seeded into 5 100+ am dates.

At this time, the culture solution was changed from non-selective medium to selective medium (DMEM
+10% dialysis FC8) Switched. Approximately 2-
After culturing for 3 weeks, the obtained colonies were detached from the dates by trypsin treatment, re-inoculated into a 48-well plate at 1 colony/well, and cultured until full size.

For human mature TGF-β1 activity in the culture supernatant (approximately 35 clones) from each well, the supernatant was treated under acidic conditions, i.e., with 0.2 M acetic acid, to pro-TGF-β1.
After cleaving the precursor portion from and obtaining mature TGF-β1, a bioassay method using MvlLu cells [Van Zoren E. J. G. et al., Biochemical Biophysical Research Communication, Vol. 141, P1229-1235.1986 (Va
n Zoelen, E.J.

J., et at: Bloches, Bloph
ys, Res, Coa+a+, 141°1229
-1235.1988). As a result, 34
Activity was observed in 19 clones. Among them, clone 723 showed stable and high TGF-β1 activity.
I got it. By selecting this clone 723 with two steps of methotrexate (MTX), a large amount of TG can be constantly produced.
We attempted to create a cell line that produces F-β1.

First, cells were cultured in the presence of 100n and viable cells were selected. Furthermore, cell line T that stably produces large amounts of TGF-β1 was selected in the presence of 500 nM MTX.
23-7-11 was established.

TGF-β1 in the culture supernatant of T23-7-11 cells was detected by Western blotting using anti-human mature TGF-β1 polyclonal antibody and SDS-PA.
When detected by GE method, TGF-β1 was observed in the main band and minor band at positions of 105 and 25 Kd, respectively, under non-reducing conditions. In other words, 105Kd is the molecular weight of a 40Kd pro-TGF-β1 dimer with sugar chains added, and 25Kd is 12.5Kd.
This is considered to be the molecular weight of mature TGF-β1 in one dimer. From this, human TGF-β1 produced from T23-7-11 is secreted from cells as a dimer via S-8M conjugation1, and most of it is in the pro form.
It was observed that some mature types were also produced.

Experimental Example 4: Recombinant human professional and mature TGF-β
723-7-11 cells established in Production Experiment Example 3 of 1, 1
．． 5 x 10 cells were seeded in an 850c# roller bottle (manufactured by Falcon), and a serum-containing medium <DMEM/F
-12 (1; 1) +5% FC8> for 3 days. After 3 days, remove the serum medium and transfer to PBSroom.
After washing with serum-free medium <E-RDF
(Kyokuto Pharmaceutical Industry).

5μg/ml bovine insulin, 20μM monoethanolamine, 50nM Na2SeO3,0.2μ
g/ml bovine protinin, 0.1% PEG 2
0000 (all Sigma) > Replaced with 200m1,
The cells were cultured at 37°C for 7 days. After 7 days, the culture supernatant IL was collected.

Experimental Example 5: Purification of recombinant human proTGF-β1 723-7-11 cell culture supernatant obtained in Experimental Example 4 40
0ml was taken as starting material. The culture supernatant was filtered using an ultrafilter membrane with a molecular weight of 10,000, such as a Filtron cassette membrane (Fuji Filter Industries) with a 0.45 d membrane.
It was concentrated to 0ml. 30 minutes at 110,000 rpm,
After centrifugation, the supernatant was diluted with 10 mM phosphate buffer (pH
7,4) (N a H2P O4N a 2 HP
Dialyzed against O4) IL three times.

The concentrate obtained by ion-exchange chromatography and dialysing was purified using 101M phosphate buffer (plI).
7.4) S-Sepharose column (5X5) equilibrated with
(A: 20% H phosphate buffer (pH 7,4), B: A + 0.5M)
After washing the column with 100% solvent A for 10 minutes, elution was performed using a linear concentration gradient method.
00% solvent B/30 ain), 11 ml of a fraction containing poTGF-β1 was obtained.

Hydrophobic chromatography The eluate obtained above was treated with 4M ammonium sulfate-100.
After adding 11ml of mM phosphate buffer, 2M ammonium sulfate-1005M phosphate buffer (pH 17)
, 4) alkyl-superose HR51 equilibrated with
5 (Pharmacia) (A: 2M ammonium sulfate-100 + gM phosphate buffer (pH 7,
4), B: 100mM phosphate buffer (pH 7
, 4) ). After washing this column with solvent A for 10 minutes, it was eluted using a linear concentration gradient method (0%-60% solvent B
/40m1n) Fractions 32-36 shown in Figure 4
2.5 ml of proTGF-β1-containing fraction was obtained.

Gel filtration chromatography Both proTGF-β1 fractions obtained were concentrated using a Centricon 10 concentrator, and then applied to a Superose 6 column (Pharmacia) equilibrated with PBS. Elution was performed using PBS and 3 ml of fractions containing the pro-TGF-β1 fraction were collected. The elution diagram and activity measurement results are shown in FIG.

Through the above operations, 2000 μg of the purified sample was obtained.

Experimental Example 6: Confirmation of homogeneity of recombinant human proTGF-β1 In order to confirm the homogeneity of human proTGF-β1 purified in Experimental Example 5 above, 5DS-PAGE (10-15%
Its purity was confirmed using gel electrophoresis using a gradient gel.

As a result, this protein was determined to be unique. In other words, as shown in Figure 6, the culture supernatant of clone 723-7-11 itself contains many impurity proteins (lane 2), but after S-Sepharose ion exchange chromatography, proteins were found at the 105 kd and 25 kd positions. Bands considered to be pro-type and mature-type dimers were detected (lane 3). The former is 40k
The molecular weight is larger than 80 kd because a sugar chain is added to the pro-TGF-β1 dimer of d. The latter is a 12.5kd mature TGF-β
1 forms 211 bodies and exhibits a molecular weight of 25 kd. These mature forms are removed by subsequent column operations (hydrophobic chromatography with alkyl-superose and gel filtration chromatography with superose 6), yielding only dimeric proTGF-β1 as a homogeneous protein. (Lane 4 and Lane 5).

Purification of Reference Side Two Recombinant Human Mature TGF-β1 0.5 L of the T23-7-11 cell culture medium obtained in Experimental Example 4 was used as a starting material.

The culture supernatant is a Filtron cassette B with a membrane of 0.45d and a molecular weight of 1oooo like CM Shira Filter Industries.
It was concentrated to 25 ml using a Kattu ultrafiltration membrane.

This concentrate was excluded from Spectrum (Spectrum Medical Sangyo Co., Ltd., Spec
trum Medlcal Industries In
Pro-TGF-β was obtained by placing it in a dialysis membrane such as (c) and performing dialysis three times against 0,2N acetic acid solution IL.
The precursor portion was cut from 1 to obtain mature TGF-β1. This concentrate of human mature TGF-β1 was prepared at 100,000 g for 30 minutes to remove precipitated proteins.
Centrifugation was performed for 0 minutes. The supernatant from which insoluble proteins have been removed is
Lyophilized.

Gel filtration chromatography The above lyophilized preparation was reconstituted with 1 ml of 0.1% TFA/20% acetonitrile, and 0.1% TFA/20%
G3000SW equilibrated with acetonitrile (21,
It was applied to a 5×600m+*) column (Tosoh ■). Elution was performed using 0.1% TFA/20% acetonitrile, and 15 ml of mature TGF-β1 containing fractions were collected.

Mature TGF-β1 eluted from reversed phase HPLC G3000SW
The fraction containing RP304C4 (4,6X250+m)
It was further purified by reverse phase HPLC using a column (Blo-Rad). Both of the above amounts were applied to a column equilibrated with 20% solvent B at a flow rate of 1 ml/sin (A
: 0.1%TFA/H20, B: 0.06%TFA
/acetonitrile).

This column was washed with 20% solvent B for 10 minutes and then eluted using a linear concentration gradient method (20%-50% solvent B/
60 minutes). The obtained TGF-β1 fractions were collected and freeze-dried. Through the above operations, 500 μg of purified standard product was obtained.

Regarding the homogeneity of this human mature TGF-β1,
Confirmed using 5DS-PAGE, 25k as a dimer
A molecular weight band of d was detected. A 12.5 kd monomer was also confirmed by 5DS-PAGE under reducing conditions.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing the nucleotide sequence and the corresponding amino acid sequence of the cloned human preproTGF-β1 gene. FIG. 2 is an explanatory diagram showing the base sequence and corresponding amino acid sequence of the synthetic probe used for cloning. FIG. 3 is a process diagram showing the construction of plasmid pECβ for human preproTGF-β1 gene expression. FIG. 4 is a graph showing a hydrophobic chromatogram (alkyl-superose) and activity measurement results. Figure 5 shows a gel filtration chromatogram (Superose 6
) and graphs showing activity measurement results. FIG. 6 is a schematic diagram of a gel electropherogram obtained by 5DS-PAGE showing the homogeneity of proTGF-β1 in each purification step. FIG. 7 is an explanatory diagram schematically showing preproTGF-β1. FIG. 8 is an explanatory diagram schematically showing fusion type TGF beta.

Claims (1)

[Claims] 1. Preparing a DNA strand containing a base sequence encoding human preproTGF-β1, creating an expression vector having this DNA strand, transforming a host cell with this expression vector, and the resulting transformation. human pro-TGF-β1 in culture.
Human proTGF-β1, characterized by producing
manufacturing method. 2. The production method according to claim 1, wherein the host cell to be transformed is a CHO cell. 3. An expression vector having a DNA strand containing a base sequence encoding human preproTGF-β1. 4. Human proTGF-β1 produced using a transformant transformed with a DNA strand containing a base sequence encoding human proTGF-β1. 5. A transformant obtained by transforming a host cell with the expression vector according to claim 3. 6. The transformant according to claim 5, wherein the host cell is a CHO cell.