JPH0771508B2 - Polypeptide and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a polypeptide having somatomedin-like activity and a method for producing the same using a microorganism transformed with a plasmid containing a gene encoding this polypeptide. .

[Prior Art] In mammals including humans, somatomedin A, IGF-I, IGE-II and the like are known as substances involved in promoting the growth of bone tissue. These substances (hereinafter referred to as somatomedin-like active substances) are released from liver tissue and directly act on bone cells to promote their proliferation. The production and secretion of these substances are considered to be regulated by growth hormone, but it is said that somatomedin-like activity is very small in growth hormone itself.

Somatomedin-like substances are useful as drugs for promoting the growth of bone tissue, but their production and secretion are extremely small,
Since the extraction is not easy, it is difficult to obtain a large amount of these from the living body, and especially in the case of humans, it is completely impossible.

Since somatomedin-like substances are polypeptides, it may be possible to produce them in microorganisms by gene recombination techniques, but these substances are relatively low molecular weight substances with a molecular weight of about 7,000 and were expressed in the microbial cells. However, the amount thereof is small, and it is very difficult to efficiently produce them due to digestion, secretion, etc. in the bacterial cells, extraction process, and purification process.

[Problems to be Solved by the Invention] Therefore, it is an important subject to obtain a somatomedin-like substance or a substance having somatomedin-like activity that can be produced in large quantities by a gene recombination technique.

[Means for Solving Problems] The present inventors have found that this polypeptide has a somatomedin-like activity for a polypeptide having an amino acid chain from the N-terminal to the C-terminal of the human growth hormone (hGH) to the 138th position. In particular, this polypeptide is transduced with a plasmid in which a corresponding portion of a DNA segment known as a synthetic gene encoding hGH and capable of expressing hGH by a recombinant technique is expressibly inserted. It is possible to express using transformed Escherichia coli, and such a plasmid has a restriction enzyme B from a plasmid containing the above-mentioned synthetic gene having a restriction enzyme SalI site at its 3'end so as to express hGH.
The present invention was completed by the discovery that it can be prepared by digestion with gl II and Sal I and readhesion with a linker.

That is, according to the present invention, the N-terminal of hGH causes 138
The present invention provides a polypeptide having an amino acid chain up to the th position (however, methionine may be added to the N-terminal thereof).

Such a polypeptide has the formula A methionine residue may be added to the N-terminal, and some changes may be made without impairing somatomedin-like activity. It may be).

A synthetic gene containing a DNA chain encoding such a polypeptide (hereinafter referred to as hGH-AB polypeptide) is known as a gene encoding the AB portion of hGH (JP-A-60-
No. 234584). This gene is a DNA chain of the following formula that encodes hGH-AB polypeptide (hereinafter referred to as hGH-AB gene)
Is included.

The hGH-AB gene can be stably retained in E. coli and inserted into a replicable plasmid such that it can be expressed in E. coli. A physical map of the plasmid pAB-1 having the hGH-AB gene inserted in this manner is shown in FIG.

The DNA sequence from the restriction enzyme Cla I site to the downstream Sal I site on this plasmid in FIG. 1 is shown in FIG.

This plasmid can be prepared from, for example, the pGH-L series plasmid disclosed in JP-A-60-234584. FIG. 3 shows the physical map of this pGH-L series plasmid, and FIG. 4 shows the restriction enzyme site of the hGH gene part in more detail.

DNA from the Cla I site of the hGH gene to the downstream Bgl II site
The sequence is exactly the same as that of pAB-1. The region from the BglII site to the downstream SalI site encodes a part of hGH called C part, and the C-terminal code is followed by a stop codon. Its DNA sequence is shown below.

Figure 5 shows the plasmids from pGH-L series, pGH-L9.
3 shows the preparation of pAB-1. As shown in this figure, pAB-1 is pGH
-L9 is double-digested with restriction enzymes Bgl II and Sal I, and if necessary purified by agarose (acrylamide) gel electrophoresis etc. to give a large DNA fragment containing the hGH-AB gene, It can be prepared by ligating with a linker consisting of a DNA chain represented by

In addition, Escherichia coli and E. coli (p.
i) pGH-L9 has been deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry, as Microbiology Research Institute No. 7606. pGH-L
As disclosed in JP-A-60-234584, No. 9 can be easily extracted and prepared from the deposited bacterial cells by culturing the deposited strain by a conventional method.

The pAB-1 plasmid, like pGH-L9, can be easily transformed into E. coli by conventional methods. pAB-1
Has a pGH-L9-derived torptophan promoter / operator system, which strongly controls the expression of the hGH-AB gene, and thus the obtained transformants were cultured and grown in a medium and induced with indole acrylic acid.
Accumulates a significant amount of hGH-AB polypeptide. The culture conditions,
The induction conditions are the same as for E. coli pGH-L9.

The hGH-AB polypeptide accumulated in the bacterial cells can be separated and recovered by lysing the bacterial cells and then recovering and activating the usual physiologically active polypeptide.

The number of bases between the translation initiation codon and the trp L SD sequence is p
E. coli with a similar plasmid but slightly different from GH-L9.
pGH-L8 (Ministry of Microbiology Research Institute No. 7605), same-L11 (No. 7607)
No.) or -L13 (No. 7608) can be used to construct a similar expression system.

[Action] hGH-AB polypeptide directly acts on bone tissue of mammals including human to promote its growth.

[Examples] The present invention will be described in more detail with reference to Examples.

The reagents indicated by abbreviations in the examples are as follows.

Standard buffer: 50 mM Tris-hydrochloric acid pH 7.5 35 mM MgCl ₂ 35 mM 2-mercaptoethanol Tris-NaCl: 10 mM Tris-hydrochloric acid buffer, 0.14 M NaCl, pH 8.

TE-sucrose: 25% sucrose (W / V), 50 mM Tris-hydrochloric acid buffer, 1 mM EDTA pH8.

Lysozyme: 10 mg / ml lysozyme, 0.25M Tris-HCl buffer, pH 8.

0.5M EDTA: pH 8.

PNase: It is dissolved in 0.04 M acetate buffer (Ph5) at a concentration of 10 mg / ml, and heat-treated at 100 ° C for 5 minutes.

Lytic Mixture: 0.1% nonionic surfactant (Triton X-100), 50 mM Tris-hydrochloric acid buffer, 62.5 mM EDTA, pH8.

Polyethylene glycol (PEG) 6000: 1st grade powder 5M NaCl CsCl: solid.

EB solution: Prepared by dissolving ethidium bromite in water to a concentration of 4.67 mg / ml.

TE: 10 mM Tris-HCl buffer, 1 mM EDTA, pH 7.4.

TE-sarkosyl: 10 mM Tris-hydrochloric acid buffer, 1 mM EDTA, 0.38% Sodium N-Lauroyl Sarkosinate, pH 7.4.

TEN-10 mM Tris-hydrochloric acid buffer, 1 mM EDTA, 0.1 M NaCl, pH 7.4.

Example 1 E. coli pGH-L9 ((Ministry of Microbiology and Culture No. 7606) was added to PBB medium (peptone 10 g, meat extract 10 g, NaCl 2.5 g, yeast extract 2 g, water 1, pH 7.0) at 37 ° C. The plasmid was amplified by aerobically culturing at 2. When the bacteria had grown to 2-5 × 10 ⁸ / ml, chloramphenicol was added to a final concentration of about 150 μg / ml, and the mixture was incubated at 37 ° C. for 8 hours as it was.

About 30 ml of Tris-N from bacterial cells (about 1 g) collected from 400 ml of culture solution
Suspend in aCl, transfer to a centrifuge tube for cooling centrifuge with a volume of 40 ml,
Precipitation was performed by centrifugation at 7,000 rpm for 7 minutes. 10 ml of TE-sucrose was added and suspended, and after notification at 0 ° C for 5 minutes, 2 ml of 0.5 mM EDTA was added, and the mixture was further left at 0 ° C for 10 minutes. 16ml of Lytic
Mixture was added all at once, and the centrifuge tube was inverted several times to homogenize the whole. Leave it at 0 ℃ for 15 minutes, then at 0 ℃ for 15,0
It was centrifuged at 00 to 18,000 rpm for 30 minutes. The supernatant was gently transferred to another centrifuge tube. About 30 ml of clear lysate was obtained.

1/10 weight of the PEG powder and 1/10 volume of 5M NaCl were added to the cleared lysate and completely dissolved with a magnetic stirrer. After leaving it at 0 ° C. overnight, it was centrifuged at 10,000 rpm for 10 minutes. After separating the precipitate from the supernatant, add it to 3 ml of T
Dissolve well in E, add a small amount of TE, and add 4.7
It was 6 ml. To this was added 5.00 g CsCl, 0.5 ml EB solution and mixed well.

Add 2 volumes of ethanol and leave at 0 ° C for 20 minutes, then 10,000
Centrifuge at rpm for 10 minutes, remove the supernatant well, and precipitate with 9 ml TE-
Dissolve well in Sarkosyl, and with a small amount of TE-Sarkosyl 9.
Adjusted to 52g. The solution was mixed with 10 g CsCl and 1 ml EB solution and centrifuged at about 20 ° C. and 36,000 rpm for 36 hours. The plasmid ccDNA band was narrowly collected under irradiation with a black lamp (360 nm).

Sepharose Cl-48 column (0.8 x 20c) equilibrated with TEN
The obtained ccDNA fraction (0.8 ml) was added to
Gel filtration was performed by EN by the free flow method.

Approximately 5 ml void as monitored by absorbance at a wavelength of 254 nm
volume, DNA fraction, RNA fraction, followed by EB and CsCl
Spilled. Collect the first peak corresponding to the DNA fraction and
It was dialyzed into E to obtain almost pure pGH-L9 plasmid DNA.

50 μg of pGH-L9 was added to 20 mM Tris HCl (pH 7.5) -10 mM β
−EtSH −175 mM NaCl −10 mM MgCl ₂ Bgl II 37.5 U, Sal
Add I175U and incubate at 37 ℃ for 17 hours to stop the reaction.
After phenol treatment, DNA is precipitated with ethanol and 5%
It was subjected to polyacrylamide electrophoresis. From the gel, extract about 4.6 Kbp DNA fragment containing hGH-AB partial gene About 20
μg was obtained.

on the other hand A BglII-SalI linker DNA fragment consisting of the following chain was chemically synthesized.

Add 350 U of DNA ligase (Boehringer Mannheim) to 1 pmol of hGH7-AB gene and 10 pmol of linker DNA fragment to give 20
The binding reaction was performed at 20 ° C for 20 hours.

After stopping the reaction, phenol treatment was performed, and DNA was precipitated with ethanol. Using 1/4 of these, E. coli HB101 was transformed by the method of Cohen et al., And cloned using ampicillin resistance and tetracycline sensitivity as indicators.
The obtained transformant E. coli HB101 pAB-1 was transformed into E. coli.
Culture, lysis, plasmid isolation and purification were carried out in the same manner as for pGH-L9 to obtain 20 μg of pAB-1 plasmid. The obtained pAB-1 plasmid was sequenced by the dideoxy method, and it was confirmed that this coincided with the physical map of FIG.

M9-0.2% Casamino Acid-0.4% Glucose-Ap (20
μg / ml) -thiamine (10 μg / ml) medium was used as the medium, and indole acrylic acid (40 μg / ml) was used at the beginning of the logarithmic growth phase.
g / ml) and transformant E. coli HB101
pAB-1 was aerobically cultured for 23 hours.

The obtained bacterial cells contain 7M urea after ultrasonic disruption and centrifugation.
Purification by 20 mM Tris-HCl (pH 7.5), 10 mM β-mercaptoethanol chromatography was performed.

The same buffer was used for elution. Next is DEAE-cellulose
Charge the column, 0 mM in 20 mM Tris-HCl (pH 7.5) containing 10 mM β-mercaptoethanol and 7 M urea.
Gradiento elution was performed with 3M NaCl. High molecular weight impurities were removed using a Sephadex G-75 column in the same buffer.

The obtained hGH-AB fraction was dialyzed against a 10 mM ammonium carbonate solution. SDS-P at each purification step
The results of analysis by AGE are shown in FIG. In the figure, lane Mr is a molecular weight marker, 1 is an ultrasonic disruption solution, 2 is a solution of a precipitate obtained by centrifugation, 3 is a fraction after Sephadex G-50 column chromatography, and 4 is DEAE-.
fraction after cellulose chromatography, 5 for S
Each fraction was dissolved in ephadx G-75 and subjected to Sephadex G-50 column crumb. In addition, red + and-indicate the case where mercaptoethanol is contained and the case where it is not contained, respectively.

Example 2 The hGH-AB polypeptide thus obtained was found to have somatomedin-like activity in some ³⁵ S sulfate uptake experiments using chondrocytes obtained from rabbits.

The cells used in this bioassay were chondrocytes obtained from some rabbits, containing Dulbeco's modif containing 0.3% FCS.
After 24 h preincubation in ied Eagle's medium (DMEM), test sample and 0.5 μci H ₂ ³⁵ S
O ₄ was added and incubation was continued for another 24 h. Proteoglycan synthesis was performed by measuring the incorporation of ³⁵ SO ₄ ²⁻ into the substance precipitated with cetylpyridinium chloride after treatment with pronase E (Table 2).

[Effect of the Invention] Unlike the conventional substances having somatomedin-like activity, hGH-AB polypeptide has a high expression rate
It can be purified easily and efficiently, and can be widely applied as a bone growth promoter.

[Brief description of drawings]

FIG. 1 shows the active ingredients of the growth promoting agent for bone tissue of the present invention.
A physical map of a plasmid that can be used to prepare hGH-AB polypeptides is shown in FIG.
Fig. 3 shows the nucleotide sequences of both ends of the B gene, Fig. 3 shows the physical map of the plasmid which is the raw material for preparing the plasmid of Fig. 1, and Fig. 4 shows the hGH gene of this raw material plasmid. , FIG. 5 is a diagram explaining the preparation of the plasmid of FIG. 1 by the method of the present invention, and FIG.
FIG. 3 is a diagram showing an SDS polyacrylamide electrophoresis pattern of hGH-AB polypeptide.

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Claims (2)

[Claims] 1. A formula A polypeptide having an amino acid chain represented by (however, a methionine residue may be added to the N-terminus) and having somatomedin-like activity. 2. A formula A bacterium transformed with a plasmid capable of expressing a gene segment having a DNA sequence represented by is cultured in a nutrient medium to express this gene, which is the inherited product of the formula: A method for producing a polypeptide, which comprises collecting a polypeptide having an amino acid chain represented by (however, a methionine residue may be added to the N-terminus) and having somatomedin-like activity.