JPH069516B2 - Method for producing β-urogastron - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing β-urogastrone, more specifically, a β-urogastrone is excreted outside a cell by culturing a transformed microorganism constructed by a genetic engineering technique. The present invention relates to a method for collecting urogastrone.

2. Description of the Related Art Escherichia coli has been most studied genetically and biochemically, and thus has been widely used for the purpose of producing industrially useful polypeptides by so-called genetic engineering techniques. However, the polypeptide produced by Escherichia coli conventionally constructed by the above-mentioned genetic engineering method usually stays in the bacterial cell, and cannot be excreted out of the bacterial cell except some low molecular weight polypeptides. It was That is, various Gram-negative bacteria such as Escherichia coli are characterized by having a double membrane structure of an inner membrane and an outer membrane, and the secretion in E. coli is produced in the cytoplasm inside the inner membrane. A precursor of a polypeptide that passes through the inner membrane, the signal peptide required for its passage is separated from the precursor, and the signal peptide is inserted in the periplasmic space, which is a space sandwiched between the inner membrane and the outer membrane. It means that a polypeptide not containing is produced and accumulated. Except for some special exceptions, it is not generally known that the polypeptide accumulated in this periplasmic space is excreted outside the bacterial cell through the outer membrane, and the polypeptide is a periplasmic space. Either remained or was metabolized here.

Therefore, conventionally, in the production of a polypeptide by culturing Escherichia coli, the desired polypeptide has been collected by physically or chemically destroying the outer membrane of the bacterial cell or the bacterial cell. However, in the case of physically destroying the bacterial cells and the like, it is unavoidable that all soluble components in the bacterial cells are extracted at the same time, which makes the subsequent purification operation complicated. Further, as a method of applying a physicochemical force only to the outer membrane to take out the components accumulated in the periplasmic space, for example, the osmotic pressure shock method [H. C. Neu and A. Heppe
l, J. B. C. , 240 , 3685-3692 (19
65)] is known, but this method cannot avoid the complicated operation of extracting the target substance.

On the other hand, the present inventors previously found that the DNA base sequence encoding the signal peptide and the D encoding β-urogastrone.
We have succeeded in constructing a vector containing a DNA base sequence encoding a fusion polypeptide directly linked to an NA base sequence and Escherichia coli transformed with this vector, completed the invention based on this finding, and filed a patent application (Patent application Sho 59-2712
No. 06), the target polypeptide was collected from the transformed Escherichia coli in the same manner as in the conventional method of this kind.

Problems to be Solved by the Invention An object of the present invention is to expel the target polypeptide from the microbial cell instead of the method of collecting the target polypeptide accumulated in the microbial cell or periplasmic space as in the conventional method. It is to provide a new method of collecting.

The inventors of the present invention constructed a secretory expression vector of β-urogastrone, a microorganism transformed with the vector,
In the process of earnestly researching the culture conditions of the microorganism and the like, surprisingly, according to the culture of Escherichia coli transformed with the β-urogastron secretory expression vector, the secreted amount of β-urogastron, that is, periplasm While the amount of β-urogastrone accumulated in the space decreases with time, it was discovered that the target β-urogastrone is discharged to the outside of the cells in parallel with the growth of the bacteria instead. did.

The present invention has been completed based on this new finding.

Means for Solving the Problems That is, in the present invention, a DNA base sequence encoding a β-lactamase signal peptide derived from Escherichia coli (hereinafter simply referred to as “signal peptide”) and a DNA base sequence encoding β-urogastrone are directly attached to each other. A method for producing β-urogastrone, which comprises culturing Escherichia coli transformed with a vector containing a waste DNA base encoding a ligated fusion polypeptide and collecting β-urogastrone discharged outside the cells. Involve

According to the method of the present invention, the target β-urogastrone is discharged to the outside of the microbial cell, so that its collection is very easy, and the target substance substantially contains impurities such as soluble components derived from the microbial cell. There is an advantage that it can be easily isolated and purified without containing it.

Hereinafter, the method of the present invention will be described in more detail.

In the method of the present invention, a vector containing a DNA base sequence encoding a fusion polypeptide in which a DNA base sequence encoding a signal peptide and a DNA base sequence encoding β-urogastrone are directly connected is used. Specific examples of the vector include plasmid pUG2
01 can be illustrated. The plasmid pUG201 is pB
Constructed using R322 as the origin vector, the promoter and ribosome binding site of β-lactamase are followed by β-lactamase.
A plasmid pGH55 having a DNA base sequence encoding a lactamase signal peptide and a DNA base sequence encoding β-urogastrone were prepared from E of pBR322.
are those constructed from ccRI and _B am HI restriction is constructed by inserting between the sites plasmid pUG3 Prefecture. Details of the construction of the plasmid pUG201 are as described in Examples below.

The above-mentioned plasmid pUG201 was constructed in E. coli HB10
This strain has been introduced into one strain and the plasmid pUG201-harboring strain has been deposited at the Institute for Microbial Technology, Ministry of International Trade and Industry, under the designation "HB101 [pUG201]".
The deposit number is Micro Engineering Research Article No. 681 (FERMBP-
681).

In the present invention, the plasmid pUGM106 may be used as the vector instead of the plasmid pUG201. The plasmid pUGM106 is
It is constructed from the above plasmids pUG201 and pGH49, and similarly, a DNA base sequence encoding a signal peptide of β-lactamase and a DNA base encoding β-urogastrone following the promoter and ribosome binding site of β-lactamase. It has a sequence directly linked to the sequence. Details of the construction will be shown in Examples below.

In the present invention, E. coli, such as K12
The strain-derived HB101 strain or JM103 strain is transformed, and this transformed E. coli is cultured.

Transformation by introduction of the above vector into Escherichia coli can be carried out according to a usual method. Specifically, for example, a method of treating Escherichia coli as a host cell at low temperature in an aqueous solution containing calcium chloride and adding a vector to the solution [E. Lederbeg, S .; Cohen, J .; Bacterol., 1
19 , 1072 (1974)] and the like.

Further, the culture of the transformant obtained as described above is carried out according to a conventional method, for example, a commonly used medium such as L broth, E medium, M9 medium or the like, or various carbon sources, nitrogen sources, vitamins. It can be carried out using a medium to which an inorganic salt or the like is added. The culturing method is also not particularly limited, and for example, it may be a solid culturing method in addition to a liquid culturing method such as an ordinary shaking culturing method or an aeration and stirring method.

According to the above culture, with or after the growth of the bacterium,
The target polypeptide accumulated in the periplasmic space of the transformant is gradually released outside the cells. The method of the present invention collects the target substance released to the outside of the cells. Collection of this target product can be carried out by separating bacterial cells and culture supernatant according to a general method and collecting the supernatant, and the culture supernatant thus obtained is further subjected to, for example, gel filtration or adsorption chromatography. It can be purified by a combination of e, high performance liquid chromatography, ion exchange chromatography, and the like.

Examples Hereinafter, examples will be given to explain the present invention in more detail. In each example, the notation of amino acids, nucleobases, and other abbreviations follow the conventions of IUPAC and IUB or symbols conventionally used in the field. Further, each method and operation used in each example are as follows unless otherwise specified.

1. Cleavage operation of DNA by restriction enzyme An aqueous solution (or buffer solution) of DNA or powder is mixed with a concentrated solution of each buffer solution shown in Table 1 below and water, and then the restriction enzyme is added, and the mixture is placed in a 37 ° C water bath. Let stand for 3 hours to react. The standard amount of the restriction enzyme to be used is 1 unit per 1 μg of DNA, and the final liquid volume should be 10 μm or more.

2. After the enzymatic reaction, this extraction method was performed to inactivate the enzyme and stop the reaction. That is, to the reaction solution, half the volume of TE saturated phenol (10 mM Tris-hydrochloric acid (pH 8.0) buffer containing 1 mM EDTA saturated with phenol) was added and thoroughly mixed. Chloroform is added to further mix, and then the mixture is centrifuged to take a emotional fluid layer containing DNA. Further 0.1
Double volumes of 3M sodium acetate buffer (pH 5.0) and double volumes of cold ethanol were added and mixed, and the mixture was allowed to stand at -20 ° C for 1 hour or longer to recover DNA as a precipitate, thereby completely removing phenol. Remove.

3. DN by DNA polymerase I (Klenow fragment)
Method for converting blunt end of A 40 mM potassium phosphate (pH 7.4), 6 mM magnesium chloride, 1 mM β-mercaptoethanol, 1 m
MATP and 1 mM each of dATP, dCTP, dGT
DNA is dissolved in an aqueous solution containing P and dTTP, and D
Add 1 unit of DNA polymerase I (Klenow fragment, manufactured by Takara Shuzo Co., Ltd.) to 1 μg of NA, and add 1 unit.
Incubate for 30 minutes at 2 ° C.

4. DNA fragment binding (circularization) operation with T ₄ DNA ligase 66 mM Tris-HCl (pH 7.5), 6.6 mM magnesium chloride, 10 mM dithiothreitol and 1 mM
In an aqueous solution in which 0.01% bovine serum albumin was added to ATP, DNA fragment and T ₄ DNA ligase (manufactured by Takara Shuzo Co., Ltd.) in an amount of 3 units per 1 μg thereof were mixed with each other.
The DNA is bound (circularized) by reacting at 2 ° C. for 5 hours or more.

5. Transformation method As a host cell, HB101 strain derived from Escherichia coli K12 strain is used.

HB101 strain was transferred to LB culture (1% bactotryptone,
Grow with 0.5% Bacto yeast extract, 0.5% sodium chloride) at 37 ° C. until absorbance at 610 nm of 0.25. Centrifuge 40 ml of this culture (6000
Rotation / minute × 10 minutes) to collect the bacterial cells, followed by cooling with ice. Wash this with 20 ml of 0.1M magnesium chloride,
Then ice-cooled 0.1M calcium chloride and 0.05M
Suspend in 20 ml of magnesium chloride solution and chill with ice for 1 hour. After centrifugation (6000 rpm / minute × 10 minutes), the cells are resuspended in 2 ml of ice-cooled 0.1 M calcium chloride and 0.05 M magnesium chloride solution. This suspension 0.2
0.01 ml of a reaction composition solution of a DNA fragment bound using T ₄ DNA ligase is added to ml and ice-cooled for 1 hour. Then, the mixture was heated in a water bath at 42.5 ° C. for 90 seconds, and LB culture was performed
8 ml is added and this is left still in a 37 degreeC water bath for 1 hour.

Next, the resulting transformant is selected with the following antibiotic resistance. That is, 0.3 ml of each solution of the reaction composition obtained above was spread on a plate medium prepared by adding 50 μg / ml of ampicillin or 20 μg / ml of tetracycline to an LB culture containing 1.5% agar. Were incubated at 37 ° C overnight,
Isolate growing E. coli colonies.

6. Isolation of plasmid A strain carrying the plasmid was treated with 50 μg of ampicillin /
ml or 500 ml of LB medium supplemented with 20 μg / ml of tetracycline, and shake-cultured at 37 ° C. until the absorbance at 610 nm becomes about 0.6. Then, 80 mg of chloramphenicol is added, and the mixture is cultured at 37 ° C. for 12 to 16 hours with shaking.
The cells are collected by centrifugation (6000 rpm / minute × 10 minutes), and washed with 0.85% sodium chloride aqueous solution.
50 mM Tris-hydrochloric acid containing 20% sucrose (pH 8.
0) Suspended in 2.5 ml buffer solution, then 0.5% 0.25 M Tris-HCl (pH 8.0) buffer solution containing 1% lysozyme
Add ml and cool on ice for 10 minutes. Further 0.25M EDT
Add 1 ml of A (pH 8.0) and cool with ice for 10 minutes. Next, 6 mM Tris-HCl (pH 8.0), 60 mM EDTA
And 4 ml of a solution of 0.1% Triton X-100.
This is subjected to ultracentrifugation (25,000 revolutions / minute × 90 minutes) to collect the supernatant. 9.0 g of cesium chloride was added to 8.2 ml of this supernatant.
To dissolve, then add 0.8 ml of 1% ethidium fromide solution. This is centrifuged (2000 rpm / min x 10 min) to remove suspended matter, and the solution is ultracentrifuged (5000
0 revolutions / minute × 15 hours). Then, the plasmid portion that emits fluorescence is separated by ultraviolet irradiation. 5-6 this with isopropanol saturated with 5M sodium chloride solution
Extract twice to remove ethidium bromide.
Finally, 10 mM Tris-hydrochloric acid containing 1 mM EDTA (p
Dialysis against (H8.0) buffer to remove cesium chloride.

7. Oligonucleotide synthesis was carried out by the solid phase synthesis method (solid phase phosphate triester method) shown below [H. Ito et al, Necleic Acids
Research, 10 , 1755-1769 (198)
2)].

That is, first, 1% cross-linked polystyrene resin S-X1 (200
~ 400 mesh, manufactured by Bio-Rad Laboratories) is reacted with a monosuccinic acid ester of 5'-O-dimethoxytrityl nucleoside to obtain a nucleoside-supporting resin. Next, the following operation is carried out using a DNA synthesizer manufactured by Birch.

40 mg of the above resin is placed in a reaction tube, and the dimethoxytrityl group at the 5'-position is eliminated by using a 1 M solution of zinc bromide in dichloromethane-isopropanol (85:15). Then the fully protected dinucleotide [C. Broka et al,
Nucleic Acids Researc, 8 , 5461-5471
(Prepared by the method of (1980)], and 50 mg of triethylammonium salt are added and condensed using a condensing agent (mesitylenesulfonyl-5-nitrotriazole).
The above operation is repeated to sequentially extend the chain length and obtain a resin carrying the protected oligonucleotide. Incidentally, in the final condensation step, 25 mg of triethylammonium salt of mononucleotide prepared by the method described in the above literature is used in place of dinucleotide, if necessary.

The protected oligonucleotide is then cleaved from the resin using 0.5M pyridine cardoximate in pyridine-water (1: 1). This is Sephadex G-5
0 column (Pharmacia Co., 2 × 100 cm), and further high performance liquid chromatography (pump; Waters 6000A type, detector; 440 type detector, column; microbonder pack C18, elution solvent; (5 →
40%) acetonitrile-0.1 M triethylammonium acetate aqueous solution). Next, a deprotection reaction is performed with 80% acetic acid, and the product is purified again by high performance liquid chromatography until a single peak is obtained. The conditions for this high performance liquid chromatography are (5 → 25
%) Acetonitrile-The same as the above except that 0.1 M triethylammonium acetate aqueous solution is used.

8. Analysis of DNA Base Sequence The analysis of the DNA base sequence was carried out by the method of Messing [M13 method, Methods Enzymol. , 101 , 20 (1
983)]. That is, first, a DNA fragment is cut out with a restriction enzyme and separated by 1% agarose gel electrophoresis. This DNA fragment is M13
mp8RF (manufactured by Amersham) is cloned as a vector. The resulting recombinant phage DNA was subjected to the method of Mandel and Higa [J. Mol. Bio
l. , 53 , 154 (1970)] by E. coli JM.
Transduce 107 strains. To 0.2 ml of this cell suspension,
25 mg / ml isopropyl-β-D-thiogalactoside 25μ and 20 mg / ml 5-bromo-4-chloro-3
40 μm of indolyl-β-D-galactoside was added. Next, after heating and dissolving this bacterial cell suspension, 50 ° C
3 ml of H-topu agar solution (1% bactotryptone, 0.8% sodium chloride and 0.5% agar) kept warm at 3. was added and solidified by adding 1.5% agar (1. 6% bactotryptone, 1% yeast extract and 0.
Layer over 5% sodium chloride) and incubate overnight at 37 ° C. Recombinant phage with the inserted DNA fragment produces colorless plaques, whereas parent strain M13mp8 produces blue plaques, so that the target recombinant phage can be easily selected.

Next, a single colorless coupler was taken out with a Pasteur pipette, and this was mixed with 0.01 ml of JM103 culture solution and 2x.
In addition to 1 ml of TY culture, shake culture at 37 ° C. for about 5 hours to grow recombinant phage. After culturing, the bacterial cells were removed by centrifugation, 0.2 ml of 20% polyethylene glycol 6000 was mixed with the supernatant, and the mixture was allowed to stand at room temperature for 15 minutes or longer, and then the precipitated precipitate was collected by centrifugation and extracted with phenol. Then, a single-stranded DNA is extracted from the phage and used as a template single-stranded DNA.

Template single-stranded DNA and primer (Takara Shuzo Co., Ltd., M1)
15 base primer of 3 [5'AGTCACGACGT
TGTA3 ']) and 0.5 pmol of each,
After heat treatment at 60 ° C. for 20 minutes, it is gradually cooled. Next, α ³² P-dCTP (manufactured by Amasyamu Co., 400 Ci /
2 mol of DNA polymerase I (Klenow, manufactured by Takara Shuzo Co., Ltd.) and thoroughly mixed, and 3.2 μ each of them was mixed with 4 kinds of dNTP-ddNTP shown in Table 2 below. Add each of the liquids to a reaction tube containing 2 μm. After reacting for 20 minutes at room temperature, 1 μ of the chase reaction solution (1 mM each of dATP, dCTP, dGTP and dTTP) is added to each, and further reacted for 20 minutes. Formamide stop solution (95% v / v formamide, 0.1% xylene cyanol and 0.1% bromophenol blue) is added in 6 μm increments, heated at 95 ° C. for 3 minutes, and then rapidly cooled. Next, 2 μm of each sample was electrophoresed by 6% or polyacrylamide gel (1800 V,
30 mA, 2-3 hours). After the migration, transfer the gel to paper (Watman 3MM) and dry with a gel dryer.
Take an autoradiogram and decode the DNA base sequence.

However, in Table 2, ddA is dideoxyadenosine, ddC is dideoxycytidine, ddG is dideoxyguanosine, and ddT is dideoxythymidine.

9. Agarose Gel Electrophoresis Guidebook by Schleif and Wensink ["Practical Methods in Molecular B
Biology ”(1981), Springer-Verlag, pp11.
4-125], the agarose gel electrophoresis and the separation of the DNA fragment from the gel after the electrophoresis are performed. As a power source for electrophoresis, Constar Power SJ1065 type manufactured by Ato Co., a 12 × 15 cm plastic water tank (with platinum electrode) as an electrophoresis tank, and agarose I (manufactured by Dojindo Laboratories) as agarose, and for electrophoresis As the buffer, 40 mM Tris-hydrochloric acid (containing 5 mM sodium acetate and 1 mM EDTA, pH 7.9) is used.

10. Polyacrylamide gel electrophoresis According to the method described on pages 78-87 and 114-125 of the above-mentioned guide, DNA fragments are separated from the gel after polyacrylamide gel electrophoresis and electrophoresis. As an electrophoretic power supply, Conto Power SJ10 manufactured by Atto
Type 65, SJ1060SD manufactured by Atto as a migration tank
Use a mold. As an acrylamide solution, acrylamide and N, N'-methylenebisacrylamide (29:
An aqueous solution of 1) is used as a polymerization accelerator to prepare N, N, N ',
N'-tetramethylene ethylenediamine is used, and ammonium persulfate is used as a polymerization catalyst. 90 mM containing 2.5 mM EDTA as a buffer for electrophoresis
Tris borate buffer (pH 8.3) is used.

Example 1 I) Construction of pGH54 and pGH55 Expression Vectors pGH54 and pGH55 (A) Construction of Intermediate Plasmid pGH53 Synthesis of Oligonucleotide Having DAN Base Sequence Encoding a Part of Signal Peptide of Escherichia coli β-Lactamase For this purpose, each of the four types of oligonucleotides having the following base sequences was synthesized by the solid-phase phosphotriester method described above.

<1> (5 ') CGCCGGCCTTTTTGCCTT
CCTGTC (3 ') <2> TTCGGCGAACTCAGCTGC
A <3> GCTGAGTTCGCGAGAAC
AG <4> GAAGGCAAAAGGCCGGC
GAT The 5'ends of the oriconucleotides <2> and <4> are respectively T ₄ polynucleotidyl kinase (manufactured by BRL).
Was used for phosphorylation. That is, 10 μg of each oligonucleotide was added to 50 mM Tris-HCl aqueous solution (10 mM magnesium chloride, 5 mM dithiothreitol, 1 mM A
Dissolve in 50μ containing TP (pH 9.5) and add T
Add ₄ units of polynucleotidyl kinase, and add 37 units.
The reaction was carried out at 30 ° C. for 30 minutes, and the reaction was stopped by phenol extraction.

As a cloning vector, plasmid pBR3
22 [Bolivar et al. Gene, 2 , 95-113 (1
977)] was used.

10 μg of the plasmid pBR322 was digested with the restriction enzyme Ps
Digested with tI (Takara Shuzo Co., Ltd.) and PvuI (NEB Co.) in a high-salt buffer, followed by 1.0% agarose gel electrophoresis to separate a DNA fragment of about 4.24 kb. .

The DNA fragment obtained above was converted into the phosphorylated oligonucleotides <2> and <4> and the unphosphorylated oligonucleotides <1> and <3 prepared above.
About 1 μg of each> were combined and subjected to a binding reaction with T ₄ DNA ligase. After completion of the reaction, the reaction composition solution was used to transform the HB101 strain derived from the Escherichia coli K-12 strain.
One strain was selected from the obtained transformants showing tetracycline resistance, and a plasmid was isolated from this strain to obtain the desired p
GH53 was obtained.

The outline of a series of operations is as shown in FIG.

The obtained pGH53 had a size of about 4.3 kb as a result of 1.0% agarose gel electrophoresis, and its DN
As a result of analysis of the A base sequence by the M13 method, pBR32
2 between the P _st I and P _vu I restriction sites were deleted,
Instead, as shown below, oligonucleotides <1>, <
It was confirmed that 2>, <3> and <4> were inserted.

The HB101 strain harboring the pGH53 has been designated as “HB101 (pGH5
3] ”is displayed with the name of Micro Engineering Lab. No. 678 (FERMBP-
678).

(B) β-urogastrone expression vector pGH54
Construction of 10 μg of pGH53 obtained in (A) above was cleaved with a restriction enzyme NaeI (manufactured by NEB) and AvaI (manufactured by Takara Shuzo Co., Ltd.) in a medium salt concentration buffer, and then 1.0% agarose gel. DNA of about 2.22 kb after electrophoresis
Fragment << A >> was separated.

This fragment contains most of the DNA sequence derived from the synthetic oligonucleotide and the replication origin of the plasmid.

pBR322 was cleaved with restriction enzymes AvaI and HindIII (both manufactured by Takara Shuzo Co., Ltd.) using a medium salt concentration buffer solution and subjected to 1.0% agarose gel electrophoresis to obtain a DNA fragment of about 1.40 kb << B >> was obtained.

This fragment contains a part of the promoter of the tetracycline resistance gene and the entire tetracycline resistance structural gene.

20 μg of pBR322 was digested with the restriction enzyme Fnu4HI (N
EB) and cut with a low salt buffer, then S
The protruding base at the end of the DNA fragment was decomposed and removed by 1 nuclease. The DNA after phenol extraction was dissolved in 1 ml of 6 mM sodium acetate and 40 mM sodium chloride 1 mM zinc sulfate buffer (pH 4.5), and 2000 units of S1 nuclease (manufactured by BRL) was added to the solution.
The reaction was carried out at 30 ° C. for 30 minutes. Then
After the phenol extraction, the DNA was cleaved with a restriction enzyme Hind III using a medium salt concentration buffer and subjected to 6% polyacrylamide gel electrophoresis to obtain a DNA fragment << C >> of about 0.28 kb.

This fragment contains the promoter of the β-lactamase, the ribosome binding site, part of the gene encoding the signal peptide, and part of the promoter of the tetracycline resistance gene.

The three fragments << A >>, << B >> and << C >> obtained above
Was ligated with T ₄ DNA ligase. After the reaction
The HB101 strain was transformed with this reaction composition solution. One strain was selected from the obtained transformants showing tetracycline resistance and the plasmid was isolated. Thus, pGH54 was obtained.

As a result of the nucleotide sequence analysis by the M13 method, pGH54 was β
-A lactamase promoter and a ribosome binding site, followed by a DN base sequence encoding a signal peptide, and NruI upstream of the 3'end of the base sequence and PvuII restriction enzyme recognition sequences downstream thereof. It was confirmed that

A schematic diagram of a series of operations is shown in FIG.

pGH54 is characterized by the size of about 3.9 kb as described above and the restriction enzyme cleavage map shown in FIG. 2, and as a result of the nucleotide sequence analysis by the M13 method, it is encoded by the nucleotide sequence shown in the following formula (1). It was confirmed to have the gene for the β-lactamase signal peptide.

ATGAGTATTCAACATTTCCGTGTCG
CCCTTTATTCCCTTTTTTTGCGGCCTT
TTGCCTTCCTGTCTTCCGCGAACTCA
GCTG (1) Escherichia coli HB101 carrying the above plasmid pGH54
The strain has been deposited under the designation "HB101 [pGH54]" as Micro Engineering Research Article No. 679 (FERM BP-679).

(C) Vector pGH55 for β-urogastron expression
Construction of pBRRH02, a plasmid in which the nucleotide sequence between the AvaI and PvuII restriction sites of pBR322 was deleted, was prepared by the following procedure. That is, 5 μg of pBR322
With the restriction enzymes AvaI and PvuII in a medium salt buffer.
(Both were manufactured by Takara Shuzo Co., Ltd.), extracted with phenol, and then blunt-ended with DNA polymerase I (Klenow fragment, manufactured by Takara Shuzo Co., Ltd.). Next, DN of about 3.72 kb was obtained by 1.0% agarose gel electrophoresis.
The A fragment was isolated and this fragment was circularized with T ₄ DNA ligase. After the reaction was completed, HB101 strain was transformed with this reaction composition solution, and one strain was selected from the obtained transformant strains showing ampicillin resistance and tetracycline resistance, and the flasmid was isolated to obtain pBRH02. The pB obtained
RH02 is different from pBR322, Pva in AvaI
I was not cut even in II.

5 μg of pBRH02 obtained above was added to the restriction enzyme Ppt.
I and BamHI (both manufactured by Takara Shuzo Co., Ltd.) were used to cleave in a medium salt concentration buffer solution, and then 1.0% agarose gel electrophoresis was performed to obtain a DNA fragment << D >> of about 2.60 kb. separated.

This fragment contains part of the tetracycline resistance gene and the replication origin of the plasmid.

limit the 10μg of pGH54 enzyme PstI and _{B am}
Cleave with HI in medium salt buffer, then 1.0%
Agarose gel electrophoresis was performed to obtain a DN of about 0.66 kb.
A fragment << E >> was obtained.

This fragment contains DNA encoding the β-lactamase promoter, ribosome binding site, and signal peptide.
The sequence and part of the tetracycline resistance gene are included.

The two fragments << D >> and << E >> obtained above were transformed into T ₄ D
Ligation was performed using NA ligase. After the reaction, HB101 strain was transformed with this reaction composition solution. One strain was selected from the obtained transformants showing tetracycline resistance and the plasmid was isolated. Thus, pGH55 was obtained.

A schematic diagram of a series of operations is shown in FIG.

pGH55 was characterized by the restriction enzyme cleavage map shown in FIG. 3, and had a size of about 3.3 kb as a result of 1.0% agarose gel electrophoresis. Also the pG
H55 is pGH as a result of the nucleotide sequence analysis by the M13 method.
Approximately 0.6 including the second Pvu II restriction site at 54
It was confirmed that the first PvuII restriction site was the same as that of the pGH54 except that it lacked a 4 kb DNA, and that the first PvuII restriction site existed near the 3'end of the NDA nucleotide sequence encoding the signal peptide.

E. coli HB101 carrying the above plasmid pGH55
The strain has been deposited under the designation "HB101 [pGH55]" as Microtechnology Research Institute No. 680 (FERM BP-680).

II) Construction of a vector having a DNA base sequence encoding a signal peptide-β-urogastrone fusion polypeptide (A) Synthesis of a DNA base sequence encoding β-urogastrone This base sequence was reported by G. Gregory. Amino acid sequence [Nature, 257 , 325-3
27 (1975)], first, a start codon before and after the DNA base sequence encoding β-urogastrone,
It was carried out by constructing the DNA base sequences shown in Table 3 below, in which a stop codon and a restriction enzyme recognition site were added. This DAN base sequence has been applied for a patent by the present inventors as Japanese Patent Application No. 59-137691.

(B) Construction of a plasmid having a DNA base sequence encoding β-urogastrone 10 μg of pBR322 was first cleaved with EcoRI (Takara Shuzo Co., Ltd.) and BamHI in a high salt buffer,
Then, 1.0% agarose gel electrophoresis was performed to isolate a DNA fragment of about 3.99 kb.

The DNA fragment obtained above and the β-obtained in (A) above
The DNA base sequence encoding urogastrone was added to T ₄
Ligated with DNA ligase. After the reaction, H in the reaction composition
The B101 strain was transformed, and one strain was selected from the obtained format-converted strains showing ampicillin resistance and a plasmid was isolated. Thus the DNA sequence encoding the β- urogastrone obtain plasmid pUG3 inserted between pBR322 EcoRI and _B am HI restriction site.

The HB101 strain harboring this plasmid pUG3 is
"HB101 [pUG3]" is displayed and the microfabricated microbe
Deposited as No. 43 (FERM BP-543).

(C) Construction of pUG201 The DNA fragment obtained by cleaving pUG3 obtained in (B) above with the restriction enzyme HinfI is inserted into the PvuII restriction site of pGH55 to encode a signal peptide-β-urogastrone fusion polypeptide. PUG201, which is a vector containing a DNA base sequence, was constructed by the following method.

15 μg of pUG3 was added to Hinf in high salt buffer.
Cut with I (Takara Shuzo Co., Ltd.), extract with phenol, and then D
The cleaved fragment was blunt-ended with NA polymerase I (Klenow fragment). Then, 6% polyacrylamide gel electrophoresis was performed to isolate a DNA fragment << F >> of about 0.43 kb.

This fragment contains DNA encoding β-urogastrone.
Among the nucleotide sequences (including the translation stop codon), the nucleotide sequences excluding the 7 nucleotides at the 5'end were included.

In pGH55, the first 7 DNA base sequences encoding the N-terminal region of β-urogastron are directly linked to the DNA base sequence encoding the β-lactamase signal peptide, and the direct connection cuts with the restriction enzyme Pvu II. Having a DNA base sequence configured as described above, 5 μg of the pGH55 in a medium salt concentration buffer,
Cleavage with PvuII gave a DNA fragment << G >> of about 3.2 kb.

This fragment carries all the genetic information of pGH55.

About 1 μg of the fragment << F >> obtained above and about 0.5 μg of the fragment << G >> obtained above were ligated with T ₄ DNA ligase. After the reaction, HB101 strain was transformed with this reaction composition solution, one strain was selected from the resulting transformants resistant to tetracycline, and plasmid pUG201 was isolated.

As a result of 1.0% agarose gel electrophoresis, pUG201 had a size of about 3.8 kb. This is BamHI
Or, when cut with Hind III, 2 kinds of DNA each
Since the fragment was obtained, it was found that the pUG201 contained the β-urogastron gene, and the size of the fragment was examined. As a result, it was found that the plasmid was the desired plasmid. Furthermore, with respect to pUG201, the nucleotide sequence of a DNA fragment including the β-lactamase promoter portion to the β-urogastron gene was examined by nucleotide sequence analysis by the M13 method. As a result, the DNA base sequence is shown in Table 4 below, and pUG201 encodes a promoter, a ribosome binding site, a β-lactamase signal peptide base sequence, and a β-urogastrone base sequence (the fusion polypeptide is It was recognized that the encoding base sequences) are arranged in this order in the exact order. Table 4 also shows the amino acid sequence corresponding to the DNA base sequence.

The above-mentioned pUG201 is held in Escherichia coli HB101, and this holding strain has been deposited at the Institute of Microbiology, Ministry of International Trade and Industry, Institute of Industrial Technology, under the designation "HB101 [pGH201]". The deposit number is Mikken Ken 681 (FE
RM BP-681).

III) Production of β-urogastrone by Escherichia coli harboring pUG201 (A) Culture of Escherichia coli HB101 strain harboring pUG201 A modified E medium having the following composition was used.

<Modified E medium (composition per 1)> Magnesium sulfate heptahydrate 0.2 g Citric acid monohydrate 2.0 g Anhydrous dipotassium phosphate 10.0 g Ammonium hydrogen phosphate sodium sodium tetrahydrate 3 0.5 g glucose 2.0 g casamino acid 1.0 g L-proline 0.23 g L-leucine 39.5 mg thiamine hydrochloride 16.85 mg tetracycline hydrochloride 20.0 mg pUG201 pre-cultured liquid 1 ml of Escherichia coli HB101 strain. 2 at 37 ° C in 200 ml of modified E medium
The cells were cultivated with shaking for 4 hours, and a fixed amount (10 ml) was collected over time to separate the bacterial cells and the culture supernatant.
-The amount of urogastrone was measured by β-urogastrone specific radioimmunoassay (RIA).

The bacterial cells and the culture supernatant were separated by centrifugation (6000 rpm / minute × 10 minutes, 4 ° C.). The obtained culture supernatant is used as the extracellular fraction.

In addition, the cells were subjected to the osmotic pressure shock method [H. C. Neu and L.L.
A. Heppel, J .; B. C. , 240 , 3685-36
92 (1965)], the perivurism fraction was extracted. In this operation, first, 1 g of wet weight of cells was suspended in 80 ml of 30 mM Tris-HCl buffer (pH 8.0) containing 20% sucrose, and 0.32 ml of 0.25M EDTA aqueous solution (pH 8.0) was added, After stirring for 10 minutes at 180 rpm at 24 ° C on a rotary shaker, centrifugation (90
The cells were collected at 80 rpm / min × 10 minutes), resuspended in 80 ml of ice-cold water, left standing in ice for 10 minutes, stirred occasionally, and centrifuged (9000 rpm / minute). Min x 10
It was carried out by separating the bacterial cells and the supernatant by the step (1). The obtained supernatant is used as a periplasm fraction.

Furthermore, the bacterial cells were washed with a buffer (10 mM Tris-HCl and 30 mM).
After washing with mM sodium chloride, pH 8.0), PBS
(20 mM sodium phosphate containing 150 mM sodium chloride, pH 7.0) Suspended in 10 ml, and crushed three times for 30 seconds each with an output of 100 W using an ultrasonic crusher (manufactured by Otake Seisakusho) model 5202). , Centrifuge (1800
(0 rpm / minute × 20 minutes, 4 ° C.) to obtain a supernatant. This is the intracellular fraction.

(B) Measurement of β-urogastrone by RIA For each fraction obtained in (A) above, β
-The presence of urogastrone was examined by β-urogastrone specific radioimmunoassay (RIA). RIA
The method is as follows. That is, an antiserum was prepared by immunizing a rabbit with purified human β-urogastrone as an antigen.
After dissolving 300 μg of β-urogastrone in 0.2 ml of distilled water, 1.5 ml of 50% polyvirpyrrolidone solution was added and stirred at room temperature for 2 hours. 2.0 ml of complete Freund's adjuvant was added to emulsify and subcutaneously injected into the chest of 3 rabbits. After repeating immunization 4 times every 2 weeks, 5 more
0 μg of antigen was intravenously injected, and after 3 days, whole blood was collected to separate serum.

Next, examine the titration curve for determining the dilution ratio of the antiserum used for the assay, the incubation time for optimizing the assay conditions, and the method for separating antibody-bound labeled antigen (bound) and free labeled antigen (free). Was added and the following measurement conditions were set.

That is, 0.5% bovine serum albumin (BSA), 140
A 10 mM phosphate buffer (pH 7.4) containing mM sodium chloride and 25 mM disodium EDTA was used as a diluting solution, and the diluting solution 400 μ, 100 μ of a measurement sample or standard human β-urogastrone and 100 μ of anti-human β-urogastrone serum were added. After incubating at 4 ° C. for 24 hours, 100 μm (about 5000 cpm) of ¹²⁵ I-labeled human β-urogastrone was added. After further incubation at 4 ° C for 48 hours, the second antibody (anti-rabbit γ-
Globulin goat serum) (1:20) 100 μ, normal rabbit serum (1: 200) 100 μ, and 10 mM PBS solution 900 μ containing 5% polyethylene glycol were added and incubated at 4 ° C. for 3 hours. Then 300 rpm
After centrifuging at 30 minutes, the supernatant was removed and the precipitate was counted. The content of human β-urogastrone immunoactive substance in the sample was determined from a standard curve obtained from standard human β-urogastrone.

The results of the above RIA are shown in FIG. In the figure, the horizontal axis represents the culture time (hr) and the vertical axis represents the extracellular β-urogastrone amount (μg / l) shown in graph (1), graph (2).
Periplasmic layer β-urogastrone amount (μg
/ L), the amount of intracellular β-urogastrone (μg / l) shown in graph (3), and 61 shown in graph (4).
The respective absorbances (OD) at 0 nm are shown.

From FIG. 5, in the main culture, the amount of β-urogastron in the priplasm layer (graph (2)) increased in proportion to the growth of the bacteria up to 6 hours, but decreased thereafter.
-It is clear that the amount of urogastron (graph (1)) increases.

(D) Purification and confirmation of β-urogastrone The β-urogastrone immunoreactive substance in the extracellular fraction 20 was subjected to adsorption chromatography using Butyl Toyopearl 650C (manufactured by Toyo Soda Co., Ltd.), DEAE-.
Anion exchange chromatograph using Toyopearl 650M (manufactured by the same company), cation exchange chromatograph using CM-Toyopearl 650M (manufactured by the same company), and gel filtration using Cefadex G-25 (manufactured by Fuarmacia). Fractionally purified by sequential chromatographic operations to obtain a single polypeptide 98 with a purity of 99% or more.
to obtain mg.

Natural β-urogastrone (manufactured by Japan Chemical Research) isolated and purified from the obtained polypeptide and human urine
Figure 6 shows the elution pattern obtained by reversed phase high performance liquid chromatography. FIG. 6 shows TSK gel-ODS-12.
22% using 0T column (manufactured by Toyo Soda Co., Ltd.)
50 mM phosphate buffer (pH 7.
0) is the result of elution at a flow rate of 1.0 ml / min. In the figure, the vertical axis represents the absorbance at 280 nm and the horizontal axis represents the retention time (minutes), and (1) is the above-mentioned value relating to the present invention. The results for polypeptide and (2) are the results for native β-urogastrone.

From the figure, it can be seen that the retention times of both are completely the same.

In addition, the polypeptide obtained above was hydrolyzed with 4M metal sulfonic acid to obtain a Hitachi amino acid analyzer model 8
The results of amino acid analysis obtained by the orthophthalaldehyde method using the No. 35 type are shown in Table 5 below.

From the table, it can be seen that the number of actually measured residues is in good agreement with the theoretical number of residues.

Furthermore, a test for confirming the cell growth promoting activity of the polypeptide obtained above was conducted as follows.

This test is performed by the method of Nakamura et al. [J. Biochem. , 94 (1
983) 1029-1035], and the high molecular weight DNA of [ ³ H] -thymidine by primary rat hepatocytes of the mature rat.
Was measured by measuring uptake into.

The results are shown in Fig. 7. In the figure, (1) shows the result of the polypeptide obtained by the present invention, and (2) shows the same test for mouse EGF (manufactured by Collaborative Research) known to have similar activity. The result.

From the above figure, the polypeptide according to the present invention is mouse EG
It is clear that it exhibits a stronger cell growth promoting activity than F.

In addition to the above, as a result of polyacrylamide gel electrophoresis, amino acid sequence analysis, early eyelid cleavage activity test of newborn mice, etc., in any of them, the polypeptide of the present invention corresponds to natural β-urogastrone. Admitted.

Example 2 I) β-urogastron expression vector pUGM106
First, from plasmids pGL101 and pBR322,
pGH49 was constructed and then with pGH49 and Example 1I.
Target pU from pUG201 obtained in (C) of I)
GM106 was constructed.

An outline of a series of operations is shown in FIG.

(A) Construction of pGH49 pGL101 is a plasmid having a size of about 2.4 kb and contains an ampicillin resistance gene and the lacUV5 promoter [C. S. Thummelet al. J. Vir
ology, 37 , 683-697 (1981)].

5 μg of pGL101 was first digested with the restriction enzyme HinCII in medium salt buffer and then in high salt buffer,
After digestion with the restriction enzyme EcoRI, a DNA fragment << H >> of about 1.94 kb was separated by 1.0% agarose gel electrophoresis. This fragment contains the lacUV5 promoter and the replication origin of the vector.

After cleaving pBR322 with a restriction enzyme ScaI and EcoRI in a high salt buffer, 1.0% agarose gel electrophoresis was performed to separate a DNA fragment << I >> of about 3.85 kb.

This fragment contains the tetracycline resistance gene and the replication origin of the vector.

The two fragments << H >> and << I >> obtained above were transformed into T ₄ D
Ligation was performed using NA ligase. After the reaction was completed, HB101 strain was transformed with this reaction composition solution. One strain was selected from the obtained transformants showing tetracycline resistance and the plasmid pGH49 was isolated.

The resulting pGH49 was characterized by the diagram shown in Figure 8 above and had a size of approximately 5.8 kb as a result of 1.0% agarose gel electrophoresis.

(B) Construction of pUGM106 10 μg of pGH49 was treated with a restriction enzyme SalI in a buffer solution (SalI buffer solution) containing 150 mM sodium chloride, 6 mM Tris-HCl (pH 7.9), 6 mM magnesium chloride and 6 mM β-mercaptoethanol. Cleavage, followed by digestion with a restriction enzyme PvuII in a medium salt buffer, followed by 1.0% agarose gel electrophoresis to isolate a DNA fragment << J >> of about 0.75 kb.

This fragment contains part of the tetracycline resistance gene along with the lacUV5 promoter.

10 μg of pUG201 in medium salt buffer
Digested with the restriction enzyme AatII. 1.5 μg of this is 1
00 mM sodium chloride, 20 mM Tris-HCl (pH
8.1), 12 mM calcium chloride, 1 mM EDTA
Bal3 of 0.03 unit in 40 μm buffer containing
Partial decomposition was carried out by reacting with 1 nuclease (manufactured by Bethesda Research Laboratories) at 37 ° C. for 20 minutes. Then, this was cleaved with SalI in a SalI buffer and subjected to 1.0% agarose gel electrophoresis to obtain a DNA fragment << K >> of about 2.8 to 2.9 kb.
Was isolated.

This fragment contained a DNA base sequence encoding a fusion polypeptide in which a β-lactamase signal peptide was linked to β-urogastrone, a replication origin region of a vector, and a part of a tetracycline resistance gene.

The fragment << J >> obtained above and the fragment << K >> obtained above
And were mixed and ligated with T ₄ DNA ligase. After the reaction was completed, HB101 strain was transformed with this reaction composition solution, and one strain was selected from the obtained transformants resistant to tetracycline, and plasmid pUGM106 was isolated.

The obtained pUGM106 was characterized by the diagram shown in FIG. 8, and had a size of about 3.7 kb from the result of 1.0% agarose gel electrophoresis, and β-lactamase had a signal peptide β-lactamase. It was found to be a vector containing a DNA base sequence encoding a fusion polypeptide in which urogastron is linked and a tetracycline resistance gene. Further, the bla promoter used for the expression of the above-mentioned polypeptide in pUG201 remained as it was upstream of the DNA base sequence encoding the above-mentioned polypeptide, and lcaUV5 derived from pGH49 was located at about 90 bases upstream thereof. It was confirmed that the promoter was incorporated from the analysis performed using the restriction enzymes HhaI and MboII.

II) Production of β-urogastrone by Escherichia coli harboring pUGM106 (A) Culture of Escherichia coli HB101 strain harboring pUGM106 HB101 strain harboring the vector pUGM106 was cultivated, and the extracellular excretion of β-urogastrone is shown below. It investigated by the method. As the culture medium, M9 medium having the following composition, to which glucose, casamino acid, proline, leucine, thiamine and tetracycline was added, was used.

<Medium (composition per 1)> Disodium phosphate dodecahydrate 13.4 g Monopotassium phosphate 3.0 g Sodium chloride 0.5 g Ammonium chloride 1.0 g Calcium chloride dihydrate 14.7 mg Magnesium chloride 6 Water salt 203 mg Glucose 5.0 g Casamino acid 5.0 g L-proline 50 mg L-leucine 40 mg Cyamine hydrochloride 1 mg Tetracycline 15 mg Inoculate a flask containing 200 ml of the above medium, and inoculate with 37 bacteria.
Shaking culture was performed at 24 ° C. for 24 hours.

Absorbance at 610 nm was measured and then centrifuged (600
(0 revolutions / minute × 10 minutes, 4 ° C.) to separate the bacterial cells and the culture supernatant, and then the intracellular fraction and the extracellular fraction were obtained by the method described above, and the amount of β-urogastrone for each of them was obtained. Was measured by radioimmunoassay (RIA). RIA
Was performed by the method described above.

The results of the above RIA are shown in Table 6. In addition, Table 6 also shows the results of the same culture of the HB101 strain carrying pUG201 as a control.

From the above table, in the pGUM106 strain, β
-The total amount of urogastrone exceeded that of the pUG201 strain, and it was confirmed that most of it was excreted out of the cells by culturing at 37 ° C for 24 hours.

(B) Purification of β-urogastrone The immunologically active substance of β-urogastron in the extracellular fraction was purified to a single polypeptide in the same manner as above.

As a result, the purified β-urogastrone is the same substance as β-urogastrone isolated and purified from human urine in all of polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, amino acid analysis and N-terminal analysis. It was confirmed.

[Brief description of drawings]

FIG. 1 is a diagram showing the steps of cloning the synthetic oligonucleotides <1>, <2>, <3> and <4> into the origin vector pBR322 to obtain the plasmid pGH53, and the characteristics of pGH53 thus obtained. The middle □ indicates the base sequence derived from the synthetic oligonucleotide. FIG. 2 is a diagram showing a step of obtaining a vector pGH54 for β-urogastron expression from pGH53 and pBR322 and characteristics of the obtained vector pGH54. In the figure, ■ represents a nucleotide sequence encoding a signal peptide. FIG. 3 shows that pBRRH02 was obtained from pBR322, and
It is a figure which shows the process of obtaining the vector pGH55 for β-urogastron expression from H02 and pGH54, and the characteristics of the obtained pGH55. FIG. 4 is a diagram showing a process of obtaining a vector pUG201 containing a DAN base sequence encoding a fusion polypeptide of a signal peptide and β-urogastrone from pGH55 and pUG3 and a characteristic of the obtained vector pUG201. The arrow indicates the gene for β-urogastron. FIG. 5 is a graph showing the change with time of the amount of β-urogastrone obtained in Example 1. FIG. 6 is a diagram showing the results of reversed-phase high performance liquid chromatography of the polypeptide obtained in Example 1 and natural β-urogastrone. FIG. 7 shows the cell proliferation promoting activity of the polypeptide obtained in Example 1. FIG. 8 shows from pGL101 and pBR322 to pGH49.
FIG. 3 is a diagram showing the characteristics of each step and each vector for obtaining pUGM106 from the obtained pGH49 and pUG201. In the figure, the black arrow indicates the lacUV5 promoter, and the open arrow indicates the bla promoter.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical labeling (C12P 21/02 C12R 1:19) (C12N 15/31 C12R 1:19) Microbial accession number FERM BP-680 Microbial consignment number FERM BP-681 (72) Inventor Kazuhide Oshida 3218-12 Sakakoshi, Ako City, Hyogo Prefecture (72) Inventor Maki Yano 3139-60 Ozaki, Ako City, Hyogo Prefecture (72) Inventor Shigeru Mihara Akaho, Hyogo Prefecture 1-7, Kotobuki-cho (72) Inventor Aizo Matsushiro 4-15-12 Aoyamadai, Suita City, Osaka Prefecture (72) Inventor Noboru Yauchihara 403-22 Kita, Shizuoka City, Shizuoka Prefecture

Claims (1)

[Claims] 1. A vector containing a DNA base sequence encoding a fusion polypeptide in which a DNA base sequence encoding a β-lactamase signal peptide derived from Escherichia coli and a DNA base sequence encoding β-urogastrone are directly linked. A method for producing β-urogastrone, which comprises culturing transformed Escherichia coli and collecting β-urogastrone discharged from the cells.