JPS6323898A - Novel polypeptide, dna coding said polypeptide and production thereof - Google Patents

Abstract

NEW MATERIAL:A polypeptide expressed by formula I [left end is amino terminal and right end is carboxyl terminal; m, n, p and q represent 0 or 1; A-F represent an amino acid (sequence) region respectively corresponding to the following amino acid number, A: number 1, B: number 2-20, C: number 21, D: number 22-237, E: number 238-298, F: number 299-363]. USE:An activator for biophylaxis such as immune response, differential proliferation, etc. PREPARATION:For example, a human leukocyte is cultured with a differentiation inducer and an mRNA fraction is separated from the cell. A cDNA library is prepared by using the mRNA as a template. Then the cDNA is cloned from this library with a colony hybridization method, etc., and the cDNA is combined with an expression vector and introduced into a host cell to transform. The transformed host cell is cultured to provide the polypeptide expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel polypeptides and DNA encoding them.
I am also inspired by their manufacturing methods.

It is well known that blood cells such as T74 cells, B lymphocytes, macrophages, and white blood cells in human terminally ill blood produce and secrete various physiologically active substances. Interleukin 2 is produced by the 1974 cell, which acts as a growth factor for self-propagation and other 1974 cells.

In addition, factors related to the proliferation and differentiation of B74 cells are produced from B74 cells and 1974 cells. It has already been revealed that macrophages produce interleukin-71, cancer necrosis factor, and the like.

In this way, the differentiation and proliferation of lymphocytes and blood cells is not only controlled by extremely sophisticated and complex mechanisms by humoral factors produced by their own cells or other cells, but also by the humoral factors produced by these cells. immune response to infection and inflammation,
For malignant tumors, I! [It is assumed that it acts as a direct or indirect modification or protective factor.]

Naturally, human leukemia cells are similar to human blood cells.
For example, the human leukemia cell line HL-G.

It has been revealed that cells (ATCC, CCL240) produce interleukin 1 and cancer necrosis factor (
Y, Furutani et al., Nucletc Ac1d
s Res, 13°5809 (1985) and Penn1ca et al., Nature, 312°7
24 (+984>).

The present inventors focused on the diverse functions of the human leukemia cell line and believed that this cell line can produce an even wider variety of physiologically active substances, and as a result of intensive research using gene recombination technology. , 1-(Identification of the hitherto unknown polypeptide produced by L-GO cells and the gene encoding it,
The present invention has now been completed.

The present invention relates to a polypeptide having an amino acid sequence represented by the following general formula.

Am-Bn-Cp-D-Eq-F However, in the above formula, the left end means the amino terminal, the right end means the carboxy terminal, rrh ns p, Q is 0 or 1
and A-F mean the following amino acids or regions homologous to the amino acid sequences in Table 1, respectively.

A Niamino acid number 1 amino acid (Met), B Niamino I! Amino acid sequence of I@ No. 2-20 (Gly-3et)
, C° Amino acid number 21 (A I a), D=
Amino acid sequence of amino acids 22 to 237 (Tyr~
Thr), provided that the amino acid (X) at amino acid number 200 means Phe or Leu.

E” Amino acid sequence of amino acid numbers 238 to 298 (As
p~Glu), F'amino acid sequence of amino acid numbers 299-363 (As
p~Thr).

In the amino acid sequence represented by the general formula A-11-C-D-Eq-F, AB is an amino acid sequence that controls the secretion of a polypeptide synthesized within animal cells to the outside of the cell,
Therefore, the polypeptide secreted extracellularly is considered to be a polypeptide having an amino acid sequence represented by the general formula CP-D-Eq-F.

The novel polypeptide according to the present invention is a polypeptide that is induced to be produced when HL-f30 cells differentiated into macrophage-like cells by the action of phorbol ester, etc. is stimulated with endotoxin, or a polypeptide containing the main part thereof. It is clear that polypeptides are deeply involved in various defense mechanisms of the living body, such as immune response, cell differentiation and proliferation, and activation of biological defense functions.

The present invention also relates to the general formula %, where AF and ffh n1p1Q have the same meanings as above.

It relates to a DNA encoding a polypeptide having the amino acid sequence represented by

The method for producing DNA and polypeptide according to the present invention will be explained below.

DNA encoding the polypeptide of the present invention can be obtained by culturing human leukemia cells with a differentiation inducer such as phorbol ester, and then culturing them with an inducer for producing the polypeptide of the present invention, and then extracting mRNA from the cells. Separate the fractions,
A cDNA library is prepared using this as a template, and from this library, a cDNA encoding the polypeptide of the present invention is extracted by the colony hybridization method.
NA can be cloned.

Next, by culturing a host transformed with an expression vector incorporating this cloned cDNA or a DNA derived therefrom, the polypeptide of the present invention can be produced in the host or in a medium.

Below is the DNA related to the present invention! ! ! The method for producing the polypeptide according to the present invention will be explained in more detail.

Production of DNA according to the present invention 1, Preparation of mRNA Human leukemia cells were prepared at 1×10′ to 5×10′ cells/1 irises.
Cells are seeded at a density of 1, and a differentiation inducer is added to the cells.

The amount of differentiation-inducing agent added depends on the type and cell type! Category i.

Although it varies depending on the culture conditions etc., it is generally about 100 to 2,000
0 ng/■1 (final concentration) is preferred. Human leukemia cells together with a differentiation inducer at 35-38°C, preferably about 37°C
.

In air containing about 5-10% carbon dioxide, temperature about 90-100
% for about 24-72 hours.

Examples of human leukemia cells that can be used here include HL-
Examples include EtO cells (^TCC, CCI, 240).

Examples of differentiation-inducing agents include phorbol esters and diterpene compounds such as meserein.

As the medium, various synthetic media suitable for culturing higher animal cells are used, such as RPMI-1640° Eagle's MEM medium, MEM medium by Dulbecco's modified method [Cell Culture Manual, edited by Kosuke Munemura, Kodansha (1982). and Ce1l and Ti5sue Culture, J.
, Paul.

E, &S, Llvjngstone Ltd, (197
0)] is listed V Rale Z.

It is preferable to add animal serum (for example, fetal bovine serum, calf serum) to the medium in an amount of about 1 to 20% of the total volume of the culture solution.

After confirming that the cells have adhered to the surface of the culture container and differentiated into macrophage-like cells, the culture solution and floating cells are removed by suction. Next, a production inducer (e.g., phorbol ester and todoxin) and a protein synthesis inhibitor (e.g., cycloheximide) that induce the production of the polypeptide of the present invention are used.
By adding and culturing for an additional 3 to 8 hours, the mRNA
is accumulated in the differentiated cells.

After culturing, for example, Chirugubi 7 (Chirgubi 7) is extracted from the cells.
gwin) et al. [Biochemistry, +
8,5294 (1979)], total RNA was extracted by
Next, this is subjected to adsorption column chromatography using oligo(dT) cellulose or poly(U) cellulose according to a conventional method, or poly(A) is subjected to a batch method.
Separate the mRNA fraction. The mRNA can be concentrated and purified by subjecting this poly(A) mRNA fraction to acid urea agarose gel electrophoresis or CyN density gradient centrifugation.

11. Cloning of cDNA Using the mRNA fraction obtained in step ① as a template, oligo(
dT) as a primer, dATP, dGTP.

mR by reverse transcriptase (e.g., avian bone threatening leukemia virus-derived reverse transcriptase) in the presence of dCTP, dTTP.
s sc DNA complementary to NA is synthesized, and then, using this 5sc DNA as a template, dsc DNA is synthesized using reverse transcriptase or Escherichia coli DNA polymerase (large fragment). d s obtained here
c DNA was extracted using the poly(dG)-poly(dC) homopolymer extension method [Nelson, T., 6. , ”Metho
ds fn Enzy*ology''.

08.4+(+979>, ^ academic Pres
For example, using the restriction enzyme Ps of plasmid pBR322 according to a conventional method such as
Incorporate into the tI cleavage site.

The obtained recombinant plasmid is, for example, 2-en (C
ohen) et al.'s method [Proc, Nat, ^cad, s
ci, Us^, feri.

2110 (+972)], for example, E, col
i A host such as the 88101 strain is introduced and transformed, and a tetracycline-resistant strain is selected to prepare a cDNA library.

In order to obtain a transformant containing a recombinant plasmid into which a cDNA encoding the polypeptide of the present invention has been integrated from a cDNA library, for example, by adding a production inducer, a gene complementary to specifically produced mRNA can be obtained. Fronny Hypligizeta test using single-stranded cDNA as a probe [11anahan D, et al.
al., Gene, 10.83 (+980)] to select recombinants containing cDNA containing a base sequence complementary to the mRNA specifically synthesized by the production cycle, and from among these, further human cancer Necrosis factor and human interleukin 1 (hereinafter abbreviated as human IL-1).

) [V, Furutant et al. Nu
cleic Ac1ds Res,, 5869(19
85), Y, Yaiada et al. J, Biotech;
3.141 (1985) as a probe, a colony hybridization test is performed to select clones having cDNA containing a base sequence that does not bind to these probes.

Some of the cloned DNA fragments obtained in this way are, for example, produced by Maxam-Gilbert (Maxam-Gilbert).
Gilbert) #: [Proc, Nat,, Ac
ad, Sci, LIS^, 74°560 (+977)
] or dideoxy method CSang using M13 phage
er,F,,et al,,Proc,Nat,^ca
d, sci, Us^.

74.5403 (+977) and Messing, J.
, Methods in Enzymology, 10
1, 20 (1983)], a cloned cDNA containing a nucleotide sequence encoding the polypeptide of the present invention, which is different from the amino acid sequences of IL-1 and human cancer necrosis factor, was obtained. Obtainable.

The base sequence encoding the polypeptide of the present invention is
NA can also be totally synthesized based on the base sequences shown in Table 1.

In addition, in the above-mentioned σ-2/G operation, DNA consisting of the base sequences shown in Table 1 is partially synthesized, and these are used as probes, and a base sequence encoding the polypeptide of the present invention is selected from among the clones that bind to this. Cloning c to correct
DNA can also be obtained.

The base and amino acid sequences of typical NAs encoding the polypeptides of the present invention are shown in Tables 1 and 3.

Production of the polypeptide of the present invention The vector for producing the polypeptide of the present invention comprises a DNA encoding the polypeptide of the present invention obtained as described above,
Alternatively, it can be obtained by incorporating the DNA encoding the polypeptide of the present invention obtained by total chemical synthesis into an appropriate expression vector according to techniques known in the art. All can be used, such as plasmids (E. coli plasmid, pBR322, etc.),
Phages (lambda phage derivatives, etc.), viruses (S
V40, etc.). These may be used alone or in combinations thereof, such as the pBR322-3V40 Vibrift plasmid. its DNA
The site of incorporation can also be arbitrarily selected depending on the purpose.

More specifically, for example, base numbers 61 to 1149 in Table 1
A translation start codon (ATG) is added upstream of a DNA fragment consisting of a base sequence corresponding to , and a translation stop codon (TAA, TAG, or TGA) is added downstream, and these are combined with an appropriate promoter and Shine-Dulcalf (SD).
Expression vectors for producing polypeptides of the invention can be constructed by subsequent ligation via synthetic oligonucleotide adapters and incorporation into vectors (eg, plasmids).

Examples of promoters include Iac, trp, t
ac.

Examples include ``Yaji'', ■Toku, PL, and SV40 Hatsuma Promoter.

Transformants are produced by introducing these expression vectors into hosts such as microorganisms, animal and plant cells, such as Escherichia coli, for example, by the method of Nien et al.14, and then culturing the transformants to achieve the desired target. A polypeptide or a polypeptide having methionine attached to its N-terminus can be produced. The product can be accumulated either within or outside the cytoplasm of the microorganism depending on the promoter used and the construction method of the expression vector. To secrete the protein to the outside of the cytoplasm, use a secreted protein gene 1, such as the alkaline phosphatase gene (dish) or the phosphate binding protein gene (dish), and insert the target polypeptide following the signal peptide-encoding region. What is necessary is to construct a gene expression vector in which the encoding DNA is linked. The transformants thus obtained were cultured under appropriate culture conditions depending on each transformant.
After culturing until the polypeptide of interest is sufficiently produced, the polypeptide is extracted from the culture. If the produced polypeptide is accumulated in the cytoplasm, for example, the host cells are destroyed by lysozyme digestion, freeze-thawing, ultrasonic disruption, French press, etc., and then the extract is collected by centrifugation or filtration. In addition, when it is accumulated in the periplasm, for example, the method of Wfllsky et al. [J
, Bacter+o1. .

127.595 (197B>).

The polypeptide of the present invention can further be used in general protein purification methods,
For example, purification can be performed by a combination of ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and the like.

When formulating the polypeptide according to the present invention, a lyophilized product is preferable, and it is preferable to add excipients and stabilizers.

EXAMPLES The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to these Examples.

(Left below) Example 1 Cloning of cDNA encoding the polypeptide of the present invention HL-60 cells were seeded in a Petri dish (diameter 8 cm) at 1 x 10 cells/10 ml/dish. The culture solution contains RPMI-164 containing 10% tallow serum.
0 medium was used, and phorbol-12-myristate-13-acetate (PMA) and vitamin A were used as differentiation inducers.
ffi was added at a final concentration of 500 ng/ml. After culturing for 2 days at 37°C in air containing 5% carbon dioxide and a humidity of 90 to 100%, the culture solution and floating cells were removed by suction. To the dish to which the differentiated cells had adhered was added 10 μg/total of entodoxy/(Escherichia coli-derived paste) and 0.5 μg 7 m of RPMI-1640 medium containing lθ% beef tallow serum as an inducer.
101 of a medium containing 1 Mg/ml of PMA and the protein synthesis inhibitor tositecycloheximide at 1 Mg/ml was added, and the cells were further cultured for 5 hours.

After culturing, the culture medium was removed by suction, and the cells remaining on the dish were treated with 0.5% sodium lauroyl sarcosinate,
5. It was dissolved in a 6M guanidinyl thiocyanate solution containing mM sodium citrate and 0.1M 2-mercaptoethanol and homogenized. This homogenate is
, on a 5.7M cesium chloride aqueous solution containing IM EDTA, and centrifuged at 26,500 rpm for 20 hours using an ultracentrifugal machine (RPS27-20 (Hitachi Koki)) to give a total R
The NA fraction was obtained as a pellet. This is 0.35M
NaCI, 20mM Tris and 20mM EDTA
was dissolved in a small amount of 7 M urea solution containing 100% urea and collected as an ethanol precipitate.

This total RNA fraction was dissolved in lomM containing 1 mM EDTA.
Tris-HCI buffer (pH 7,4) (hereinafter referred to as TE
(referred to as liquid) 211 and heated at 65°C for 5 minutes.

After adding NaC+ solution to this to make it 0.5M,
Poly(A) mRNA was obtained by applying the column to an oligo(dT) cellulose column equilibrated in advance with a TE solution containing 0.5 M NaCI and eluting the adsorbed poly(A) mRNA with the TE solution.

■ Synthesis of c DNA Using the poly(A) mRNA obtained in section (1) as a template, the poly(A) mRNA obtained in section (1) was used as a template.
HoNman) method CGene, 25, 263
cDNA was synthesized according to (+983). The poly(A) mRNA (6 µg) was dissolved in 6 µβ of distilled water, and 0.6 µg of the poly(A) mRNA was dissolved in 6 µβ of distilled water. A 100 mM aqueous methylmercury hydroxide solution was added thereto, and the mixture was allowed to stand at room temperature for 10 minutes. Then 20 units of RNAse inhibitor (RNasin ■).

Prowega Biotec products) as gold products 00m
M 1.7μ of 2-mercaptoethanol solution! was added. After leaving at room temperature for 5 minutes, add 10mM MgC
I2, 1.25mM dGTP, 1.25mM dAT
P, 1.25mM dTTP, 0.5mM,
dCTP, 0.17 μM α-”P-dCTP (specific activity, 750 Ci/■ole>, 4 μg oligo(dT)
+2-+a, 32μ containing 120 units of avian osteogenic leukemia virus-derived reverse transcriptase! 50mM T r
i s -HCI (f)H8,3) Add W new solution,
After reacting at 42'C for 60 minutes, EDTA was added to stop the reaction. Phenol/Li0O form mixture (1:
1), and add ammonium acetate to the aqueous layer at a final concentration of 2.
．． The reaction product (sscDNA-mRNA complex) was precipitated with ethanol. This s sc DNA-mRNA complex was dissolved in 100 μl of a reaction buffer having the following composition.

Reaction buffer composition: 5 mM Mgc+2. 10mM (NH4)2sO4
, 100mM KCI, 0.15mM β-nicotinamide adenine dinucleotide, 40μM dGTP,
40 μM dATP.

40μM dTTP, 40μM dCTP, and 5μM
20mM Trfs-HCI containing 1.25 units of E. coli ribonuclease H, E. coli DNA polymerase at position 24111 (pI (7,5))
Warm me.

The lysate was reacted at 12°C for 60 minutes, 2.5 units of colon + 1 DNA ligase was added thereto, and the solution was further incubated at 22°C.
The reaction was carried out at C for 60 minutes. After stopping the reaction by adding EDTA, extraction was performed with a phenol/chloroform mixture in the same manner as above, and the reaction product (dscDNA) was extracted with ethanol.
) was precipitated and collected.

(3) Oligo (dC) tail addition cDNA H preparation dscDNA obtained in section ① was added to reaction buffer 1 with the following composition.
00μ! Dissolved in and reacted at 37°C for 30 minutes, d
An oligo (d C) tail was added to the s c DNA.

Reaction buffer composition: 2mM COCl2.0.2mM dithiothreitol.

0, ImM a-”P-dCTP (specific activity I Ci/
smote) and 10 units of terminal deoxynucleotidyl transferase]00 mM sodium cacodylate (pH 7,2).

The reaction was stopped by adding an aqueous EDTA solution, extracted with a mixture of Fer/-Hol/chloroform, and the oligo(dC)-tailed dsc DNA was precipitated with ethanol and collected. This was mixed with 1mM EDTA and +00mM Nac.
10mM Tr j s -HC containing + (+)H
7,4) Dissolved in a buffer solution to a concentration of 2 μg/■1.

(4) Recombinant plasmid production oligo (dG)
Added pBR322 (Bethesda Res, Lab
s.

lnc, 1) and the oligo (dC) obtained in section (3)
Add dscDNA I, 5ml 1mM EDT
A and 10mM Tris containing 100mM NaCl
-HCI (p) (7,4) in buffer, 1.
After dissolving and mixing to contain 5 μg and 0.09 μg,
Annealing was performed by heating at 65°C for 10 minutes, at 57°C for 2 hours, and further at 45°C for 2 hours, and the recombinant plasmid solution was isolated.

■ Selection of transformants Using the recombinant plasmid solution obtained in section (4),
E, col+IIBI01 strain was transformed. That is,
E, col 11111101 strain was mixed with L-broth (composition: 710 g of tryptopone per 11, 5 g of yeast extract, N
aCl 5 g.

Glucose 1g, PH7,2) in 2Qml, 37
Cultivate at °C until the absorbance (000n-) becomes 0.5,
The bacterial cells were weighed by centrifugation, and 10 m containing 50 mM CaCI2 was added.
M Tris-1TCI FB new liquid PH7
, 3) There was a scuffle at 10m1.

The collected cells were suspended in the same buffer solution 21 and incubated at 0°C for 5 minutes. Add and mix 0.11 of the above recombinant plasmid solution to 0.21 of this suspension, let it stand at 0°C for 15 minutes, and then hold it at 42°C for 2 minutes. 0.51 of Shiichi Gloss having the same composition as above was added and cultured with shaking for 1 hour. A portion of this culture solution was spread on an L-gloss agar plate to which tetracycline/(15 μg/al) was added in addition to the above composition, and cultured at 37°C for about 12 hours. Tetracycline-resistant plants were selected and c. D.N.
A library was created.

(6) The cDNA library obtained in Cloning Section 0 was subjected to a colony hybridization test using the plus-minus method. The following two types of probes were created. That is, poly(A) mRNA obtained in step C1)
Synthesize “P W m single-stranded cDNA using ” as a template,
This was used as an induction plus probe. Separately, using poly(A) mRNA prepared by the method described in step (1) as a template from untreated HL-80 cells, P-labeled single-stranded cDNA prepared in the same manner was used as an induced minus probe. Clone 7, which had a cDNA containing a base sequence that specifically bound only to this induced plus probe, was selected.

Next, this il! Human IL from selected clones
In order to exclude cDNAs encoding IL-1 and human cancer necrosis factor, colony hybridization was performed using a single ML DNA of cDNA encoding human IL-1 and human cancer necrosis factor labeled with 52p as a probe. Clones that bind to these probes were excluded by a hybridization test.

In order to further sort the selected clones,
EcoRI, EcoRV, PvuII, RsaI
, 5acII.

A restriction enzyme map was created using several restriction enzymes such as sph I, and clones that produced a common DNA fragment of approximately 350 bp by simultaneous digestion with EcoRV and sph I were selected.

Among the selected clones, there are two that have a cleavage recognition site by EcoRI and one that does not! Since type R was recognized, the cloned cDNAs containing the largest cDNAs (ptlLGI9 and p)ILG26)
I selected.

■ Determination of base sequence of cloned cDNA Cloned cDNA isolated and purified from the clone selected in section (6)
The nucleotide sequence was determined by the dideoxy method using MLI phage. M 13mp18 and M 13mp+9
(Pharmacia P-L Iliochemical
ALS) was used as a cloning vector, and MI3 Sequencing/Gig Kit (Amersham Intern) was used as a cloning vector.
'M13 Cloning and Sequence Sync Handbook' (manufactured by ANonal plc)
The test was carried out according to Sham International plc).

As a result, the obtained cloned cDNA pHLG19
and P) The base sequence of the polypeptide coding region of ILG2G and the amino acid sequence determined from the base sequence are shown in Table 21 and Table 3, respectively.

(The following is a blank space) Example 2 Production of polypeptide of the present invention-1 (1) Construction of expression polypeptide for production General formula C-D
An expression plasmid for producing the polypeptide of the present invention represented by -E-F (where X in the formula is Phe) was constructed as shown in FIG.

That is, the cloned cDNA obtained in Example 1-+61
Restriction enzymes 4 and 4 were reacted on pHLGI9 to obtain D corresponding to base numbers 90 to 912 in Table 2.
The NA fragment was flattened. This DNA fragment was synthesized using the following formula % formula %[] and the following formula 5'-ACTGGTCTGCTACTACACCAG
[b1]3'-AGACGATGATGTGGT
The oligonucleotide adapters denoted by C are sequentially
4. Linked using DNA ligase. This DNA fragment is called a Cc-d-e] fragment.

On the other hand, plasmid PCT-1 [1kehara, M, e
tal,.

Proc, Nat, Acad, Sci, USA 81,
5950 (1984) restriction enzyme 11pa) and Aat
A DNA fragment of approximately 380 bp containing part of the TRP and bromocou regions was excised by the action of IT, and an oligonucleotide adapter represented by the following formula 5 synthesized by a conventional method was added to this DNA fragment with T4DN.
The ligation was performed using A ligase.

This DNA fragment was previously made with :A [c - d - e]
The fragments were ligated using T4 DNA ligase to form a DN
I got fragment A. This DNA fragment was attached to the promoter [c-d
-e] fragment.

Clo/conjugated cDNA, restriction enzyme Sa to pHLGI9
c II and Ava I were reacted to base number 91 in Table 2.
A DNA fragment corresponding to about 470 bp downstream from 3 was isolated, and an oligonucleotide adapter of the following formula 5 [1] synthesized by a conventional method was added to this DNA fragment to T4DN.
The ligation was performed using A ligase.

This DNA fragment was combined with the previously prepared promoter [c-
d - e] fragment using T4 DNA ligase, and the resulting DNA fragment is referred to as promoter [polypeptide coding region co-fragment.

Separately, a large NA fragment (approximately 3.7 kb
p) was separated by 0.7% agarose gel electrophoresis.

Both ends of this DNA fragment were treated with DNA polymerase I (Klenow fragment) and dGTP, dATP, dC.
T.P.

Make blunt ends using dTTP, and connect both ends with T4 DNA.
The conjugation was performed using ligase. This plasmid vector is called pBR56. Furthermore, this pBR5O vector was treated with restriction enzymes ^at[ and old ndlll to isolate and purify a large NA fragment (approximately 3 ikbp).

An expression plasmid for producing the polypeptide of the present invention was constructed by ligating the previously prepared promoter [polypeptide coding region 111r fragment to this DNA fragment using T4 DNA ligase. This expression plasmid was named p)IDG-↓.

(2) Production of polypeptide The expression plasmid obtained in section (1) was introduced into E. coll IIBIO+ by the following method to obtain a transformant. That is, E. coli HIIIOI was mixed with L gloss (composition: 710 g of tryptopone per 11, 5 g of yeast extract,
51 (15 g of NaC, 1 g of glucose; pH 7,2) and cultured overnight at 37°C. One portion of the bacterial cell suspension was inoculated into 1001 broth and cultured at 37°C until the turbidity (absorbance G50 nm) reached 0.6.

After standing in ice water for 30 minutes, the bacterial cells were collected by centrifugation.
This was suspended in 50mM CaCl2 at 0℃ for 6 hours.
It was left standing for 0 minutes. Next, the bacterial cells were collected by centrifugation,
l0ml of 50mMcacI2 containing 20% glycerin/
resuspended in.

this! ``!! Add the expression vector pHDG-↓ described in the suspension, and place it in ice water for 20 minutes and at 42°C for 1 minute.

After incubating for 10 minutes at room temperature, I, 11 broth (composition: per 11, 710 g of trypto, 7 yeast extract,
5 g and NaCl IOg, pH 7,5) were added and shaken at 37°C for 60 minutes. That bacterial body! ! ! A portion of the suspension was plated on an LB agar plate containing 25 μg/l anbinli/, and after culturing at 37°C overnight, anbinlin-resistant clones were selected to obtain transformants.

This transformant was incubated overnight at 7°C in LB broth. 11 of the cell suspension was added to 100 ml of improved M9 medium (composition: 1.5% Na*HPO4, 12Hz 0.0.3
%KH2PO4゜0.05%NaCl, 0.1%NH
aC+, 2 B/l Vitamin I31° 0.5% Casamino Acids, 2 mM Mg5Oa, O, ImM C
aC12.

0.5% glucose) and incubated at 37°C for 1 hour, then indole-3-acrylic acid at a final concentration of 20 μg.
/■l, and after continuing the culture for an additional 24 hours, the bacterial cells were collected by centrifugation. 0 of 1011 bacterial cells
．． 50 m containing 1% lysozyme and 30mM NaC+
Resuspend in MTris-HCI (+)) (8,0) buffer, let stand at 0°C for 30 minutes, then transfer to dry ice/
After repeated freezing in an ethanol bath and thawing at 37°C, bacterial cell residue was removed by centrifugation to obtain a clear extract.

This extract was analyzed by 5DS-polyacrylamide gel electrophoresis, and it was confirmed that Dalton's target polypeptide with a molecular weight of about 40 was contained.

Example 3 Production of the polypeptide of the present invention-2 An expression plasmid for producing the polypeptide of the present invention represented by the general formula D-E-F (wherein, X in the formula is Phe) was prepared in Example 2. - Constructed according to the method shown in (1). However, instead of the synthetic oligonucleotide adapter [a+], a synthetic oligonucleotide adapter represented by the following formula 5% [1] was used.

The polypeptide was produced according to the method of Ki α in Example 2-■. As a result, 5DS-polyacrylamide gel electrophoresis analysis confirmed Dalton's target polypeptide with a molecular weight of about 40.

Example 4 Production of the polypeptide of the present invention-3 An expression plasmid for producing the polypeptide of the present invention represented by the general formula C-D-F (wherein, X in the formula is Leu) was prepared in Example 2. 2 (+1). However, cloned cDNA, pHLG19
Instead of the cloned cDNA obtained in Example 1-(61)
1pHLG2[i was used.

The polypeptide was produced according to the method described in Example 2-Q). As a result, the desired polypeptide with a molecular weight of approximately 34 was confirmed by 5DS-polyacrylamide gel electrophoresis.

Example 5 Polypeptide of the present invention! ! ! Construction-4 An expression plasmid for producing the polypeptide of the present invention represented by the general formula DF (where X is Leu) was constructed according to the method shown in Example 3. however,
In place of the cloned cDNA, pt-ILG19, the cloned cDNA obtained in Example 1-(6)
, pHLG2G was used.

The polypeptide was produced according to the method described in Example 2-1). As a result, Dalton's target polypeptide was confirmed to have a molecular weight of about 34 by 5DS-polyacrylamide gel electrophoresis analysis.

Table 1 (In the table) <+tphe or Leu, R means T or C. ) Table 1 (continued) ^1aAlaThr Table 2 GIyLysVilThrl 1aAspsarSet
TyrAsp I l@AlaLys11asarGl
r+H1sL@uAtpPne1tsuQ100 Table 2 (continued) PheGIy^rlssrPhsThrLsu^1iS
++rSerGluThr to IyVall: Iy to a
ProllosarGly2711
+I mouth ProG4yl
leProGlyArgPh@ThrLysGluAl
iGly'rl+rLeuA! aTyrTyrGluI
1ecys29・
)08L@uLysAipArgCl
nL@uA I mG lyA l ass LVa
1TrpA I aLeuAspLeuAspAg p
PheClnko5Iko5O Ali^1nThr Table 3 Table 3 (continued)

[Brief explanation of drawings]

FIG. 1 shows the construction process of the expression plasmid pHDG-4. Synthetic oligonucleotide adapter [a
], [b], [c], and [d] are Example 2-(1
) Patent applicant featuring the synthetic oligonucleotide adapter described in Dainippon Pharmaceutical Co., Ltd.

Claims (4)

[Claims] (1) A polypeptide having an amino acid sequence represented by the following general formula. Am-Bn-Cp-D-Eq-F However, in the above formula, the left end means the amino terminal, the right end means the carboxy end, m, n, p, q mean an integer of 0 or 1, and A- F means a region corresponding to the following amino acid or amino acid sequence in Table 1, respectively. A: Amino acid sequence of amino acid number 1, B: Amino acid sequence of amino acid number 2 to 20, C: Amino acid sequence of amino acid number 21, D: Amino acid sequence of amino acid number 22 to 237, however,
Amino acid number 200 (X) is Phe or Le
means u. E: Amino acid sequence of amino acid numbers 238-298, F: Amino acid sequence of amino acid numbers 299-363. (2) DNA encoding the polypeptide according to claim 1. (3) A trait expression vector incorporating the DNA according to claim 2. (4) A transformant into which the vector according to claim 3 has been introduced.