JPH03123487A - Production of new polypeptide - Google Patents

Abstract

NEW MATERIAL:DNA containing a DNA coding a polypeptide having EAQ at N-terminal, consisting of 60 amino acids and expressed e.g. by formula. USE:Production of a polypeptide produced by cancer cell and utilizable for the investigation of the proliferation mechanism of cancer and the development of a remedy for cancer. PREPARATION:A cDNA is produced e.g. by culturing a cancer cell such as human mammary cancer cell MCF7, human gastric cancer cell MKN-45 or KATO-III in a medium, collecting the proliferated cells, extracting the whole RNA by guanidine-hot phenol process, etc., separating mRNA by oligo-dT cellulose column chromatography and using the mRNA as a template for the production of cDNA with a reverse transcriptase. A cDNA library is prepared from the cDNA by conventional method, E.coli is infected with the cDNA, the infected E.coli is screened to select a strain capable of producing polypeptide and the DNA is separated from the cell to obtain the objective DNA coding a polypeptide expressed by formula, etc.

Description

[Detailed description of the invention]

The present invention involves cloning a cDNA encoding a polypeptide produced from a certain type of cancer cell. The present invention relates to a method of using the same to produce polypeptides produced by original cancer cells using genetic engineering techniques. Proteins and polypeptides secreted by certain cells can be isolated and purified using various methods, such as gel filtration, adsorption chromatography, electrophoresis, and electrophoresis. Although it is done by painting method. When the amount of bioactive substance present is small, its isolation is difficult. In order to solve this problem, we attempted to produce this polypeptide in large quantities using genetic engineering techniques.
It has become possible to produce the present polypeptide, which reacts equally with the polypeptide produced by the original cells in an immunoassay. On the other hand, it is known that cancer cells secrete various polypeptides such as growth factors, and there is interest in the relationship between these polypeptides and cancer. For example, MCF7 of human breast cancer cells is expressed by TGF (trans
forming growth factor) a
, β, γ [Dickson et al., CancerRes, 46.1707
-1713. (1986)), insulin-like growth factor [Haff et al., Cancer Research (Can
cer Res, ) 46.4613-4619. (1
986)) and platelet-derived growth factor [Blausert (B)
Proceedings of the National Academy of Sciences USA (Pro, Na
tl, Acad. Sci, USA) 84.5763-5767 (1
987)) are known to be secreted. On the other hand, one of the present inventors has discovered that MCF7 cells secrete various growth factors and EG, which is a type of growth factor.
F (Epidermal growth fact
In order to investigate the relationship between Er A (Enzyme Immuno As5ay or
) and established MCF7 and MKN-45 in human gastric cancer cells.
As a result of trying to measure the culture solution of KATO-nl,
It was discovered that a new substance different from EGF was produced. It is thought that this was detected because other antibodies were mixed in with the antibody against EGF in the human EGF measurement system described above. In order to learn about the relationship between this new polypeptide and cancer, we purified this polypeptide and clarified its structure.
Biophys, Res. Comm,) 155366 (1988)), and a patent application has been filed based on this (Japanese Patent Application No. 1-35111).
issue). This polypeptide has EAQ at the N-terminus and consists of 60 amino acids, and this polypeptide is derived from human breast cancer cells MCF7. Alternatively, it is a novel polypeptide produced by human gastric cancer cells MKN-45 or KATO-■, and this polypeptide has an isoelectric point of 4.
3. The molecular weight is 6,661 daltons, and its amino acid sequence is EAQTETCTVAPRERQNCGFPGVTPS
QCANKGCCFDDTVRGVPWCFYPNTI
DVPPEEEECEF. Furthermore, regarding the mRNA induced by estrogen in human breast cancer cells, Chambon et al.
on) have elucidated its complete base sequence, and from this base sequence the existence of a polypeptide (9140 Daltons) consisting of an 84 amino acid sequence has been predicted. Then, the signal peptide sequence in this 84 amino acid sequence is predicted, and whether the polypeptide actually secreted is composed of 58 amino acid sequences (6450 Daltons) or 63 amino acid sequences (6970 Daltons). [Chambon et al., Nucleic Acids Research (Nucleic Ac1
ds Research) 12. No. 6 (19
84) 2861-2878, and Chambon et al., DNA (4) 11-21 (19
85)]. However, this is just a prediction, and until the amino acid sequence of such a secreted polypeptide could be determined, it had not been isolated and purified. The problem that Ming is trying to solve is to isolate and purify polypeptides secreted by human breast cancer cells such as MCF7, and to use them to investigate the relationship between these polypeptides and cancer, etc. Although it has been desired in the field, it is difficult to isolate and purify small amounts of polypeptide, and it is also difficult to stably extract large quantities of this polypeptide from natural sources.
It has been desired to provide a method for producing large quantities of this polypeptide. In order to make a prediction, the present inventors have developed a method for producing this polypeptide in large quantities using mRNA obtained from human breast cancer cells such as MCF7.
Complementary DNA (cDNA) is synthesized from cDNA, and based on this, an expression vector containing DNA having a base sequence encoding the present peptide is manufactured, and this is inserted into E. coli, yeast, animal cells, etc., which is so-called genetic recombination. The present invention provides a method for producing the present peptide in large quantities. The present invention is characterized in that (i) the N-terminus is EAQ and the amino acid is 60
(ii) The DNA is used as an expression vector for E. coli, yeast, or animal cells to express a polypeptide having EAQ at the N-terminus and consisting of 60 amino acids. (iii) a transformant retaining the recombinant DNA, (iv) culturing the transformant, and producing a polypeptide having EAQ at the N-terminus and consisting of 60 amino acids in the culture. The present invention relates to a method for producing the polypeptide, which is characterized by producing and accumulating the polypeptide, and collecting the polypeptide. Expression vectors containing DNA having a base sequence encoding a polypeptide in the method of the present invention include, for example,
(i) Various cells producing the present peptide, such as M CF 7
Metsenger RNA (mRNA) is isolated from cells, etc., and single-stranded complementary DNA (cD
NA), then synthesize double-stranded DNA, (iii) integrate the complementary DNA into a phage or plasmid, and (
iv) Transforming a host with the obtained recombinant phage or plasmid, (V) culturing the obtained transformant,
Isolate a phage or plasmid containing the desired DNA from the transformant by an appropriate method 1, for example, by hybridization with a DNA probe encoding a part of the present peptide or by immunoassay using an anti-water peptide antibody, (vi) the recombinant DNA;
It can be produced by cutting out the desired cloned DNA from the following and (■) ligating the cloned DNA or a portion thereof downstream of a promoter in an expression vector. The mRNA encoding the present peptide can be expressed in various cells producing the present peptide, such as human breast cancer cells MCF7. Or human gastric cancer cells MKN-45 or KATO-
It can be obtained from Ill et al. As a method for preparing RNA from these peptide-producing cells, the guanidine thiocyanate method [(J.M., Chirgwin et al.,
Bio-chemistry
, 18.5294 (1979)). Using the thus obtained mRNA as a template, using reverse transcriptase, for example, Okayama () 1. [Molecular and Cellular Biology (Molecular and Cellular Biology (1,
cDNA is synthesized according to the following methods (Biology) 2.161 (1982) and 3.280 (1983)), and the obtained cDNA is incorporated into a plasmid. Examples of plasmids that incorporate cDNA include pB R322 derived from Escherichia coli [gene, 295
(1977)), ρBR325 (Gene, 4, 1.21
(1978)), pU C12 [Gene, Mon. J,, 25
9 (1982)), pU C13 (Gene, U piece 259
(1982)), PUBIIO (Biochemical and Biophysical Research Communication) derived from Bacillus subtilis
ical Re5earch Communicati
on), Dan 2678 (1983): l, but any other species can be used as long as it can be replicated and propagated within the host. Also c
Phage vectors that incorporate DNA include, for example, λgtll (Young and Davies (Young, R.
, , and Davis, R.,) Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl., Acad., Sci., U.S.A.) ,),
80.1194 (1983)), but other species can also be used as long as they can proliferate within the host. As a method for incorporating into a plasmid, for example, the method described by T. Maniatis et al., Molecular Cloni
ng) =1- Spring Harbor Laboratory (Co1.dSpring Harbor La-
laboratory), pp. 211-268 (1982)
Examples include the method described in . Furthermore, as a method for incorporating cDNA into a phage vector, for example, the method of Hyunh, T.V. et al.
Cloning, A Practical Appr.
49 (1985)). The plasmid thus obtained can be used in a suitable host, such as a bacterium of the genus Escherichia. It is introduced into bacteria of the genus Bacillus. Examples of the Escherichia genus bacteria include Escherichia coli K 12D.
H1 (Procedure of the National Academy of Sciences)
Acad, Sci, U, S. A, ) 1160 (1968)), M2O3 (Nucleic Acids Research, (Nucleic Ac1d)
s Research), 9309 (1981)), J
A221 (Journal of Molecular Biology)
iology)), 1205]7 (1978)], H
B 101 (Journal of Molecular Biology Month 1459 (1,969)), C600 (Genetics), Blood 440 (19
54)). Examples of the Bacillus genus include Bacillus subtilis M I
114 (Gene. 255 (1983)), 207-21 (Journal of Biochemistry).
Biochemistry% 87 (1984)). As a method for transforming a host with a plasmid, for example, T. Maniatis et al. Molecular Cloning (Molecular C)
loning), Cold Spring Harbor Laboratory
Examples include the calcium chloride method and the calcium chloride/rubidium chloride method described in J. Laboratory, p. 249 (1982). Furthermore, when using a phage vector, it can be introduced, for example, into propagated E. coli using an in vitro packaging method. This peptide cDNA containing this peptide cDNA
The A library can be obtained by the method described above, but this peptide cDNA can be obtained from the MCF7 cDNA library.
Methods for cloning A include colony hybridization or plaque hybridization using oligonucleotides chemically synthesized based on the amino acid sequence of this peptide as probes [T. Maniatis et al., Molecular Cloning ( Molecular Cloning
) Cold Spring Harbor Laboratory (C
old Spring Harbor La-bora
(1982)). The peptide cDNA thus cloned can be used as a plasmid, for example, ρBR322゜pUc, if necessary.
l, 2. pUcl3. pUcl8. pUcl9.
pUcl18. Polypeptide cDNA can be obtained by subcloning into pUcl19 or the like. The base sequence of the DNA obtained in this way was determined by, for example, Maxam-Gilbert.
(Maxam, A, M, and G11ber
t, w, Proceedings of the National Academy of Sciences of the New Year.
Nis Ni (proe, Natl, Acad, S
ci, U.S.A.), 74, 560 (1977))
Alternatively, the dideoxy method (Messing, J. et al., Nucleic A
c1ds Research) 9309 (1981))
cDNA (Formula I) that coats the present peptide is obtained from the known amino acid sequence. The DNA encoding the present peptide cloned as described above can be used as it is, depending on the purpose, or after being digested with restriction protein if desired. An expression vector can be obtained by cutting out a region desired to be expressed from the cloned DNA and ligating it downstream of a promoter in a vehicle (vector) suitable for expression. The DNA has AT as a translation initiation codon at its 5' end.
G, and a T as a translation stop codon at the 3' end.
It may have AA, TGA or TAG. These translation start codons and translation stop codons are created using appropriate synthetic N
It can also be added using an A adapter. Furthermore, in order to express the DNA, a promoter is connected upstream thereof. Vectors include the above E. coli-derived plasmids (e.g., p, BR322, pBR325, pUc12, pUc
13), yeast-derived plasmids (e.g., YIp, YEp, Y
Rp, YCp) or bacteriophages such as λ phage, retroviruses, and animal viruses such as vaccinia virus. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. When the host for transformation is Escherichia, the trp promoter, flac promoter, rec
A promoter, λPL promoter, QPP promoter, etc., when the host is Bacillus, 5POL promoter, SP○2 promoter, penP promoter, etc., and when the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH.
Promoters and the like are preferred. In particular, it is preferable that the host is a bacterium of the genus Escherichia and the promoter is the trp promoter or the PL promoter. When the host is an animal cell, a promoter derived from SV40, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, etc. can be used. Note that the use of enhancers for expression is also effective. A transformant is produced using the thus constructed vector containing the DNA encoding the mature peptide of the present peptide. Examples of the host include bacteria of the genus Escherichia, bacteria of the genus Bacillus, yeast, and animal cells. Specific examples of the Escherichia genus bacteria and Bacillus genus bacteria include those mentioned above. Examples of the yeast include Saccaron+yces cerevisiae.
e) A H22. Examples include AH22R, NA37-11A, and DKD-5D. Examples of animal cells include monkey cells CO5-7, Ve
ro, Chinese hamster cells CHO; Examples include mouse L cells and human FL cells. To transform the above-mentioned Escherichia bacteria, for example, the procedures described in Proceedings of the National Academy of Sciences (Proc. Natl., Acad. Sci, LISA), 69.2110 (1972), Gene, 17, 107107 (19 etc.) To transform Bacillus bacteria, for example, Molecular and General Genetics (Molecular Genetics)
cular & General Genetics)
, 168, 111 (1979). To transform yeast, for example, the Proceedings of the National Academy of Sciences (
Proc, Natl, Acad, Sci, USA),
75.1929 (1978). To transform animal cells, for example, virology (
It is carried out according to the method described in J. Virology) Noboru, 456 (1973). In this way, a transformant transformed with an expression vector containing the DNA encoding the mature peptide of the present invention is obtained. When culturing a transformant whose host is a bacterium of the genus Escherichia or Bacillus, a liquid medium is suitable as the culture medium, which contains a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of carbon sources include glucose, dextrin,
Soluble starch. Examples of nitrogen sources such as sucrose include inorganic or organic substances such as ammonium salts, nitrates, corn steep liquor peptone, casein, meat extract, soybean meal, and potato extract; examples of inorganic substances include calcium chloride, diphosphate, etc. Examples include sodium hydrogen and magnesium chloride. Additionally, supplements, vitamins, growth promoting factors, etc. may be added. The pH of the medium is preferably about 5-8. As a medium for culturing Escherichia bacteria, for example, M9 medium containing glucose and casamino acids [Miller (Mi
+Journal of Experiments
In Molecular Genetics (J. urnal of Experiments in M
olecular Genetics). 431-433. Co1d Spring Harbo
r Laboratory, New York 197
2) is preferred. If necessary, a drug such as 3β-indolyl acrylic acid can be added here to make the promoter work efficiently. When the host is Escherichia, the culture is usually about 15-4
The reaction is carried out at 3° C. for about 3 to 24 hours, and aeration and stirring can be added if necessary. When the host is Bacillus, the culture is usually about 30-40'
C for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, the medium may be, for example, Burkholder (Burkholder).
) Minimal medium (Bostian, K. L., et al. [Procedure of the National Academy of Sciences (Proc. Natl. Acad.
, USA) 77.4505 (1980)]. The pH of the medium is preferably adjusted to about 5-8. Cultivation is usually carried out at about 20° C. to 35° C. for about 24 to 72 hours, with aeration and stirring added as necessary. When culturing a transformant whose host is an animal cell, the medium may be, for example, ME containing about 5 to 20% fetal bovine serum.
M medium [Science 122. 501 (1952)], DMEM medium [Virology, 8,396 (19
59)), RPMI 1640 medium [Journal of the American Medical Association (The Journal of the American Medical Association)
canMedical As5ocation) 1
99, 519 (1967)], 199 Medium [Pro-ceeding of the Society for the Biological Medicine (Pro-ceeding of
the 5ociety for the biol
(1950) 73.1 (1950
)) etc. The pH is preferably about 6 to 8. Cultivation is usually carried out at about 30° C. to 40° C. for about 15 to 60 hours, with aeration and stirring added as necessary. The mature peptide of the present invention can be separated and purified from the above culture by, for example, the following method. When extracting mature peptides from cultured bacterial cells or cells, after culturing, the bacterial cells or cells are collected by a known method and suspended in an appropriate buffer. A method can be used as appropriate, such as destroying bacterial cells or cells by ultrasonication, lysozyme and/or freeze-thawing, and then centrifuging or filtrating to obtain a crude extract of the precursor protein or mature peptide of the present peptide. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When a precursor protein or mature peptide is secreted into the culture solution, after the completion of the culture, the bacterial bodies or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The precursor proteins and mature peptides contained in the culture supernatant or extract thus obtained can be subjected to an appropriate combination of known separation and purification methods. These known purification methods mainly include methods that utilize solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and 5DS-polyacrylamide gel electrophoresis. Methods that utilize differences in molecular weight, methods that utilize differences in charge such as ion exchange chromatography, methods that utilize specific affinity such as affinity chromatography, and methods that utilize differences in hydrophobicity such as reversed phase high performance liquid chromatography. Method 1: A method that utilizes a difference in isoelectric points, such as isoelectric focusing method. The precursor protein and mature peptide of the present peptide thus produced can be measured by enzyme immunoassay using a specific antibody. "Saku" and 4 rolls

[In bacterial cells or cells that have been infected or transformed with the DNA of the present invention, it is possible to produce and purify a large amount of pre-ovoid protein or mature protein of the present peptide. This polypeptide is produced and secreted by certain types of cancer cells, and is predicted to be related to cancer.
In turn, it is thought that it will be useful in providing therapeutic drugs for cancer. In addition, this polypeptide is a known pancreatic spasmolytic enzyme secreted from the pancreas.
zyme, PSP) [Jorgensen
regulatory peptides (Regul nsen), etc.
atory Peptide) 3, 207-219
(1982)), it is presumed that this substance has an effect of suppressing intestinal peristalsis, and is also presumed to have a muscle contraction and relaxation effect. In the specification and drawings of the present invention, when bases, amino acids, etc. are indicated by abbreviations, IUPAC-IUB Co++
+m1sion on Biochemical No.
It is based on the abbreviation by menclature or the abbreviation commonly used in the field, and examples thereof are shown below. In addition, when an amino acid can have optical isomers, unless otherwise specified, the L-isomer is assumed to be indicated. DNA: Deoxyribonucleic acid cDNA: Complementary deoxyribonucleic acid A: Adenine T: Thymine G Nigeanine C: Cytosine RNA: Ribonucleic acid mRNA: Metsenger liponucleic acid dATP: Deoxyadenosine triphosphate dTTP: Deoxythymidine triphosphate dGTP: Deoxyguanosine triphosphate dCTP phosphate: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS Sodium nidodecyl sulfate G Nigericin A: Alanine V: Valine L: Leucine ■: Isoleucine S: Serine T: Threonine C Nidistine M: Methionine E : Glutamic acid D : Aspartic acid K : Lysine R : Arginine H : Histidine F : Phenylalanine Y : Tyrosine W Nitributophane P Nibroline N : Asparagine Q : Glutamine fruit] The present invention will be explained below with reference to Examples. However, the present invention is not limited thereto. Transformant Satucharomyces obtained in the Examples described below
Saccharomyces cere
visiae) AH22R/pGLD906-10 was granted accession number F to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, on February 21, 1999, under the Budapest Treaty.
The microorganism was deposited as ERM BP-2301, and since February 28, 1999, the microorganism has been transferred to the Fermentation Research Institute (IF).
It has been deposited with O.O. under accession number IF0 10465. m Preparation of MCF7 cell cDNA library MCF7 cells 1 were cultured in monolayer in Eagle's minimal medium containing 10% fetal bovine serum, and RNA was extracted from 10' cells using the guanidine/hot phenol method [Maniatisti (Ma
Niatis, T.) et al., Molecular Cloning Laboratory Manual (M.
oning-A 1 laboratory manual
), pp. 194-195, 1982), and poly(A) RNA was purified from this RNA by oligo dT cellulose column chromatography (Ibid., pp. 194-195, 1982).
pp197-198). cDNA was prepared using this poly(A) RNA as a template by the method of Okayama et al., and a cDNA library was prepared. Preparation of a DNA probe encoding a part of this peptide Amino acid sequence from the 9th Val to the 15th Gin Val-Ala-Pro-Arg-Glu-Ar
Among the DNA sequences encoding g-Gin, the most frequently used codons were reported by Lathe (R.) [
Journal of Molecular Biology (J, Mo1
．． A DNA probe having the following sequence was synthesized, selected according to Biol, Keishin, 1-12 (1985). ”GTG GCCCCCCGT GAAAGA
The 5' end of this DNA probe was P-phosphorylated using T4 polynucleotide kinase and used to screen a cDNA library. Isolation of cDNA of this peptide and determination of its nucleotide sequence Escherichia coli C600hfl was infected with the above-mentioned cDNA library and plated to allow colonies to appear. Colonies were transferred onto a nylon membrane and hybridized with the 31P-labeled DNA probe described in the previous section. Hybridization was performed at 42°C. Several clones with positive hybridization were isolated, and one of them, No. 52, cD
The base sequence of the NA portion was determined. The cDNA portion contained in this plasmid was 1.8 Kbp. This cDN
The base sequence of part A was determined using Sanger's method.
Proceedings of the National Academy
of Science (Proc, Natl, Acad, S
ci, USA) 74.5463-5467 (1977)
) was determined. This base sequence and the amino acid sequence of the precursor protein deduced from it are shown in Formula I. The region enclosed by a loco is the present peptide. Formula ■ A TCCCTG ACT CGG GGT CGCC
TT 22MetAla 2 23 TGG AGCAGA GAG GAG GC
A ATG GCC463Thr Met Glu A
sn Lys Val Ile Cys 1047
ACCATG GAG AACAAG GTG A
TC, TGC-70+1 Ala Leu Val
Leu Val Ser Met Leu 187
1 GCCCTG GTCCTG GTG TCCA
TG CTG %TCT GCCTGCATCCTG
ACG GGG TGCCGT CCCCAG CA
CGGT GAT TAG TCCCAG AGCTC
G GCT GCCACCTCCCACCGGA CAC
CTCAGA CACGCT TCT GCAGCT
GTG CCT CGG CTCACA ACA CA
GATT GACTGCTCT GACTTT GAC
TACTCA AAA TTG GCCTAA AAA
[
After digestion with Zen Shuzo Co., Ltd., a 0.36 Kb DNA fragment was separated using 1.5% agarose gel electrophoresis. Add XhoI linker-d to this DNA fragment (2 μg)
(CCTCGAGG) (Zen Shuzo Co., Ltd.) was added, and 50 μQ of the anti-solution (66 mM Tris
-)1cQ, pH7,6/6.6mM MgCQ2/
The mixture was reacted at 14° C. for 16 hours in cod dithiothreitol (10.1 mM ATP). Next, 30 units of restriction enzyme xhO1 [Nirabon Gene Co., Ltd.] was added, and 37
C. for 2 hours, and both ends of the DNA fragment were trimmed. 0.5 μg of this DNA fragment and yeast expression vector pGLD906-1 (Japanese Patent Application Laid-Open No. 61-43991)
9.4Kb D obtained by digesting with restriction enzyme 5alI
200 units of 0.1 gg of NA fragment (7) T 4.
Using DNA ligase, prepare the above reaction solution (50μQ
) and connect in E. coli DHI (Molecular Cloning (Mole)
cular cloning, Co1d Spring
g Harbor Lab. (1982) was transformed. From the obtained ampicillin-resistant transformant, plasmid pG
LD906-1o was isolated (Figure 1). Using 103 μg of plasmid pGLD906, protoplast method [Innen et al.
Proceedings of the National Academy
of Science (Proc, Natl, Acad, S
ci, USA) 75.1927 (1978)].
s cerevisiae) A H22R- (Miyanohara et al., Proceedings of the National Academy of Sciences (Proc, Natl, Acad)
, Sci, USA) 80°1 (1983)). As a result, the transformant AH22R7pGL D906-1 was able to grow in a medium containing no leucine.
0 was isolated. Peptide production of the present peptide using the 7s mother as a host Many transformants obtained in Example 4 S, cere
viciae AH22RVpGLD906-10 were selected and their ability to produce these peptides was examined by the following method. Kitano (Bio/Technology (BIO/TEC)
NOLOGY). 5, 281 (1987)) was dispensed into test tubes, inoculated with the transformant, and cultured with shaking at 30°C for 3 days. 1 mQ of the same medium was transferred to a test tube containing 10 mQ of the same medium, and cultured with shaking at 30°C for 1 day. Next, transfer the 3 mfl to 200 mQ containing 30 mQ of the same medium as before.
The cells were transferred to a Q flask and cultured at 30°C for 2 days. The obtained culture solution was centrifuged at 3,000 rpm for 10 minutes to separate the supernatant and bacterial cells. Bacterial cells were prepared using the method of Rose et al.
), 78.2460 (1981)) to obtain an extract. That is, the bacterial cells collected from 10 mQ of culture solution were once added to SM buffer (85 mM NaCQ, 1
mM g SO4, 20mM Tris-HCQ
, pH 7,4), and then frozen at -80°C. Add 1 mQ of disruption buffer (10
0mM Tris-HCQ, pH 8, 0, 20%G
lycerol, 1 m M P M SF,
1 m MDTT) and 2 g of glass beads were added and crushed by strong stirring using a portex mixer. This was centrifuged and the supernatant was used as a bacterial cell extract. Quantification of this peptide was performed by EIA shown below. Plate sensitized IgG fraction containing anti-hEGF (purified from rabbit serum)
200μg/mQ was added to 50mM Tris-HCI (pH 8).
. 37℃,
After reacting for 3 hours, the solution was discarded and buffer A was further added to perform blocking. Then, it was left to stand at 4°C overnight. The composition of Buffer A is as follows. 0.1M phosphate buffer (pH 7,0) 0.1%
Bovine serum albumin (BSA) 0.3M NaCl 0.1% NaN. 1mM MgC1□ Using this assay system, an assay was conducted as follows. Immediately before use, the assay plate was washed once with 200 μQ/well of buffer A. 100 μQ/well of buffer A and 100 μQ of sample were added (duplicate). This was allowed to react at 4°C overnight. Then, the cells were washed four times with 200 μQ/well of washing buffer. To this thing, F
a b '-HRP conjugate (stock solution diluted 1000 times with washing buffer) ■00μQ/
The wells were added and reacted at 37°C for 4 hours. Thereafter, the cells were washed 4 times with 200 μQ/well of washing buffer and 0°6% HPPA (3-(p-hydroxyphenyl)propionic acid) dissolved in 0°1M phosphate buffer γ-(pH 7,0). ] at 100μQ/well and 0.0
Add 100 μQ/well of 15% H2O2 and incubate at 37°C for 1 hour.
After reacting for more than 1 hour, add 0.1M glycine-NaOH (p
H10,3) The reaction was stopped with 50 p Q/υell. Transfer this to DYNATECH MICROFLUOR (white, for fluorescence enhancement), and
ader) was used to measure fluorescence (365nI11 excitation,
415 nm fluorescence measurement). In addition, the following was used as a control. Negative: H20 (0) positive (pos
tive): 1.0Mg/+n in O,lN sulfuric acid
Qkinin (100) As a result, it was found that the transformant produced this peptide both inside and outside the bacterial cell. Inside the cell Outside the cell B (crushed liquid only) O L AH22R-No, 1 54.1 202
2 71 No, 3 52.0 3 JJ No, 5 54.8 4 71 No, 7 66.1 2305
/L No. 9 59.8 2276
For the purpose of expressing the present polypeptide in animal cells such as CO37 and CH○ cells, a plasmid as shown in the figure below is Plasmid No. 52 was purified, and after cutting with Pstl, T
After treatment with 4 polymerase and ligation of a BglII linker, it was integrated into the Bglll site of pT B 551 to obtain pTs6002 (Fig. 2). This plasmid was introduced into CO37 of animal cells, and the production of this polypeptide was examined by EIA. As a result, it was confirmed that this polypeptide was secreted into the culture medium outside the cells. We also confirmed its presence within cells. Extracellular (pm/ml) Intracellular (pg/ml) specimen 1 33,5 10.82 3
8.5 37.53 44.0
30.04 36.5
51.0 Furthermore, CHO cells (
Chinese hamster
ll) contains 1020.3750.170 in the extracellular fluid.
A cell line was obtained that produced 0.2150.1070 pg/ml of the present polypeptide. Yeast transformant AH22RVρGLD 906-1
0 was cultured in the presence of 3SS-cysteine, and the supernatant was
Add the antibody used in EIA for measuring this polypeptide,
After standing at 37°C for 1 hour and overnight at 4°C, Protein A Sepharose was added to collect the antigen and antibody complexes, followed by 20% polyacrylamide gel electrophoresis, and after drying, autoradiography was performed. As a result of the analysis, a single band was identified at a molecular weight of approximately 8,000 daltons. MCF71 cells were similarly cultured by adding 353-cysteine to the culture medium, and the supernatant was analyzed by polyacrylamide gel electrophoresis in the same manner as described above (4th
figure). The present polypeptide expressed in this manner can be purified in large quantities and subjected to examination of its physiological activity. Yeast obtained in Example 7 above (A H22R-/p GL
D 906-10) Dialyze the culture supernatant IQ with 1% acetic acid (
Spectra/Por3 (registered trademark) and lyophilized with 0.05 M ammonium acetate (pH 5,5).
Dissolved in 25mQ. Precipitate at 300°C at 4°C
Orpm, and removed by centrifugation for 10 minutes. Then DE
AE-Sephadex A=
25 column chromatography (Figure 5), Sephadex G-50, gel filtration method using Superfine (
Figure 6), reversed-phase high performance liquid chromatography (HPL)
C) (Fig. 7), EIA positive fraction No. 7 was used for determining the amino acid sequence. For DEAE-Sephadex A-25 column chromatography, the column is 1.6 cm in diameter x 43
C! II length, floor volume 100m Q, 0.8
Elution was performed with a linear gradient of 0.05 M to 2 M ammonium acetate (pH 5,5) at a flow rate of m QZ min, and fractionation was performed using a Pharmacia FPLC system at 10 m Q /fraction. A Absorbance at 280 nm of these fractions. The conductivity and EIA (enzyme immunoactivity) were measured.
The results are shown in the figure. Freeze-dry the fractions 21 to 30 corresponding to this peptide, and 1
% acetic acid 10mQ and proceeded to the gel filtration step. For gel filtration, Sephadex G-50 Superfine with a diameter of 2.5C1ll x 108cm long and a bed volume of 500mQ was used, elution was performed with 1% acetic acid at a flow rate of 0.2mu/min, and the fraction was 4mΩ/fraction. It was fractionated at a ratio of Absorbance at 280 nm, conductivity, and EIA were measured, and the results are shown in FIG. Fraction number 89 ~
Freeze-dry the 101 part and divide this into 10
% TFA and proceeded to the reverse phase HPLC step. For reverse phase HPLC, diameter 4.6nvnX 250
mm length column (Ii!akopaksacosil
IOC1,, '71 registered trademark), 0-50% CH3
EIA measurements were performed after a CN-containing 0.1% TFA linear gradient was run at a rate of 1.0 m Q 7 min and fractionated at a rate of 1.5 m Q /fraction. The results are shown in FIG. The sample (fraction number 7) was freeze-dried, dissolved in 20% acetonitrile, subjected to Edman degradation, and analyzed according to the standard method. As a result, the N-terminal sequence was expressed as a single letter for each amino acid. It was EAQTETCTVAP. This sequence corresponds to residues 25 to 35 of a novel polypeptide secreted by human breast cancer cells MCF-7 cells, which was discovered by some of the present inventors.
It was found that the peptide is the same as the peptide secreted by -7 cells (see Japanese Patent Application No. 1-35111).

[Brief explanation of the drawing]

FIG. 1 is a construction diagram of the present peptide expression vector using yeast as a host, and FIGS. 2 and 3 are construction diagrams of the present peptide expression vector using animal cells as a host. FIG. 4 is an electropherogram related to the identification of this polypeptide produced in yeast and MCF7 cells. Figures 5, 6, and 7 are diagrams showing the purification process of the polypeptide obtained by the present invention, in which Figure 5 is column chromatography, Figure 6 is gel filtration, and Figure 7 is is a diagram showing the results of reversed phase high performance liquid chromatography.

Claims (11)

[Claims] (1) DNA containing EAQ at the N-terminus and encoding a polypeptide consisting of 60 amino acids
. (2) The DNA according to claim 1, wherein the polypeptide has an isoelectric point of 4.3. (3) The DNA according to claim 1 or 2, wherein the polypeptide has a molecular weight of 6,661. (4) Amino acid sequence is EAQTETCTVAPRERQNCGFPGVTPS
QCANKGCCFDDTVRGVPWCFYPNTI
The DNA according to claim 1, which is a polypeptide consisting of DVPPEEEECEF. (5) The DNA according to claim 1, which is cDNA synthesized from messenger RNA (mRNA) derived from human breast cancer cell MCF7 or human gastric cancer cell MKN-45 or KATO-III. (6) The DNA according to claim 1, 2, 3, 4 or 5,
A recombinant DNA constructed to express a polypeptide having EAQ at the N-terminus and consisting of 60 amino acids in an expression vector for E. coli, yeast, or animal cells. (7) The recombinant DNA according to claim 6, which contains a signal sequence that causes the polypeptide to be secreted into the culture medium in yeast. (8) A transformant carrying the DNA according to claim 6 or 7. (9) The transformant host is E. coli, yeast, or MCF7, which is a human breast cancer cell, or MK, which is a human gastric cancer cell.
The transformant according to claim 8, which is an animal cell other than N-45 or KATO-III. (10) culturing the transformant according to claim 8 or 9;
A method for producing a polypeptide, which comprises producing and accumulating a polypeptide having EAQ at the N-terminus and consisting of 60 amino acids in a culture, and collecting the polypeptide. (11) A method for producing the polypeptide according to claim 10, which is modified by site-specific mutation.