JPH0334994A - New polypeptide - Google Patents

Abstract

NEW MATERIAL:An immunoreactive polypeptide containing 60 pieces of amino acid, EAQ as N-chain end, amino acid sequence expressed by the formula, etc., and having 4.3 isoelectric point and 6661 molecular weight. USE:A reagent for elucidation of reproducing mechanism of cancer or a remedy for cancer. PREPARATION:For instance, MCF-7 cell as a breast carcinoma of human is cultured in a medium and dialyzed with buffer solution, then subjected to anion- exchange chromatography, thus eluted with a solution having linear concentration gradient by means of ammonium sulfate buffer solution and ammonium acetate buffer solution and positive fraction in enzyme immunoassay method is collected, then purified by subjecting to gel-filtration column chromatography and reversed phase high-performance liquid chromatography to afford the aimed polypeptide expressed by the formula, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel polypeptide 1 produced from certain cancer cells, and particularly to polypeptide 1 whose N-terminus is EAQ and consists of 60 amino acids. .

It has been reported that transformed cells react to substances produced by themselves, thereby allowing them to proliferate [Spora M, B, etc., Nature] 31.3.745-747. (1
,985)).

This mechanism is called autocrine, and there has been recent interest in this mechanism due to its association with cancer and the like.

Among cancer cells that can be considered a type of transformed cell, for example, MCF7 of human breast cancer cells is a transforming growth factor (tranformed cell).
sforming groity factor)
rt, β, γ [Djckson et al., Cancer Res, 46.
1707-1713. (1986)), insulin-like growth factor [Haff et al., Cancer Res, 46.46] 3-461
．． 9. (1986)) and platelet-derived growth factor [Brousert et al., Proceedings of the National Academy of Sciences USA (P.
ro, Natl, Acad, Scj, USA)
84.576:3-5767 (1987)] is known to secrete @.

For estrogen-induced mRNA in human breast cancer cells, Chambon et al.
et al. have elucidated its complete nucleotide sequence, and from this nucleotide sequence polypeptides 1 to (9) consisting of 84 amino acid sequences
The existence of 140 Daltons has been predicted.

Then, the signal peptide sequence in this 84 amino acid sequence is predicted, and the actual secreted polypeptide is determined.
A sequence consisting of 58 amino acids (6/150 Daltons) or a sequence consisting of 63 amino acids (69
70 Dal 1 Heng) [Chambon et al., Nucleic Assists Research
Acjds Research) 12, No. 6
(1984) 2861-2878, and Chambon et al., DNA (4) II-21
(1985)). However, this was only a prediction, and such secretory polypeptides had not been isolated and purified until the amino acid sequence could be determined.

Problems to be Solved by the Invention It is an object in the field to isolate and purify polypeptides secreted by MCF7 and other human gastric cancer cells, and to provide them for investigation of the relationship between these polypeptides and cancer, etc. It was desired.

As shown above, MCF7 cells secrete various growth factors and EGF (Epider+na), which is a type of growth factor.
In order to investigate the relationship between the
A (Enzy+ne I+nmuno As5ay)
We established MCF7 and MKN-45 of human gastric cancer cells.
As a result of trying to measure the culture solution of KATO-
It was discovered that a new substance different from EGF was produced. It is thought that this was detected because, in addition to the antibody against EGF, another antibody was mixed in the human EGF measurement system described above.

In order to understand the relationship between this new immunoreactive polypeptide and cancer, the present inventor purified this polypeptide 1~ and clarified its structure.

In the present invention, the N-terminus is EA, Q, and the amino acid is 6
This polypeptide is a novel polypeptide produced by human breast cancer cells, MCF7, or human gastric cancer cells, MKN-45 or KA'J''0.
has an isoelectric point of 4.3, a molecular weight of 6,661 Daltons, and its amino acid sequence is E A Q T E T CT V A P R E R
Q N CG F P GVTPSQCANKGCCF
DDT″ V RG V PWCFYPNTID
VPPEEEECEF.

The polypeptide of the present invention is MCF7 or MKN-45.
Alternatively, these cancer cells are produced by culturing KATO-1H, and these cancer cells are cultured using a conventional culture method. Although the culture may be serum culture using a serum-containing medium or serum-free culture, serum-free culture is suitable for the purpose of isolating the polypeptide of the present invention.

Although the culture method is not particularly limited, monolayer subculture is usually used. The culture medium used is a medium commonly used for higher animal cell culture [for example, Dulbecco, Eagle,
Minimum essential medium (DMEM) and ham (l
lam) F-10 medium, etc.], and the medium is 0.05 to 5 mQ, preferably 0.1 to 5 mQ per 105 cancer cells.
Use 0.2mf1. The culture period is 2 to 10
The appropriate time is 2 to 3 days for serum-free culture, and 3 to 4 days for serum culture. The culture temperature is usually 37°C±1°C.

The polypeptide of the present invention can be isolated and purified using conventional peptide isolation and purification methods such as extraction, ion exchange or gel filtration column chromatography, high performance liquid chromatography, and electrophoretic recrystallization. Ion exchange or reverse phase column chromatography and high performance liquid chromatography are particularly suitable for the purposes of the invention.

The polypeptide purified in this way consists of 60 amino acids, and has the ability to bind human 1 to PS2 polypeptide 1, which is thought to be secreted by breast cancer cells, as determined by Chambon et al.
PS2 pre-waste protein consisting of 84 amino acids encoded by DNA [DNA (4,)l]
~21 (1985)).

However, as mentioned above, Chambon et al.
Amino acids were only estimated from NA, and polypeptides were not isolated and purified as in the present invention. The novel polypeptides obtained in the present invention are immunoreactive with anti-PS2 antibodies.

Furthermore, Chambon et al. estimated that the mature polypeptide is obtained from this precursor by enzymatic action at positions 22 and 27, yielding 58 or 63 amino acids.
The polypeptide 1 actually obtained in the present invention consists of 60 amino acids obtained by enzyme action at position 25, which is 3 fewer than that estimated by Chambon et al., or 2 amino acids.
There are many polypeptides, and in the present invention, the novel polypeptide 1- is isolated and purified.

The assay system used for detecting the polypeptide of the present invention was manufactured as follows.

200 μg/mQ of the IgG fraction (purified from rabbit serum) containing the plate-sensitized anti-hEGF antibody was mixed with 50 mM 1-Lis-HCI (
The mixture added to pl(8,0) was dispensed into a 96-well plate (manufactured by Sumitomo Bakelite) at 100 μQ/well.

After reacting at 37°C for 13 hours, the solution was discarded and buffer A was further added to perform blocking. And another 4 degrees
It was allowed to stand overnight at C. The composition of Buffer A is as follows.

0.1M phosphate buffer (pH 7,0) 0.1%
Bovine serum albumin (BSA) 0.3M NaCl 001% NaN3 1 mM MgC]2 Using this assay system, an assay was conducted as follows.

Assay plate 1 - Washed once with 200 μQ/well of buffer A immediately before use. 100 μQ/well of buffer A and sample] 00 μα was added (dupljcate).

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) 100 μQ
/well was added and reacted at 37°C for 4 hours. Thereafter, the cells were washed 4 times with wash buffer 7-200 ILQ/1yell, and 0°6% HPPA [3-(p-high1) dissolved in 0°1M phosphate buffer (pH 7,0)
~ Roxyphenyl) propionic acid] was added to 100 μQ/well and O, O1, 5% I-I, 02 was added to 100 μQ/well, reacted at 37°C for over 1 hour, and 0.1 M glycine-NaOH (p Hlo, 3) was added. 50/LQ/1u
e1. ] to stop the reaction. This thing is DYNAMI M Tsuku DYNATEC)! Transfer to MICROFLUOR (white, for fluorescence enhancement), and transfer to Immunoreader (Immun).
(365 nm excitation, 415 nm fluorescence measurement). In addition, the following was used as a control.

Negative (negatl, ve): H2O (○) Positive (
posjtive): 1.0μH in O,lN sulfuric acid
/nutkinin (100) Action/Effect The polypeptide of the present invention is produced and secreted by certain types of cancer cells, and is predicted to be related to cancer.

For example, by examining the concentration and distribution of the peptide within cells and tissues, it becomes possible to diagnose cancer and understand the progress of cancer. Furthermore, it is thought that it will be useful in elucidating the cancer growth mechanism and, ultimately, in providing therapeutic drugs for cancer. This polypeptide also contains the known pancreatic spasmolytactic enzyme (Panc), which is secreted from the pancreas.
Reactive spasm.

1ytic enzyme, PSP) [Jorgensen et al., Regul, atoryPeptj, de)
3, 207-2] 9 (1982)), it is presumed that this substance has an intestinal peristalsis suppressing effect, and is also presumed to have a muscle contraction/relaxation effect. According to the present invention, the polypeptide can also be produced and purified in large quantities by genetic engineering techniques.

In the present specification and drawings, when amino acids, etc. are indicated by abbreviations, IUPAC-IUB Comm1sj
on on Biochemjcal, Nomenc
] ature or the abbreviations commonly used in the field, examples of which are given below. In addition, when an amino acid can have optical isomers, unless otherwise specified, the L-isomer is assumed to be indicated.

G Nigericin A: Alanine ■: Valine L: Leucine T: Isoleucine S: Serine T: Threonine C Nidistine M: Methionine E: Glutamic acid D = Aspartic acid K: Lysine R: Arginine ■■: Histidine 1-F: Phenylalanine Y: Tyrosine W: 1 Helyptophan P Nibroline N: Asparagine Q: Glutamine I Particularly, the present invention will be explained with reference to Examples below, but the present invention is not limited thereto.

1. Purification of new protein (a) Culture of MCF-7 cells Plastic Alanine (17
MCF-7 cells were incubated with 10% calf serum, insulin (12X10-6M), β-estradiol (
10-11M), glutamine (0,3mg/+n
Dulbecco Eagle Minimum Essential Medium (DMEM) containing Q), penicillin (50 units/mQ), and Leptomycin (0.05/mQ).
) at 37°C under the conditions of 5% CO2/95% Aj2-r. After the cells have expanded sufficiently, wash the cells with phosphate buffer and use clarifier-free medium (DME).
M and HAMF], 2 mixed at a ratio of 1:1). The medium contains β-estradiol (10'M). After culturing for 24 to 48 hours, collect the medium,
Centrifuge at 5,000 rpm for 30 minutes.

(b) DEAE-Sephadex
The sample obtained by anion exchange chroma 1-graph (a) using A-25 was immersed in a dialysis membrane (Spec 1-Labor, 5pec
trapor 3 ) against 0.05 M ammonium acetate buffer (pI-T5.5). A flow rate of 50 mQ/
The column was run under certain conditions. Wash the column with the same buffer and add 500% each of 0.05M ammonium sulfate buffer (pH 5.5) and 2M ammonium acetate buffer (pH 5.5).
Elution was performed with a linear concentration gradient using mQ. Enzyme immunoassay (IEIA)
) The positive fractions were collected and lyophilized. Figure 1 shows the results.

(c) The lyophilized powder obtained by gel filtration (b) was redissolved in 10 mQ of 1% acetic acid, and a Sephadex G-50 Superfine column (inner diameter 2
．． 5 cm, length 100 r+n) at a flow rate of 12 mQ/hour. After elution with the same 1% acetic acid, EIA-positive fractions were collected and dried under reduced pressure. The results are shown in FIG.

(d) The vacuum-dried product obtained by reverse-phase high performance liquid chromatography (c) was dissolved in 100 mD of 10% trifluoroacetic acid, and μ Bondapak C1
, 8 columns at a flow rate of 0.7 mQ/min. Elution from the column was performed using a linear concentration gradient of 0 to 50% acetonitrile containing 0.1% TFA over 100 minutes. Protein detection from the column was performed at 210 nm.
The results are shown in FIG.

(e) Determination of amino acid sequence The amino acid sequence of the purified peptide was determined using an automatic amino acid sequencer manufactured by Applied Biosystems.

Determined by Model 129A and Edman decomposed P
EAQTETCTV when showing T H amino acid from the N-terminus
APRERQNCGFPGVTPSQCANKGCCF
DD (Figure 4). Furthermore, the part following this amino acid and the part before the above amino acid predicted from the gene (DNA) are shown below.

WCFYPNTIDVPPEEECEF The boxed area is the part whose amino acid sequence was previously determined.

(f) The recovery rate of this peptide during m production is 2↓, 8
%. The purified polypeptide consists of 60 amino acids and was found to be part of the 84-amino acid protein encoded by the PS2 cDNA determined by Chambon et al. does not actually purify the protein to the point where the amino acid sequence can be analyzed as in the present invention, and only cD
It merely shows the amino acid sequence deduced from NA. Furthermore, Chambon et al. proposed that enzymes act at positions 22 and 27 to obtain mature polypeptides from this precursor.
It is estimated that 8 or 63 amino acids can be obtained, but the polypeptide actually obtained by the present invention consists of 60 amino acids obtained by the action of signal peptidase at position 25, which is in line with the estimate of Chambon et al. It has three fewer polypeptides or two more polypeptides than that of the polypeptide of the present invention.
(see figure).

[Brief explanation of drawings]

Figures 1, 2, and 3 are diagrams showing the method for purifying the polypeptide of the present invention, and the diagrams are anion exchange chroma 1 to 1.
Graphie, Figure 2 shows gel filtration, and Figure 3 shows reverse phase high performance liquid chromatography 71-16. FIG. 4 is a diagram showing how the amino acid sequence of the polypeptide of the present invention was determined. FIG. 5 is a diagram comparing the position where the signal peptidase acts to obtain the mature polypeptide in the present invention and the enzymatic cleavage site estimated by Chambon et al. for the precursor of the polypeptide of the present invention. be.

Claims (5)

[Claims] (1) An immunoreactive polypeptide whose N-terminus is EAQ and consists of 60 amino acids. (2) The polypeptide according to claim 1, which has an isoelectric point of 4.3. (3) The polypeptide according to claim 1 or 2, which has a molecular weight of 6,661. (4) The polypeptide according to claim 1, 2 or 3, which is produced by MCF7, which is a human breast cancer cell, or MKN-45 or KATO-III, which is a human gastric cancer cell. (5) Amino acid sequence is EAQTETCTVAPRERQNCGFPGVTPS
QCANKGCCFDDTVRGVPWCFYPNTI
5. The polypeptide of claim 1, 2, 3 or 4, consisting of DVPPEEEECEF.