JP2770926B2 - Human placental angiogenesis inducer capable of promoting capillary endothelial cell protease synthesis, DNA synthesis and migration - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION   This application is based on the capillary endothelial cell protease of Moscatelli et al.
Human placental blood can promote DNA synthesis and migration
U.S. filed December 17, 1985, entitled Tube Formation Inducer
This is a continuation application of National Patent Application No. 809,873. Background of the Invention   Angiogenic factors have various properties, namely, (1) endothelial cells.
Vesicle proliferation; (2) increased endothelial cell protease synthesis; (3)
Promoting endothelial cell migration toward the location of the protein; and
(4) a protein capable of inducing capillary proliferation in vivo
Is defined. Particularly classified as an angiogenic factor
Substances affect the DNA synthesis of endothelial cells
Has the ability to promote tearing, and therefore the rate of endothelial cell proliferation and blood vessels
It has been observed to increase the rate of formation.   In connection with this property, angiogenic factors are
Has the ability to increase protease synthesis. These professionals
Plasminogen activator (PA) and the like
And collagenase. Specifically, angiogenic factors
Promotes the synthesis of PA and latent collagenase;
Rasmin preenzyme is a broadly specific protease activity
Plasmin, which activates latent collagenase
Converted to sex collagenase. Active plasmin and
The two enzymes of activated collagenase are found in most of the surrounding tissues.
Can degrade most proteins, and therefore capillary endothelium
Increases erosion of various tissues such as vesicles. Further angiogenic factors
The offspring are chemotactic, especially in certain cells such as capillary endothelial cells.
That is, the angiogenic factor
Induce them to migrate in the direction of those factors.   Isolating angiogenic factors with these properties in mind
Can increase the vascular supply to organs
It was thought that a therapeutic agent could be created. For example
Blood supply to the heart interrupted as a result of certain myocardial infarction
Promote renewal of feeding or revascularization in chronic atresia
It is desirable to promote growth. In addition, induction of angiogenesis
Factor ulcers, surgical incisions and especially elderly and
It can promote the recovery of delayed healing wounds in diabetic patients. Further
Applying this substance to burns will increase the speed and extent of healing
Can be Therefore, suitable for human therapeutic applications
The purified angiogenic factor was searched. Further promotes angiogenesis
Blood supply to cancer cells by studying evolving substances
Believe that there is a way to block cancer and starve cancer
Some scientists do.   A class of proteins already called "angiogenesis-inducing factors"
Are these proteins identified as nonhuman animals?
Have been isolated. Blood vessels isolated from non-human animals
Formation-inducing factors can have side effects on immune response to heterologous proteins
Not suitable for use as a therapeutic in humans due to its potency
it is conceivable that. Moreover, these individual non-human proteins
Whether it has the above four characteristics as an angiogenesis inducer
Whether the observed properties are between protein combinations
It has not been confirmed whether it is due to interaction.   In fact, each of the alleged endothelial cell mitogens
Seed proteins fall into two classes: heparin-cef
Hull eluted with 1 M NaCl from allose and has an acidic pI
Cell growth factor-like molecules; and heparin-sepharose
Fibroblast growth factors that bind more strongly and have a basic pI
It is a child-like molecule. Furthermore, the present inventors have recently
Biochemistry, Vol. 24, 5480-5499 by Vallee et al.
In a paper published on pp. 1985.
(Angiogenin) "
Believe that the factor exists. Angiodienin is true
It has the properties of an angiogenesis-inducing factor but has mitogenic effects
It is distinguished in that it does not have a business.   The present inventors have studied based on these various studies.
Hey, FGF_basicIsolated from human placental tissue that can be classified as
A single molecule of substantially the same nature as
Shape, that is, mitogenic, chemotactic and capillary in vivo
Ability to promote proliferation and promote protease synthesis
A human angiogenesis-inducing factor with high quality was discovered. More books
The present inventors have found that this angiogenic factor is substantially more effective than human placental tissue.
An attempt was made to isolate it in a partially purified state. This isolated
The amino acid sequence of the angiogenic factor was determined. this
Determination of the amino acid sequence allows the synthesis of the angiogenic factor
Chromosome or cDNA sequences useful for recombinant DNA methods
DNA probes can be obtained for use. Summary of the Invention   The present invention relates to angiogenesis-inducing factors in general, and more specifically to FGF
_basicAngiogenic factors classified as
You. In particular, the present invention contemplates that it can be isolated from human placental tissue.
Substantially equivalent, providing mitogenic and chemotactic performance
Induces protease synthesis to promote capillary growth in vivo
FGF that can be awakened_basicAngiogenic factors
I do.   Specifically, the present invention provides (1) Natural isolated from human placental tissue (postpartum)
Purified with substantial homology to angiogenesis-inducing factor
Blood consisting of human or synthetic single-chain polypeptide proteins
An angiogenesis-inducing factor (bFGF)
Indicates mitogenic activity, chemotactic activity,
Selected from the group consisting of
SD having at least one active site exhibiting reduced activity
Has a molecular weight of about 18,700 daltons as determined by S-PAGE,
And the following amino acid sequence (2) or further above
An additional amino acid is added to the N-terminus of the angiogenesis-inducing factor of (1).
The present invention relates to an angiogenesis inducing factor and a method for producing the same.
Here, the angiogenesis-inducing factor of the above (2) was measured by SDS-PAGE.
Having a molecular weight greater than about 18,700 daltons
Including peptides.   It is an object of the present invention to provide an angiogenic factor having these properties.
To provide the offspring in a purified state. Further, the present invention
The purpose of this is to use the amino acid sequence of
Is to decide. Furthermore, the purpose of the present invention is to
Mitogenesis with the ability to promote the synthesis of thease and
Valuable as a pharmaceutical preparation exhibiting chemotactic properties
FGF_basicMay be provided in purified form.   Other objects and advantages of the present invention are set forth in the following description.
And are apparent from the description, or
Learned from. These objects and advantages are particularly
Realized by the methods and means as set out in the claims,
Can be achieved.   In order to achieve the object and in accordance with the object of the present invention,
Mitogenic activity, chemotactic activity, ability to promote protease synthesis
Activities selected from the group consisting of
Angiogenesis with at least one active site
Inducers are revealed. Its blood of human or synthetic origin
Tube formation inducer is FGF_basicClassified as human placental tissue
Natural angiogenic factor and substantially homogeneous, which can be isolated from
It has sex.   Angiogenic factor itself can promote protease synthesis
It is preferable to use the “Protea
The term "ase" includes active forms and precursors. like that
Precursors include latent or procollagenase
You. Further within the scope of the present invention,
An angiogenic factor that does not directly promote growth
It is also possible. But their angiogenic factors
Provokes a biological response and thus promotes protease synthesis
You. Therefore, the angiogenesis-inducing factor of the present invention can be directly or
Immediately promotes protease synthesis.   Particularly preferred angiogenic factors according to the present invention include the following:
Has a core amino acid sequence:   Furthermore, a peptide having the following sequence
Present in the peptide.   And in the sequences shown here, Kazuko has been completely identified.
Indicates the presence of no single amino acid residue. Another especially
Preferred angiogenic factors have the following sequences:  Amino acids represented by the above-mentioned abbreviations are the following "preferred"
Description of Embodiments ".   Further, to achieve the objectives and in accordance with the present invention,
Of a natural angiogenic factor that can be isolated from placental tissue
Qualitatively purified are disclosed. Still further, the purpose
At least for the purposes of the present invention.
One active ingredient, angiogenesis induction described by the present invention
Disclosed are pharmaceutical compositions containing the factor.   The above general description and the following detailed description are exemplary and
The present invention is for explanation only and is in the scope of the claims.
Needless to say, it does not limit the light. Description of the preferred embodiment   The presently preferred embodiment of the present invention will be described.
It explains the basics of the invention in conjunction with the description and examples that follow.
is there.   As mentioned above, the present invention relates to isolated blood in purified form.
It relates to a tube formation inducer. Preferably the angiogenesis of the invention
Inducers are natural vascular forms that can be isolated from human placental tissue
Substantially the same as the induction factor, immunologically equivalent and
Single polypeptide chains which are preferably bioequivalent
It is a protein. Used in the specification and claims.
"Biologically equivalent" means that the composition of the invention
As, but not necessarily the same as,
Mitogenic activity and chemotactic performance
And can induce protease synthesis
Means   Used throughout the following specification and claims
“Substantially the same” has been reported to date and has been purified.
Compared to substantially identical angiogenic factors
Also means higher homology with natural angiogenesis inducer
I do. Preferably the degree of homology is at least 50%, preferably
60%, more preferably 75% or more, especially preferred
Protein is 85% or 90% homologous to the native protein
It is. The degree of homology described above, especially as a reference
Atlas of Protein Sequences and Structure,
Vol. 5, p. 124 (1972), National Biochemical Rese
arch Foundation, Washington, D.C., M.O.Dayhoff in
100 to add to the array as described by
Introduce 4 gaps in amino acid length and compare
Identical amino acids are aligned and found in the shorter of the two sequences.
Calculated as percent of amino acid residues.   The angiogenesis inducing factor of the present invention described here is
An isolated or synthetic polypeptide. "Go
The term “growth” polypeptide has never been
An amino acid sequence that is not isolated in purified form.
U. Using this definition, "synthesis" means that
Together with polypeptides created by recombinant DNA methods
Include those synthesized in whole or in part in a test tube.
In particular, the preferred sequences described below and one or two
Synthetic polypeptides with different amino acids are designed.   A preferred angiogenic factor according to the present invention is a human placenta group
Found in woven extracts and isolated for the first time in purified form
Was. For the purposes of the present application, the angiogenesis inducers disclosed herein
"Pure form" or "purified"
`` Shape '' refers to the expression of proteins other than angiogenic factors.
It does not contain qualitatively. Preferably the blood of the present invention
The tube formation inducer is at least 50% pure, preferably 70%
% Purity, preferably 80% or 90% pure
Degrees.   Furthermore, the angiogenesis-inducing factor of the present invention is useful for various tumors and tumors.
And normal cells. As an example, SK-Hepl
Vesicles, HeLa cells, K562 cells and human fetal lung fibroblasts
There is.   The angiogenesis-inducing factor of the present invention comprises the following steps:
Can be isolated purely from human placental tissue by the method:
Collection of placental tissue; (b) for fractionation of proteinaceous substances in the tissue
Isolation of an angiogenesis-inducing substance from human placental tissue by
(C) identification of a fraction having angiogenesis-inducing factor activity; and
And (d) enrichment of the fraction showing angiogenesis-inducing factor activity.   In a preferred embodiment, the protein present in human placental tissue
Heparin Affinity Chromatography, I
On-exchange chromatography and optionally gel filtration chromatography
Fractionation is performed by a combination of matographies. State here
Angiogenic factors are placental protein-specific monoclonals.
It can also be isolated using antibodies. In this example, the antigen is
Matri containing monoclonal antibodies against disc protein
Bind to fox (resin) and wash the resin with buffer
Removes non-antigenic proteins. Next higher pH or lower
PH buffer, high ionic strength buffer or chaotropic
By changing the temperature of the stick agent alone or in combination
The antigen is removed from the antibody.   Angiogenesis-inducing factor is used for the fraction thus obtained.
Search for the presence of activity. Preferably a suitable endothelial cell culture
Cultivation, preferably a mammalian capillary endothelial cell
Incubate in the presence of offspring
Measuring PA and intracellular PA
Do this by assessing the effects on collagenase synthesis
Was. Cells generated by angiogenic factors are generated
ELISA or RIA assay or immunization
It is measured by an immunological method such as a sedimentation method.   The mitogenic activity of the angiogenic factor is preferably
Appropriate endothelial cells, preferably mammalian capillary endothelial cells.
Tube formation inducer and radiolabeled nucleotide, preferably
Is¹²⁵I-deoxyuridine iodide (¹²⁵In the presence of I-dU)
It is measured by incubating. Trichlor
Incorporated in acid-insoluble fraction¹²⁵The amount of I-dU is
Measure as an indicator of degree. Chemotaxis of angiogenic factors
Performance depends on suitable endothelial cell culture, preferably mammalian capillary
Incubate endothelial cells in the presence of angiogenic factors
And in a suitable tank, preferably a modified Boyden chamber.
The determination is made by measuring the mobility of the cells.   As described above, the present inventors used angiogenesis-inducing factor in human embryos.
Isolate in a previously unpurified form from disc tissue
Succeeded. Isolation of the protein in purified form
Is the correct sequencing of this protein and an angiogenic factor
It is essential for the development of a pharmaceutical composition containing   Preferred angiogenic factors of the present invention are the following core amino acids:
Having a no acid sequence: A peptide having the following sequence exists outside the core sequence. Also
Another particularly preferred angiogenic factor has the following sequence:
Do:   The abbreviations are, for example, Biochemistr by A.L.Lehninger.
y, 2nd edition, Worth Publishers, Inc., New York (19
1975), compared to the standard amino acid sequence abbreviation described on page 72.
Respond. The claimed activity of the angiogenic factor is
15, 16, 17 or 18 N-terminal amino acids
It is believed that removing the acid residue will not be affected.
ing. Therefore, all of these omitted sequences are included in the present invention.
I will. In addition, the N-terminal amino acid of the native polypeptide
It is also planned to extend up to 110 amino acids to the acid
You.   Also, the C- or N-terminal of the angiogenesis-inducing factor of the present invention.
It is also conceivable to add a polypeptide chain to the peptide. Especially,
The polypeptide chain can be attached to either end by protein fusion technology.
Are combined. These chlorinated polypeptides are
Responsible for enhancing the efficacy of tube formation inducers. For example other
By fusion with the polypeptide, the polypeptide is brought to low pH or
Will be able to maintain activity at elevated temperatures or
Polypeptides have longer circulating lifetimes, higher against degradation
Resistance or through the small intestinal epithelium
Increases the permeability.   However, due to these changes, the amino acid sequence
Causes immune response side effects in organisms receiving formation inducers
Should not appear to be such side effects
The damage from the inducer is such that the benefits cannot be guaranteed.
Can get. Bioactive molecules can cause such immune response side effects.
The method of determining whether or not to
You.   The angiogenesis-inducing factor of the invention and the invention disclosed herein
Analogs have mitogenic or chemotactic properties.
Pharmaceutical product forms capable of promoting improved protease synthesis
Applied to humans and animals. At least one activity
Contains one of the angiogenesis-inducing factors of the present invention as a component
Pharmaceutical compositions may be suitably pharmaceutically acceptable depending on the intended dosage form.
Carriers, diluents, fillers, binders, and other additives
Contains excipients. Active protein in the digestive tract for oral administration
Measures must be taken to prevent the decomposition. Therefore, enteric coating
The form is considered as one form suitable for oral administration. Non-tradition
If oral administration is employed, the preparation may be water or saline or
Or other pharmaceutically acceptable solutions. In general
Preparations for parenteral administration are ultimately identical to body fluids
It is preferable to include a sufficient concentration of sodium chloride to achieve osmotic pressure.
New A pharmaceutical preparation containing the angiogenesis-inducing factor of the present invention
Injection or local treatment for the treatment of wounds, surgical incisions or skin ulcers
Topical administration, such as local administration, is also contemplated. More myocardial infarction
Sustained release for administration to resume blood supply to the post-occlusion heart
Sexual intercalating agents are also conceivable.   Dosages appropriate for the treatment of each of the above-mentioned diseases and the above-mentioned deli
The calculations required to determine the appropriate amount to use for the Barry method are routine.
It is performed by a person skilled in the art in the usual manner,
Unnecessary due to the size of the assay and the assays disclosed herein.
This is the range that can usually be done without experimentation. Calculated
Dosage should be combined with the appropriate dose response data to determine the effective dose.
Determined by using established assays
You.   Specific teachings of the teachings of the present invention
Use of ordinary skill in light of the teachings contained herein
Within the ability of the person. Examples of products of the invention and
Typical methods for their isolation and production are shown in the following examples.
You. Example 1 Purification of human angiogenesis-inducing factor from placental tissue A. Protein purification   The postnatal term placenta is frozen at -20 ° C. Frozen placenta
Into small pieces, and use the electric food chili par (General Slicing,
Mill in Balden, New York)
Modify. All operations after homogenization are performed at -4 ° C.
Do. Place the homogenized placenta in cold 20 mM Tris, pH 7.
Dilute with 5,3mM EDTA, 50W (Model 185 sonicator,
Branson Sonic Power Co., Plain, New York
Sonicate for 10 minutes with view. Usually 1 kg frozen embryo
Obtain 2 liters of processed material from the board.   The sonicated product was adjusted to pH 4 with hydrochloric acid, and the pH was adjusted to 2 minutes at this pH
After uvating, neutralize with NaOH. NaCl to a final concentration of 0.5M
And centrifuge at 10,000 xg for 60 minutes.
You. The supernatant was washed with 0.5 M NaCl / 3 mM EDTA 120 mM Tris, pH
Heparin-Sepharose equilibrated in 7.5 (Pharmacia
A, Piscataway, New Jersey)
Place on 85 × 153mm. Wash column with same buffer, 2M
  Elute with NaCl / 3 mM EDTA / 20 mM Tris, pH 7.5. Elution
Dilute the solution with 3mM EDTA / 20mM Tris, pH 7.5 to reduce conductivity to 2
4 mmho and a second heparin-sepharose column (1
6 × 190mm). Column is 3mM EDTA / 20mM Tris,
pH 7.5, wash with 0.7M NaCl solution in the same buffer NaCl
Elute with a gradient from 0.7 to 2M.   The protease inducing activity was measured for each fraction,
The third heparin-sepharose column (12 x 75m
m). The column is first loaded with 3 mM EDTA / 20 mM Tris, p
H7.5, 0.8M NaCl solution in medium, then 0.1M sodium phosphate
Washed with 0.2M NaCl solution, pH 6.0, 0.1M Na phosphate
Elute with a 0.2 M NaCl solution in thorium, pH 6.0. third time
20 times the active fraction from the Heparin-Sepharose column
Dilute with volume of 0.1 M sodium phosphate, pH 6.0.   Centrifuge the diluent at 10,000 xg for 30 minutes and then
9 × 72 mm CM-Sephadex C-50 equilibrated with the buffer
Place on column (Pharmacia). Column is 0.1M phosphoric acid
Dissolve sequentially in 0.15M, 0.5M and 2M NaCl solution in sodium
And measure the protease-inducing activity of each fraction.   CM-Sephadexcala containing protease-inducing activity
0.5M NaCl eluate from the system
Concentrate on a loin column. This column is 60 mM sodium phosphate
Wash sequentially with 0.5 ml of 2M NaCl solution in pH 6.0, pH 6.0
U. All activity eluted in the first 1 ml. This eluate
With 2M NaCl, 60mM sodium phosphate, pH 6.0
At a flow rate of 0.5 ml / min.
Almasia).   Angiogenesis-inducing factors as claimed in the present invention are
Isolated from the effluent. This angiogenic factor is
Also called "protein". B. Confirmation of protein properties and angiogenesis inducing factor activity 1.NaDad SO_Four−PAGE   NaDod SO_FourUsing polyacrylamide gel, 3% above
Prepare a layered gel and a 10-18% gradient degraded gel, especially
Nature 277, 680-685 adopted here as reference
Electrophoresis according to the method of Laemmli described in (1970)
You. The active fraction from the column is NaDod SO_Four-Molecular weight by PAGE
There were 18,700 single bands. 2.Protein measurement   Protein concentration was determined using Bi using bovine serum albumin as a standard.
o-Rad Protein Assay (Bio-Rad Laboratories, Calif.
(Ritzchimond, Lunia). Of this protein
Information on the sequence is provided in Example 4. 3.mitotic activity¹²⁵Removal of I-deoxyuridine
Inset   Bovine capillary endothelium from the adrenal cortex of a freshly slaughtered one-year-old cow
(BCE) cells, Proc. Na, incorporated herein by reference.
Reported in tl.Acad.Sci.USA 76, 5217-5221 (1979)
And isolation by the method of Folkman et al. Especially here
J. Cell Biol. 95, 974-981
Mouse as described by Gross et al.
10% (v / v) supplemented with medium conditioned with 180 cells
v) Cells in Alpha Minimum Essential Medium (MEM) with calf serum
Grow until confluent. Confluence
Medium, the medium should contain 5% calf serum supplemented conditions.
Replace with fresh MEM medium.   A confluent culture of BCE cells was added to 5% M calf serum.
Maintain in EM medium for 7 days. Then, adjust the concentration of the medium to various concentrations.
5% calf serum-containing MEM medium containing placental angiogenesis-inducing factor
Change to earth. After 20 hours, the medium was supplemented with 5% calf serum and 0.
3 μCi / ml¹²⁵I-deoxyuridine iodide (2000 Ci / mmo
l, New England Nulear, Boston, Massachusetts)
Replace with Dulbecco's Modified Eagle Medium containing Mark
After incubation for 16 hours in a culture medium, the cells were cooled in cold phosphate buffer.
The label is stopped by washing with a salt solution. Cells are cold 5% tric
Incubate with chloroacetic acid (TCA) for 30 minutes and add 5% TCA
After washing twice with distilled water, it is incorporated into the acid-insoluble matter.   ¹²⁵I-Deoxyuridine iodide is measured. 4.Migration measurement   The migratory measurement is performed by the Pro, which is hereby incorporated by reference.
c. Natl. Acad. Sci. USA 79: 5597-5601
Gelatin and fibronect according to Castellot's method
PVP-free polycarbonate coated with tin and having a pore size of 5 μm
-200μ blind using a carbonate filter
Wells (Nuoleopore, Pleasant, Calif.)
In). In MEM medium containing 0.5% fetal bovine serum
Add 10-fold diluted purified protease inducer under the wells
Put in layers. Next, insert a filter and add 0.5% of 200μ
5 × 10 in MEM medium supplemented with fetal calf serum^FourBCE cells
Add to upper well. After incubating at 37 ° C for 4 hours
Remove the medium in the upper layer of the well and remove the fine surface from the upper surface of the filter.
Gently remove the cells with a cotton swab. Next, remove the filter
And dried at room temperature, and the light-Giemsa dye (Baker Chemical
 Co., Philadelphia, New Jersey)
You. The total number of cells on the lower surface of the filter was determined by light microscopy (40
(0x magnification). 5. Induction measurement of PA and collagenase   Maintained in MEM containing 5% calf serum for at least 2 days
Confluent cultures of cultured BCE cells with 5% calf serum and
And replace with a new MEM multiple containing the substance to be tested. 37
Collect the medium after incubation for 24 hours at
Moscatelli in Cell. 20; 343-351 (1980).
Assay collagenase as described by
You. All collagenases are latent and detect activity
Activated with trypsin. From this same culture
The cell monolayer was washed twice with cold phosphate buffered saline and
0.5% (v / v) Twiton X-1 in sodium, pH 8.1
00 solution and described by Gross et al. (Supra).
PA activity is measured as described above. As a result of the experiment,
PA amount is proportional to the amount found in the conditioned medium
I can tell you. One unit of protease-inducing activity is PA or
And half of the maximal induction of collagenase synthesis
Is defined as the amount required for Example 2 Purification of angiogenic factors from human placental tissue   A second heparin-sepharose according to the method of Example 1
Obtain the eluate to be applied to the column. Column is 3mM EDTA / 20mM
Tris, pH 7.5, wash with 0.95M NaCl solution in the same buffer
Elute with 2M NaCl solution.   2M eluate against 0.2M NaCl / 20mM MES, pH 6.0
Dialyze. Centrifuge dialysate at 100,000 xg for 60 minutes
Excluding insolubles, Fast Protein Liquid Chromatography
Apply to Mono-S column using (FPLC) system. Mosquito
The ram was washed with 0.2 M NaCl / 20 mM MES, pH 6.0, then 20 mM M
Elute with a 0.2-2M NaCl gradient in ES, pH 6.0. For each fraction
The protease-inducing activity is measured. Protease induction
Conductivity eluted between 0.45 and 0.6M NaCl. Example 3 Purification of angiogenic factor from hepatoma cells   All purification steps except FPLC are performed at 4 ° C. SK-
Hep-1 cells (American Type Culture Collection [ATC
C] Cells from confluent monolayer with accession number No. HTB52)
Was collected by centrifugation at 400 xg for 10 minutes.
You. Suspend cell pellet in 10 volumes of PBS / 0.5M NaCl
The Branson Sonicator (Plainville, New York)
Sonicate at 5 watts for 3 minutes using (U). Lottery
The product is centrifuged (10,000 xg, 1 hour) and the supernatant is
Collect Pellet re-stain in equal volume of PBS / 0.5M NaCl
Perform sonication after turbidity and centrifuge. Combine the supernatant
Heparin-equilibrated with 28 x 75 mm PBS / 0.5 M NaCl
Sepharose column (Pharmacia, New Jersey)
State, Piscataway). Column is 0.5M NaCl / 3mM
  After washing with EDTA / 100 mM Tris, pH 7.5,
Elute with a 0.5-2M NaCl gradient. PA induction for each fraction
The activity is measured, and the activity images are collected and the conductivity becomes 20mmho.
Dilute up to 3 mM EDTA / 20 mM Tris, pH 7.5.   The active substance is then added to 0.5 mM in 3 mM EDTA / 20 mM Tris, pH 7.5.
  Second heparin sepharose equilibrated with NaCl solution
Apply to a column (10 x 75 mm). First column is 0.5M NaCl
And then wash with 3 mM EDTA / 20 mM Tris, pH 7.5
Elute with a 0.9-2M NaCl gradient in the buffer. Three active fractions
Load the second heparin-Sepharose column (7 x 85 mm)
After washing with 0.5 M NaCl in 20 mM MES, pH 6.0, the NaCl was
Elute as 2M. Third Heparin-Sephaloska
Active fraction from lamb diluted 1:10 with 20 mM MES, pH 6.0
To remove insoluble material by centrifugation at 100,000 xg / hour.
Good.   The Mon was equilibrated with 0.2 M NaCl / 20 mM MES, pH 6.0.
Load on o-S FPLC column (Pharmacia). column
Was washed with 0.2 M NaCl and eluted with NaCl gradient (20 mM ME
S, pH 6.0, 0.2 to 2M in). PA for each fraction
The inducing activity was measured, and the active fractions were collected to determine the purity of NaDod SO._Four
Test by -PAGE. Example 4 Of placental angiogenesis-inducing factor (PAF) isolated from human placenta
Determination of amino acid sequence A. Lys-C peptide   According to the method of Example 2 described above, 20 mM MES, pH 6.0, 0.5 M Na
Obtain purified PAF dissolved in Cl. Natural protein as below
Digest with endo-proteinase Lys-C: 2 nmo
le natural protein at 350 μM 20 mM MES, pH 6.0, 0.5 M N
Dissolve the reaction solution containing aCl in 2M NH_FourHCO_ThreeAdd 15μ
And adjust to pH 8.7. 1.17 units end type proteiner
Lys-C (Boehringer) was added and the mixture was added at 37 ° C for 7 hours 3
Perform digestion for 0 minutes. Next, add 2-mercaptoethanol
Add to a final concentration of 1% (v / v),
Incubate for 15 minutes. Trifluoroacetic acid (TF
A) to a final concentration of 0.1% (v / v)
Reversed-phase high-performance liquid chromatography using Synchrom RP8 column
Fractionate by chromatography (HPLC). 0.1% TFA for peptide
Acetonitrile gradient in aqueous solution and then 0.1% TFA (60
Elute from the column with 0-60% acetonitrile per minute)
You. Elution of peptide from column is A₂₁₅And A₂₈₀Track with
And collect the appropriate fractions manually.   Automated edging of peptides eluted with 19% acetonitrile
Sequencing by Mann decomposition showed the following sequence:   Amino acids shown in this sequence and in the following sequence
Residues are residues that have not been identified.   Sequencing of peptides eluted with 19.8% acetonitrile
The result was the following array:   Others eluted with 17.5% acetonitrile from the same digest
The peptide had the following amino acid sequence:   Automated edging of peptides eluted with 26% acetonitrile
The following amino acid sequence was given following Mann degradation:   Two more peptides eluted at 16% acetonitrile
Was obtained as a mixture and was re-purified before sequencing. Get
The peptide mixture was dried under vacuum and 100 μl of 50 mM
Re-suspend in Tris-HCl, pH 8.5, 8M urea. 20nmole
Dicitritol (DTT) is added at 37 ° C for 15 minutes.
Reduce existing SS bonds. Then 60nmole^ThreeH-Iodine
Carboxymethylate the peptide by adding acetic acid, room temperature
Incubate in the dark for 20 minutes. 60nmole DDT
And incubate for 30 minutes at room temperature.
Was adjusted to 0.1% TFA and H using an Altex C-3 reverse phase column.
Refractionate by PLC. Peptide is 0.1% TFA soluble from column
Solution and subsequent acetonitrile gradient (0-60% over 120 minutes)
(Acetonitrile) (flow rate 1 ml / min).   Automated Edma analysis of peptides eluted with 13% acetonitrile
The following sequence was shown after digestion: B.SMP-peptide   Digest native PAF protein with mouse mandibular gland protease
To obtain more peptides.   2 nmole in 350 μM 20 mM MES, pH 6.0, 0.5 M NaCl
25μM 1M TANCO in solution containing protein_Three, PH 9.0,
Adjust to pH 8.0 by addition. Mandibular gland protease (3.6μ
g) is added and digestion is carried out at 37 ° C. for 24 hours. 90nmole D
Add TT and incubate at 37 ° C for 30 minutes
You. 360nmole^ThreeAdd H-iodoacetic acid to add peptide
Perform ruboxyl methylation and incubate at room temperature in the dark for 20 minutes.
Make a basis. Add more 360 nmole of DTT
Adjust the reaction to 0.1% (v / v) in TFA and add
The peptide mixture is fractionated by RP-8 HPLC.   One peptide mixture eluting with 12% acetonitrile
And dried to 100 μl of 50 mM Tris-HCl, pH
HPL using Altex C-3 column after resuspension in 0.8M urea
Refractionate with C and elute as described for Lys-C peptide purification.
It eluted in the same manner as the kejur.   14.8% acetonitrile (a) and 15.3% acetonitrile
Automated Edman analysis of the two peptides eluted with Lil (b)
The solution yielded the following amino acid sequence:   Natural PAF digested with S. aureus protease (V8)
Several peptides were obtained. 500 μM 20 mM Tris-HCl,
1 mole of protein dissolved in 2M NaCl (pH 7.5) is reverse phase RP-8
Demineralize by HPLC using a column. Protein-containing desalted fraction
Dry, resuspend in 50μM 50mM acetic acid, pH 4.0, 1μ
Add g of V8 protease. Digest at 37 ° C for 18 hours
U. Peptides were applied to RP-8 reverse phase columns as described above.
Fractionate by HPLC.   Automated Edma analysis of peptides eluted with 17% acetonitrile
The following amino acid sequence was obtained upon degradation:  20% acetonitrile eluting peptides from V8 digests
Sequencing gave the following results:   Sequence V8 peptide eluted with 21% acetonitrile
And got the following results:   The above amino acid sequences are summarized as follows:
The core sequence of fibroblast growth factor is obtained:   Further isolation of other peptides for automated Edman degradation
I did. Their amino acid sequences are outside the above core sequence.
Was.   13% acetonitrile in fraction of mandibular gland digest (see above)
The peptide eluted with ril showed the following amino acid sequence:   Lys-C peptide eluted with 20% acetonitrile
The amino acid sequence of was: Example 5 Properties of angiogenesis-inducing factors suitable for clinical application as therapeutic agents
Identification   Placenta-derived factors isolated by the methods of Examples 1 and 2.
And factors isolated from liver cancer cells become angiogenic factors
Demonstrate all three expected properties in a test tube
I did it. First, at a concentration ranging from 0.1 to 10 ng / ml,
Is a synthesis of PA and latent collagenase in BCE cells
To promote. PA has broad specificity for the proenzyme plasminogen
Can be converted to plasmin, a new protease
You. Plasmin activates latent collagenase
Can be converted to ze. Therefore, the effect of low concentrations of this factor
Skin cells are small enough to degrade most proteins in surrounding tissue
Produces at least two proteases, resulting in cells
Break into the weave.   Purified molecules depend on PA and collagenase in BCE cells
To promote it. All collagenases are in active form. G
After the treatment with lipsin, cholase-degrading activity was detected. PA
And latent collagenase are both promoted in a correlated manner.
1/2 maximal enhancement at 1 ng / ml concentration of protease inducer
To be observed. PA and collagen in untreated cells
The amount of zeolites varies from experiment to experiment, and therefore the rate of promotion varies
I do. Very high concentration of protease inducer
Promotes PA synthesis as well as chemotaxis and mitogenesis
Also decrease. Induces BCE cells to PA and collagenase
Concentrations of Protease Inducer and 24 Hour Incubate
Then, the morphology of the cell is extended from a typical boulder type spindle
Change to type.   Second, this factor has chemotactic performance against BCE cells
Is shown. Therefore, in the test tube, the direction of this factor is
Go to Concentrate this factor in the 0.001 and 0.1 ng / ml range.
BCE cell chemotaxis when added in a blind well
Promoted in the tank. At higher concentrations, chemotaxis is not seen.
I can't. Increase in cell migration from upper tank to lower tank
Observed only when there is a higher concentration of
This indicates that true chemotaxis is taking place. Chemotaxis
May not account for more than 25% of the observed increase in motility
I got it.   Third, this factor is mitogenic for BCE cells
Is shown. Add protease-inducing factor to BCE cell culture
Depends on the amount added¹²⁵I-iododeoxyuri
Fig. 4 shows promotion of incorporation of gin into DNA. Protease induction
This inducing activity decreases with increasing factor concentration.¹²⁵I−
Enhancement of iododeoxyuridine uptake by PA and cola
Observed at the same concentration as where the genease induction is possible. Placenta
By the original coarse sonicated material¹²⁵I-iododeoxyuridine
Enhanced incorporation of DNA into DNA conflicts with other mitogenic activities
The present inventor has already confirmed that this is the case. Accordingly
This factor is truly mitogenic. Purified
A single molecule induces PA and collagenase in BCE cells,
Has the ability to promote cell replication and its motility
Conceivable. Example 6 Angiogenic activity   For measurement of angiogenesis, Auat. Rec. 199, 33-42.
(1981) using the method of Dunn et al.
In an experiment, the angiogenic factor of Example 2 was found on the avian ciliary allantois.
At a dose of 65 ng, 91% of the eggs promoted angiogenesis. Example 7 a) N-terminal amino acid sequence of PAF   Human placental blood as described above (see Examples 1 and 2)
Purify tube forming factors. 20 mM MES buffer, pH 6.0, and
Purified protein dissolved in NaCl and 500 mM NaCl
Desalting by high-performance liquid chromatography using a medium. twenty five
Automated Edman degradation of desalted proteins from 0 to 500pmole
Run on an ABI470A gas phase protein sequencer. Obtained
High-performance liquid chromatography using PTH amino acids
Identify with.   From these experimental results, the N-terminal amino acid sequence of PAF is as follows.
Determined as follows:   Further, the N-terminal amino acid sequence of PAF described above is Example 4, A
23% acetonitrile in the chromatographic system described in
By automated Edman degradation of Lys-C peptide eluted in
Even confirmed. b) C-terminal amino acid sequence of PAF   C-terminal from Lys-C digest of PAF as described in Example 4.
PAF peptide was isolated. This peptide is 22% acetonitrile
Elute with rill. Can this peptide be used for automated Edman degradation?
The following amino acid sequence is shown:   AILFLPMSPSAKS (I) Example; (ii) N-terminal amino acid sequence of PAF; (iii)
C-terminal amino acid sequence of PAF; and (iv) cDNA data
The total amino acid sequence of PAF is
RU: 1.PAF type 1 2.PAF type 2: N-terminal block PAF   Automated Edman degradation of purified and natural PAFs
In many experiments, some portion of the charged protein (50-80)
%) Did not decompose in the Edman treatment.   A portion of the PAF protein molecule exists with the N-terminus blocked.
Was considered to be present.   The nature of the blocking group is a purified amino-terminal peptide
Made it clearer. Enzymatic degradation of PAF (see Example 4)
Amino-terminal peptide obtained from
Identify. Also filter paper electrophoresis or thin layer chromatography
Chem. Ber. 87, 1103-1, which is adopted as a reference.
Written by F. Reindel and W. Hoppe on page 107 (1954)
Identified by staining with the chlorine / O-tolidine reagent
You. (The blocked peptide is a lysine residue on the side chain.
Other than weak coloration by nin
Not detected with hydrin. )   In addition, small N-terminal block PAF peptides are acidic
Narita with PAF digest from thermolysin or pepsin
(1975) by Protein Sequence Determination,
30-103, Springer-Verlag, Berlin, Heidelibe
Lug, New York, Dowex50, x as described in
20 (H⁺Type) or I. Klun
By Coll. Czech. Chem. Comm. 44, 145-14
Sulfoethyl-Seph as described on page 7 (1979).
It can be isolated by Adexus chromatography. This
These references are included as references.   The structure of the blocked short peptide is
Determined by method and method. For example, G. Allen's Seq
uencing of Proteins and Peptides; North Holland Pub
lishing Company, Amsterdam, New York, Ohio
Tuxford (1981) or listed there
The literature has adopted these references as references in this application.
You. These procedures and methods involve blocking the N-terminal block.
Peptide carboxypeptidase, proglutamic acid
Digestion with minopeptidase, hydrazinolysis, mass fraction
Analysis, nuclear porcelain resonance, gas chromatography and Boisse
Natl. Acad. Sci. USA 82, 8448-8452 (198
5 years). 3. PAF type 3: shortened and elongated PAF   Bovine kidney fibroblast growth factor (FGF) is
It is known that many amino acids are deficient (A. Ba
Ird et al., Regul. Pept., printing: 1985) (D. Gospodarowic)
z, Meth. Enzymol Printing: 1986). G. Neufeld and D.G
ospodarowicz, J. Biol. Chem. 260, 13860-13868 (1
985), shortened fibroblasts as indicated by
The growth factor retains the binding ability of the FGF receptor,
This indicates that the N-terminus of this protein is a cell surface receptor for FGF.
Does not play a significant role in interacting with
You. Therefore, both the shortened and elongated PAF
It is expected to retain Putter binding capacity. Biological activity of PAF
N-terminal maximal deletion and elongation with retention still determined
It has not been. Example 8: cDNA clone of PAF   SC-HEP-1 cells in 10% fetal bovine serum, non-essential amino
Cultivation in Eagle's medium, the minimum essential with acid and Pen-Strep
I do. RNA is Molecular C
loning: A Laboratory Manual. (Cold Spring Harbor La
boratory, New York, 1982, pp. 191-193).
isolated using the NP-40 lysis method described by S et al. P
oly (A)⁺Proc.Natl.Acod.S using mRNA as literature
ci.USA 69, 1408 (1972)
Oligo dT chromatography using the method of P. Leder
-(BRL). G adopted here as a reference
ene, 25, pp. 263-269 (1983).
As described by an, first strand synthesis with oligo dT and
Using second strand synthesis involving RNase H-DNA polymerase,
8 μg of double-stranded cDNA was obtained from 5 μg of mRNA. This operation
Amersham's reagent was used. The following reactions are specifically described
Unless otherwise specified, follow the manufacturer's instructions. 10 cDNA
Blunt ends using T4 DNA polymerase (Amersham)
It has become. The EcoR I site contains 400 units of EcoR I methylase (New
 England Biolabs) and 100 mM S-adenosylmethy
Protected with onin. Equal amount of EcoR I linker (New Englan
d Biolabs, 8 base pairs) with 1 unit of T4 DNA ligase (Prom
ega Biotec). Excess linker is 200 units of E
Digest with coR I (New England Biolabs) and remove
g of this cDNA EcoRI digested alkaline phosphatase
Processed lambda gt-10 DNA (Vector Cloning Systems) 1
Ligation with μg. The DNA is packaged in vitro (V
ector Cloning System), plate on E.coli C600HFLa
8.2 × 10^FiveTransformants were obtained. Oligonucleotide probe design   Mixing of two types for isolation of SK-HEP-1 cDNA clone
Sequence oligonucleotide probes were used. probe
Pools all DNA sequences corresponding to the determined amino acid sequence.
Made up of The probe is described herein
Sequences in the core amino acid sequence of PAF were selected. probe
# 5 is the amino acid sequence DNA encoding Ile-Lys-Gly-Val-Cys-Ala
17-mer consisting of 192 sequences created to bridge
Is:   X = A, T, G or C   N = A or G   Y = T or C   Z = T, C or A Probe # 8 is an amino acid sequence   DN encoding Tyr-Cys-Lys-Asn-Gly-Gly-Phe,
Created to hybridize with A, and from 256 types of sequences
Is a 20 mer.   Both probes are combined on an Applied Biosystems DNA synthesizer.
Done. All are purified by gel and T4 polynucleotides
[Gamma-³²P] ATP (Am
ersham) 4-6 × 10⁶Radiolabel to specific activity of cpm / pmol
Was.   Hybridization temperatures have been specifically adopted in the literature.
S.V.Suggs et al., (Developmental Biology Using Purfi
ed Genes: Edited by D.D.Brown and C.F.Fox, pp. 683-693: 19.
1982, Academic Press, New York)
Tm value calculated for most of the AT
2 ° C. lower than the temperature. Calculated for last wash
We performed at the Tm according to the value. (That is, hybridization
Temperature 2 ° C higher than the   cDNA library 150mm Luria Bertoni agar
E. coli to a concentration of 50,000 plaques per plate
  Break with C600HFLa strain and NZCYM upper agar (0.7%)
I did it. Phage DNA is two nitrocellulose files
Trishua to Tar (Schleicher and Schuell, BA85)
Science adopted in the literature, Volume 196, pp. 180-182
(1979) by Benton and Davis
And prepared for hybridization. Filter
ー is 6 × SSC (20 × SSC is 3M NaCl, 0.3M sodium citrate)
Um, pH 7.5), 2x Denhardt's solution (100x Denhardt's)
The solution was 2% Ficoll, 2% polyvinylpyrrolidone and
And 2% BSA), 0.1% SDS, 0.05% sodium pyrophosphate
In a solution containing yeast and 100 mg / ml yeast tRNA at 48 ° C for 2 hours.
Perform rehybridization. Probe # 8 to 0.
Add 2 pmol / ml and perform hybridization for 16 hours.
Now. After hybridization, filter the following
Washes as follows: 15 minutes at room temperature in 6 × SSC and 0.1% SDS
3 times each and the last one at 50 ° C for 8 minutes. Then filter
Dry the Kodak XAR5 film and "Lightning
24 hours at -70 ° C using a “plus” amplification screen
Perform tradographies. Positive with two filters
Plaques are suspended for purification. Those plaques
A secondary selection was performed using probes # 5 and # 8,
Induction of angiogenesis in plaque hybridized with probe
Factors were selected as candidates for coding.   Advanced Bacterial Genetic especially adopted as literature
s: A manual for Genetic Engineering (Cold Pring Hob
ar Laboratory, New York: 1980) in R.W.Davis,
In accordance with the method described by D. Bostein and J.R. Roth
Therefore, the plate lysis method and formua
DNA was prepared by mid extraction. EcoRI digestion of this DNA
Release 1.1 kb insert on 1% agarose gel
Was. This insert is described in Maniatis et al., Supra, pages 173-178.
5% acrylic for subcloning as described
Purified from luamide gel. Bacteria off insert
Ligation to EcoR I digest of ARG M13mp19RF DNA
Sa, described in J. Mol. Biol. 94, 441 (1975).
sequenced using the nger dideoxynucleotide method
Was. As a result of analyzing the obtained sequence, the temporary structure of PAF was
I got a good reading.   Protein sequence data (see Example 7) and reported
FGF information (Esch et al .; Proc. Natl. Acad. Sci. USA 82, 65
07-6511: 1985), some active forms of PAF have been purified.
It seems to have been. PAF Type 1 (see Example 7)
Translation starts from this DNA somewhere 5 'of AGTMAA
And translation of AG binding by a process that has not yet been elucidated
Generated by later cutting. PAF3 is a consensus
Start site (M. Kozak, Microbiol. Rev. 47, 1-45:
(1983) translation started with the MAA sequence
Purify by onion decomposition removal. PAF3 is another machine
It is also purified by translation initiation from a functional start site. So
Such parts are easily understood by those skilled in the art.
Thus, it is particularly clear from the description herein. In addition,
Post-translational processing of the translated primary product also occurs,
Not required. PAF2 is still a type 1 or 3
Free amino-terminal amino acids
It is caused by the occurrence of a group block. Example 9: PAF expression   The principle of PAF expression is as follows. Lambda gt10
The 1.1 kb EcoR I fragment isolated from the
Subclone to 9. This fragment contains the entire PAF coding sequence.
Including. Two small fragments of the subclone are used in the expression system
Useful for constructing One is from 367 bp Ava I to BamHI.
This is the GTMAA residue of type 1 protein from placenta
PAF of amino acid residues from 17 to 137, counting from 1 to 5
Contains an array that encodes The other is from 405bp Nco I
The fragment up to BamHI, which is the PAF from 4 to 137 amino acids.
It contains a sequence that encodes a noic acid residue. Of these restriction fragments
Complete the coding sequence with synthetic adapters at both ends,
Prepare translation start, end or coupling sequence
Sequences necessary for ligation with a suitable expression vector are also added.   PAF isolated from human placenta has a sequence that begins with GTAMM.
I do. G from cDNA clone isolated from SK-Hep-1 cells
At least 100 amino acids upstream of the TMAA sequence or M
Suggests that other forms of PAF that start with AA exist
You. Yeast (S. cerivisiae) and bacteria using placenta
(E. coli). Other SK-Hep-1 types and amino acids
The truncated form of the non-terminal is expressed by modification of the method described below.
And the method is obvious to those skilled in the art.
You. The method is based on the amino terminus of the cDNA fragment (Nco I site or
Is the Ava I site) for ligation to the expression vector.
Changing the adaptor. Another way
As the nucleotides-supported from the GTMAA type shown below
Defect displacement processing ("Nucleic Research
 Future Development ", K. Mizobuchi, I. Watanabe and
And J.D.Watson, Academic Press, New York, 41
9-436: M. Inouye, K. Nakamura, S. Inouye
And described by Y. Masui) plus truncated expression
You can build a mid.   Use the following adapters with an Applied Biosystem DNA synthesizer
Synthesize and purify the gel. 5 'end is T4 polynucleotide
Phosphorylates with dokinase (Pharmacia). Pair complement
Anneal the oligonucleotide as follows.
Purify the heavy chain adapter. Equivalent amount of nuclear oligonucleotide
To 50 mM NaCl, 10 mM Tris, pH 7.5 and 1 mM EDTA
Added. The solution is heated in a boiling water bath. continue
Remove the water bath from the heat and cool to room temperature in 2 hours. Use
A list of adapters used and their descriptions. Example 9: Construction of yeast expression plasmid   pUC8 is digested with HindIII to obtain a polylinker derived from pUC8.
Lack of restriction sites from Hind III to Sma I
Plasmid pGS185 and construct it with a Hind III site.
III / Sma I adapter (Amersham,
Tag No. DA106) and Sma I digested and diluted solution (1 ng / m
l) Ligation inside and concatenation to form E.coli JM83
Change quality. Plasmid DNA isolated from the transformant
digest with coR I, Sma I or Hind III to get the correct plasmid
Identify the code. Thus, the EcoR I site and Sma I
Plasmid that has a site but lacks a Hind III site.
The transformants possessed were identified.   The EcoR I fragment containing the yeast MF1 gene was cloned into J. Kurgan and I.H.
Described by erskowitz in Cell, 30: 933 (1982)
By gel electrophoresis from plasmid pCY17
Purify and ligate to pGS185 cut with EcoRI. This line
Transform E.coli HB101 using the reaction mixture
Then, a transformant resistant to ampicillin is selected. Transformation
Isolate plasmid DNA from the replacement and digest the DNA with EcoRI.
To verify the presence of the correct insert. This is the plasmid pGS2
85.   Plasmid pGS285 is completely digested with Hind III and diluted
(1 nm / ml) and linked to Kurjan and Hersknwitz described above.
Of the four Hind III sites in the MF1 gene
Remove the pieces. Select the correct configuration as described above
I do. This is plasmid pGS385.   Due to site-specific mutations, pGS385 to MF
Exclude one gene, purify by gel (1.5 kb), digest with EcoRI
Ligated with M13np18RF. Using ligation reaction solution
To transform E. coli 71-18,
The clone inserted directionally³²P] Labeled MF1 gene
And hybridized. Zoller for MF1 array
And Smith (Methods in Enzymology, 100, 1983)
Year, Academic Press Inc., page 468)
GTA TCT TTG GAT using standard methods for in vitro mutation
Change from AAA AGA to GAT AGC TTG GAT AAA AGA.
-Factor gene mutation, MF-H sequenced by dideoxy method
Confirm by setting.   The RF type of the M13pm18 clone was digested with EcoRI and MF-H
The gene was removed and the 1.5 kb EcoRI fragment was
Purified by electrophoresis and ligated with EcoRI cut pGS185.
You. E. coli HB using the obtained ligation reaction solution
101, and the plasmid containing the MF gene is stored.
Include colonies with MF-H genetics³²1.5kb of P label
Hybridize with the EorRI fragment and identify. like that
The plasmid is named pGS286.   The PAF gene is inserted into pGS286 as follows. Adap
Litters # 1 and # 2 with the PAF Nco I / BamHI fragment
You. The reaction mixture is electrophoresed on a polyacrylamide gel,
The PAF DNA with the added peptide is identified by increasing MW.
The correctly ligated DNA fragment was eluted from the gel, Hind II
Ligation with pGS286 digested with I / Sal I. E.coli HB101
Transformed with ligation reaction solution, ampicillin resistant
Select colonies. Plasmid with correct insertion
Is transformed into [γ-³²P] ATP and T4 polynu
Radiolabeled by incubating with nucleotide kinase
Hybridize with adapters 1A and 2A and identify.
The plasmid thus constructed and isolated was transformed into pGS286-
Named PAF. This plasmid has the MFH gene
PAF at the Hind III site in the "prepro" region of the FH gene
It is fused with a gene. Such constructs are introduced into yeast
A.J.Brake et al., 1981 (PNAS: USA 81, 4642)
Page), synthesis of heterologous proteins, processing
It is known to direct signaling and secretion.   The EcoR I fragment containing the fusion of the MF H gene and PAF
GS286-PAF. This fragment was digested with EcoR I and digested.
Isolate by polyacrylamide gel electrophoresis. T4
T4 DNA ligator after blunt ends with DNA polymerase
Connect Pst I adapter (Pharmacia)
You. This fragment was then transformed into the vector pC1 / 1 (A.J.Brake et al., 1981).
Year, PNAS USA 81: 4042).
E.coli HB101 is transformed with this reaction solution and TET^rRoller
Choose a knee. Hybridization with PAF gene
To identify the correct construct. S.ce
rivisiae DBY 746 (Yeast Gentic Stock Center, Cali
Toh-E and Wickner (J.
Bacteriology, 145, 1981, pp. 1421-1424)
2 micron DNA plasmid deleted as described
And introduced according to standard transformation protocols.
Transformants expressing PAF were purified with purified anti-PAF IgG.
Select according to the reactivity of the nature. Example 10: E. coli periplasmic secretion   PAF expression in a form suitable for transport to E. coli periplasm
To control, the following control factors were used: transcription
T on plasmid pCJ-1 for high level initiation
ac promoter; plasmid pCJ-1 for transcriptional regulation
Lac operator on the chromosome of E. coli JM107
Lac repressor (lac I) present. PAF periplasm
Omp A leader peptide to PAF for transport to
The encoding DNA contains the C-terminal Ala of the former peptide and PAF1 type
Ala-Gly bond in the primary product
Is cleaved by E. coli leader peptidase and matures
Link to produce PAF.   The E. coli secretion vector is constructed as follows. Adap
Litters # 5 and # 4 to the Ava I / BamHI fragment of PAF
You. Elute the correct size DNA from polyacrylamide
And Omp A leader DNA and EcoR I / Pst I digested M1
Connect with 3mp19RF. Ligation of E.coli JM-107
Transform with the reaction solution. Transformants containing PAF gene
Detection by restriction enzyme mapping and sequencing by the dideoxy method.
Confirm the sequence of the construct by determination. Contains PAF gene
Digestion of the EcoR I / Pst I fragment with EcoR I and Pst I
And RF DNA by elution from polyacrylamide gel
From This was linked to EcoR I / PstI digested pCJ-1.
Then, E. coli JM107 is transformed with the reaction solution. Raw PAF
Growing colonies on Tet plates and affinity
Select by immunoscreening with purified anti-PAF IgG
You. Example 11: Intracytoplasmic expression of E. coli   Controls PAF expression to remain in the E. coli cytoplasm
The following operating factors were used for this: Plasmid pCJ-
1. lac promoter on plasmid pCJ-1
Lac rep on the chromosome of Pererator and E. coli JM107 strains
Letsser (lac I^q); Consensus Shine-Dalgar
Sequencer; translation coupler for initiating high-level translation
Opm A leader peptide fragment used as a primer. Translation
The pulling sequence encodes the translation initiation region of the Opm A gene
DNA, the first 8 amino acids of the Omp A leader peptide
Acid, the consensus Shine-Dalgarno sequence described above and
And a translation terminator. Translation coupling sequence
Is between the lac operator and the translation initiation site of the PAF gene.
Insert it over the latter. (The characteristics of the translation coupler are E.co
Incorporation into the DNA sequence shown with the adapter for secretory expression of li
It is rare. )   The PAF gene is translated into the pCJ-1 plasmid as follows:
Captured with puller. Adapter # 3 and Omp
  A translation coupler from the M13mp19 construct described in Example 10
Bind to the PAF Ava I / Pst I fragment. This fragment is
Purify from acrylamide gel. Then this fragment is M13 m
Ligation to EcoR I / Pst I digest of p19 RF and ligation reaction
Are used to transform E. coli JM107 cells. PAF gene fusion
Plaques containing the compound are selected by restriction enzyme mapping.
You. The EcoR I / Pst I fragment containing the PAF gene fusion was
Eluted from acrylamide gel, EcoR I / Pst I of pCJ-1
Ligation with digests and transformation of E. coli JM107 cells with reaction
Replace. Select colonies showing tetracycline resistance
And PAF production was immunized with affinity-purified anti-PAF IgG.
Perform epidemiological screening.   Many modifications and variations of the methods and products of the present invention are made.
Variations will be apparent to those skilled in the art. Follow
Accordingly, the present invention is directed to the appended claims and their equivalents.
Such modifications and variations of the present invention are provided so far as
And mutations. Example 12: Intracytoplasmic expression in E. coli   The construct for secretion of M13 mp19 described in Example 11 was
Digest with Nco I and elute large fragments from gel. next
Adapter # 6 is ligated to the Nru I / Nco I cut DNA. E.co
The li JM107 strain is transformed with the reaction solution. PAF gene fusion
Plaques Containing Drosophila Confirmed by Dideoxy Method Sequencing
I do. The EcrR I / Pst I fragment containing the PAF gene fusion was
Elution from acrylamide gel, digestion with EcoR I / Pst I
And ligated with pCJ-1 and transformed E. coli JM107 cells with the reaction solution.
Convert. Select colonies showing tetracycline resistance
And PAF production with affinity-purified anti-PAF IgG.
Perform immunoscreening. Adapter # 6 Example 13: mRNA identification   Isolate total RNA from the following cell lines: SK-HEP-1 human
Hepatoma cells, human embryonic lung (HEL) bacteria, RPMI 7272 human black
Tumor cells and sarcomas 180 cells. Next, Poly A Plus Minor
Smut messenger RNA (mRNA) to oligo (DT) cellulose
Isolate using chromatography. Each of the above cells
15 μg of Poly A plus or minus RNA
By electrophoresis of toagarose / formaldehyde gel
To separate. Transfer these RNAs to zeta probe membrane
You. To human basic FGF from human hepatoma cell SK-HEP-1
Nitto cDNA probe (FGF15, EcoRI insert)
Specific activity 8.64 × 10 by the translation method⁸cpm / μg
So that³²Label with P-DCTP.³²P-BFGF cDNA Pro
50% hybridization to RNA
Formamide, 6 × SSC, 2 × Denhardt's solution, 1 par
St. SDS, 0.05% sodium pyrophosphate
And in 175 μg / ml tRNA at 42 ° C. for 16 hours. Zetap
Lobe membrane 0.5 × SSC / 1% SDS, 0.2 × SS
One each for C / 1% SDS and 0.1 × SSC / 1% SDS
Wash with a tube at 65 ° C. for 15 minutes, and further add 0.1 × SSC / 1%
Wash with SDS at 70 ° C for 15 minutes. Next, dry the membrane and
Tradiography at -80 ° C for 1 and 3 days
U. SK HEP-1 cells, HEL cells and RPMI 7272 cells
Above 8.0KB, 4.3KB, 2.3KB and 1.0KB respectively
Contains 4 types of RNA that hybridizes with the above cDNA probe
did. cDNA probes are compatible with any RNA from mouse sarcoma 180 cells
I did not hybridize.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 ADA A61K 37/02 ADA (31) Priority claim number 895,829 (32) Priority date August 12, 1986 ( 33) Priority country United States (US) (72) Inventor Rifkin, Daniel Bee United States of America 10021 New York, New York, E. Sixty Sixth Street 444 (72) Inventor Sommer Andries United States 80301 Boulder, Colorado 4145 (56) References Bioscience Reports, vol. 5, p. 738-790 (1985) Biochemistry, vol. 20, p. 5480-5499 (1985)

Claims (1)

(57) [Claims] (1) An angiogenesis-inducing factor (bFGF) consisting of a purified human or synthetic single-chain polypeptide protein exhibiting substantial homology to a natural angiogenesis-inducing factor that can be isolated from human placental tissue (postpartum). The angiogenesis-inducing factor has at least one active site exhibiting an activity selected from the group consisting of mitogenic activity, chemotactic activity, protease synthesis promoting ability and a combination thereof; Has a molecular weight of about 18,700 daltons as determined by and has the following amino acid sequence: (2) or an angiogenesis-inducing factor having an additional amino acid at the N-terminus of the angiogenesis-inducing factor of (1). 2. Amino acid sequence The angiogenesis-inducing factor according to claim 1, comprising: 3. The angiogenesis-inducing factor according to claim 2, which has an additional amino acid at the N-terminal of the amino acid sequence according to claim 2. 4. The angiogenesis-inducing factor according to claim 1, wherein the amino acid sequence of the protein differs from the sequence according to claim 2 by one amino acid residue. 5. The angiogenesis-inducing factor according to claim 1, wherein the amino acid sequence of the protein differs from the sequence according to claim 2 by two amino acid residues. 6. The angiogenesis-inducing factor according to claim 2, wherein the N-terminus of the protein is blocked. 7. The angiogenesis-inducing factor according to claim 1, which is isolated in a substantially purified state from human placenta (after delivery). 8. (1) An angiogenesis-inducing factor (bFGF) consisting of a purified human or synthetic single-chain polypeptide protein exhibiting substantial homology to a natural angiogenesis-inducing factor that can be isolated from human placental tissue (postpartum). The angiogenesis-inducing factor has at least one active site exhibiting an activity selected from the group consisting of mitogenic activity, chemotactic activity, protease synthesis promoting ability and a combination thereof; Has a molecular weight of about 18,700 daltons as determined by and has the following amino acid sequence: (2) or a method for obtaining an angiogenesis-inducing factor having an additional amino acid at the N-terminus of the angiogenesis-inducing factor according to the above (1), wherein (a) an angiogenesis-inducing factor (B) isolating an angiogenic factor from the tissue by fractionating the proteinaceous substance in the tissue, and (c) identifying a fraction having angiogenic activity ;
And (d) concentrating the fraction exhibiting angiogenesis-inducing factor activity. 9. 9. The method according to claim 8, wherein the tissue capable of producing an angiogenesis-inducing factor is human placental tissue. 10. (1) An angiogenesis-inducing factor (bFGF) consisting of a purified human or synthetic single-chain polypeptide protein exhibiting substantial homology to a natural angiogenesis-inducing factor that can be isolated from human placental tissue (postpartum). The angiogenesis-inducing factor has at least one active site exhibiting an activity selected from the group consisting of mitogenic activity, chemotactic activity, protease synthesis promoting ability and a combination thereof; Has a molecular weight of about 18,700 daltons as determined by and has the following amino acid sequence: (2) or a method for producing an angiogenesis-inducing factor having an additional amino acid at the N-terminus of the angiogenesis-inducing factor according to the above (1), comprising: Expressing the angiogenic factor, and (b) isolating and purifying the expressed angiogenic factor, in any order. 11. Prior to performing steps (a) and (b), (1) isolating a DNA sequence encoding an angiogenesis-inducing factor; (2) inserting the DNA sequence into a vector that can be expressed in a host microorganism; 3) The method according to claim 10, comprising transforming a host microorganism capable of expressing an angiogenesis-inducing factor with the vector containing the DNA sequence. 12. The method according to any one of claims 10 or 11, wherein the host microorganism is transformed with pGS286-PAF. 13. 12. The method according to any one of claims 10 or 11, wherein the host microorganism is E. coli. 14． The claim 10 or the claim wherein the host microorganism is yeast.
11. Any one of the methods of clause 11.