JPH10191989A - Production of betacellulin compounds - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject substance useful for treating wounds, ulcers, diabetes (complications), etc., by refolding the inactive betacellulin expressed in a procaryotic cell host by a genetic engineering method in a redox buffer. SOLUTION: This method for producing an active betacellulin comprises expressing the inactive betacellulin (BTC) in a procaryotic cell host by a genetic engineering method, solubilizing the cell with a degenerating agent (e.g. guanidine), and subsequently diluting the degenerating agent with a redox buffer solution containing a mercapto group-free amino acid (e.g. arginine) or a reduction type glutathione and an oxidation type glutathione, etc., and having a pH of 7-9 up to a concentration for inactivating the degenerating agent to refold the betacellulin. Thus, the active betacellulin useful for treating wounds, ulcers, diabetes, diabetic complications (atherosclerosis, diabetic retinopathy), etc., can efficiently by obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to betacellulin (BT) expressed in prokaryotic cells using genetic engineering technology.
C) Refolding of pharmacologically active BTC
And a method for producing the same.

[0002]

2. Description of the Related Art BTC stands for Hanahan (Science,
259, pp. 1604-1607, 1993), which is a protein belonging to the epidermal growth factor (EGF) family consisting of 80 amino acids and having a molecular weight of about 9000, which was found in β-cell tumors. It has a growth promoting effect on epithelial cells, 3T3 cells and the like. It is also known that BTC is produced using prokaryotic or eukaryotic cells by genetic engineering techniques (JP-A-6-87894).

[0003]

When BTCs are expressed in prokaryotic cells, the yield of pharmacologically active BTCs is low.
When eukaryotic cells such as Chinese hamster cells (eg, CHO) and monkey cells (eg, COS-7) are expressed as a host, pharmacologically active BTC can be obtained (Japanese Patent Laid-Open No. 6-8 / 1994).
7894) However, the medium for eukaryotic cells is expensive and the expression level of the protein is low, so that production on an industrial scale cannot be said to be appropriate. Therefore, there is a need for an industrially advantageous method for producing pharmacologically active BTCs.

[0004]

Means for Solving the Problems In order to solve these drawbacks, the present inventors have made intensive studies to utilize the high productivity of prokaryotic cells and to provide an efficient activation method (regeneration method). As a result, the first attempt to refold in redox buffer in a method of activating and then expressing BTCs in prokaryotic cells using genetic engineering technology has led to unexpected industrial activation. The inventors have found that the present invention can be performed efficiently, and have completed the present invention. That is, the present invention provides an active BTC characterized in that BTCs expressed in a prokaryotic host by genetic engineering are refolded in a redox buffer.
And a method for producing the same.

[0005] The BTCs used in the present invention include known BTCs.
It has the same action as TC, and includes BTC derived from mammals, a protein having Met added to its N-terminus (hereinafter sometimes referred to as Met-BTC), and among others, FIG. Human BTC having the amino acid sequence of positions 2 to 81 of SEQ ID NO: 1) and Met
1 (SEQ ID NO: 1) to which a protein having the amino acid sequence of positions 1 to 81 is added. Furthermore, BT
A fragment thereof may be used as long as it has the same action as that of C. For example, a fragment having the amino acid sequence from the third (Gly) to the 81st (Tyr) in SEQ ID NO: 1 or a smaller fragment from the 30th (Ser) to 81
And those having the amino acid sequence of the (Tyr). Further, BTCs may be used as salts, and salts with inorganic acids such as hydrochloric acid, hydrogen bromide, nitric acid, sulfuric acid, phosphoric acid,
Salts with organic acids such as acetic acid, phthalic acid, fumaric acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, alkali metal salts such as sodium salt and potassium salt, calcium salts, etc. And pharmaceutically acceptable salts and hydrates of alkaline earth metal salts and ammonium salts.

The prokaryotic cells used in the present invention include Es
Escherichia bacteria such as cherichia coli (Escherichia coli), Baci
Bacillus species such as llus subtilis (Bacillus subtilis), Serratia
Examples include bacteria of the genus Serratia such as marcescens (Seratia), among which Escherichia coli and the like are preferable. Transformation, culture and the like of these prokaryotic cells can be carried out according to a conventional method, in addition to the methods described below (see JP-A-3-204897). For example, an expression type vector containing a cDNA having a nucleotide sequence encoding BTCs in the method of the present invention includes, for example, (i) separating messenger RNA (mRNA) from BTC-producing cells, and (ii) single-stranded from the mRNA. CDNA and then double-stranded DNA, (ii)
i) incorporating the complementary DNA into a phage or plasmid, (iv) transforming a host with the obtained recombinant phage or plasmid, and (v) culturing the resulting transformant. For example, a phage or plasmid containing a target DNA is isolated by hybridization with a DNA probe encoding a part of BTC or by an immunoassay using an anti-BTC antibody, and (vi) the target DNA is isolated from the recombinant DNA. The cloned DNA can be cut out, and (vii) the cloned DNA or a part thereof can be ligated downstream of a promoter in an expression vector.

[0007] mRNA encoding BTCs is, for example, a pancreatic tumor cell (eg, ATCC accession number CRL105).
85, CRL10875).
As a method for preparing mRNA from pancreatic tumor cells, the guanidine thiocyanate method [(JMChirgwin et al., Biochemi.
stry), Vol. 18, p. 5294 (1979)]. Using the mRNA thus obtained as a template and reverse transcriptase, for example, the method of H. Okayama et al. (Molecular and Cellular Biology, Vol. 2, 161) P. (1982) and the same volume 3, 280 p.
Year)], and the obtained cDNA is incorporated into a plasmid. Examples of a plasmid incorporating the cDNA include, for example, pBR322 derived from Escherichia coli (Gene, Vol. 2, p. 95 (1977)), pBR32
5 [Gene, vol. 4, p. 121 (1978)], pUC
12 [Gene, 19, 259 (1982)], p.
UC13 [Gene, 19, 259 (1982)
Year)], pUB110 derived from Bacillus subtilis [Biochemical
Biochemical and Biophysical Research Communicati
ons), Vol. 112, p. 678 (1983)], etc., but any other ones can be used as long as they are replicated and propagated in the host.
As a phage vector into which cDNA is incorporated, for example, λgt11 [Young and R.
and Davis, R.), Proceedings of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci., US
A.), Vol. 80, p. 1194 (1983)], etc., but any other one can be used as long as it can be propagated in the host. Examples of a method for incorporating the plasmid into a plasmid include, for example, Tea Maniatis (TM
aniatis) et al., Molecular Cloning (Molecular)
Cloning) Cold Spring Harbor Laboratory, page 239 (1982), and the like. As a method of incorporating cDNA into a phage vector, for example, the method of Hyunh (TV) [D.N.
A cloning, A Practical Approach, Vol. 1, p. 49 (1985)].

[0008] The plasmid thus obtained can be used in a suitable host such as a bacterium belonging to the genus Escherichia,
It is introduced into Bacillus sp. Examples of the above Escherichia bacteria include Escherichia coli (Escheria coli).
ichia coli) K12DH1 [Proceding of the National Academy of Science (Proc.
Natl. Acad. Sci. USA) 60, 160 (196
8 years)], JM103 [Nucleic Acids Research, Vol. 9, p. 309 (19)
1981)], JA221 [Journal of Molecular Biolog
y)], 120, 517 (1978)], HB1
01 [Journal of Molecular Biology, Vol. 41, p. 459 (1969)], C600 [Genetics, Vol. 39, p. 440 (19)
1954)], MM294 [Nature, 21
7, 1110 (1968)].
As the Bacillus genus, for example, Bacillus subtilis MI114 [Gene, 24
Volume, 255 pages (1983)], 207-21 [Journal of Biochem.
istry), Vol. 95, p. 87 (1984)]. Methods for transforming a host with a plasmid include:
See, for example, T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory.
ing Harbor Laboratory), page 249 (1982), the calcium chloride method or the calcium chloride / rubidium chloride method. When a phage vector is used, it can be introduced into, for example, propagated Escherichia coli using an in vitro packaging method. A BTC cDNA library containing a cDNA encoding BTCs can be obtained by the above-described method or the like, but can also be purchased as a commercial product.

[0009] The cDNA encoding the BTCs cloned in this manner may be used, if necessary, with plasmids such as pBR322, pUC12, pUC13 and pUC1.
8. BTC cDNA can be obtained by subcloning into pUC19, pUC118, pUC119 and the like. The nucleotide sequence of the cDNA obtained in this manner is used, for example, by the Maxam-Gilbert method [Max
am, AM and Gilbert, W., Proceedings of the National Academy of Sciences
Acad. Sc of Proc. Natl. Acad.
i., USA), vol. 74, p. 560 (1977)] or the dideoxy method [Messing, J. et al., Nucleic Acids Research, Vol.
09 (1981)], and the presence of BTC cDNA can be confirmed by comparison with a known amino acid sequence. As described above, cDNA encoding BTCs
Is obtained. BTC cloned as described above
The cDNAs encoding these can be used as they are, or digested with restriction enzymes or exonucleases as desired.

Next, a region to be expressed is excised from the cloned cDNA, and ligated downstream of a promoter in a vehicle (vector) suitable for expression to obtain an expression type vector. The cDNA may have ATG at the 5 'end as a translation initiation codon, and may have TAA, TGA or TAG at the 3' end as a translation stop codon. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. To express the DNA, a promoter is connected upstream thereof. As the vector, the above-mentioned plasmid derived from Escherichia coli (eg, pBR322, pB
R325, pUC12, pUC13) and Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194).

[0011] The promoter used in the present invention may be any promoter suitable for the host used for gene expression. When the host for transformation is Escherichia, T7 promoter, trp promoter, lac promoter, re
When the host is a bacterium belonging to the genus Bacillus, the cA promoter, the λPL promoter, the lpp promoter, etc.
O1 promoter, SPO2 promoter, penP promoter and the like are preferable. In particular, it is preferable that the host is a genus Escherichia and the promoter is a T7 promoter, a trp promoter or a λPL promoter. The use of enhancers for expression is also effective.
A prokaryotic cell transformant is produced using the vector containing the cDNA encoding the mature peptide of BTCs constructed as described above.

In order to transform the genus Escherichia, for example, Processing of the National
Academy of Science (Proc.Natl.Acad.Sci.U
SA), 69, 2110 (1972) and Gene (Ge)
ne), vol. 17, p. 107 (1982). For transformation of Bacillus sp., For example, Molecular & General Genetics, 16
8, p. 111 (1979). In this manner, c encoding BTCs
A prokaryotic cell transformant transformed with the expression vector containing DNA is obtained. When Escherichia is used as a host and T7 promoter is used as a promoter, in order to improve the expression efficiency of the T7 promoter,
A T7 lysozyme expression plasmid may be co-present in addition to the expression vector containing the cDNA encoding BTCs.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for culturing, and a liquid medium necessary for growth of the transformant is contained therein. A carbon source, a nitrogen source, an inorganic substance and the like are contained. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8. As a medium for culturing Escherichia, for example, glucose,
M9 medium containing casamino acids [Miller, Journal of Experiments in Molecular Genetics
Genetics), pages 431-433, Cold Spring Harbor L
aboratory, New York 1972], LB medium and the like.
If necessary, an agent such as isopropyl-β-D-thiogalactopyranoside (IPTG) or 3β-indolylacrylic acid can be added to the promoter to make it work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
When the host is a bacterium belonging to the genus Bacillus, culturing is usually performed at about 30 to 40 ° C.
For about 6 to 24 hours, and if necessary, ventilation or stirring can be applied.

When BTCs form inclusion bodies in host prokaryotic cells, after culturing, cells are collected by a method such as centrifugation, the cells are disrupted, and the inclusion bodies are solubilized using a denaturing agent. Thereby, BTCs can be extracted. The disruption of the cells can be carried out in a conventional manner, for example, by sonication. PH value around neutral as a suspending medium (pH 6.5-
It is preferable to use a suitable buffer (for example, phosphate buffer or the like) adjusted to 7.5). At this time, EDTA may be added to promote cell disruption. After crushing the cells in this way, the insoluble components (inclusion bodies) are separated by centrifugation or filtration by any method.
In order to remove prokaryotic cell-derived proteins as much as possible, washing with, for example, water or a phosphate buffer is preferable, but washing with about 4 M urea may be used in some cases. As a denaturing agent for solubilizing the obtained precipitate (pellet) using a denaturing agent, a known denaturing agent, in particular, guanidine or urea can be used. The denaturing agent is usually used as an aqueous solution, and the concentration of the denaturing agent in the aqueous solution is 4 to 8 mol / liter, preferably about 6 to 7 mol / liter for guanidine.
For liter and urea, it is 5 to 9 mol / l, preferably about 8 mol / l. Guanidine is usually used as an acid addition salt of guanidine such as guanidine hydrochloride.

When the BTCs do not form inclusion bodies in the host prokaryotic cells, after culturing, the cells are collected by a method such as centrifugation, and then the cells are solubilized using a denaturing agent, or the cells are disrupted. BTCs can be extracted by solubilizing with a post-denaturing agent. Examples of denaturing agents used for solubilizing the collected cells include guanidine. The denaturing agent is usually used as an aqueous solution, and the concentration of the denaturing agent in the aqueous solution is usually 4 to 8 mol / l, preferably about 6 to 7 mol / l for guanidine. Guanidine is usually used as an acid addition salt of guanidine such as guanidine hydrochloride. The disruption of the cells can be carried out in a conventional manner, for example, by sonication or French press. As a denaturant used for solubilization of the disrupted cells, a known denaturant, particularly guanidine or urea, can be used. The denaturant is usually used as an aqueous solution, and the concentration of the denaturant in the aqueous solution is 4 to 8 mol / l, preferably about 6 to 7 mol / l for guanidine, and 5 to 5 mol / l for urea.
９9 mol / l, preferably about 8 mol / l. Guanidine is usually used as an acid addition salt of guanidine such as guanidine hydrochloride.

Either solubilize the inclusion bodies as described above, directly solubilize the cells with a denaturant, or solubilize the cells with a denaturant and then remove impurities by centrifugation or the like. After subjecting the obtained supernatant to a purification step or a step of removing the N-terminal Met as necessary, BTCs can be refolded (activated, regenerated).
The refolding is performed by adding a redox buffer to the purified BTCs or diluting the supernatant containing the BTCs with the redox buffer. "At this time, for example, it is preferable to include an amino acid having no mercapto group in the redox buffer." When the supernatant containing BTCs is diluted with the redox buffer, the concentration of the denaturing agent may be increased. It is desirable to dilute to an inactive concentration at a suitable neutral pH, for example when the denaturant is guandinine, the concentration of guanidine in the diluent is 0-2.0 mol / l, preferably about 1 mol / l Until the following, when the denaturing agent is urea, the concentration of urea in the diluent is 0 to 4.0.
It is desirable to dilute to less than about 2 moles / liter, preferably about 2 moles / liter.

The amino acid having no mercapto group to be added to the redox buffer upon refolding may be any amino acid having no mercapto group, as long as the object of the present invention is achieved. , Aspartic acid, valine, lysine, alanine, citrulline and the like are preferable, and arginine and the like are BT
It is particularly preferable in view of the yield in refolding of Cs, and the addition concentration of the amino acid in the redox buffer is 0.1 to 1.0 mol / L, preferably 0.1 to 1.0 mol / L.
0.5 mol / liter. As the redox buffer, the oxidized form and the reduced form are generally preferably organic substances, such as a buffer containing oxidized glutathione (GSSG) and reduced glutathione (GSH), cysteine and cystine, or cysteamine and cystamine (eg, phosphate). Buffer, acetate buffer, citrate buffer) and the like, among which GSSG
And phosphate buffer containing GSH are particularly preferred. The concentration of the oxidizing and reducing agents in the redox buffer is generally between 0.01 and 100 mmol / l and between 0.01 and 100 mmol / l, respectively.
More specifically, when using GSSG and GSH,
The concentration of GSSG in the redox buffer is 0.1 to 1
0 mmol / l, preferably 0.1 to 1.0 mmol / l, and the concentration of GSH is 0.1 to 10 mmol / l, preferably 0.2 to 2.0 mmol / l. The temperature for the refolding is 0
-30 ° C, preferably 4-15 ° C, pH is 7-9, preferably pH 7.5-8.5. The time required for refolding is usually 0.5 to 7 days, preferably 1 to 3 days.
Days.

Note that, after solubilization and before refolding, known and commonly used purification steps such as extraction, salting out, dialysis, distribution, crystallization, recrystallization, gel filtration, and chromatography can be introduced. Preferably, it can be purified by, for example, applying to Sephadex G-25 (Pharmacia Biotech Co., Ltd.) in a 0.1 mol / liter phosphate buffer. Separation of the denaturing agent is optionally 0.1 mol / l
It is also possible by dialysis against a phosphate buffer. The purification step can also be performed following refolding. Generally, such purification includes, for example, extraction, salting-out, dialysis, distribution, crystallization, recrystallization, gel filtration, chromatography, and the like. Preferred examples include dialysis and, for example, SP-Sepharose (Pharmacia Biotech Co., Ltd.) , SP-5PW (Tosoh Corporation) or
Examples include a purification method by ion exchange chromatography via DEAE-5PW (Tosoh Corporation), for example, reverse phase chromatography using ODP-50 (Showa Denko Corporation).

BTCs obtained by expression in prokaryotic cells by genetic engineering may be BTCs with no Met added at the N-terminus, but are usually Met-BTCs with Met added at the N-terminus. is there. The obtained BTCs are Me
In the case of t-BTC, before or after refolding, the N-terminal Met residue may be removed to obtain BTC without Met added to the N-terminal.
The removal of the N-terminal Met residue can be performed, for example, by reacting Met-BTC with α-diketones and then hydrolyzing. α-diketones are Met-BT
Any compound can be used as long as it can promote the transamination reaction of C, for example, a compound represented by the formula R ¹ —CO—CO—R ² [wherein R
¹ represents a lower alkyl or a phenyl group (preferably hydrogen or methyl, more preferably hydrogen) which may be substituted with hydrogen or a carboxyl group, and R ² may be substituted with a hydroxyl group, a lower alkoxy group or a lower alkyl. Shows a good amino group (preferably a hydroxyl group). Or a salt thereof. In the above formula, the lower alkyl group represented by R ¹ includes methyl,
1 to 6 carbon atoms such as ethyl, propyl, i-propyl, butyl, i-butyl, sec-butyl, t-butyl, etc.
And a lower alkoxy group represented by R ² includes methoxy, ethoxy, propoxy, i-propoxy, butoxy, i-butoxy, sec.
And alkoxy groups having about 1 to 6 carbon atoms, such as -butoxy and t-butoxy. Examples of the amino group optionally substituted with lower alkyl represented by R ² include:
Examples thereof include an amino group which may have one or two lower alkyl groups represented by R ¹ described above. Further, examples of the salt include salts with inorganic acids, salts with organic acids, alkali metal salts, and alkaline earth metal salts ammonium salts similar to the above-mentioned BTC salts. Specific examples of the α-diketones include glyoxylic acid, pyruvic acid, oxalacetic acid, phenylglyoxylic acid, 2-oxoglutaric acid, and the like. Among them, glyoxylic acid is preferably used.

The transamination reaction between Met-BTC and α-diketones is usually carried out in an amount of 10,000 to 10,000 mol (preferably 2000 to 40 mol) per mol of Met-BTC.
00 mol) of about 0 to 70 ° C.
The reaction is preferably performed at (preferably about 20 to 40 ° C.) for about 5 minutes to 2 hours (preferably about 15 minutes to 1 hour). The pH of the reaction is adjusted to about 2 to 9, especially about 4 to 7, especially about 5 to 6,
The reaction is preferably allowed to proceed under conditions where Met-BTC is not denatured. In order to adjust the pH of the reaction solution, for example, a buffer such as a phosphate buffer, an acetate buffer, and a citrate buffer may be used. In order to promote the transamination reaction, it is preferable to react α-diketones in the presence of a transition metal ion.
It is preferable to use about 0.001 to 0.1 mol (preferably 0.01 to 0.05 mol) of transition metal ion. As the transition metal ion, for example, copper ion, cobalt ion, nickel ion, iron ion, zinc ion, aluminum ion, manganese ion, gallium ion, indium ion, magnesium ion, calcium ion and the like can be used. Copper ions, cobalt ions, nickel ions and the like, particularly copper ions, are preferably used. These transition metal ions are usually added to the reaction solvent as a salt with an inorganic acid such as sulfuric acid, nitric acid, hydrochloric acid, bromic acid, carbonic acid, perchloric acid or a salt with an organic acid such as acetic acid or citric acid. In particular, copper sulfate and copper acetate, particularly copper sulfate, are preferably used. The α-diketones are preferably reacted in the presence of a base, and usually 0.1 to 2 mol (preferably 0.5 to 1.1 mol) per 1 mol of the α-diketones.
It is preferable to use about 0 mol) of a base. Examples of the base include organic amines such as alkylamines such as triethylamine and tributylamine, and aromatic amines such as N, N-dimethylaniline, pyridine, lutidine, collidine, 4- (dimethylamino) pyridine and imidazole. Although it can be used, among them, aromatic amines,
In particular, pyridine is preferably used. Furthermore, in the above-mentioned transamination reaction, it is preferable to react α-diketones in the presence of a transition metal ion and a base, and practically, three components of a transition metal ion, a base and α-diketones (for example, , Copper sulfate, pyridine and glyoxylic acid) are added to an aqueous solution containing 0 to 4 mol / l of urea containing Met-BTC to allow the transamination reaction to proceed.

The diketone [CH ₃ -S- (CH ₂ ) ₂ -CO-CO-BTC] obtained by the transamination reaction can be used for purifying peptides or proteins, for example, extraction, salting out, partitioning, Recrystallization,
Although it can be isolated and purified from the reaction solution by chromatography or the like, it can be directly subjected to the next hydrolysis reaction. Examples of the base used for the hydrolysis reaction include alkylamines such as triethylamine and tributylamine, N, N-dimethylaniline, pyridine, lutidine, collidine, 4- (dimethylamino) pyridine,
Aromatic amines such as imidazole, o-phenylenediamine, tolylene-3,4-diamine, 3,4-diaminobenzoic acid, 2,3-diaminophenol, 4-chloro-
Diamines such as o-phenylenediamine (preferably aromatic diamines, especially o-phenylenediamines), thiosemicarbazides such as thiosemicarbazide, acetone thiosemicarbazide and phenylthiosemicarbazide, and seleno such as selenosemicarbazide and acetoneselenosemicarbazide Semicarbazides and the like can be used. Among them, diamines and thiosemicarbazides are preferably used, and o-phenylenediamine is particularly preferably used. The amount of the base is usually about 1 to 10,000 mol, preferably about 5 to 1 mol per mol of the diketone compound.
00 to 2000 moles. The hydrolysis reaction is usually carried out at about 0 to 70 ° C (preferably about 20 to 40
° C) for about 1 to 50 hours (preferably about 10 to 2 hours).
5 hours). In the reaction, it is preferable to use a buffer as a solvent.
Examples thereof include a phosphate buffer, an acetate buffer, and a citrate buffer. Among them, an acetate buffer is preferable. The pH of the reaction is preferably adjusted to about 2 to 9, especially about 3 to 7, especially about 4 to 6, and the reaction is allowed to proceed under conditions where the resulting BTC is not denatured.

The BTCs thus obtained can be purified by known purification means, for example, extraction, salting out, dialysis, distribution, crystallization, recrystallization, gel filtration, chromatography and the like.
It can be isolated and purified from the reaction solution, but preferred examples are, for example, SP-Sepharose (Pharmacia Biotech), SP-5PW (Tosoh) or DEAE-5PW (Tosoh) Exchange chromatography via OD, OD
A purification method such as reverse phase chromatography using P-50 (Showa Denko KK) may be used. The BTCs obtained in the present invention have the same action as known BTCs,
It can be used in the same manner as a known method of using BTC.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION In the specification and drawings of the present invention, when bases and amino acids are indicated by abbreviations, IUPAC-
This is based on the abbreviation by IUB Commision on Biochemical Nomenclature or the abbreviation commonly used in the art, examples of which are described below. In addition, when an amino acid can have an optical isomer, the L-isomer is indicated unless otherwise specified. cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid EDTA: ethylenediaminetetraacetic acid SDS: sodium dodecyl sulfate DTT: dithiothreitol Gly (G): glycine Ala ( A): Alanine Val (V): Valine Leu (L): Leucine Ile (I): Isoleucine Ser (S): Serine Thr (T): Threonine Cys (C): Cysteine Met (M): Methionine Glu (E) : Glutamic acid Asp (D): Aspartic acid Lys (K): Lysine Arg (R): Arginine His (H): Histidine Phe (F): Phenylalanine Tyr (Y): Tyrosine Trp (W): Tryptophan Pro (P): Proline Asn ( ): Asparagine Gln (Q): glutamine Asx: Asp + Asn Glx: Glu + Gln following Reference Examples and Examples The present invention will be described more specifically, the present invention is not intended to be limited thereto.

[0024]

【Example】

Reference Example (Production of Human Met-BTC) According to the method described in Examples 4 to 6, 8 and 13 of JP-A-6-87894, human Met-BTC was produced by the following method.
Was manufactured.

Reference Example 1 (Escherichia coli human BTC cDNA)
Construction of Expression Plasmid) A 0.6 Kb EcoRI-BamHI fragment encoding human pro-BTC (1-147 amino acid residues) was ligated to the plasmid pTB described in Example 5 of JP-A-6-87894.
Isolated from 1515. A synthetic adapter having an ATG translation initiation codon (SEQ ID NO: 2: 5'-TATGGATGGG-3 '; SEQ ID NO: 3'-AATTCCCATCCA-3') was ligated to the EcoRI site of the 0.6 Kb fragment, and the resulting 0 .6Kb NdeI-Bam
The HI fragment was ligated with the T7 promoter (Gene, 56, 1).
25 (1987)).
3c to construct plasmid pTB1505. 80 amino acid residues of human BTC (JP-A-6-878)
10 (1) of FIG. 10-1 to FIG.
(To Tyr))
Plasmid pTB1505 as a template and two oligonucleotides as primers (SEQ ID NO: 4: 5'-AT
ACATATGGATGGGAATTCCA-3 ′; SEQ ID NO: 5: 5′-CCGGATCCTAGTAAAAC
PCR (polyAAATCCAACTCT-3 ')
merase chain reaction). The product is NdeI
And BamHI, and fractionated by 2.0% agarose gel electrophoresis to isolate a desired 0.25 Kb DNA fragment. This 0.25 Kb NdeI-BamHI fragment was inserted into T4DN downstream of the T7 promoter of pET-3c.
Ligation was performed using A ligase to obtain plasmid pTB1516 (see FIG. 13 of JP-A-6-87894).

Reference Example 2 (Human Met-BT in E. coli)
Expression of C) E. coli MM294 was lysogenized with lambda phage (Studier, Supra) recombined with the T7 phage RNA polymerase gene. Then, the plasmid p
LysS is introduced into E. coli MM294 (DE3),
E. coli MM294 (DE3) / pLysS was obtained. The plasmid pTB1516 obtained in the above Reference Example was added to the cells.
Was introduced and E. coli MM294 (DE3) / pLysS,
pTB1516 was obtained. 50 μg of the transformed cells
Containing 1 liter of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 1 / ml ampicillin and 15 μg / ml chloramphenicol2
The cells were shake-cultured in a liter flask at 37 ° C. for 8 hours. The obtained culture solution was used in 19 liters of the main fermentation medium (1.
68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.05% magnesium sulfate, 0.02%
Antifoam, 0.00025% ferrous sulfate, 0.0005%
Transplanted into a 50 liter fermenter containing thiamine hydrochloride, 1.5% glucose, 1.5% casamino acid)
Aeration and agitation culture was started at ℃. The turbidity of the culture is about 500
At the time of the Kret unit, 100 mg / liter of isopropyl-β-D-thiogalactopyranoside (I
(PTG) was added, and the culture was further continued. After 7 hours, the culture was terminated. The culture termination solution is centrifuged to about 340
g of wet cells were obtained and stored frozen at -80 ° C.

This transformed E. coli MM294 (DE3)
/ PLysS, pTB1516 has accession number FERM
Deposited with BP-3836 at the National Institute of Advanced Industrial Science and Technology (NIBH), Ministry of International Trade and Industry, and accession number IFO
No. 15282 has been deposited with the Fermentation Research Institute (IFO).

Reference Example 3 (Escherichia coli human BTC50cD
Construction of NA Expression Plasmid) pET3c (Rosenberg et al., Gene, 56)
Volume, page 125 (1987), by Ndel and BamHI
To obtain a fragment of about 4.6 k containing the T7 promoter and the ampicillin resistance gene. Downstream of the Ndel site, the ATG translation initiation codon, M of SEQ ID NO: 1
et-BTC (referred to as BTC50cDNA) from Arg ³² genes encoding Tyr ⁸¹ in the fragment with a stop codon and BamHI site, pET3 of Nd
The fragment cleaved with el and BamHI was ligated with T4 DNA ligase to obtain plasmid pTB1976 (FIG. 4).

Reference Example 4 (Human Met-BT in E. coli)
Expression of C50) Plasmid pTB1976 was introduced into E. coli MM294 (DE3) / pLysS, and E. coli MM294 (DE3) was introduced.
/ PLysS, pTB1976. This transformed cell Escherichia coli MM294 (DE3) / pLysS, pTB1
976 has 1 as accession number FERM P-16505
It was deposited with the Ministry of International Trade and Industry on November 6, 997 at the Institute of Biotechnology and Industrial Technology, and with the deposit number IFO 16122 on October 16, 1997 at the Institute of Fermentation (IFO). E. coli MM294 (DE3) / pLysS, pTB19
76 was cultured with shaking at 30 ° C. for 16 hours in 1 liter of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 50 μg / mL ampicillin. The obtained culture solution was added to 20 L of a main fermentation medium (1.68).
% Sodium monohydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.024% magnesium sulfate, 0.02%
It was transplanted to a 50 L fermenter containing Newpole LB-625, 0.0005% thiamine chloride, 1.5% glucose, and 1.5% casamino acid), and aeration and stirring culture was started at 37 ° C. When the turbidity of the culture solution reaches about 500 Klet units, 5.95 mg / L of isopropyl-β-D
Thiogalactopyranoside (IPTG) was added, and the culture was further continued. After 4 hours, the culture was centrifuged to about 2 hours.
00 g of the cells were obtained and stored frozen at -80 ° C.

Example 1 (Activation of Met-BTC) To 10 g of the cells obtained in Reference Example 2, 100 mM Tris /
HCl, 7 M guanidine hydrochloride, 1 mM EDTA (p
H8.0) 20 ml was added to dissolve the cells, and then 0.1 ml was added.
120 ml of M phosphate buffer (pH 6.0) was added, and centrifugation (10000 rpm, 1 hour) was performed. 0.1 M arginine, 50 mM Tris / HCl, 1 m in the supernatant
M EDTA, 0.5 mM GSSG, 1 mM GSH
(PH 8.0) 5 liters were added and activated at 4 ° C. overnight.

Example 2 (Purification of Met-BTC) An SP-Sepharose column prepared by adjusting the pH of the regenerating solution having been activated in Example 1 to pH 6.0 and equilibrating with a 100 mM phosphate buffer (pH 6.0). (25mm ID x 120m
mL), and then 500 mM NaCl / 100
Elution was performed with mM phosphate buffer (pH 6.0). Fractions containing BTC were pooled, followed by ODP-50 (21.2) equilibrated with 0.1% trifluoroacetic acid (TFA).
5mmID × 300mmL, 5μ) (Showa Denko KK)
5m with a step gradient of 20-60% B (B = 80% acetonitrile / 0.1% TFA) for 60 minutes
Elution was performed at a flow rate of 1 / min. After pooling fractions of Met-BTC, lyophilization was performed to obtain about 10 mg of Met-BTC.

Example 3 (Characteristic determination of Met-BTC) (a) Analysis using SDS polyacrylamide gel electrophoresis The BTC obtained in Example 2 was sample buffered with 10 mM DTT [Laemmli, Nature, 22].
7, 680 (1979)], heated at 95 ° C. for 1 minute, and electrophoresed on Multigel 15/25 (Daiichi Pure Chemicals). Run the gel after electrophoresis with Coomassie Brilliant
As a result of staining with blue (Coomassie brilliant blue),
A single band protein was observed at about 16 Kd. From this, it was found that the purified Met-BTC was almost single and formed a strong dimer even under the reduction condition with DTT (FIG. 2).

(B) Analysis of amino acid composition
300E). As a result, Me was added to the N-terminal.
The amino acid composition was consistent with the amino acid composition deduced from the nucleotide sequence of the BTC cDNA to which t was added (Table 1).

Table 1: Base sequence of BTC per mole of amino acid residues predicted from the number of amino acid residues per 1 mol of--------------------------------------------------------------------------------------- Asx 7.07 Thr ¹⁾ 5.9 6 Ser ¹⁾ 5.3 5 Glx 9 Asx 7.07 Thr ¹⁾ 5.9 6 Ser ¹⁾ 5.3-5 Glx 9---------------------- .29 Pro 4.04 Gly 6.97 Ala 4.04 Cys ²⁾ D. 8 Val 4.04 Met 0.90 Ile 2.02 Leu 3.13 Tyr 4.04 Phe 3.03 His 2.62 Lys 5.35 Arg 7.17 Trp2 ⁾ N . D. 0-------------acid hydrolysis (6N HCl-1% phenol, 110
C, 24 and 48 hours of hydrolysis) 1) Extrapolated value at 0 hours 2) Not detected Analysis was performed using about 20 μg.

(C) Analysis of N-terminal amino acid sequence The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystem Model 477A). As a result, it was consistent with the N-terminal amino acid sequence of BTC deduced from the nucleotide sequence of cDNA, except that Met was added to the N-terminal of the obtained BTC (Table 2).

TABLE 2 Base sequence of BTC detected Residue No. 1-------------------------BTC (Pmol) amino acid predicted from PTH ¹⁾ -amino acid Amino acid------------------------Met (563) 2 Asp ( 271) Asp 3 Gly (490) Gly 4 Asn (465) Asn 5 Ser (217) Ser 6 Thr (304) Thr 7 Arg (184) Arg 8 Ser (122) Ser 9 Pro (268) Pro 160 G Glu 11 Thr (155) Thr 12 Asn (120) Asn 13 Gly (114) Gly 14 Leu (213) Leu 15 Leu (235) Leu 16 N. D. Cys 17 Gly (165) Gly 18 Asp (94) Asp 19 Pro (134) Pro 20 Glu (37) Glu ------------------------------------------------ -------- 1) Analysis was performed using 1 nmol of phenylthiohydantoin.

(D) C-terminal amino acid analysis The C-terminal amino acid was determined using an amino acid analyzer (Beckman System 6300E). Obtained Met-BT
C was consistent with the C-terminal amino acid deduced from the nucleotide sequence of the cDNA (Table 3).

[Table 3] The analysis was performed using 15 nmol of a gas-phase hydrazine decomposition method (100 ° C., 3.5 hours).

Example 4 (Measurement of Met-BTC activity) BALB / c of purified Met-BTC obtained in Example 2
3T3 A31-714 Clone 4 (International Journal of Cancer, 12, 463)
Page (1973)), the BTCI described in Example 13 of JP-A-6-87894 was used.
Purified product).

Example 5 (Removal of N-terminal Met) 10 mg of Met-BTC with Met added to the N-terminal was
After dissolving in 4 ml of M urea solution, 50 mM copper sulfate 0.5
ml, glyoxylic acid 0.25 g, pyridine 0.5 ml
(PH 5.0) was added and reacted at 25 ° C. for 1 hour. After the completion of the reaction, the reaction solution was equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0) and a Sephadex G-25 column (25 mm ID ×
(600 mmL), and the solution used for equilibration is 6 ml.
/ Min and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M was added to this fraction.
After adding the sodium acetate solution, o-phenylenediamine was added to a concentration of 40 mM, degassed, sealed with nitrogen gas, and reacted at 37 ° C. for 15 hours. After completion of the reaction, the reaction solution was equilibrated with a 50 mM phosphate buffer (pH 6.0) using a Sephadex G-25 column (25 mm ID).
X 600 mmL) and the buffer used for equilibration was
The mixture was developed at a flow rate of ml / min, and the BTC fraction without Met added to the N-terminus was pooled. After adjusting the pooled BTC fraction to pH 6.0, 100 mM phosphate buffer + 200
SP-5P equilibrated with mM NaCl (pH 5.0)
W (7.5 mm ID x 75 mm L), adsorbed on Tosoh Corporation), and then 0.8 ml / for 30 minutes with a step gradient of 0 to 100% B (B = 100 mM phosphate buffer + 200 mM NaCl, pH 9.0). Elution at a flow rate of BT
The C fraction was pooled, and the BTC fraction was further 0.1% T
ODP-50 equilibrated with FA (10 mm ID × 250
mmL), after adsorbing to Showa Denko KK
Eluted with a step gradient of% B (B = 80% acetonitrile / 0.1% TFA) for 40 minutes at a flow rate of 2 ml / min. B
After pooling the fractions of TC, lyophilize,
About 760 μg of BTC was obtained.

Example 6 (Characterization of BTC) (a) Analysis using SDS polyacrylamide gel electrophoresis The BTC obtained in Example 4 was sample buffered with 100 mM DTT in Sample buffer [Laemmli, Nature, 2
27, 680 (1970)], heated at 95 ° C. for 1 minute, and electrophoresed on Multigel 15/25 (Daiichi Pure Chemicals). Run the gel after electrophoresis with Coomassie Brilliant
As a result of staining with blue (Coomassie brilliant blue),
A single band protein was observed at about 16 Kd. From this, it was found that the purified BTC was almost single and that a strong dimer was formed even under the conditions of reduction with DTT (FIG. 3).

(B) Analysis of Amino Acid Composition The amino acid composition was determined using an amino acid analyzer (Beckman
300E). As a result, the BTC cD
This was consistent with the amino acid composition deduced from the nucleotide sequence of NA (Table 4).

Table 4: Base sequence of BTC per mole of BTC amino acid number of residues Predicted value based on ------------- Asx 7.07 Thr ¹⁾ 5.9 6 Ser ¹⁾ 4.8 5 Glx 9 Asx 7.07 Thr ¹⁾ 5.9 6 Ser ¹⁾ .19 Pro 4.04 Gly 7.17 Ala 4.04 Cys ²⁾ D. 8 Val 4.64 Met 00 Ile 1.9 2 Leu 3.03 Tyr 4.04 Phe 3.03 His 2.42 Lys 5.15 Arg 7.07 Trp ²⁾ D. 0-------------acid hydrolysis (6N HCl-1% phenol, 110
C, 24 and 48 hours of hydrolysis) 1) Extrapolated value at 0 hours 2) Not detected Analysis was performed using about 20 μg.

(C) Analysis of N-terminal amino acid sequence The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystem Model 477A). As a result, it was consistent with the N-terminal amino acid sequence of BTC deduced from the nucleotide sequence of the obtained BTC cDNA (Table 5).

Table 5 Base Sequence of Detected BTC Residue No .:--------------------Detected (Pmol) Amino acid predicted from PTH ¹⁾ -amino acid Amino acid -1 Asp (271) Asp 2 Gly (432) Gly 3 Asn (249) Asn 4 Ser (95) Ser 5 Thr (150) Thr 6 Arg (98) Arg 7 Ser (89) Ser 8 Pro (255) Pro 9 Glu (177) Glu 10 Th ) Thr 11 Asn (139) Asn 12 Gly (187) Gly 13 Leu (227) Leu 14 Leu (281) Leu 15 N. D. Cys 16 Gly (126) Gly 17 Asp (70) Asp 18 Pro (100) Pro 19 Glu (44) Glu 20 Glu (75) Glu −−−−−−−−−−−−−−−−−−−−− 1) Analysis was performed using 1 nmol of phenylthiohydantoin.

(D) Analysis of C-terminal amino acid The C-terminal amino acid was determined using an amino acid analyzer (Beckman System 6300E). The obtained BTC is cD
This was consistent with the C-terminal amino acid deduced from the nucleotide sequence of NA (Table 6).

[Table 6] The analysis was performed using 15 nmol of a gas-phase hydrazine decomposition method (100 ° C., 3.5 hours).

(E) Biological activity of BTC According to the method described in Molecular Cell Biology, vol. 8, p. 588 (1988), BALB / c 3
Activity was measured using T3 A31-714 clone 4 (International Journal of Cancer, vol. 12, p. 463 (1973)), and purified BTC was used.
Has a cell growth promoting activity equivalent to that of a standard product (purified BTCI described in Example 13 of JP-A-6-87894).

Example 7 (Activation of Met-BTC50) To 200 g of the cells obtained in Reference Example 4, 100 mM Tris / HCl, 7 M guanidine hydrochloride, 1 mM EDTA
(PH 8.0) After adding 400 ml to dissolve the cells,
Centrifugation (10000 rpm, 1 hour) was performed. 0.2 M arginine, 50 mM Tris / HCl, 1
mM EDTA, 0.5 mM GSSG, 1 mM GS
H (pH 8.0) (10 L) was added and activation was performed at 4 ° C. overnight.

Example 8 (Purification of Met-BTC50) The regenerated solution that had been activated in Example 7 was desalted and concentrated using a Pellicon cassette system (PTGC membrane, Millipore ultrafiltration membrane, molecular weight cut off 1000). Thereafter, the pH was adjusted to 6.0. This desalted solution was added to a 100 mM phosphate buffer (pH 6.
0) SP-Sepharose column (40 mm) equilibrated
ID × 100 mmL) and 200 mM NaCl
After washing with a / 100 mM phosphate buffer (pH 6.0), elution was carried out with 700 mM NaCl / 100 mM phosphate buffer (pH 6.0). The fractions containing BTC50 were pooled, diluted 3 times with distilled water,
M NaCl / 100 mM phosphate buffer (pH 6.0)
SP-5PW (55 mm ID x 300 mm)
L), A = 200 mM NaCl / 100 mM
Phosphate buffer (pH 6.0), B = 300 mM NaC
1/100 mM phosphate buffer (pH 9.0)
Elution was performed with a step gradient of １００100% B for 100 minutes at a flow rate of 35 ml / min to obtain a BTC50 fraction. ODS-120T further equilibrated with 0.1% TFA (21.
5mmID × 300mmL, 5μ) (Tosoh Corporation), and then adsorbed on a step gradient of 20-50% B (B = 80% acetonitrile / 0.1% TFA) for 50 minutes, 5ml /
At a flow rate of 1 min. After pooling the fractions of Met-BTC50, lyophilization was performed and Met-BTC5
0 about 100 mg were obtained.

Example 9 (Characterization of Met-BTC50) a) Analysis using SDS-polyacrylamide gel electrophoresis 100 mM of Met-BTC50 obtained in Example 8 was used.
Sample buffer with DTT added (NOV
EX) and heated at 95 ° C for 1 minute.
Electrophoresis was performed using E-PAGE MINI GEL (TEFCO). The gel after the electrophoresis is treated with Coomassie Brilliant Blue (Co
omassie brilliant blue), a single band of protein was observed, and the purified product was almost single (FIG. 5).

B) Analysis of amino acid composition The amino acid composition was determined using an amino acid analyzer (Beckman System 6300E). As a result, the amino acid composition was consistent with the amino acid composition estimated from the nucleotide sequence of BTC50 cDNA having methionine added to the N-terminus (Table 7).

Table 7: Base sequence of BTC50 per 1 mol of BTC50 per 1 mol of amino acid residues Predicted from the number of amino acid residues (Table 7)------------------------ value to be ^{-------------------------------- Asx 2.1 2 Thr 1) 1.1} 1 Ser 1) 1.82 Glx 5.05 Pro 1.9 2 Gly 4.14 Ala 2.0 2 Cys ²⁾ 6 Val 3.74 Met 0.90 Ile 1.9 2 Leu 0.91 Tyr 3 .84 Phe 3.13 His 2.02 Lys 3.84 Arg 6.06 Trp ²⁾ 0------------------------------------------------------------------------------ ------- Acid hydrolysis (6N HCl-4% thioglycolic acid, 1
(Average value of hydrolysis at 10 ° C for 24 and 48 hours) 1) Extrapolated value at 0 hour 2) Not detected

C) N-terminal amino acid sequence analysis The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, except that methionine was added to the N-terminus of the obtained BTC50, it coincided with the N-terminal amino acid sequence of BTC deduced from the nucleotide sequence of cDNA (Table 8).

[Table 8]------------------------------------------ PTH ¹⁾ -Amino acid Predicted amino acid (pmol)---------------------1 Met (388 ) 2 Arg (192) Arg 3 Lys (736) Lys 4 Gly (761) Gly 5 His (350) His 6 Phe (555) Phe 7 Ser (214) Ser 8 Arg (309) Arg 9 ND Cys 10 Pro (231) Pro 11 Lys (223) Lys 12 Gln (179) Gln 13 Tyr (158) Tyr 14 Lys (202) Lys 15 His (72) His 16 Tyr (113) Tyr 17 N.D.Cys18 85) Ile 19 Lys (84) Lys 20 Gly (75) Gly-------------------1 nmol Using Analysis was carried out. 1) Phenylthiohydantoin

D) C-terminal amino acid analysis The C-terminal amino acid was determined using an amino acid analyzer (Beckman System 6300E). Obtained Met-B
TC50 coincided with the C-terminal amino acid deduced from the nucleotide sequence of the cDNA (Table 9).

Table 9: C-terminal amino acid recovery rate BTC50 (%)----------------------------- -------- Tyr 55.2

Example 10 (Measurement of Met-BTC50 activity) BALB / C of Met-BTC50 obtained in Example 8
3T3 A31-714 Clone 4 (International Journal of Cancer, 12 , 463 (1
973)) had an activity equivalent to that of the standard product.

Example 11 (Preparation of BTC50) Met-BTC504 having methionine added to the N-terminus
0 mg was dissolved in 3.85 ml of a 3M urea solution.
A mixed solution of 0.4 ml of 1M copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine was added and reacted at 25 ° C. for 1 hour. After the completion of the reaction, the reaction solution was equilibrated with 2.5 M urea + 20 mM phosphate buffer (pH 6.0) and a Sephadex G-10 column (25 mm ID ×
(600 mmL) and 3 ml of the solution used for equilibration
Per minute, and the diketone fractions of Met-BTC50 were pooled. Subsequently, an equal amount of 4M acetic acid-
After the addition of a 4M sodium acetate solution, o-phenylenediamine was added to a concentration of 40 mM, degassing and nitrogen gas sealing were performed, and the reaction was carried out at 37 ° C for 15 hours. After completion of the reaction, the reaction solution was passed through a Sephadex G-10 column (25 mm ID × 600 mmL) equilibrated with 2.5 M urea + 20 mM phosphate buffer (pH 6.0), and 3 ml of the buffer used for equilibration was used. The fractions were developed at a flow rate of / min, and the BTC50 fractions without methionine added to the N-terminus were pooled. After adjusting the pooled BTC50 fraction to pH 6.0,
100 mM phosphate buffer + 200 mM NaCl (pH
5.0) SP-5PW (21.5 mm ID)
× 150mmL) and then 0-100% B (B =
100 mM phosphate buffer + 300 mM NaCl, pH
Elution was performed with a step gradient of 9.0) at a flow rate of 6 ml / min for 50 minutes, and the BTC50 fractions were pooled. Furthermore, BT
ODP-50 in which the C50 fraction was equilibrated with 0.1% TFA
(10 mmID × 250 mmL, Showa Denko KK), and eluted with a step gradient of 20-50% B (B = 80% acetonitrile / 0.1% TFA) for 50 minutes at a flow rate of 2 ml / min. After pooling the fractions of BTC50, lyophilization was performed to obtain about 4.7 mg of BTC50.

Example 12 (Characterization of BTC50) a) Analysis using SDS polyacrylamide gel electrophoresis The BTC50 obtained in Example 11 was replaced with 100 mM DTT.
Buffer (NOVEX)
And heated at 95 ° C for 1 minute, then PEPTIDE-PA
Electrophoresis was performed using GE MINI GEL (TEFCO). Run the gel after electrophoresis with Coomassie Brilliant Blue
(E. brilliant blue), a single band of protein was observed, and the purified product was almost single (FIG. 6).

B) Analysis of amino acid composition The amino acid composition was determined using an amino acid analyzer (Beckman System 6300E). As a result, BTC50
(Table 10).

Table 10: Base sequence of BTC50 per mole Predicted from the number of amino acid residues value ^{------------------------------ Asx 2.1 2 Thr 1) 1.0} 1 Ser 1) 1.8 2 Glx 5.05 Pro 1.9 2 Gly 4.14 Ala 1.9 2 Cys ²⁾ 6 Val 3.84 Met 00 Ile 2.02 Leu 0.91 Tyr 3.84 Phe 3. 13His2.12Lys4.04Arg6.06Trp2 ⁾ 0------------------------------------------------------------------------------------------------ --- Acid hydrolysis (6N HCl-4% thioglycolic acid, 1
(Average value of hydrolysis at 10 ° C for 24 and 48 hours) 1) Extrapolated value at 0 hour 2) Not detected

C) N-terminal amino acid sequence analysis The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, it was consistent with the N-terminal amino acid sequence of BTC50 deduced from the nucleotide sequence of the obtained BTC50 cDNA (Table 11).

[Table 11]-------------------------------------------- PTH ¹⁾ -Amino acid Predicted amino acid (pmol)---------------------1 Arg (140 Arg 2 Lys (1144) Lys 3 Gly (1242) Gly 4 His (292) His 5 Phe (992) Phe 6 Ser (425) Ser 7 Arg (407) Arg 8 ND Cys 9 Pro (559) Pro 10 Lys (597) Lys 11 Gln (505) Gln 12 Tyr (428) Tyr 13 Lys (539) Lys 14 His (115) His 15 Tyr (408) Tyr 16 N.D. Cys 17 Ile (386) (368) Lys 19 Gly (341) Gly 20 Arg (183) Arg −−−−−−−−−−−−−−−−−−−−−−−−−−−−. 1.5nmol analysis was performed using a. 1) Phenylthiohydantoin

D) C-terminal amino acid analysis The C-terminal amino acid was determined using an amino acid analyzer (Beckman System 6300E). BTC50 obtained
Was consistent with the C-terminal amino acid deduced from the nucleotide sequence of the cDNA (Table 12).

Table 12: C-terminal amino acid recovery rate BTC50 (%)------------------------------ −−−−−−−−−−−−−−− Tyr 86.8 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

E) Biological activity of BTC50 Purified product is Molecular Cell Biology, 8, 58
8 (1988), according to the method described in BALB / C3T.
3 A31-714 Clone 4 (International Journal of Cancer, 12, 463, (1
973), and was confirmed to have an activity equivalent to that of the standard product.

[0056]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 81 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys 1 5 10 15 Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg 20 25 30 Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile 35 40 45 Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val 50 55 60 Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Leu Phe 65 70 75 80 Tyr 81.

SEQ ID NO: 2 Sequence length: 10 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TATGGATGGG10.

SEQ ID NO: 3 Sequence length: 12 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AATTCCCATC CA12.

SEQ ID NO: 4 Sequence length: 22 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATACATATGG ATGGGAATTC CA22.

SEQ ID NO: 5 Sequence length: 28 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGGATCCTA GTAAAACAAG TCAACTCT 28.

[0061]

The active BTCs thus obtained are used for the treatment of wounds and ulcers, and for the treatment of diabetes, various diabetic complications (eg, atherosclerosis, diabetic retinopathy). be able to. In the present invention, inactive BTCs expressed in prokaryotic cells can be efficiently activated using genetic engineering, and pharmacologically active BTCs having the above-mentioned actions can be prepared in large quantities.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of Met-BTC.

FIG. 2 shows SDS polyacrylamide gel electrophoresis of purified Met-BTC. Lane 1 shows the molecular weight marker,
Lane 2 shows purified Met-BTC.

FIG. 3 shows SDS polyacrylamide gel electrophoresis of purified BTC. Lane 1 shows the molecular weight marker and lane 2
Indicates purified BTC.

FIG. 4 shows a construction diagram of plasmid pTB1976.

FIG. 5 shows SDS polyacrylamide gel electrophoresis of purified Met-BTC50.

FIG. 6 shows SDS polyacrylamide gel electrophoresis of purified BTC50.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:19) (72) Inventor Nori Nishimura 1-54-16 Yamato-nishi, Kawanishi-shi, Hyogo Prefecture

Claims (7)

[Claims] 1. A method for producing activated betacellulin, which comprises refolding betacellulin expressed in a prokaryotic host by genetic engineering in a redox buffer. 2. The method according to claim 1, wherein the redox buffer is a buffer containing reduced glutathione and oxidized glutathione. 3. A redox buffer having a pH of 7 to 9.
The method according to claim 1, wherein 4. The method according to claim 1, wherein the redox buffer contains an amino acid having no mercapto group. 5. The method according to claim 4, wherein the amino acid having no mercapto group is arginine. 6. The betacellulins are genetically engineered in a prokaryotic host, the cells are solubilized with a denaturing agent, and then a pH 7-9 containing an amino acid without a mercapto group.
2. The method according to claim 1, wherein the denaturing agent is diluted with a redox buffer to an inactive concentration. 7. The method according to claim 6, wherein the denaturing agent is guanidine.