JPH0475584A - Human prourokinase-like polypeptide and production thereof - Google Patents

Abstract

PURPOSE:To collect human prourokinase-like polypeptide by culturing Escherichia coli transformed with a plasmid containing DNA segment to code human prourokinase-like polypeptide. CONSTITUTION:DNA fragment to code human prourokinase-like polypeptide is recombined into a manifestation type plasmid. A recombinant (host cell) transformed by transduction of the manifestation type plasmid is cultured to accumulate the objective substance in the host cell or the culture solution. When Escherichia coli is used as the host cell, the objective substance is recovered as an insoluble aggregate after cell grinding usually by ultrasonic wave or Gorlin homogenizer. The aggregate is dissolved in an aqueous solution of guanidine hydrochloride, etc., treated by air oxidation in the presence of a thiol compound and purified by a method such as fractionation precipitation by salting-out with ammonium sulfate. In this way, human prourokinase-like polypeptide can be produced economically and advantageously.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for producing a human prourokinase-like polypeptide having the ability to covalently bind to a blood clot, using genetic engineering techniques, and the peptide has the ability to covalently bind to blood clots. It has a function as an agent.

[Conventional technology]

Human urokinase is an enzyme that exists in trace amounts in human urine and has the effect of activating inactive plasminogen into plasmin, and the generated plasmin can dissolve blood clots. For this reason, human urokinase is widely used clinically as a therapeutic agent for thrombosis.

However, human urokinase has a low affinity for blood clots and is an active type, so it activates not only plasminogen present at the thrombus site but also plasminogen in the circulating blood, releasing a large amount of plasmin into the circulating blood. generate. Plasmin produced in excess in the circulating blood causes functional changes in platelets. Blood Vol. 68, p. 275, 198
(1986), it degrades fibrinogen, blood coagulation factors V and II (Blood Vol. 68, p. 1280, 1986), and there is a risk of causing side effects such as systemic hemorrhagic tendency, leading to death. (Journal of American Bow League Cardiology)
erican ColCo11ea Cardiolo
gy, Vol. 1O, p. 970, 1987) An ideal antithrombosis drug would attack thrombus components, but would not consume circulating blood platelets and blood coagulation factors, causing disruption of the coagulation system, and would not cause the desired blood flow. It has an intermediate half-life. In recent years, human prourokinase and tissue plasminogen activator (hereinafter abbreviated as tPA) have been used as second-generation thrombosis therapeutics.
is under development. These activate plasminogen in a thrombus site-specific manner. Therefore, even when administered in large amounts, there is little risk of causing side effects such as systemic bleeding tendency. However, human prourokinase has increased thrombophilicity compared to human urokinase, which is still insufficient. In addition, tPA has a large amount of inhibitor (plasminogen activator inhibitor) in the blood, has a short half-life in the blood, and has no therapeutic effect unless administered in large doses, and after arterial recanalization due to thrombolysis. Reocclusion often occurs (circulation: C1rculation Vol. 73, 34)
7 pages 1986, New England Journal of Medicine: New England Jou
RNA of Medicine Vol. 317, p. 581, 1987). As an ideal therapeutic agent for thrombosis, a substance with increased thrombophilic affinity of human prourokinase that is not irreversibly inactivated by plasminogen activator inhibitor I is envisioned, and attempts are being made to create such a substance. .

The first is based on the recognition that the high thrombophilic affinity of tPA lies in the first half of the N-terminal side of the amino acid sequence. It is a substance with a chimeric structure with the amino acid sequence of the latter half of the CI side of human prourokinase, which has the following, and has been created by genetic recombination technology. (Journal of Biological Chemistry)
al of Biological Chemistry
y Volume 262, Page 10855, 1987, Thrombosis and Hemostasis
d Haea+ostasis Vol. 54, p. 893, 1985) However, contrary to expectations, this substance does not have as high an affinity for blood clots as tPA.

The second is N, which shows plasminogen's affinity for blood clots.
It is a substance with a chimeric structure in which a sequence consisting of about 560 amino acids on the ffA side is chemically combined with the same amino acid sequence on the latter half of the C-terminal side of human prourokinase as described above (Biochemistry Vol. 25, p. 3603). (1986). The third is a substance with a chimeric structure that chemically combines an antibody against fibrin, which is a major component of blood clots, and the amino acid sequence of the latter half of the C-terminal side of human prourokinase (Clinical Research Abstract). : C11nical Re5
Each Abstract Volume 36 265A,
(1988). It is expected that the second and third substances have improved thrombophilia, and as a result, the therapeutic effect on thrombosis will be improved. However, it is difficult to economically produce these second and third substances.

Furthermore, urokinase derivatives have been reported that chemically cross-link fibrinogen to urokinase and bind to fibrin via fibrinogen. (PCT/5
U84100008: WO Furthermore, human blood coagulation box x
■An oligopeptide that enzymatically forms a covalent bond with fibrin through the action of a factor was introduced (PCT/US 881).
02276, WO39100051), it has been suggested that the oligopeptide can be bound to urokinase to improve thrombophilia. However, according to research conducted by the present inventors, it was found that even when the oligopeptide is bound to urokinase, the thrombophilicity of urokinase is not significantly improved.

[Problem to be solved by the invention]

The present invention provides a human prourokinase-like polypeptide that has better thrombophilic affinity than human prourokinase, and also provides a method for economically producing the polypeptide.

[Means to solve the problem]

The present invention provides a human prourokinase derivative (hereinafter referred to as the present human oligopeptide) which has a structure that condenses with a thrombus to form a covalent bond through the enzymatic action of human blood coagulation factor This is a human pro-urokinase-like polypeptide (hereinafter sometimes abbreviated as the human pro-urokinase-like polypeptide of the present invention) attached to the N-terminus of a pro-urokinase derivative (sometimes abbreviated as a pro-urokinase derivative).

Next, the present invention will be explained in detail.

(A) About this oligopeptide There is a substance in the human body that has the ability to covalently bond with fibrin, which is a major component of blood clots. The present inventors have developed these representative substances, G2. As a result of a detailed study of the amino acid sequences of antiplasmin, fibronectin, and fibrinogen γ chain, we found that the amino acid sequences of more than 10 N-terminals were:
・・G1n0OVa1 (or Leu) OPro (or G
ly) --- structure (O represents an amino acid), and has the ability to form a covalent bond with fibrin etc. through the enzymatic action of a peptide having this amino acid sequence or blood coagulation factor XIII (active form). Based on this knowledge, the present invention was completed.

The present oligopeptide may be any oligomer having a structure that condenses with a blood clot to form a covalent bond through the action of the enzyme of blood coagulation factor XI (active form).
・・Gl口○○Val (or Leu)○Pr0 (or G
ly) ..., and from the spirit of the present invention, the oligopeptides are not limited to the N-terminal oligopeptides of each of fibrinogen γ chain, G2, antiplasmin, and fibronectin. Preferred specific examples of the present oligopeptide are as follows. Can be mentioned.

This oligopebuti F name Amino acid sequence A
(Met, )Asn, Gln, Glu
, Gln, Vat, Ser.

Pro, Leu Dinghr, Leu, Leu, LysF
(Net, )G lr+, Ala
, G In, G In, Met, va IG
In, Pro, G In, Ser, Pro,
Va 1. A la.

Val, Lys (Mel)Ala, Gln, Lys, Mel Val
,GlnPro,Leu,Thr,Leu,Leu,L
ys(Met,)Ala, Gln, Lys, MetJa
l, GlnPro, 'GIn, Tbr, Leu, Leu
, Lys.

(Met,) GIn, Glu, GIn, Val, Ser
, Pro.

Gin, Thr, Leu, Leu, Lys.

(Met,) Asn, Gln, Asp, Gin, Val
, Ser.

Pro, Gin, Thr, Leu, Leu, Lys(M
eL, ) Asn, Gln, Glu, Asn, Val
, 5erPro, Gln, Thr, Leu, Leu,
Lys.

(Met,) Asn, Gln, Ser, Met, l/a
1. Ser.

Pro, [;]n, Thr, Leu, Leu, Lys.

(Met) may be present in some cases.
喀V＋t■.

i s nin (B) Regarding the present human pro-urokinase derivative, such a derivative does not need to contain the entire amino acid sequence of human pro-urokinase, but contains a region necessary and sufficient for the enzymatic function of plasminogen activation to be exerted. Any amino acid sequence may be used as long as it contains the amino acid sequence (for example, a human prourokinase amino acid sequence from serine at position 1 to glutamic acid at position 3, or from serine at position 1 to glutamic acid at position 143 is deleted), or a part of an amino acid. is changed in a desirable manner [for example, one or both of lysine at position 135 and arginine at position 156 is changed to an amino acid other than a basic amino acid (e.g. glutamine), and 1
A human proturokinase derivative in which phenylalanine at position 57 is changed to an acidic amino acid (eg, aspartic acid) may also be used.

(C) About the human pro-urokinase-like polypeptide of the present invention The human pro-urokinase-like polypeptide of the present invention can be produced in many types by combining the present human pro-urokinase derivative and the present oligopeptide, but a preferred example is a naturally occurring human pro-urokinase-like polypeptide. Shown in parallel with the amino acid sequence of prourokinase. However, the amino acid sequence differs from the amino acid sequence of natural human prourokinase and the surrounding amino acid sequences are
Indicated by letter abbreviation.

... RP RF K l' I G G ...
Human prourokinase-like polypeptide of the present invention “Name AHUK (q, q) (M)NQI!QVSPLTLIJLSNELHQVP
...-GQKPSSPPEELKFQC... R
PQPKIIGG... AP(IK(q, q) (M)NQEQVSPLTLLK[, FIQVP -
--GQKPSSPPEELKFQC... RPQF
KIIGG... APUK(k, q) (M)NQEQVSPLTLLKL)IQVP -・
-GKKPSSPPEELKFQC... RPQFK
IIGG... APIIX (k, d) (M)NQEQVSPLTLLKLHQVP ---
GKKPSSPPEF! LKFQC... RPRDK
, IIGG... 8PUK(k,k) (M)NQEQVSPLTLLKLHQVP ---
GKKPSSPPEELKFQC... RPRFKI
IGG...FP[JK(4,4) (M)QAQQMVQPQSPVAVKL)IQVP
-・-GQKPSSPEELKFQC... RPQ
FKIIGG... V, PUK(k, q) (M)AQKMVGPLTLIJL)IQVP --
-GKKPSSPPEELKFQC... RPQFK
IIGG... VfPIIK(k, Q) (M)AQKMVGPQTLLJLI(QVP --
-GKKPSSPPEELKFQC... RPQFK
I IGG...ACUK(-,Q)
(M)NQEQVSPLTLLKLKFQ
C... RPQFKIIGG... pcUK(-, q) (M)QA
QQMVQPQSPVAVKLKFQC... RPQ
FKIIGG... (M) is an optionally present methionine (D) Regarding the gene system and production method of the human prourokinase-like polypeptide of the present invention, the DNA segment encoding the human pro-urokinase-like polypeptide of the present invention is a human Thrombus (main component fibrin, etc.) due to the enzymatic action of blood coagulation factor x (active type)
It consists of a DNA encoding the amino acid sequence of an oligopeptide (this oligopeptide) having a structure that forms a covalent bond with a DNA that encodes the amino acid sequence of this human prourokinase derivative.

The DNA encoding the amino acid sequence of this oligopeptide consists of consecutive codons encoding each amino acid.
All codons for each amino acid can be used, but it is preferable to use codons that can be easily expressed in the host used, and to use codons that do not form a folded wall structure at the Metzenger RNA level by contiguous codons. DNA segments that are desirable for use and designed in such a desirable manner can be easily produced synthetically.

Preferred examples of DNA segments encoding the present oligopeptide are described below.

Name of this oligopeptide ^ Amino acid sequence (Met) Asn, Gl++, G
lu, GlnJal, Ser.

DNA ATG, AAC, CAG, GAA,
CAG, GTG, TCTPro, Leu, Thr, Le
u, Leu, LysCCG, TTG, ACT, T
TG, CTT, AAG.

F Amino acid sequence (Net) GIn, Ala, GI
n, Gln, MeL, VaDNA ATG,
CAG, GCA, CAA, CAG, AT[;, GTTG
in, Pro, Gln, Ser, Pro, Val
,Ala,Val,LysCAA,CCT,CAG
, TCA, CCG, GTT, GCT, GTT
, A.A.C.

vl Amino acid sequence (Met,)Ala, GIn,
Lys, Met, Val, G1nDNA AT
G, GCA, CAA, AAA, ATG, GTT, CA
GPro, Leu, Thr, Leu, Leu,
LysCCG, CTG, ACC, TT, CTT
, AAG■, amino acid sequence (Met, )A la
, G In, Lys, Met, Va 1. G
InDNA ATG, GCA, CAA,
AAA, ATG, GTT, CAGPro, GIn
,Thr,Leu,LeulysCCG CAA,AC
T, CTT, CTT, AAGVJ amino acid sequence
(Met), Gln, Glu, Gin, Val
, Ser, Pr.

DNA ATG CAG GAA CAG
GTG TCT CCGGln, Thr, Leu,
Lea, Lys.

CAG ACT TTG CTT AAGV,
7 Me/acid sequence (Met), Asn, Gln, Asp,
GIn, Val, Ser.

DNA ATG AACCAG
GACCAG GTG TCTPro, Gln,
Thr,Leu,Leu,LysCCG CAG
ACT TTG CTT AAGVs7 amino acid sequence (Met), Asn, Gln, Glu, Asn, V
al, Ser.

DNA ATG AACCAG
GAA AACf, TG TCTPro, GIn
, Thr, Leu, Leu, Lys.

CCG CAG ACT TTG CTT
AAGV, amino acid sequence (Net), Asn, Gi
n, Ser, Mel Vat, Ser.

DNA ATG AACCAA
TCT ATG GTG TCTPro, Gl
o, Tbr, Leu, Leu, Lys.

CCG CAG ACT TTG CTT
AAG (Met) is an optional methionine. The DNA segment encoding the human pro-urokinase derivative is the DNA encoding the natural human pro-urokinase.
Obtained from A. In that case, the DNA encoding natural human prourokinase may be used as it is, but it will be even better if it is changed to a codon that can be easily expressed in the host used or a codon that does not form a folded structure at the Metsenger RNA level. Favorable results are obtained.

Furthermore, when using human prourokinase with a deletion from serine at position 1 to glutamic acid at position 3, a larger amount is produced and accumulated in E. coli than when using human prourokinase without the deletion.

Preferred specific examples of the present human prourokinase derivative are as follows: (1) N of the natural human prourokinase amino acid sequence
Those in which the codon encoding the amino acids at positions 2 to 8 from the end are the following codons (in the case of human prourokinase in which the amino acids at positions 1 to 3 are deleted, the amino acids from Leu at position 4 to Pro at position 8) Codons encoding) 1, Asn", Gfu"Leu', His', GIn"
, Val'. “Pro”.

AAC, GAG, CTC, CAC, CAG, G
TT, CCG (2) In the case of a human prourokinase derivative with deletion of serine at position 1 to glutamic acid at position 143, the codon encoding leucine/lysine corresponding to positions 144 and 145 is the codon 2 2, Le
, l (4, LysIts CTC, AAG f3) In the case of a human prourokinase derivative in which arginine at position 156 is changed to glutamine, the codon encoding the amino acid from glutamine at position 156 to glycine salt at position 162 is codon 3. , 01n”', Phe”
', Lys"', Lle1s', Lle"'CAG,
TTT, AAA, ATC, ATT.

GlyilIl, GlyilIl GGC, GGC.

A DNA fragment encoding a human prourokinase-like polypeptide of the invention is recombined into an expression plasmid. The specific DNA base sequence and corresponding amino acid sequence are shown in Figure 5 (FcUK(-, q) and Figure 6 (AHUK).
(Q, Q)), Figure 7 (8 PUK (k, k)) and Figure 8 (APUK (k, q)) are shown. Recombinant (host cell) transformed by introduction of the expression plasmid. By culturing E. coli, the target substance accumulates in the host cells or culture medium. When E. coli is used as the host cell, the target substance is usually recovered as an insoluble aggregate after disrupting the bacterial cells using ultrasound or a Gorlin homogenizer. be done.

After dissolving this aggregate in an aqueous solution such as guanidino hydrochloride or urea, it is subjected to treatment such as air oxidation in the presence of a 1,000-ol compound to reproduce the original three-dimensional structure of the target substance. The product is then purified using techniques such as fractional precipitation using ammonium sulfate salting out, hydrophobic chromatography, and metal chelate chromatography, or other commonly used biochemical purification techniques may also be used.

In this way, the human prourokinase-like polypeptide of the present invention can be produced economically and advantageously.

〔Example〕

Next, the present invention will be specifically explained with reference to Examples.

The conditions for using each restriction enzyme, the method for isolating DNA fragments, and the synthetic double-stranded DNA used in Examples are described below.

(11 Reaction DNA (plasmid or DNA fragment) for each restriction enzyme 1 μg
10 units of each restriction enzyme was added to 50 μl of each of the following reaction solutions containing the following, and the mixture was incubated at each Shimotomi temperature for 2 hours.

When partial digestion was performed, the amount of restriction enzyme added was 1 to 2 units, and the incubation time was 0.5 to 1 hour.

蕊＜＜＜＜Rococo Mountain-! Niyama≦Otsu44-Jnot2)
T, DNA blunting reaction using DNA polymerase Add 50 μl of the following reaction solution containing 1 to 2 μg of linear DNA.
05-1 Uni-nod T, add DNA polymerase,
It was kept warm at 37°C for 1 hour.

[Composition of reaction solution]

67mM) Lis-HCl (pH 8,8) 6.7mM M
gCl2.16.6u M(N)11)2 SO2,1
0mM β-mercaptomercaptoethanol μV ethylenediaminetetraacetic acid, 0.0167% bovine serum albumin,
330 μM dCTP, 330 μM (IATP,
330 μM dGTP and 330 μM dTTP
o(3) DNA blunting reaction using Frenow fragment 50 μl of the following reaction solution containing 1 to 2 μg of linear DNA
Add 05 to 1 units of Flenow fragment to
It was kept warm at 25°C for 1 hour.

[Composition of reaction solution]

67mM potassium phosphate buffer (pi47.4), 6.
7mM MgCL, 1mM β-mercaptoethanol, 33μM dATP, 33μM dTTP, 3
3 μM dGTP and 33 μM dCTP.

(4)'T, DNA binding reaction using DNA ligase To 75 μl of a DNA solution containing the DNA fragments to be ligated (approximately 1 μg), add 60 μl of rDNA liger 7-yonkinot manufactured by Takara Shuzo (Kiki), 60 μl of solution A, and 75 μN of solution B.
(T, containing DNA ligase) and heated at 16°C.
It was kept warm for 30 minutes.

(5) Isolation of DNA fragments After digesting each plasmid with restriction enzymes, the desired DNA fragments were separated by agarose electrophoresis, and the DNA fragments recovered by electroelution were subjected to phenol-chloroform extraction and ethanol precipitation. It was purified and used. Each of the constructed plasmids was used to transform Escherichia coli JM103, and the transformants were examined by rapid isolation using alkaline lysis to obtain clones containing the desired plasmid.

(6) Synthetic double-stranded DNA used Synthetic double-stranded DNA used to create the target plasmid
The oligomers of each single-stranded DNA were synthesized by the amidite method, and each pair of isolated DNA was purified and dried using an OPC cartridge (manufactured by Applied Biosystems) to 20mM).
Liss hydrochloride, 10mM MgCL buffer (p)17.
6>, heated at 95°C for 2 minutes, cooled slowly to room temperature, and then annealed by holding at 12°C overnight to obtain the following double-stranded DNAs.

Synthetic double-stranded DNA name HL-A 5 -TTAAGCTGAGCA
ACGAGCT-31(L-B3-C
GACTCGTTGC-5CUK-A 5-C
AAGTTTCAGTGTGG-3CtlK-B
3-TCGAGTTCAAAGTCACACC-5A
P-A 5-CATGAACCAGGAAC
AGGTGTCTCCGTTGACTTTGCTTAA
GC Ding-3 AP-B 3--TGCAGTACTTGGTCCT
TGTCCACAGAGGCAACTGAAACGAA
T-5 FN-A 5-CATGCAGGCACAACAG
ATGGTTCAACCTCAGTCACCGGTTG
CTG-3 FN-B 3 -GTCCGTGTTGTCTA
CCAAGTTGGAGTCAGTGGCCAACGA
CAATT-5 V, -A I-CATGGCACAAAAATGG
TTCAGCCGCTGACCTTC-3 Vl-B 3-CGTGTTTTTTACC
AAGTCGGCGACTGGAACCAATT -
': V, -A V, -8 s -A s -B V, -A 4-B s-A s -B V, -A s B 5- -CATGGCACAAAAAAATGGTTCA
GCCGCAAACTCTC-3 3--CGTGTTTTTTACCAAGTCGGCG
TTTGAGAAGAATT -5 5--CATGCAGGAACAGGTGTCTCCG
CAGACTTTGCTTAAGCT -3 3' -TGCAGTACGTCCTTGTCCAC
AGAGGCGTCTG, ItAACGAAT-5
-5'-CATGAACCAGGACCAGGTGT
CTCCGCAGACTTTGCTTAAGCT -
3 3'-TGCAGTACTTGGTCCTGGTC
CACAGAGGCGTCTGAAACGAAT
-5 5--CATGAACCAGGAAAACGTGTCT
CCGCAGACTTTGCTTAAGCT -3 3--TGCAGTACTTGGTCCTTTTGCA
CAGAGGCGTCTGAAACGAAT -5 5--CATGAACCCAATCTATGGTGTCT
CCGCAGACTTTGCTTAAGCT -3 3--TGCAGTACTTGGTTAGATAACCA
CAGAGGCGTCTGAAACGAAT -5 TUKIA 5 UKI-B TUK2-A 5 UKIB CRO5D-A CRO3D-B Example 1 Plasmi Digested oligo with Blasmi Sac I Plasmid Plasmid GCCTCAGTTTAAAATCATTG GCGGC
G-3AGTCAAATTTTAGTAACCGC
CGCTTA -5GCCTCGCGATAAAATC
AT-dingGGCGGCG -3AGCGCTATTTT
AGTAACCGCCGCTTAA-5CTAGATA
AGGAGGTGAAAAACCATG-33-TATT
CCTCCACTTTTGGTAC-5 plasmid pA
CUK(-,q)15, plasmid pFCUK(-,
q)15, plasmid pV+PUK(kq)15 and plasmid IIV2PUK(k,Q)15.

DepHLIK (Q, q) 10 was digested with restriction enzyme Bal I and then isolated with an approximately 3.9 kb DNA fragment and mer CIJ.
K-A/E was ligated with DNA ligase and pCUK (
-, Q) 10 was obtained.

DopcUK(-,q)10 was digested with restriction enzyme Aat II, and then an approximately 3.9 kb DNA fragment isolated and mer AP-
A/B was ligated with T and DNA ligase to create pACUK(-,
q) Obtained 10.

A DNA fragment obtained by digesting p9420 with the restriction enzyme Nco I and treating it with alkaline phosphatase was combined with the plasmid p9420.
After digesting ACUK(-,Q)10 with restriction enzyme Bsp)I, the isolated DNA fragment containing the urokinase gene was digested with T.
, plasmid pAcLI ligated with DNA ligase
K(q)15 was obtained.

Plasmid pACUK(-,q)15 was digested with restriction enzyme Aat
The approximately 5.4 kb DNA fragment isolated after digestion with II and Aft m and plasmid p9410 were digested with restriction enzyme Aa.
Approximately 0.5 kb isolated after digestion with f 11 and Nco I
DNA fragment and synthetic oligomer FNA,/B
ligated with T and DNA ligase to create plasmid pFcU.
K(-,q)15 was obtained.

Plasmid pActl[k, q)/s was digested with restriction enzyme Aa.
After isolation after digestion with j ir and Afl I [approximately 5.8
1 tll DNA fragment and plasmid p9410 with restriction enzymes Aat II and Nc.

The DNA fragment of approximately 0.5 kb isolated after digestion with a plasmid pV, PUK (k, q) 15 was ligated with a synthetic oligomer Bi^/B using T and DNA ligase. In addition, plasmid pACUK(k, q)15 was digested with restriction enzyme A.
Approximately 5.8 isolated after digestion with at II and Afl II
A DNA fragment of approximately 0.5 kb and plasmid p9410 was isolated after digestion with restriction enzymes Aat II and Neo I.
kb DNA fragment and synthetic oligo 7-V2-A/B
, ligated with DNA ligase to create plasmid pVzPUK (k
q) 15 was obtained.

Example 2 Construction of plasmid pAPUK(q,q)15 and plasmid pFPIJK(Q,q)15.

Plasmid pHUK(q, q)10 was digested with restriction enzyme AaL.
The approximately 4.3 kb DNA fragment isolated after digestion with Sac II and Sac T and the synthetic oligomer AP-A/B were combined with T, DN
Ligate with A ligase and plasmid pAPUK(Q, q)
Got 10.

Plasmid pAPUK (q, q) 10 was isolated after digestion with restriction enzymes Afl I and Bam HI.
kb DNA fragment and plasmid pACUK(-,q)
15 was digested with restriction enzymes Aft II and BamHI, and the isolated DNA fragment of approximately 5.1 kb was ligated with T4 DNA ligase to obtain plasmid pAPUK(Q, Q)15.

Plasmid pAPUK (q, q) 15 was isolated after digestion with restriction enzymes Aat I [and Afl m].
A DNA fragment of approximately 0.kb and plasmid p9410 were isolated after digestion with restriction enzymes Aat I and Neo I.
The 5 kb DNA fragment and synthetic oligomer FN-A/E were ligated using T4 DNA ligase to create plasmids pF and PUK (
q, q)15 was obtained.

Example 3 Plasmid pAHUK(Q, q)15. Plasmid pAPtlK(k, k)15. Plasmid pAPUK(k, Q)/'5 plasmid IIAPU
Creation of K(k, d)15.

Plasmid pAPUK(Q, q)15 was digested with restriction enzyme Af.
Approximately 4.9k isolated after digestion with tII and BamHI
DNA fragment of b and plasmid pHUK(q, q)1
A DNA fragment of approximately 1.4 kb isolated after digestion of 0 with restriction enzymes BamHI and 5acI and a synthetic oligomer HL-
Plasmid p ligated with A/B using T and DNA ligase
AHUK (Q, q) 15 was obtained.

Escherichia coli HBIOI containing plasmid pAHUK(q,q)15 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 2467 (FERM BP-
2467).

Plasmid pAPUK(q, q)15 with restriction enzyme)1
5.2 isolated after digestion with indI [[ and Pst I
The 1.1 kb DNA fragment and the approximately 1.1 kb DNA fragment isolated after digesting plasmid pMU74H with restriction enzymes HindI and Pst I were ligated with T+ DNA ligase to obtain plasmid pAPUK(k, k), /s. .

Escherichia coli HBIOI containing plasmid pAPUK (k, k > 15) was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as a gift to the Institute of Microbiological Technology, No. 2465 (FERN BP-
2465).

Plasmid pAPUK(k, k)15 was digested with restriction enzyme Ba
Approximately 5.5 kb isolated after digestion with I and BamHI
and an approximately 0.75 kb DNA fragment isolated after digesting plasmid pCUK(-,q)10 with restriction enzymes Bat I and Bam HI.

Ligate with DNA ligase to create plasmid pAPtlK (k,
q) 15 was obtained.

Escherichia coli HBIOI containing plasmid pAPUK (k, Q) 15 was donated to the Institute of Microbiology, Agency of Industrial Science and Technology, as part of the Fiber Science and Technology Research Institute No. 2466 (FERM B
P-2466).

Plasmid pAPUK(k, k)15 was digested with restriction enzyme Ba
Approximately 5.5 k isolated after digestion with I and Bag) II
DNA fragment of b and plasmid pHUK (q, d)1
0 was digested with restriction enzymes Bat I and Ba+m) II, and the isolated DNA fragment of approximately 0.75 kb was ligated with T4 DNA ligase to obtain plasmid pAPUK(k, d) 15.

Example 4 Expression of human prourokinase-like polypeptide gene by E. coli.

Plasmids obtained according to Examples 1.2.3 and 4 pA)IUK(Q, Q)15, pAPUK
(Q, q)15, pAPliK(k, q
)15, pAPUK(k,d)7'5, pAPUK
(k,k)15, pFPUK(q,q)15, pA
cUK(-, q)15, pFCLiK(-, q'),
15, p ■IP L K (k, q'),
'5 and pV+PUK(k,Q)15 were each transformed into E. coli strain JM103+ according to the conventional method. The resulting recombinant microorganisms were incubated aerobically in 50 rnl of L-broth at 37°C, and when the absorbance at 600 nm was approximately 10, 0.5 i of 00 mM isopropylthiogalactopyranoside (IPTG) was added. was added and cultured for an additional 4 hours to express each human prourokinase-like polypeptide gene.

Example 5 Extraction and purification of gene products from E. coli.

100-I for each Log of wet bacterial cells obtained in Example 4
O + nM glycine-NaOH buffer y- (pH 9,0,
NaCl0. IM, EDT^5IIM)
After adding 50 μg of Norizozyme and stirring for 1 hour while cooling with water, the bacterial cells were disrupted by ultrasonication. Immediately after disrupting the bacterial cells, the precipitate was collected by centrifugation at 4° C., 112 OO rpm, for 30 minutes.

The precipitate is lomM green-NaOH buffer y-(p
After washing several times with H9,0), 500rnl of 0.1M
50 in glycine-NaOH buffer (p) 19.0)
0-8M guanidine hydrochloride was added and dissolved. Add ionized water and 1M Green-NaOH buffer to the intestinal solution (
pH 9,0) Add EDTA and reduced glutathione to make 4L. Final concentration: 10M guanidine hydrochloride, 0.04M guanidine.

EDTA lmM, reduced glutathione 0.2mM
), - Leave at room temperature overnight and refold (refo
lding) was performed. The refolded solution was concentrated using an ultrafiltration device (molecular weight cut off: 6000). Then, a 25-55% ammonium sulfate saturated fraction was collected as a crude product by ammonium sulfate salting out. The crude product was added to 50mM phosphate buffer (pH 7,5,0,5M guanidine hydrochloride, 0.6
After dissolving M in ammonium sulfate (containing ammonium sulfate), insoluble matter was removed by centrifugation, and the supernatant was adsorbed onto a phenyl sepharose (manufactured by Pharmasha) column equilibrated with the same buffer. It was eluted with a buffer, and the eluted fraction containing the target product was collected. This fraction was adsorbed on a Zn chelate (Cu chelate for ACllK (-, q) and FCUK (q)) Sepharose (manufactured by Farmanya) column, and 50 mM phosphate buffer (pH 6,0,0,5M
After washing thoroughly with 20mM acetic acid buffer (containing NaCl), elute with 20mM acetic acid buffer y-pH5.4 (containing 105M NaCl), and concentrate the eluted fraction containing the target product using an ultrafiltration membrane PM-10 (manufactured by Amicon). If necessary, the active form of the human prourokinase-like polypeptides of the present invention was removed by desalting (if necessary), and the corresponding human prourokinase-like polypeptides of the present invention (A) IoK (q, q) and APUK (Q, Q
), APUK(k, q), APUK(k, d), A
PUK (k, k), FPUK (q, q), ACUK
(-, q), FC[IK(q), V+PUK(k,
Q) and V2PUK (k, Qllllllll sound products.

Reference example 1 Plasmid pMtlT90pml and p94
20 creation.

Plasmid pPLL was treated with restriction enzymes Aat IF and Xba.
DN containing prelilac p10 isolated after digestion with I
The A fragment and plasmid pMJR1560 (manufactured by Amajam) were digested with restriction enzyme Pst I and then treated with DNA polymerase. Next, the approximately 3.4 kb DNA fragment containing the 1acl' gene isolated after digestion with the restriction enzyme AaL II and the synthetic oligo 7-CroS, /D-A/E were combined with T.
, and ligated with 1lNA ligase to obtain plasmid p9410.

pMUT8 containing the native human prourokinase gene
After digesting L with the restriction enzyme Nco I, the isolated DNA fragment T containing the hector fragment was ligated with DNA ligase to obtain plasmid p6514.

After digesting plasmid p6514 with the restriction enzyme EcoRI and supplementing the terminal complementary DNA with Flenow fragment, T
4 DNA ligase to obtain plasmid p6516.

Plasmid p6516 was digested with restriction enzyme Ba! A DNA fragment of approximately 4.2 kb isolated after digestion with Ill I was supplemented with complementary DNA at the end using Frenow fragment, and then subjected to T4DNAIJ.
Plasmid p6610 was obtained by ligating with gauze.

Buthisumi F derived from plasmid pMUT8L
Approximately
．． A 7 kb DNA fragment and a ca.
．． The 6 kb DNA fragment was ligated with T, DNA ligase to obtain 90 pml of plasmid pMUT.

Plasmid p9410 was digested with restriction enzyme Kpn J, treated with T4 DNA polymerase, and further digested with restriction enzyme 1Aa.
A DNA fragment containing Prelyrank p/o was isolated after digestion with L, and 90 pml of plasmid pMLIT was digested with restriction enzyme Pst I, treated with T, DNA polymerase, further digested with restriction enzyme AaL II, and isolated.
The approximately 3.5 kb DNA fragment containing cplo was converted into T, DNA
Plasmid p9420 was obtained by ligation with ligase.

Reference Example 2 Creation of plasmids p) tUK(q, d) 10 and p) tUK(q, q) 10.

Plasmid pM containing the human prourokinase gene
After digesting UT8L with the restriction enzymes Axy I and HindI, an approximately 4.4 kb DNA fragment was isolated, supplemented with terminal complementary DNA with Frenow fragment, and then ligated with T4 DNA ligase to obtain plasmid pMtlT4)1.

Plasmid 11M1] T4H was digested with restriction enzymes Ace m and Nar I, and then an approximately 3.1 kb DNA fragment was isolated, and plasmid pMUT90pIIll was digested with restriction enzymes Ac.
The approximately 1.3 kb DNA fragment isolated after digestion with cm and Nar I was ligated with 74 DNA ligase to create plasmid p.
6028 was obtained.

Plasmid p6028 was digested with restriction enzymes Eco47 II and Hind■, and a DNA fragment of approximately 3.7 kb isolated was supplemented with terminal complementary DNA using Flenow fragment and ligated with T4 DNA ligase to obtain plasmid p6N17.

Plasmid p6N17 was digested with restriction enzymes Dra II and E.
About 3.7 kb DNA fragment isolated after digestion with coR1 and synthetic oligomers TUKI-A/B and TUK2-A/
Each of B was ligated with T4 DNA ligase to create plasmid p.
7214 and plasmid p7224 were obtained.

Plasmid pMUT90 pml was digested with restriction enzyme Ban I [
An approximately 3.8 kb DNA fragment isolated after digestion with plasmid p? 214 and plasmid p7224 were each isolated after digestion with the restriction enzyme Ban I [about 0.5
The kb DNA fragments were ligated using 74 DNA ligase and plasmid pi (UK(Q,Q)10 and plasmid pFI
[1K(q, d)10 was obtained.

Reference Example 3 Urokinase-like polypeptide A with heftite added to the N-terminus that has the ability to bind to fibrin
Preparation of TUK (q, q) 100,000 units of the purified preparation of the human prourokinase-like polypeptide AHUK (q, q) obtained in Example 5 was added to 50 mM lithium-hydrochloric acid buffer containing 1μI of O, 1M NaC]. The mixture was dissolved in a solution (pH 7.5), 0.015 cu of serum plasmin (manufactured by Mitri + Aji Co., Ltd.) was added, and the mixture was incubated at 37°C for 1 hour. Next, 10 μg of trypsin inhibitor (manufactured by Sigma) was added to stop the reaction, and after adsorption onto a henzamine sepharose column, 2 (1 mM
Elute with acetic acid buffer (pH 4, 5), concentrate and desalt the eluted fraction using ultrafiltration membrane PM-10 (manufactured by Amicon),
Urokinase-like polypeptide ATUK (Q,Q) was obtained. As a result of amino acid sequence analysis near the N-terminus, this purified specimen is a prourokinase-like polypeptide AHUK (Qq).
It was determined that MiB is a urokinase-like polypeptide with a sequence identical to that near the N-terminus of .

〔Effect of the invention〕

It is presumed that the human prourokinase-like polypeptide of the present invention covalently binds to and activates blood clots through the enzymatic action of blood coagulation factor Since it does not separate from blood clots, it can be an ideal thrombolytic agent that has excellent thrombus specificity, has few side effects such as systemic bleeding tendency, and is not inhibited by butysminogen activator inhibitors. It can be produced economically using genetic engineering techniques.

Test Example 1 Binding ability test for fibrin, the main component of blood clots.

The fibrin salt binding ability of each of the purified human prourokinase-like polypeptides of the present invention (hereinafter abbreviated as test samples) was compared with tPA, human prourokinase, and urokinase-like polypeptide ATUK (q, q). The binding ability test was conducted using the following three methods.

(Reagent) 1) Tris-HCl buffer; 50mM Tris, 3
8 mM NaCl, 0. 0 l
9'o Triton X-100゜pH7
,5.

2) Fibrinogen solution, plasminogen-free human fibrinogen 10/ml Tris-HCl buffer manufactured by Nogma 3) Thrombin solution, human thrombin 100 manufactured by Sigma
4) Plasmin solution, Mitori + Aji Co., Ltd. Plasmin 1
5 C U 2' m I H r 0 05) Trypnone inhibitor solution; Trypnone inhibitor 5■/III 820 manufactured by Nogma Co., Ltd. 06) Hirudin, Hirudin manufactured by Sigma Co., Ltd.

7) Test sample solution, test sample 5000 international volumes/1 Tris-HCl buffer ■. Binding ability test method during fibrin clot formation process under water cooling! In a 5 ml Elapen tube, add 75 ul of fibrinogen solution, 0. 1M, CaCl2 aqueous solution 5μm, 1OOμl of each test sample was taken, Tris-hydrochloric acid buffer was added to this to make the solution 297μm, and the mixture was well mixed. After mixing, 3μm of thrombin solution was added and 37μl of each test sample was taken.
Fibrin salts were generated by treatment in a water bath at °C for 60 minutes. Immediately centrifuge at 16,000 rpm at 4°C.
After washing the precipitate on a Trisha sofa, INIH's hirudin and 5-15 μm plasmin solution were added to dissolve the precipitate and activate the test sample. After adding trypunin inhibitor in an amount 5 times that of plasmin to suppress plasmin activity, the amount of the test sample was measured from the decomposition activity of the synthetic substrate S-2444 manufactured by Kabi Co., Ltd. and was used as the amount of bound test sample.

■ Binding ability test after fibrin clot formation Same method as method I, except that after forming fibrin salt without adding the test sample, add 100 μm of test sample solution and 200 μm of Tris-HCl buffer and treat in a water bath at 37°C for 60 minutes.■ The amount of the test sample in the centrifuged sediment treated in the same manner as above was measured.

(Supplementary) In the above test method, when the test samples were human prourokinase and APUK (k, k), the tests were conducted in the presence of hirudin to prevent decomposition and inactivation of the test samples by thrombin.

The binding ability of each test sample to fibrin salt is shown in the following table.

The amount of tPA and APUK (q, q) released from the fibrin salt after binding during the fibrin clot formation process was measured over time. After 2 hours, tPA is 2
There was 3% withdrawal, and it was observed that the withdrawal gradually progressed after that, and the withdrawal amount for APUK (q, q) after 2 hours was 8%.
Subsequent withdrawals were not allowed.

Test Example 2 Lytic activity test for fibrin, the main component of blood clots.

The fibrin lytic activity of each of the purified human prourokinase-like polypeptides of the present invention (hereinafter referred to as test samples) was measured using standard urokinase (distributed by the National Institutes of Health).

Test method Bovine fibrinogen (Seikagaku Corporation) 2% and CaCl
O-IM phosphate buffer containing 210mM (pH 7,4
) 025% agarose aqueous solution 5- dissolved in 5- by heating
and bovine thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) solution (100 activity units/++/) 0.1- were added thereto, and after mixing, the mixture was poured into a Petri dish with a diameter of 9.0 mm to prepare a fibrin plate.

5 μm containing 1 unit of hirudin on this fibrin plate.
Each test sample was spotted and incubated at 37°C for 18 hours, after which the activity value was calculated from the size of the lysis circle. The fibrinolytic activity of each test sample relative to the unit absorbance at 280 nm is shown in the table below in international units.

APUK(k,d) 8.7
x 10'APUK(k,k)
9.2 x 10'V2PUK(k,q)
1 0.4 x 10'

[Brief explanation of drawings]

FIG. 1 is a production system diagram of Examples 1 and 2, FIG. 2 is a production system diagram of Example 3, FIG. 3 is a production system diagram of Reference Example 1, and FIG. 4 is a production system diagram of Reference Example 2. Figures 5, 6, 7 and 8 show the human prourokinase-like polypeptide PCUK(-, q) of the present invention, respectively.
), AHUK (q, q), APUK (k, k), and the DNA base sequence and corresponding amino acid sequence (-letter abbreviation) encoding APUK (k, q). Applicant (430) Agent of Nippon Soda Co., Ltd. (71)
25) Yokoyama Yoshima (964B>East)
Written by Yu Kai Figure 5-1 Figure 5-2 TCrAGATAAGGAGGTGAAAACCAIN
fAGGCACAACAGAMQAQQMVQPQSP
V SHRECQQPHYYGSEVTTKMLTGCTG
TT
AAAATCATAVKLK
FQCGQKTLRPQFKIICAADPQWKTD
SCQGDSGGPLTGGCGGCGAATTCAC
CACCA
TCTACAGGAGGCAGGEFTTIENQP
WFAAIYRRHVC5LQGRMTLTGIVSW
GRGCCCGGGGGGGGCI'CTGTCACCr
T'CAGCC[A
TRGGSVTYVCGGSLISPCWVIALKD
KPGVYTRVSHFLPWIRCAGCGCCAC
ACACTGCTTCATTGATrACCCAAAG
AAGGAGGACTACATCGTCTACC']℃
GGSATHCFIDYPKKEDYIVYLGSHI
KEENGLAL Tatami TI'TTGCAGTAGAGTCATCTCCATC
AC; CrGrAAGAR3RLNSNTQGEMKF
EVENLICCTACACAAGGACTACAGC
GCIY;A, CA
'ITGCCTTα? 11uAALHKDYSADT
LAHHNDIALKAGAGCIY;GGAATA
TAGGC'rcrGCACAGATGAGA A IR5KEGRCAQPSRTIQTICLCAATA
GCITTACCCTCA1040CAATAGCIT
TACCCTCACAAGCTrPS 56
0 570 580 590
600AG A GA ATTCTA CC
GACTATCTCTATCCCGGAGCA GCT
GAAAATGAσrGTTGTGAAα:TGATE
NSTDYLYPEQLKMTVVKLIFigure 6-1Figure 6-2MNQEQVSPLTLLK CTGTGACTGTCTAAATGGAGGAACA
TGLSNELHQVPSNCDCLNGGTCGCA
GCACTGTGAAAATAGATAAGTCAAAA
ACCTGCTATGAGGCGAATGGTCA σ
1TTTACCGQHCEIDKSKTCYEGNGH
FYRCσRo'CAGCAAAα7ACCATGCα:
:ACAGAT(TGALQQTYHAHR5DALQ
LGLGK)1TAATT
ATGTGCA
απGGGCCTNYCRNPDNRRRPWCYVQ
VGLKLLVQECMVHDCADGQKPSSTC
CTCCAGAAGAATTAAAATTTCAGTG
TGGCCAAAA to ACTCI℃AGGCCTCAG
'mAAAATPPEELKFQCGQKTLRPQF
KICATTIII; GCα; CC; AATrCACC
ACCA
ππACAGGAGIGGEFTTIENQPWFAA
IYRRLV to SLQGRMTLTGIVSWGRGFigure 6-3Figure 7-1 C A L K D K P G ■ Y T R ■ S H F L P W ■ H N Q E Q ■ S P L T L L K R S H T K E E N G L A L TTTrGCAGTAGAGTCATCTCCAEAG
CTGTAAGAAGAGCTGGGAATATAGG
CTCTGCACAGATGA■ D K S K T C Y E G N G H F Y R G K A S CGACAATAG (71TACCCTCACAAGC
TrN P D N R R R P W C Y ■ Q ■ G L K L L ■ L K F Q C G Q K T L R P R F K ■ ■ G G E F T T ■ E N Q P W F A A ■ Y R R H R G G Figure 7-2 Figure 7-3 TACCCTCACAAGCTT Figure 8-1 Figure 8-2

Claims (4)

[Claims] (1) A human prourokinase-like polypeptide in which an oligopeptide having a structure that forms a covalent bond with a blood clot through the enzymatic action of human blood coagulation factor XIII is attached to the N-terminal side of a human prourokinase derivative. (2) An amino acid sequence of a human prourokinase-like polypeptide to which an oligopeptide having a structure that forms a covalent bond with a thrombus as described in claim 1, or a polypeptide having an amino acid sequence substantially the same as this A DNA segment that encodes a peptide. (3) DNA segment according to claim 2,
A plasmid containing the control regions for its expression and the necessary DNA sequences for replication in E. coli. (4) Production of a human pro-urokinase-like polypeptide, which comprises culturing Escherichia coli transformed with the plasmid of claim 3, and collecting the human pro-urokinase-like polypeptide from the cultured cells. Method.