JPH0361484A - Novel tissue plasminogen activating factor - Google Patents

Abstract

PURPOSE:To efficiently produce t-PA different from natural or other modified t-PA by transforming a host cell with a manifestation vector containing DNA coding t-PA replaced Lys<415> to other amino acid. CONSTITUTION:Host cell is transformed using a manifestation vector containing DNA in which Lys<415> is replaced by other amino acid and coding natural or other modified tissue plasminogen activating factor (t-PA). Next, said transformant is cultured in a medium and produced t-PA is collected from the cultured substance. The used cells can include microorganism such as Escherichia coli, Bacillus subtilis or yeast, zootic cell system or cultured plant cell, etc. Resultant t-PA is able to be purified by dialysis, gel filtration or column chromatography, etc., and useful as fibrinolytic agent for the treatment of vascular disease.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a novel tissue plasminogen activator.More specifically, the present invention relates to a novel tissue plasminogen activator. It has a strong activity of converting into plasmin, which produces soluble products.
D encoding the amino acid sequence of a novel tissue plasminogen activator, thus useful as a thrombolytic agent.
The present invention relates to NA, a method for producing the same, and a pharmaceutical composition containing the same.

The complete amino acid sequence and structure of natural human "tissue plasminogen activator" (hereinafter referred to as "rt-p"), which is derived from human melanoma cells (Bowes) The DNA sequence encoding it has already been revealed by recombinant DNA technology [Nature 301
．． 214 (1983)]. Native t-PA is a single-chain serine protease that is converted by plasmin into a dimirrolic form of heavy and light chains linked by a single disulfide bond. The light chain (L) is the protease domain and thus contains the active site of the enzyme. The heavyweight (H) has a finger domain (F), a four-length factor domain (E), and two kringles with three disulfide bonds (ie, kringle 1 and kringle 2 domains; 1 and 2).

Therefore, natural t-p^ has five functional domains F1E,
[European Patent Application Publication No. 0198920 and Proc, Natl.

^cad, sc1. l]5A834670 (1988)
reference].

Natural t-p^ is rapidly inactivated by fast-acting plasminogen activator inhibitor (FAI) [see, e.g., Churchill Livingstone, Haemcstasis and Thrombosis].
patients with myocardial infarction have twice the FAI concentration of healthy controls, indicating that myocardial infarction is often caused by acute coronary thrombosis. There is a report that. Therefore, for the treatment of thrombosis, particularly myocardial infarction, it is desired that the thrombolytic agent be less inactivated by FAI. natural t-P
As a result of various studies regarding the modification of A, the inventors of the present invention discovered that Ly at position 415 of the amino acid sequence of natural t-PA
We found that s plays an important role in the interaction between t-PA and PAI.

Structure and Effects of the Invention Therefore, the present invention provides a novel t-PA that differs from natural t-PA or other mutant t-PAs by having Lys415 constituted by other amino acids. The new t-P8 has a higher PA compared to the natural t-P8.
It is difficult to be inactivated by I.

Other amino acids can include acidic amino acids (e.g., glutamic acid, aspartic acid), neutral amino acids (e.g., leucine, glutamine), and basic amino acids other than lysine, and preferred other amino acids include acidic amino acids and neutral amino acids. It is a sexual amino acid.

Other mutant t-PAs include natural t-PAs other than Lys415.
By substituting one or more of the constituent amino acids of PA with another amino acid, by deleting any one or more of the constituent amino acids of natural t-P^, and/or by substituting one or more of the constituent amino acids of PA with another amino acid; a) Includes t-PA prepared by adding an acid.

In this specification, the numbering of the constituent amino acids to natural or novel t -P is based on the Pennica
[Nature 301.214
(1983)], for example Tyr
” means tyrosine at position 2 of the amino acid sequence of natural t-PA.

A preferred example of the novel t-PA of this invention can be represented by the following amino acid sequence: H1-A2-415
-R2-A417-428- R3-A43O-fi2
7 (T) (wherein R1 is Ser- or G1y8l
ArgSer-R2 is -Glu-R3 is -Lys- or -Glu-A2-415 is Tyr2 to 6ly of natural t-PA
A417-4215 is the same amino acid sequence as His417 to Leu42a of natural t-PA, A43O-827 is the same amino acid sequence as Glu430 to A of natural t-PA.
082? Shows the same amino acid sequence as before. ) In this specification, for convenience, the following code name is used for the novel t-PA: P41B In the above amino acid sequence (I), R1 is -Glu-, Ser-R3 is -Glu-, and R2, A"ls, A"'-"'oJ:biA43O-I
I2γ is each as defined above.

aP415-1 In the above amino acid sequence (I), R1 is Ser - R3 is -Lys -, R2, A2-415. A417-42el and A-430-+121 are each as defined above.

aP415-2 In the above amino acid sequence (1), R1 is Ser - R2 is 10In- R3 is -Lys-, and A2-418, ^417-428 and A43O-52
7 are each as defined above.

aP415-4 In the above amino acid sequence (I), R1 is Ser - R2 is -Leu - R3 is -Lys-, and ^2-4111. A41γ-42'ts J: HiA
43'-"7c each # As defined above.

The novel t-PA of this invention is produced by recombinant DNA technology and polypeptide synthesis.

That is, the novel t-PA of this invention is based on the novel t-P
It can be produced by culturing host cells transformed with an expression vector containing DNA encoding the amino acid sequence of A in a medium, and collecting the novel t-PA from the culture.

The details of the above manufacturing method will be explained below.

The host cell may be a microorganism [bacteria such as Escherichia coli (Esc
herichia colt), Bacillus subtilis
ussubtilis, etc.), yeasts (e.g. baker's yeast (Saccharomyces ceravlsi)
ae), etc.], including animal cell threads and cultured plant cells. Preferred examples of microorganisms include bacteria, especially strains belonging to the genus Escherichia (e.g. E, colt.
HBIOI ATCC33694, E, coli H
BIOI-16FERM BP-1872E, col
i 294 ATCC31446, E, coltχ
1776 ATCC 31537, etc.), yeasts, especially strains belonging to the genus Saccharomyces [e.g.
romycescerevisiae CRF 01
(see European Patent Application Publication No. 0225286)], animal cell threads [eg, mouse 929 cells, Chinese hamster overly (CHO) cells, etc.].

When bacteria, especially Escherichia coli, are used as host cells, the expression vector usually contains at least a promoter, an initiation codon, and a DNA encoding the amino acid sequence of the novel t-PA.
, a stop codon, a terminator region, and a self-replicable unit. When yeast or animal cells are used as host cells, the expression vector contains at least a promoter, an initiation codon, a signal peptide, and a novel t - DN encoding the amino acid sequence of PA
A and a stop codon, and further enhancer sequences, 5°- and 3- of native t-PA.
° − non-coding regions, splicing junctions,
A polyadenylation site and a self-replicable unit can also be inserted into the expression vector.

Preferred promoters include conventional promoters [
For example, the PL-promoter and trp for E. coli
- Promoter, TRPI gene AD for baker's yeast or DHII gene and acid phosphatase (po
05) Gene promoter, HTL for blowhole animal cells
V-promoter, SV40 early and late promoter, LTR-promoter, mouse metallothionein I (MMT)-promoter and cotton cininium promoter, etc.].

A preferred start codon is the methionine codon (ATG
).

Signal peptides include signal peptides such as natural t-PA.

DNA encoding the signal peptide or the amino acid sequence of the novel t-PA can be obtained, for example, by partial or total DNA synthesis using a DNA synthesizer and/or by transfection [e.g., E, colt LE 392λ0 (pT
PA21), E, colt JA221 (
pTPA25)AT(:C39808,E, colt
JA 221 (pTPA102) ATCC3981
0°E, coliJA 221 (pTPA102)
(Lys 277-”l1e) ATCC39811
, E. coli JM 109 (p51H
) FEBM P-9774゜E, coli
JM 109 (pN53) FERM P-9775
1 [e.g., pTPA2
1, pTPA25, pTPA102, pTP^102
(Lys 277-11e), p51H, pN53]
The complete NA sequence encoding native or mutant t-PA inserted into a suitable enzyme (e.g., restriction enzyme, alkaline phosphatase, polynucleotide kinase, D
It can be prepared by a conventional method such as treatment with NA ligase, DNA polymerase, etc.). In a preferred embodiment of this invention, the preparation of DNA encoding the novel t-PA is carried out in accordance with Veils et al.
33, 659-663 (1986) and/or flick (Frltz).
DNA Cloning 1, 151 (1985), IR
This can be done by oligonucleotide-directed mutagenesis as described in L Press.

Examples of stop codons include commonly used stop codons (eg, TAG, TG^, etc.). The terminator region may be a natural or synthetic terminator (
For example, synthetic fd phage terminator 1, etc.) can be mentioned.

As a self-replicating unit, all the DNA belonging to it
A DNA sequence that autonomously replicates in a host cell, including natural plasmids, artificially modified plasmids (eg, DNA fragments prepared from natural plasmids), and synthetic plasmids.

Preferred examples of this plasmid include plasmid pBR322 or an artificially modified version thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) when using Escherichia coli as the host cell, and when using yeast as the host cell. Examples include yeast 2μ plasmid or yeast chromosomal DNA, and when mammalian cells are used as host cells, for example, plasmid, pR5Vneo ATCC37
198, plasmid pSV2dhfr ATCC371
45, Plasmid pdBPV-MMTneo ATC
(:37224, plasmid psV2neo ATCC
37149 is mentioned.

An example of the enhancer sequence is the SV40 enhancer sequence (72b, A).

Examples of the polyadenylation site include the polyadenylation site of SV40.

As a splicing junction, for example, S
One example is the V40 splicing junction.

The promoter, initiation codon, DNA encoding the amino acid sequence to the new t-P, termination codon and terminator region are continuously arranged in a circular form from upstream to downstream together with an appropriate self-replicable unit (plasmid), as appropriate. Using a different NA fragment (e.g. linker-1 and other restriction sites, etc.), a conventional method (e.g. restriction enzyme digestion, T4D
An expression vector can be prepared by ligation using NA ligase). When a mammalian cell line is used as a host, the DNA encoding the enhancer sequence, the 5°-noncoding region of the promoter native t-PA cDNA, the start codon, the signal peptide, and the amino acid sequence of the novel t-PA, The expression vector may be prepared by ligating the stop codon, 3°-non-coding region, splicing junction and polyadenylation site sequentially and circularly from upstream to downstream into a suitable self-replicable unit using the techniques described above. .

The expression vector is introduced into host cells. Introduction is the usual method [
For example, transformation (including transfection),
microinjection, etc.] to obtain transformants.

In order to produce novel t-PA using the production method of this invention, the expression vector-containing transformant thus obtained is cultured in an aqueous medium.

The medium contains a carbon source (e.g. glucose, glycerin,
mannitol, fructose, lactose, etc.) and inorganic or organic nitrogen sources (e.g. ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactodributone, beef extract, etc.)
Contains.

If desired, other ingredients [e.g., inorganic salts (e.g., I! sodium or potassium phosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (e.g., vitamin Bl), antibiotics ( For example, ampicillin, kanamycin), etc.] may be added to the medium. Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) supplemented with fetal bovine serum and antibiotics is often used to culture mammalian mrra.

Cultivation of transformants (including transfectants) is
Generally, pi(5,5 to 8.5, preferably pi(7 to
7,5), and may be carried out at 18 to 40°C (preferably 25 to 38°C) for 5 to 50 hours.

When the novel t-P^ produced in this way is present in the solution fraction of the culture, a culture filtrate (supernatant) is obtained by filtration or centrifugation of the culture, from which natural or synthetic Conventional methods commonly used for protein purification and isolation (e.g., dialysis, gel filtration, anti-t-
It can be purified and isolated by affinity column chromatography using a PA monoclonal antibody, column chromatography using an appropriate adsorbent, high performance liquid chromatography, etc.). When the produced new t-P^ is present in the cells of the cultured transformant, the cells are collected by filtration or centrifugation, and the cell wall and/or cell membrane is disrupted, for example, by treatment with ultrasound and/or lysozyme. and debris. The debris can be dissolved in a suitable aqueous solution (eg, 8M urea aqueous solution, 6M guanidium salt aqueous solution), and the new t-PA can be purified from the solution by the conventional method as described above.

New t-p^ produced with E. coli needs to be refolded. Refolding can be performed by conventional methods.

The novel t-P^ of this invention is useful as a thrombolytic agent for the treatment of vascular diseases (eg, myocardial infarction, stroke, heart attack, pulmonary embolism, etc.). New t-PA of this invention
can be mixed with a pharmaceutically acceptable carrier and administered parenterally to mammals, including humans, in the form of a pharmaceutical preparation such as an injection.

Pharmaceutically acceptable carriers include peptides or proteins (
For example, various organic or inorganic carrier materials commonly used in the preparation of pharmaceutical compositions containing serum albumin, etc.) may be included.

The dosage of the novel t-PA of this invention will vary depending on various factors such as the type of disease, the weight and/or age of the patient, as well as the type of route of administration;
- The optimal dosage of PA is usually selected within the range of 0.1 to tomg/kg/day for injection or infusion.

The above daily dose may be divided and given to the patient every few hours.

The following examples are intended to illustrate the invention, but are not intended to limit it.

All enzymes used in the examples (e.g., restriction enzymes, alkaline phosphatase, DNA polymerase, DNA ligase, etc.) are commercially available, and the conditions for using these enzymes can be found, for example, in the instructions attached to the commercially available enzymes. It will be obvious to those skilled in the art upon reference.

Bacteriophage Lambda Charon 4
A human genome library containing a partial digest of human DNA cloned into the EcoR1 site of A-[T, available from Maniatis, Mani
atis et al., Ce1l 15.687-701 (19
78)] using a DNA probe obtained by 32p nick translation using Brak hybridization (Wahl et al., Proc. Na
tl, Acad, Sci, USA■, 3683-
3687 (1979)]. Plasmid pTPA102 (Lys277-11
e) [Transformed strain E containing it, colt JA
221 (pTPA102) (Lys277-1ie)
Mutant t-PA (L
ys277-Tie) expression vector, 232bp Ps of PCT International Publication No. WO361015313 reference
By using the tI-RsaI fragment as a probe, 3f! The overlapping recombinant λ18A1. λ19A2 and λ21A2 were isolated. λ1 containing the transcription start site of the t-PA gene and the 5′-noncoding sequence of the t-PA gene.
9 A 2 h) et al.'s 1.5 Kb Eco RI −
A second plaque hybridization was performed using the old Ram fragment as a probe. This hybridization yielded λ132 Al and λ110 At containing the first two exons of the t-PA gene.

Mapping of inserted DNA sequences was performed by restriction enzyme analysis, and their DNA sequences were analyzed using the M13 sequencing kit (
It was partially determined by the dideoxynucleotide chain termination method using Amarjam.

The sequences and mapping of their inserted DNA were performed using published t-PA genome clones [Degen et al., J.
, Biol, Chem, 261.6972-698
5 (1986)].

Chi total RNA from melanoma cell culture
rgwin et al., Biochemistry 18.52
94 (1979). Human melanoma cells (Bowes), 3%
(V/V) fetal bovine serum and 10 mM) IEPS
Dulbecco's Modified Eagle Minimum Essential Medium (DM) supplemented with
EM) Culture 1. to a confluent monolayer in 10 rotating bottles each containing 400 m4. Ta.

To actively produce t-PA, human melanoma cells were cultured for 18 hours in 400+nJ2 each of DMEM containing 668 μg of the protease inhibitor aprotinin.

Cells were collected by centrifugation and treated with 6M guanidinium isothiocyanate, 5mM sodium citrate (pH 7.0).
, 0.1v2-mercaptoethanol and 0.5% N
-Resuspended in 50ajZ of sodium lauroyl sarcosinate. Cells were dispersed by homogenization. The homogenate was placed in a Beckman SW50, 1 polyaromer tube with 0.1 M EDTA (pH 7.5).
Cushion 3 ll1a of 5.7M cesium chloride inside
12 at 30,00 rpm, 20℃
Centrifuged for hours.

The supernatant was discarded, and the RNA pellet was soaked in 1 ml of water. RNA was precipitated by adding 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of ethanol. mRNA was purified from total RNA YA products using oligo-dT cellulose column chromatography [
Aviv et al., Proc. Natl. Acad, S.
ci, 69.1408 (1972)]. 4 X
Typical yield from 109 cells is 11 mg of total RNA.
, about 900 μg of poly(A) plus mRNA.

This poly(A) plus mRNA was extracted using the 5'-primer extension method [Lawn et al., Nucleic Ac1ds
Res, 9.8103-6114 (1981)]
was used for the preparation of double-stranded cDN^. mRNA 1
80 μg and the sequence 5′-GATCACTTGGTAA
5 μg of Brimer with GA-3° was added to 0.1 M [
It was heated at 90°C in 1200μ°C for 5 minutes, and then slowly cooled to 42°C. Using annealed mRNA,
50IIIM Tris H (:1 (pH 8,3), 10eM
Mg (:h, 10mM DTT, 4mM sodium birophosphate and 4 fl of each 1mM deoxyribonucleotide triphosphate (dATP, dTTP)
The first cDNA was incubated at 42°C for 60 minutes using 200 units of reverse transcriptase in 400 μL of %dcTP, dGTP).
Synthesized over a period of minutes. The mixture was added to 201111+-Lis-〇 by 1. (+)17.5), 5 mM MgCl2
．． 101M (N)14) 2504.100mM KC
I, Q, 18mM NAD.

RNase H at 50 ug/mfl BS^2IIlu at 10 µ/mu, DNA Polymerase I at 50 µ/mA and 50! The cells were treated with E. coli DNA ligase at 12° C./ml for 1 hour and at 22° C. for 1 hour.

74 DNA polymerase 15 in the reaction mixture! # added. After incubating for 10 minutes at 37°C, the reaction mixture was extracted once with phenol/chloroform and the aqueous solution was precipitated with 1 volume of 4M ammonium acetate and 4 volumes of ethanol. Double-stranded cDNA was extracted with 2QQ mM potassium cacodylate, 25 mM Tris-)ICI (po
6.9), 1mM dCTP, 0.1mM DT
T, 30 min at 37°C with 10 units of terminal transferase in 200 μA of 1 mM GaCl2.
The reaction mixture was extracted with phenol/chloroform for a period of minutes and the aqueous solution was precipitated with 0.1 volume of 3M sodium acetate (pH 5,2) and 2 volumes of ethanol. cd with tail
NA, 10mM Tris-HCI (pH 8,0),
'1mM EDT^, 0.15M NaC1600μ
The mixture was annealed with 660 ng of deoxy (G)-tailed puc9 (Pharmacia) for 1 hour at 58° C. in fl. The annealed DNA was used to transform compatible E. colt DHl. Approximately 17,000 transformants with ampicillin-resistant phenotype were obtained. This 5°-brimer extended cDNA library was used as a DNA library obtained by 32p nick translation.
Colony piperidization (
Grustein et al., Proc. Natl, ^cad
, Sci, 72.3961-3985 (1975)
[Reference]]. 1, Skb EcoRI-Ram) II fragment from t19A2 as a probe, the 5°-non-coding sequence and signal sequence of the t-PA cDNA were isolated from one of the eight colonies that gave a positive piperidization signal. The plasma containing pM1200 was isolated. cDNA of 9MI200
The length of the DNA piece was approximately 170 bp, and the base sequence of the DNA was determined by the dideoxynucleotide chain termination method (the cDNA sequence is shown in Figure 4).

E. coli JA221 (pT)'A102) (
Lys277-11e) One loop of a slant culture of eight TCC3981+ was inoculated into L-pros containing 50 μg/+n+2 ampicillin at 1°C, and 600 n
It was incubated with shaking until the optical density at m (0,0,) was approximately 0.6 absorption units. 0. When D was approximately 0.6, chloramphenicol powder was added to the medium to a final concentration of 100 μg/mfl, and incubation was continued overnight.

B5 Plus Yodo pTPA102 (L 5277-H
e) Isolation of U, co from the culture broth obtained in Example 3A
lt JA221 (pTPA102) (Lys27
7-11e) cells were collected.

Cells were collected by centrifugation at 4000g for 10 min at 4°C, and the supernatant was discarded. Cell pellets, 5 mg
Solution 1 containing lysozyme (50mM glucose, 25mM Tris-1 (C1(pl(8,0),
Aqueous solution containing 10mMEDTA) 20mj! The mixture was resuspended and left at room temperature for 5 minutes. Next, lysozyme-treated UJJ cells were treated with solution 2 (0.2N, NaOH and 1%
40 mjZ (aqueous solution containing SDS) was added, and the solution was gently mixed by inversion. The mixture was left on ice for 10 minutes.

Wood vinegar 11J111.5mu and water 2B, 5mjZ 5M
Water-cooled acetic acid prepared by adding potassium acetate solution (pH 4,8) 60 mJZ! 0 mL of XX liquid was added to the mixture obtained above and mixed by inversion. Place the solution on ice for 10 minutes.
Leave it for a minute, then turn it on. Seiko No. 4N-II (
or its equivalent), 20. OOQrpm, 4
Centrifuged for 20 minutes at °C. Host DNA and debris formed a pellet at the bottom of the tube. Approximately 72 ajl of supernatant liquid was collected, 0.6 volume of isopropanol was added, mixed and left at room temperature for 15 minutes. Plasmid DNA was collected by centrifugation at 11,000 g for 15 minutes at room temperature. The supernatant was discarded and the DNA pellet was washed with 70% ethanol at room temperature and decanted. Dry the pellet in a vacuum dessicator,
TE buffer [10mM Tris-)ICI (p)17.
5) and 1mM EDTA] 8 rails.

18 g of CsC was added to the DNA solution. 10mg/r
An aqueous ethidium bromide solution of nIL was added to each 10 mfl of G5C1-DNA solution. The final density of the solution is approximately 1.55
g/mfL, ethidium bromide concentration is approximately 6GOμg/1
It was nfL. Transfer the solution to a Beckman VTi65 centrifuge tube, fill to the top, seal, and incubate at 50,000 r.
p.m.

Centrifugation was performed at 20°C for 12 hours. After centrifugation, two DNA bands were visible under normal light. A syringe with a #21 hypodermic needle was inserted into the side of the centrifuge tube to collect the lower DNA band. Ethidium bromide was removed by extraction with the collected [)NA warm solution 5M NaCl saturated isopropanol several times. T E 11 CsC by dialysis against venom
1 was removed. Plasmid pTPA102 (Lys27
7-41Le) About IIIg was obtained, which was stored at 4°C.

Isolated plasmid pTPA102 (Lys277-” l1
e) Approximately 50 μg was added to 100 mM NaCl, 50 mM Tris-MCI (pH 7,5), 10 mM MgCh
, restriction buffer A consisting of 7mM 2-mercaptoethanol at 100μC. 3 depending on # position
Digestion was performed at 7°C for 2 hours. DNA was concentrated by ethanol precipitation and electrophoresed on a 0.8% agarose gel.

A 1974 bp Bgl II fragment (its DNA sequence is shown in FIG. 4) was visualized and recovered in the same manner as in Example 5A.

B, Ligation and E, Transformation of colt Dt+1 approximately 0.8 μg of pMI2Qo into restriction enzyme buffer at 40 μC, 1130 units at 37 °C for 2 hours.
Linear DNA was prepared by time digestion. The linearized plasmid was precipitated with 2.5 volumes of ethanol in the presence of 0.3 M sodium acetate, and the DNA was collected by centrifugation. DNA pellet, 94μ square of water, alkaline phosphatase solution (approximately 250$101; Takarashu Co., Ltd.)
1 μC and 1M Tris-HCl (p) 18.0)
Resuspended in 5 μm and placed at 37° C. for 1 hour. DNA
, a (1:1) mixture of phenol and chloroform and a (24:1) mixture of chloroform and isoamyl alcohol.
) mixture, precipitated with ethanol, and diluted with 20μ of water.
Ushichu (resuspended. This dephosphorylated pMI 2007
ail (approximately 200 ng) was added to 1974 bp raw 11 fragment 2 μi (approx.
330mM Tris-)ICI (pH 7,5), 33
11M MgC1z, 50mM 2-mercaptoethanol, 2.51M AYP] was added to 3[mu]C, T4[IN^ligase l[mu]C and water 2[mu]C. The reaction mixture was incubated at 14°C overnight.

In the same manner as in Example 5D, ε, coli D) 11 was transformed with the Ligacon mixture, and the resulting E,
calf DHI (pMI 205) transformants were identified by their ampicillin resistance phenotype and by analysis of their plasmid DNA with several restriction enzymes. From one of the transformants, plasmid pMI 205 was transformed into jIL! as in Example 3. i! Shita.

1凰shoj Plasmid pR5Vneo ATCC No. 3719
Approximately 13 μg of 8 was added to restriction enzyme ■ in restriction buffer at 50 μ℃.
■II! 30 units and restriction enzyme Bam HI 30
Digestion was performed at 37°C for 2 hours depending on the vehicle position. Digested plasmid DNA was run on a 1% agarose gel, and the gel was stained with ethidium bromide and visualized with long-wave UV light. The large) lindlll -Ram HI restriction fragment was excised and frozen at -80°C for 20 minutes. Freeze-thaw method (Thu
ring et al., 1975, Anal, Bioche
m, 66, 213).
IINA was collected by precipitation with 2.5 volumes of ethanol-0.3M sodium acetate and centrifugation. The pellets were dried in vacuo and resuspended in 20 AtfL of dry distilled water.

B, approximately 2.1 kb 1 of plasmid MT205 (ind
lI[-JLI of BamHT fragment! I! Approximately 30 μg of plasmid 9MI205 was incubated at 37°C with 30 units each of restriction enzymes III and MI in restriction buffer A.
completely digested. This resulted in two DNA fragments with sizes of 2.7 kb and 2.1 kb. DNA was precipitated with ethanol prior to running on a 1% agarose gel. The approximately 2.1 kb old ndlll-BamHI band was visualized, excised, and collected as in Example 5A.

C. Each of the fragments 1109n obtained in Ligation Examples 5A and 5B was treated with 66 mM Tris-HCl (pH 7.5), 6. '6
Reaction mixture containing mM MgCl□, 10 mM 2-mercaptoethanol and TP to 0.5.11M 20
During the μ-day, T4 DNA ligase (350,000 cells/m
Ligation was carried out at room temperature for 4 hours using 1 μC (1 L; Takara Shuzo Co., Ltd.).

D, E, Transformation of colt HBIQI) Using the resulting ligation mixture, v.

Simanis, ONA Cloning Volume 1, IR
L Breath, Oxford, District of Columbia (
Ampicillin 5
Plate containing 0 μg/n+JL (1% Bactodributon, 0.5% yeast extract, 0.5% sodium chloride, 1%
．． 5% agar, p) 17.5) HBIOI
was transformed.

Transformants were determined by their ampicillin resistance phenotype as well as by 2. Okb Raw I! Identification was made by analysis of restriction enzyme cleavage patterns of plasmid DNA, including the presence of fragments. Using the resulting cells, plasmid psT104 was isolated from -QL in the same manner as in Example 3.

X-hang Banhei plasmid 5T105N Otsuki A, 5V2dhfr approximately 0.9kb Bgl II
- Single plasmid psV2dhfr A with BamHI cleavage
Approximately 50 μg of TCG No. 37145 was added to 100 μg of restriction 111 buffer A, restriction enzymes BQ IT and BamH.
Complete digestion was carried out using 30 units each of I for 2 hours at 37°C. DNA was concentrated by ethanol precipitation and electrophoresed on a 1% agarose gel. A fragment of approximately 0.9 kb was visualized and recovered as in Example 5A.

B, Ligecon and E, colt) IBIO
Approximately 0.6 μg of hardened plasmid ps7104 in restriction buffer A40Jlf1. medium, 37 by 30 units of BamHI
Linear DNA was prepared by digestion for 2 hours at °C. The linearized plasmid was precipitated with 2.5 volumes of ethanol and 0.3M sodium acetate, and the DNA was collected by centrifugation.

The DNA pellet was mixed with 94μ of water, 1μ of alkaline phosphatase solution (250 units/mJL; Takara Shuzo Co., Ltd.) and 5μ of IM Tris-) ICI (pH 8,0).
A and placed at 37°C for 1 hour. DNA,
After extraction with a (1:1) mixture of phenol and chloroform and a (24:1) mixture of chloroform and isoamyl alcohol, it was precipitated with ethanol and resuspended in water at 20 μC. This dephosphorylated psT1047 u fl
, (approximately 200 ng) of BBI I obtained in Example 6A
I-Bam old fragment 2μft (approx. 10100n, 5x
Ligation buffer (containing TP) 3μ℃, T4
DNA ligase was added to a mixture of 1 μC and water at 2 μC. The reaction mixture was incubated at 14°C overnight.

E. coli HBIOI was transformed with the ligation mixture in the same manner as in Example 5D, and the resulting transformant E. coli HBIOI (psT10
5) were identified by their ampicillin resistance phenotype as well as old ndlll-BamHI restriction enzyme analysis of their plasmid DNA. Plasmid psT105 was isolated from one of the transformants in the same manner as in Example 3.

C, Isolation of psT105 Narl-5acT fragment Approximately 10 μg of plasmid psT105 was treated with 19 mM t-Lis-)ICI (p)17.5), 10 mM MgCh
, $11 Limited Paper Street Solution C50μ containing 1mM DTT
In fi, 20 units of restriction enzyme NarI and 5 acl
Complete digestion was carried out at 37° C. by 10 units. The digested plasmid DNA was run on a 1% agarose gel, the gel was stained with ethidium bromide, and the DNA bands were visualized under long-wave ttV light. The large Narl-5acl fragment was excised and recovered as in Example 5A.

D, approximately 0.9 kb NarT- of plasmid TPA21
Approximately 10μ of plus pTPA21 containing the DNA sequence encoding the 5acI fragment Karakori native t-p^ and part of the 3°-noncoding region.
g was completely digested with 20 units of restriction enzymes NarI and 110 units of 5ac in restriction buffer C at 37°C. After concentrating the DNA by ethanol precipitation, it was applied to a 1% agarose gel. Approximately 0.9 as in Example 5A
The kb NarI-5acI band was visualized, excised, and collected. (The DNA sequence of the Narl-5acl fragment is shown in Figure 8).

E, ligation and E, coil HBOI
About 11 of each of the above fragments obtained in Examples 6C and 6Ill in the form of
00n was added to T 4 DNA ligase (350,000 units/mu; Takara Shuzo Co., Ltd.) in a 2oμp reaction mixture.
The mixture was ligated for 4 hours at room temperature. Using the ligation mixture, as in Example 5D, the bivalent HB
IOI was transformed. Transformants were identified by their ampicillin resistance as well as restriction enzyme analysis of plasmid DNA. Using the obtained cells, plasmid psT105N was produced in the same manner as in Example 3! # Released.

After pre-incubation of 20 μg of psT105N in Restriction Buffer A at 50 μC with 15 units of restriction enzyme in order to achieve only partial digestion as examined by gel electrophoresis analysis. Digested for 4 minutes at 37°C. Concentrate the DNA by ethanol precipitation and add 150mM NaCl,6
mM Tris-HCl (pH 7,9), 6mM MgC
12, 6mM 2-mercaptoethanol and 100
Restriction enzyme 5alI containing μg/mfl BSA
Resuspend in buffer at 50 μC and add 20 units of 5alI.
added. The reaction was carried out at 30°C for 2 hours. DNA was concentrated by ethanol precipitation and 30mM sodium acetate (p
H4,6), 50mM NaC11mM ZnC1z
and resuspended in mung bean nuclease buffer containing 5% glycerol. The reaction mixture was incubated at 37°C for 30
After incubation for minutes and extraction with a (1:1) mixture of phenol and chloroform and a (24:1) mixture of chloroform and isoamyl alcohol, the IIINA fragments were precipitated with ethanol and resuspended in water at 20 μC.

B. Approximately 50 ng of the above DNA fragment obtained in Ligation Example 7A was added to 66 m
M)-Lis-)1(:l (1187,5), 6.6m
Reaction mixture 2 containing M MgCL , 10 mM 2-mercaptoethanol and 0.5a+M ATP
Self-ligation was carried out for 4 hours at room temperature using 350 units of ligase (Takara Shuzo Co., Ltd.) in Jju.

C,E, Colt 88101 liver transfer I Using the above ligation mixture, as in Example 5D,
The HBOI was transformed twice.

Transformation mixture with ampicillin 50μg/ml! Containing I
, plated. Transformants were identified by their ampicillin resistance phenotype as well as live 11-5alI restriction enzyme analysis of their plasmids. Plasmid psT106 was isolated from one of these transformants in the same manner as in Example 3.

Example 8 Plasmid pdBPV-MMTneo ATCC No.
Approximately 1 μg of 37224 was added to 8 am HI buffer for 7 nights (150
mM Na(:1, 6mM Tris-H(:1,
pt(7,9,6mM JCh) in 50 u fL;
Restriction enzyme: Complete digestion was carried out at 37°C using HIIO.

Ban H1 digested pdBPV-MMTn[l was precipitated with ethanol and resuspended in water at 20[mu]C.

B, ligation and E, calf) 1B+0
Transformation of about 1 μg of DNA obtained in Example 8A above was transformed into Example 5C.
Self-ligated in the same way. E, colt as in Example 5D using the ligation mixture.
) 18101 was transformed. Transformants were identified by their ampicillin resistance phenotype and restriction analysis of plasmid DNA. Plasmid pMMTneo was isolated from one of the transformants in the same manner as in Example 3.

Example 9 Approximately 5ug of plasmid pMMTneo was engineered! IMi venom (150[QM NaCl, 10mM f-Lis-HCl
, p) 17.5.10mM MgCl2.10mM2-
Mercaptoethanol) 100 μm medium, restriction enzyme engineering 11
Complete digestion was performed at position 504 at 37°C. DNA was precipitated with ethanol.

B. Klenow blunt-ending of BgI IT terminus.
mMt-Lis-HCI (pH 7,2), 10mM MgS
O4, 0.1mM DTT, 50 ttg/
muBs8, 0.5 mkldATP, 0
．． Klenow fragment 1 in a mixture of 5mM dGTP, 0.5mM dCTP and 0.5mM TTP! #
The ends were made blunt depending on the position. The reaction mixture was incubated at 20° C. for 30 minutes and then the reaction was stopped by heat inactivation of the enzyme. DNA was recovered by ethanol precipitation.

C. Isolation of MMT promoter-containing fragment Above Example 9B
The DNA obtained with restriction buffer A50, uJ! The mixture was digested with 50 units of restriction enzyme Ram Hl at 37°C for 1 hour. The DNA was concentrated by ethanol precipitation to 4.3 kb.
JILi fragments were collected in the same manner as in Example 5A.

D, 3.0kb t-PA cDNA
Approximately 10 μg of isolated plasmid psT108 of Hin
d III buffer (60mM NaC1, 7mM Tris-H (:l, pH 7,5, 7mM MgC12)
Complete digestion was carried out at 37°C with 1100 units of restriction enzyme old ndll in 200μj2. The DNA was ethanol precipitated and blunt-ended as in Example 9B. DNA was recovered by ethanol precipitation and added to Baff1) II buffer 501. Resuspend in tl, restriction enzyme Bam HI2
Digestion was performed at 37°C for 1 hour in the zero position. Concentrate the DNA by ethanol precipitation for approximately 3. Fragment of Okb as in Example 5A! #Removed and collected.

E, ligation and E, colt HBOI
Each of the fragments obtained in Examples 9C and 9D't' above was about 11
00n as T 4 DNA in reaction mixture 207111
Ligation was performed using ligase 1754 for 4 hours at room temperature. The ligation mixture was used to transform E. coli HBIOI as in Example 5D.

Transformants were identified by their ampicillin resistance phenotype and restriction enzyme analysis of plasmid DNA. Using the obtained cells, plasmid psT112 was isolated in the same manner as in Example 3.

Approximately 5 μg of plus psT112 and limited Pi poison solution 5
20 each of restriction enzymes Baff1 old and 5all in 0μu
Double digested into 20 single cells. After phenol-chloroform extraction, DNA was precipitated with ethanol.

! The lam Hl-5al I digestion product was blunt-ended with 2 units of DNA polymerase ■ Klenow fragment as in Example 9B. DNA was recovered by ethanol precipitation. Approximately 1100 n of blunt-ended DNA was self-ligated as in Example 5C. E, as in Example 5D using the ligation mixture.
colt HBIOI was transformed. Transformants,
Their ampicillin resistance phenotype and plasmid DNA
It was identified by restriction enzyme analysis of A. From one of the transformants, plasmid psT118 was generated in the same manner as in Example 3.
was isolated.

The plasmid psT105N, 5 mM MgCh, 2
mM 2-mercaptoethanol and 0. O1%BSA
5IIIM Tris) ICI containing (pH 7,1)
Restriction enzyme Bbel (Takara Shuzo Co., Ltd.) at 10μ℃
Digestion was carried out at 37°C for 4 hours at about 54°C. DNA was recovered by ethanol precipitation, 10 mM MgC1z,
10mM 2-mercaptoethanol and 0. O1
10mM Tris-)IC1 (pH 7) containing %BSA
, 5). Suspended [lNA, restriction enzyme
Digestion was performed at 37° C. for 5 hours with maI at 20. The reaction mixture was extracted with phenol-chloroform (1:1),
The DNA in the aqueous layer was dissolved in 3M sodium acetate (pH 5,2).
．． 1 volume and 2 volumes of ethanol. The recovered DNA was washed with 75% ethanol, dried in vacuum,
Dissolved in TE buffered venom μ°C.

B. Ligation and E. Transformation of coll JM103 Approximately 1 μg of M13mp19 RF DNA (available from Pharmacia) was digested with the restriction enzymes Bbel and xIIlaI as in Example 11A. BbeI
-XmaI DNA fragment end 110 fragments total 1100n) -ris-)
, 20 mW I) Ligation VI consisting of TT, t mM ATP and 50 μg/mfL BSA
psT105 using iT4 DNA ligase (Takara Shuzo Co., Ltd.) at 350 degrees Celsius in AjM solution at 20μ℃.
1 μg of the BbeI-XmaI fragment of N was ligated overnight at 4°C. The ligation mixture was used to transfect competent E, coII JM103 (available from Pharmacia) cells, and the transfectants were transfected with the substrate X-gal (5-bromo-4-chloro-3-indolyl- β-galactoside) and their colorless plaque phenotype and their phage RF DN
It was identified by restriction enzyme analysis of A. The resulting phage M13mp19fu7 was isolated from the transfectant and used to isolate M13mp19fu7 RF DNA and single 1iDN^.

E, overnight culture of coiled JM103 (this overnight culture was inoculated from a minimal medium stock to ensure retention of ProAB-bearing F shrimpsomes) 1
M
A single plaque of 13mp19fu7 was taken. The culture was incubated at 37°C with shaking for 6 hours. Cells were removed by centrifugation and the phage-containing supernatant 111Qfl was diluted with 2XYT broth (Bactodributon 16
g/u, NaC110g/J2, pH7,2-7,4)
Overnight culture of 400m flooded Oyohitan, Kasagai JM103 4
mJ2 was inoculated. The culture was incubated for 6 hours with shaking at 37°C, 8,900g, 100g at 4°C.
Centrifuged for minutes. M13mp19fu7 from Beret
Phage RF DNA 1. single-stranded DNA can also be isolated from the supernatant.

B, Isolation of M13mp19fu7 RF DNA Infected cell pellets were treated with 5011 IM glucose containing 5 mg/mu lysozyme, 25 mM Tris-HCl (p
na, o), 8 of 10 mM EDTA (pH 8,0)
Resuspended in mj2 and left at room temperature for 5 minutes. next,
1611IJi in 0.2N NaOH and 1% SDS (
was added to the Lysozyme 38-embedded cells, and the solution was mixed by inversion. The mixture was incubated on ice for 10 minutes. Add water-cooled 5M potassium acetate (pH 4,8) to the lysed cell mixture.
Added 0 ml. The solutions were mixed by inversion and incubated on ice for 10 minutes. The 5M potassium acetate solution is made by adding 28.5 mfl of water and 60 mj of 5M potassium acetate.
2 by adding 1.5 mfl of glacial acetic acid. The resulting solution is 3M for potassium and 5M for acetate.

The lysed cell mixture was prepared using Tommy Seiko no.

20 in a 4N-II (or equivalent centrifuge)
Centrifugation was performed at 4°C for 20 minutes at 000 rpm. Cell DN
A! 5 and debris formed a pellet on the bottom of the tube. 35 mJ2 of supernatant was collected, 6 volumes of isopropanol were mixed with this supernatant, and the resulting solution was left at room temperature for 15 minutes. Plasmid DN^ was harvested by centrifugation at 11.000 g for 15 minutes at room temperature. The supernatant was discarded and the DNA pellet was washed with 70% ethanol at room temperature. The ethanol wash was decanted and the pellet was dried in a vacuum dryer.

The pellets were then resuspended in 3.8 ml of TE buffer.

To this DNA solution was added 14 g of CsC and 648 μC of a 10 mg/mfl ethidium bromide solution in TE buffer. Transfer the solution to a Beckman VT165 centrifuge tube, fill to the top, seal, and incubate at 50,000 rpm at 20°C.
The mixture was centrifuged for 12 hours. After centrifugation, the lower DNA band of the two bands visualized in long-wave UV light was collected using a syringe with a #21 hypodermic needle inserted along the side of the centrifuge tube. Ethidium bromide, 5MN
It was removed by extraction four times with aC1 saturated isopropanol.

The aqueous layer was dialyzed overnight at 4°C against 2°C TE buffer consisting of 10mM Tris-)ICfl(987,5) and 1mM EDTA. Obtained M13mp19fu7
The FR DNA solution was used in subsequent examples.

C, Isolation of M13mp19fu7-gi[)NA Culture supernatant obtained in Example 12A 400 tn, Q, 20%
8 of polyethylene glycol and 2.5M NaCl
Added 0m12. The solution was shaken and allowed to stand for 1 hour, 89
Centrifuged for 30 min at 00g, 4°C. Discard the supernatant and add pellets to 10mM)-Lis-○CI (pi47
．． 5) and 20 mIL of 1 mM EDTA.
Shut up. Dissolved phenol in TE buffered toxin 20mj2
The mixture was extracted for 10 minutes at 9000g and centrifuged at 20°C for 10 minutes.

The aqueous layer was collected and precipitated by adding 3M sodium acetate (pH 5,2) and 50 mj2 of ethanol.The recovered M13mp19fu7-end strand DNA was washed with 75% ethanol, dried in vacuo, and added to 1 ml of TE buffer. Resuspend.

1ri M13mp19fu7 RF DNA 20ug, 6
6mM Tris-1 (CI (pH 7,4) containing 200mM MgCh, 6IIIM 2-mercaptoethanol and 100 ttg/tall BSA
4 at 37°C with 150 units of restriction enzyme 5ac in μ°C.
I spent time. DNA was recovered by ethanol precipitation,
A 7.9 kb fragment was isolated by gel filtration using Sephacryl S-1000 at 10 mjZ. 7.9kb 5
Eluates containing the acI fragment were identified by 0.8% agarose gel electrophoresis, collected, and ethanol precipitated. This DNA was resuspended in 50 μL of water.

M13mp19fu7-end strand DNA 60 μg and M1
3. 7.9kb 5acI fragment of mp19fu7 20μ
g were mixed and precipitated with ethanol. mixed DNA,
Resuspend at 200μC in 150mM NaCl and 15mM sodium citrate. This DNA solution was boiled for 7 minutes, transferred to a 65°C water bath for 10 minutes, and cooled with water. The resulting gap DNA was isolated by gel filtration using Sephacryl S-1000. Eluates containing gap DNA were identified by 1% agarose gel electrophoresis and ethanol precipitated. TEiJ gap DNA
It was resuspended in 50 μA of tl solution and used as a template in the next mutagenesis example.

Part B5, snow sequence 5'-CATGAG(:CTCCTCGAGC(
:GCTCCGAATAGAAAGGAGAC^^GG
CCT(:ATGCTCGCCGTAGCC-3° (
61mer) was synthesized.

This synthesis was performed using a DNA synthesis apparatus (Applied Biosci, Stems, model 381A), and the oligonucleotide was purified by HPLC. This is the 41st
It was designed to replace both lysine residues at positions 5 and 429 with two glutamic acid residues to create an additional restriction enzyme site.
could be easily detected.

2μg of 61M oligonucleotide was added to 10mM MgC.
70 mM t-Lis-HCl (pH 7,6) containing 12 and 5 mM dithiothreitol ([1TT) 2
Phosphorylated in 0 μu with 10 units of T4 polynucleotide kinase for 1 hour at 37°C.

1100n of gap DNA and 20 ng of phosphorylated 61-mer oligonucleotide were combined with 5
Annealed at 65°C for 7 minutes in 11M Tris-)101(p)17.5) 10μ°C, then cooled to 4°C. The annealed DNA was treated with 80 mM KCI, 3
0mM Tris-HCI (pH 7,5), 15mM
MgCh, 2mM DTT, 0.5mM ATP.

The cells were embedded with 50 nM of each of the four deoxyribonucleotide trisphosphates at 40 .mu.C using 1 unit of DNA polymerase ■Klenow fragment and T4 DNA ligase at position 1004. The mixture was incubated at room temperature for 4 hours and used to transform competent E-, colt JM103. Phage RF DNA was prepared from 48 of the transfectants and digested with restriction enzyme Xhol. Using a phage containing an XhoI restriction enzyme site in its RF DNA, M13mp19fu
8 RF DNA l-! lit, ta.

20 μg of M13mp19fu7 was added to 6mM NaC.
6mM containing 1, 6mM MgCh, 6mM 2-mercaptoethanol and 100 μg/m BSA
)-Lis-)ICI (pH 7,4) Digested in 200[mu]C with 50 units of restriction enzyme history I for 3 hours at 37[deg.]C. 5M NaC 14μ℃ and restriction enzymes! 50 ml was added to the reaction mixture and the mixture was incubated at 37° C. for 3 hours. DNA was recovered by ethanol precipitation and purified by gel filtration substantially according to the procedure of Example 13A.
The kb5caI-hepal fragment was isolated.

-Book 6-I M l 3 rap 19 fu 7
and M13mp19fu7 7.8kb 5caI-A
Example 13 Gap DNA substantially from paI fragment
Prepared and isolated according to procedure A.

B. Tokenchisen and Tachimatatatansho DNA Synthesizer (Abride Biosystems, Inc., 3)
81 A type) to synthesize oligonucleotides, and H
Purified by PL (: sequence 5°-CAAGGCC
TCATGCTCG(:CGTAGCCGGAAAGC
Using this oligonucleotide primer with TCACACTCCGTC-3° (43 bases), natural t-P
Substitution of glutamic acid residue at position 415 of A
Nota. Oligonucleotide-directed mutagenesis using this 43-base oligonucleotide and the gap DNA described in Example 14A was performed substantially according to the procedure of Example 13B.

Using the resulting mutagenesis mixture, E. coilJM
103 was transfected, and the desired mutant strain was identified by restriction enzyme mapping. M13mp19st105 RFDN prepared substantially according to the procedure of Example 12
One of the two 5acl restriction enzyme recognition sites was absent in A.

Example 15 IL isolation of fragments Approximately IQμg of M13+np19fu8 RF DNA was
5mM MgCl2.2mM 2-mercaptoethanol and 5mM containing 100μg/mA BSA
Digestion was performed with 25 units of restriction enzyme BbeI in 50 μA of Tris-HCI (pH 7,1) at 37° C. for 4 hours. D.N.
A was recovered by ethanol precipitation and 10 mM Mg
Ch, 8mM 2-mercaptoethanol and 100
10mM)-Lis-○CI (p)17.5) containing μg/IIIL of BSA and resuspended at 50 μC.

The suspended DNA was digested at 37°C with the restriction enzyme SmaI at 25°C.
Digested for 4 hours. The digested DNA was concentrated by ethanol precipitation and then applied to a 1.0% agarose gel containing 0.5 μg 7 ml ethidium bromide. 1189
The bp BbeI-5maI band was visualized under long-wave UV light, cut out, and frozen at -80 °C for 20 min. T
huring et al., 1975, ^na1. Bioche
DNA was eluted from the gel fragments by the freeze-thaw method described in J. M., 66, 213, and 0.3 M sodium acetate (pH 5
, 2) with 2.5 volumes of ethanol. After centrifugation, the DNA pellet was washed with 75% ethanol, dried in vacuo, and resuspended in 10 mJZ of TE11 street solution.

B, 5.9kb BbeI- of plasmid psT118
5mal fragment! Approximately 10 μg of P-separation plasmid psT118 was added to 5 mM Mg.
Cl, 2mM 2-mercaptoethanol and 0.2mM 2-mercaptoethanol.
5mM Tris-HCl (pH
7,1) Restriction enzyme BbeI20 in 50 u, Q
Digestion was performed for 4 hours at 37°C. DNA was recovered by ethanol precipitation and treated with 10 mM Mg (:h, 6+n
M2-mercaptoethanol and 0. 10mM f-Lis-)ICI (p)I7.5 containing O1% BSA
) Resuspended in 50 μC. The suspended DNA was treated with the restriction enzyme SmaI20. Digested with iIL for 3 hours at 37°C. The digested plasmid DNA was run on a 1% agarose gel and the large Bbel-5mal fragment was excised and recovered as described in Example 5A.

about 110 each of the short negative fragments described in Examples 15A and 15B above.
0n each, 10mM MgCh, 10mM dithiothreitol, 1mM ATP and 50μg/rai
50 dt-Lis-HCl (pH
7,8) In 20+i, T4DN^ligase35
Ligation was performed using 0 units at 16° C. for 4 hours. Using a ligation mixture, substantially V, Simanis,
DNA (:loning, Volume 1, IRL Breath,
Plates containing 50 μg/mi ampicillin (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, p. 17) according to the transformation procedures of Oxford, Washington, District of Columbia, 1985.
．． 5) E. calf HBOI was transformed with the above.

Transformants were identified by their ampicillin resistance phenotype and restriction M element analysis of plasmid DNA. Using the obtained cells, substantially following the procedure of Example 3,
Plasmid pFu160 was isolated.

1091118 Construction of plasmid psT135 M13mp19st105 118 from RF DNA
Isolation of the 9bp Bbel-5maI fragment was performed essentially as taught in Example 15A.

B, ligation and E, colt) IBIO
About 110 pieces each of the fragments described in Examples 16A and 15B above
0n each, substantially according to the procedure of Example 15C.
Reigate. E using a ligation mixture substantially similar to Example 15CIC.

E. coli HBIOI was transformed.

Transformants were identified by their ampicillin resistance phenotype and restriction enzyme analysis of plasmid DNA. Using the obtained cells and substantially following the procedure of Example 3, plus 1195 cells and 135 cells were produced! # Released.

Example 17 Construction of plasmid psT145 A, +523 bp from plasmid 5T135 B
III-5maI bus Zdo psT135 approximately 10μg
, 100 mM NaCl゜10 mM MgCl2
．． 10mM 2-mercaptoethanol and 0.01%
10mM)-Lis-○CI containing BSA (pH 7,
5) Digested with 1120 units of restriction enzyme BQ at 37°C for 2 hours at 50μ°C. DNA was recovered by ethanol precipitation, 20 mM KCI, 6 mM M
gCl2.6mM 2-mercaptoethanol and 0
．． 10mM Tris-HCl containing 01% 8SA (
p) 18.0) resuspended in 50 u 11. Suspension D
NA was digested with the restriction enzyme SmaI for 4 hours at 37°C. The digested DNA was run on a 1% agarose gel and the 1523 bp fragment was excised and recovered as described in Example 5A.

Plasmid psT112 approx. 107. +g, 100 m
M NaC1゜10mM MgC1z, 10mM2-
Restriction enzyme LL! 10mM)-Lis-CI (pH 7,5) containing mercaptoethanol and 0.01% BS8 in a 50μ square. 2 at 37℃ by 1120 car position
The time-digested DNA was recovered by ethanol precipitation and
01M MCI, B mM MgCh, 6mM 2-
10mM) lis-)ICI containing mercaptoethanol and 0.01% BS^ (p) 18.0) 50
resuspended in u It. The suspended DNA was treated with restriction enzyme Sm
Digested with a120 units for 4 hours at 37°C. Digestion DN
A was run on a 0.8% agarose gel, and a 5.8 kb fragment was excised and recovered as described in Example 5A.

C, ligation and E, colt HBOI
1100 nm each of the fragments described in Examples 20A and 20B above.
each with 10 mM MgCh, 10 mM dithiothreitol, 1 mM ATP and 5 ug/ml of B.
50n1M Tris[1 (pH 7,8) containing SA
Ligate using 350 units of T4 DNA ligase in 20 u fL. Using a ligation mixture substantially in accordance with Example 15C, the HB10
1 was exchanged in the trait.

Transformants were identified by ampicillin resistance phenotype and restriction enzyme analysis of plasmid DNA.

Using the obtained cells, plasmid psT145 was produced substantially according to the procedure of Example 3! # Released.

DNA synthesizer (Applied Biosystems).

381A type) to synthesize oligonucleotides, and
Purified by PLC. Array 5°-GCCTCATG
CAAGCCGTAGCCGGAAAAGCTCACAC
Using an oligonucleotide primer having TC-3° (35 bases), the lysine residue at position 415 of natural t-PA was replaced with a leucine residue. Using this 35-base oligonucleotide and the gapped DNA described in Example 14A, M13mp19st1 was prepared in the same manner as in Example 13B.
28 RF DNA was obtained.

Example 1 from M13m919ST126 RF DNA
After digestion with BbeI and ±■ restriction enzymes as described in Section 5A, a 1189 bp [INA fragment was obtained. This DNA fragment 1
100n and Bb of psT118 prepared in Example 15B.
5.9kb DNA cut with eI-5maI restriction enzymes
NA fragment 1100n' is ligated as described in Example 15C. With this combined reaction solution,
is transformed by the method of Example 15C. After selecting the transformants for ampicillin resistance, plasmid DNA was extracted and the restriction enzyme cleavage pattern was analyzed to determine the plasmid ps.
Transformants with T180 were identified. Plasmid psT180 was isolated from this transformant by the method shown in Example 3.

DNA synthesizer (Abride Biosystems, Inc., 3)
81 A type) to synthesize oligonucleotides using)
IPLCl: was purified. Array 5°-GCCTC
ATGCTGGCCGTAGCCGGAAAGCTCA
Using an oligonucleotide primer having CACT-3° (34 bases), the glutamine residue was substituted for the 415-position lysine residue of natural t-PA. This 34 base oligonucleotide and the gap [l] described in Example 14A
M13+++p in the same manner as Example 13B using NA
19st128 RF DNA was obtained.

Example 1 from M13mp19ST128 RF DNA
After cleavage with Bbel and Sma I restriction enzymes as described in 5A, a 1189 bp DNA fragment was obtained. this DNA
Fragment 1100n and psT118 prepared in Example 15B
5.9 kb (
7) Combine DNA fragment 1100n using the method described in Example 15C. In this combined reaction solution, E, con
Transform HBLol using the method of Example 15C. After selecting the transformants for ampicillin resistance, plasmid DNA was extracted and restriction enzyme cleavage patterns were analyzed to identify transformants having plasmid ps7182. Plasmid ps was obtained from this transformant by the method shown in Example 3.
T182 was separated by 1L.

A, Culture of fully loaded Bi and Ranma hair L-929 cells (A-Ding CC# CCL, -1)
cultured with Dulbecco's modified Eagle's essential medium containing kanamanoicin and 10% (v/v) fetal bovine serum.
It was maintained at 37°C in a 5% GO□ atmosphere. These cells were plated at a cell density of 5 x 10' per 10 cm vetri dish the day before transformation to achieve 50-60% confluency on the day of transformation. Transformation 3
The medium was changed before the time. Two 10c++ Petri dishes of cells were used for each transformation.

B. Preparation of DNA Using plasmid DNA, Gorman method [DNA (: IonLngll, 143 (
1! L-929 cells were transformed by the calcium phosphate technique as described in [15], IRLpressl. Expression plasmid (1) FU 160 psT
145, psT180 or psT182) 30μ
g and plasmid ps2neo ATCC No. 371
49 3 μg was added to 186 μX of 2M CaCl2 and 1.5 μg of water.
3 mJ2 was added. DNA solution 1.5 mj2 2
X 1ees (1,63%NaC1,1,19%t
(epes, 0.04%Na2HP04pH7,12
) It was added dropwise to 1.5 mal while bubbling.

The mixture was left at room temperature for 30 minutes before contacting the cells.

Transfection of C1 Cells 0.6 mJZ of the DNA solution prepared in Example 22B was added to a 10 cm vetri dish containing L-929 cells with gentle stirring, and heated at 37°C in a CO2 stream for 18 hours.

Cells were i) washed twice with MEM. Complete fresh growth medium containing 10% FC5 was then added and the cells incubated at 37 °C in a CO2 atmosphere.
It was heated for 24 hours. Cells were trypsinized for 300
μg/ff1il of geneticin (geneti
cin.

G418) and 1:10 subculture in selection medium consisting of DMEM containing 10% FC5.

Cells expressing phosphotransferase (neo gene product) can survive in the selective medium and form colonies. The medium is changed every 3-4 days, and colonies are
Isolated after 2-14 days.

6418-resistant colonies were picked by gentle trypsinization in small cylinders, grown to culture clumps, and tested for secretion of mutant t-PA. , cells were grown to approximately 3 x 105 cells in multi-well plate dishes with a diameter of L7 cm containing 3 mal of medium. The medium was removed and washed with PBS. 0 cells
．． 04mM ZnSO4, 1mM Sodium Butyrate, 2%
1 ml of induction culture medium consisting of DMEM containing FC5
The mutant t-PA in the medium was cultured at 37°C for 24 hours.
Indirect absorption spectrometry using 2251 [Trombosi
sResearch 31, 427 (1983)].

Mutant protein ap415, ap41B-1, ap41
5-2 and ap41B-4 were each plasmid pFU160.95145.5-2 obtained in Example 22. psT1
80 or psT1112 was purified from the induction culture medium of L-929 clones transformed (transfected). The first step in purifying the mutant protein was to use a monoclonal anti-t-PA antibody-conjugated Sepharose 4B column (1,6c
m x 3 cm) [The monoclonal anti-t-p antibody (preparation thereof was described, for example, by Kais Tom et al., Throa+bosis Research) according to the manufacturer's instructions.
40.9l-99 (1985)) coupled to CNBr-activated Sepharose 4B]. column(
0.7 x 2.5 cm), 0.01% Tween 80
and 10 KILI/n+jl of aprotinin were equilibrated with 0, IM Tris-1 (CI).
' Washed with solution a. Detachment is 0. O1 containing 1% Tween 8o and 10KID/mA of aprotinin,
1M glycine hydrochloride (p) 12.5). The desorbed fraction was immediately treated with 1M Tris-)ICI (p) 19.0)
and those containing t-FA activity were pooled. The next step of purification was 0.05M Tris-HCl (p)18 containing 0.15M NaCl,O,005% Tween 80 and 10KIII/ml aprotinin.
．． Benzamidine Sepharose 4B (
Affinity chromatography on a Pharmacia column (0.45 x 3 cm). The fractions pooled in the first step were applied to a benzamidine Sepharose column. The column was washed with IMNaCl, 0.05M Tris-HCl containing 0.005% Tween 80 and 10 Ill/ml aprotinin (p) 18.0).
Wash with 10 column volumes and wash M! containing 1M arginine! ? This was done using Solution I. The fractions containing t-PA activity were pooled and 0.1M (NH4)2CO3,0,15
Dialyzed against MNaCl, 0.05% Tween 8o.

The yield of the mutant protein in this operation was determined by the Lowry method.

The results are shown in the table below.

Example 24 Plasminogen activator activity of mutant t-PAs ap418 and ap415-1 was determined by indirect spectrophotometry [
Thrombosis Research 31,42
7 (1983)]. Briefly, various amounts of each mutant t-PA were dissolved W in 50+ nM Tris-1 (C1 (pH 8,8)) containing 0.1 M sodium chloride and 0.01% (V/V) Triton X-100.
iman et al.'s method [(:lin, Chim, Act
a127, 279 (1983)], 0.2 μM human plasminogen (Green Cross, Osaka), 0.9 mM HD-Val-Leu-Lys.
-p-nitroanilide, 28CI (S-2251%H
The 0 reaction mixture was added to a mixture of AC:I (EM) and 70 ug/vaIt of solubilized fibrin and incubated for 3 hours at 37°C. Absorbance at 405 nm was measured using a microplate reader (Titertic Multiscan, Flow Laboratories, USA) in comparison to a blank without activator. With reference to the standard curve of natural t-P8 [international standard (WHO)], the amount of sminogen activator [international units (II) of t-PA] for mutant t-P was determined. The protein concentration of mutant t-PA was determined by the Lowry method. The results are shown in the table below.

(Left below) B, Plasminogen activator inhibitor (FAI) E
, endotoxin injection of colt LPS (1 μg/
kg) After 3 hours, FA was obtained from endotoxin-treated rabbits.
T-rich plasma was obtained. PAN in FAI-rich plasma
The level was 120 u/m Jl. 5p
The method of ei'ser et al. [Thrombos Res
, 44.503 (1986)] and improved the PA
The inhibition of PA activity of t-PA by I was measured. t-P
A 50μ color was added to 50μ square of buffer or FAI dilution to give a final t-PA concentration of 2 u/in. After 10 minutes of incubation at room temperature, each sample was
．． This was acidified by adding 80 μ2 of IM Acetate Buffer (p) 13.9) and incubated for 10 minutes at room temperature. Thereafter, assay Mi venom was added to the sample to give a final amount of 300 .mu.C. Using this sample at 20 .mu.C, residual activity was measured by indirect absorption spectrometry. The results are shown in the following table.

Expression plasmid pFI+160. obtained in the above example. p
ST145, psT180, and psT182 were each inserted into Escherichiacolt HBIOI, and the following transformed strains obtained were transferred to Kogyo 1-1-3 Higashi, Yatabe-cho, Tsukuba-shi, Ibaraki-ken, Japan 305, one of the international depositary institutions based on the Budapest Treaty. To the Institute of Microbial Technology (FBI), Agency of Technology, on June 23, 1988 and 1989.
It was deposited on June 14th.

(Margin below)

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the construction of a novel t-PA expression vector. FIG. 2 shows a restriction site/function map of plasmid pMI200. FIG. 3 shows the DNA sequence of the cDNA insert of plasmid 9MI200. Figure 4 shows plasmid pTA102 (Lys277-1
1e) shows the DNA sequence of the Bgl II DNA fragment (1974 bp). FIG. 5 shows a restriction site/function map of plasmid pMI205. FIG. 6 shows a restriction site/function map of plasmid psT104. FIG. 7 shows a restriction site/function map of plasmid psT105. Figure 8 shows the Narl-SacI fragment (~0.9kb+)
) shows the DNA sequence of FIG. 9 shows a restriction site/function map of plasmid psTIQsN. Figure 1O shows the restriction site/function map of plasmid psT10B. Figure 11 shows the restriction site of MMTneo.
A functional map is shown. FIG. 12 shows a restriction site/function map of plasmid psT112. FIG. 13 shows a restriction site/function map of plasmid ps'r1ta. FIG. 14 shows a restriction site/function map of M13mp19fu7 RF DNA. FIG. 15 shows a restriction site/function map of M13mp19fu8 RF DNA. FIG. 16 shows a restriction site/function map for M13mp19st105 RF ON. FIG. 17 shows a restriction site/function map of plasmid pFu180. FIG. 18 shows a restriction site/function map of plusyodo psT135. FIG. 19 shows a restriction site/function map of plasmid psT145. Figure 20 shows M13mp19st126 RF DNA
Shows the restriction site/function map of . FIG. 21 shows a restriction site/function map of plasmid psT180. FIG. 22 shows a restriction site/function map to M13mp19st128 RF DN. FIG. 23 shows a restriction site/function map of plasmid ps7182. FIG. 24 shows the DNA sequence and the corresponding amino acid sequence of the mutant t-PA cDNA encoding aP41B shown in FIG. 17. FIG. 25 shows the DNA sequence and the corresponding amino acid sequence of the mutant t-PA cDNA encoding aP415-1 shown in FIG. 19. FIG. 26 shows the DNA sequence of the mutant t-PA cDNA encoding aP415-2 shown in FIG. 21 and the corresponding amino acid sequence. Figure 27 shows the DNA sequence and the corresponding amino acid sequence of the mutant t-apa cDNA encoding aP415-4 shown in Figure 23. Figure 1 (1) Figure 1 (2) Figure 1 (3) Figure 3TTCAGAAGAGGAGCCAGATCTTACC
A8GTGATCCCCDhlalQal(1glya
l to a + gperly + Qlllval (IJI Figure 8 (2) Pro^spTrpThrGlucygGlu10 coRI Shisha 5nal Figure 24 (1) 51jT^^0GGACGCTGTI;^^CC^^T
CFigure 24 (2) Figure 24 (3) CCTC^^^^CC^^^TOAC+^τCCCGC
CTCT Ding C Ding TCTT＾rgPro Kushichu・Example＾＾＾＾To＾^＾CCATGTCTCAATAGT＾
^^^G^^^C^^C^-3' Fig. 25 (1) 51, -, TT^^, GGACGCTC, TG^^CC
^^TCSerNelleLeul 1eGIYLy
iYalTyrThrAlaGIn^5nPrOSer
81aGlnAla cut=ly Figure 25 (2) Figure 25 (3) CGACC to TGACCA QAACACCCGACT
CCTCAAAA to CAAA Ding GAGATCCC to CC
TCTTCTTCTTT＾rzPro中◆◆ To CAGAACCAC1'GCAAAGGCGCAcT
CCTTCTCTACAGACTTCDingCCAG8CC
CACCACACCGC8G^^CCGGGACGAG
ACCCchohachiCACOAOAGG(+^^CiAricho0
CATTTTCCCCACATACTTCCCATT^^
^^To TG^^GCATGTCTCA^Ding^G-Ding^^^
^G^^^C^^GAGATCT -3 Figure 26 (1
) 5- Ding T^^GGCACCCTII: TC to CC^
ToTC Figure 26 (2) Figure 26 (3) CCACCGT (iAccAGG^^CACCCGAC
TCCTC^^^^^GC^^^TGAGATCCCC
CTCTTCTTCTT^rgPro◆-◆ ■ CAC^^0ACACTGC^^^CGCCCAGTG
CTTCTCTACAGAC ding TC ding CCAG 8 CCC
ACCAC8C (CGGGACGAC to 20C〇^^
To ACCCTAC to GGAGAGGG^GAGTCCA
TTTTCCCAGATACTTCCCATTTTGG
AAGTTTTCAGCACTDingGGTCTGATTT
CAGG＾Ding＾CTCTCTCAOATGGOAACA
C8 TGAA Ding GCACACTA (iCCTCTCCA
にG^^TGCCTCCTCCCTGGGCAG^^G
To TGGCCATGCCACCCTGTTTTCGC?
,,l ccec＾＾CCTCCdingGACCYGTCA
CCGTGACC人GCTTTGGG^^8CAGGAC
To CAC^^^^Ding C^^^^OC Jinding GTCTC^8T
Enter AGT^^^To G^^C^^G^-3 Figure 27 (1) 5 - AAGCA to TTAAGGCACGCTGT
ATC Figure 27 (2)

Claims (1)

[Claims] 1) Natural or other mutated t-PA in which Lys^4^1^5 is replaced by another amino acid
t-PA, which is different from t-PA. 2) t-PA according to claim 1 having the following amino acid sequence: R^1-A^2^-^4^1^5^-R^2-A^4^
2^6-R^3-A^4^3^0^-^5^2^7(
I ) (where R^1 is Ser- or GlyAlaAr
gSer-, R^2 is -Glu-, -Gln- or -
Leu-, R^3 is -Lys- or -Glu-, A^2^-^4^1^5 is the same amino acid sequence as Tyr^2 to Gly^4^1^5 of natural t-PA, A ^4^1^7^-^4^2^5 is Hi of natural t-PA
Same amino acid sequence as s^4^1^7 to Leu^4^2^8, A^4^3^0^-^5^2^7 is Gl of natural t-PA
It shows the same amino acid sequence as u^4^3^0 to Pro^5^2^7. ) 3) R^1 is Ser-, R^3 is -Lys- or -G
lu-, R^2, A^2^-^4^1^5, A^
4^1^7^-^4^2^8 and A^4^3^0^-
t-PA according to claim 2, wherein ^5^2^7 are each as defined in claim 2. 4) DNA encoding t-PA as defined in claim 1. 5) DNA encoding the amino acid sequence of t-PA as defined in claim 2. 6) An expression vector containing the DNA defined in claim 4. 7) An expression vector containing the DNA defined in claim 5. 8) A host cell transformed with the expression vector defined in claim 6. 9) A host cell transformed with the expression vector defined in claim 7. 10) A claim characterized in that a host cell transformed with the expression vector containing the DNA encoding t-PA of claim 1 is cultured in a medium, and the produced t-PA is collected from the culture. 1. Method for producing t-PA. 11) A host cell transformed with the expression vector containing the DNA encoding the amino acid sequence of t-PA of claim 2 is cultured in a medium, and t-PA is extracted from the resulting culture.
3. The method for producing t-PA according to claim 2, which comprises collecting t-PA. 12) A pharmaceutical composition comprising the t-PA of claim 1 and a pharmaceutically acceptable carrier.