JPH0286777A - Siminan pancreatic elastase i - Google Patents

Abstract

PURPOSE:To obtain a siminan pancreatic elastase I, consisting of a special amino acid sequence, capable of specifically hydrolyzing elastin accumulated in blood vessels and expected to be useful as a preventive or treating agent for arteriosclerosis. CONSTITUTION:Animals producing elastase I in the pancreas in animal of species related to humans are searched for to mass-produce the elastase I hardly present in human pancreas. As a result, it has been found that production is carried out in Macaca irus. It can be clarified that the amino acid sequence of the simian pancreatic elastase 1 is expressed by the formula. The simian pancreatic elastase 1 is mass-produced by using the amino acid sequence according to genetic engineering.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a DNA encoding monkey pancreatic elastase 11, a recombinant DNA expression vector containing the DNA, and a host transformed with the recombinant DNA expression vector. It is.

The present invention makes it possible to obtain a large amount of monkey pancreatic elastase I that specifically hydrolyzes elastin accumulated in blood vessels, and is expected to be used as an agent for preventing or treating arteriosclerosis.

Prior Art Elastase is a type of serine protease, an enzyme that hydrolyzes elastin, a fibrous insoluble protein. Elastin is a component of the connective tissue, key, aortic integument, and cervical cord of higher animals, and is slightly degraded by pepsin and trypsin.

In the process of researching arteriosclerosis, Balo' et al. discovered the decomposition of elastin fibers in blood vessel walls, and in 1949 they deduced the existence of an enzyme that decomposes the elastin fibers. (Ba1o', J e and
Banga, I*: Sehweiz Z, P
Athol e Baeteriol m, 12゜3
50 (1949) ml, followed by Banga et al.
In 2012, he discovered an enzyme in the pancreas that specifically degrades elastin, purified it into crystalline form, and named it elastase.
Banga, I*: Acta
Phyaiol jar Acad *Set
, Hung.

3.317 (1952)).

Elastase has been confirmed to exist in the pancreas of most animals, including humans, monkeys, cats, and domestic rabbits. In addition, a correlation between human elastase activity and age has been confirmed, with elastase activity in the pancreas and plasma significantly decreasing in men over 40 years old and women over 60 years old [Loev
@n, W, Aa, andBaldwin
, M.M.:Garontologia, 17
, 170 (1971)).

In patients with atherosclerosis, pancreatic elastase activity is
It was significantly decreased or disappeared compared to that of healthy subjects (Ba1o', J, and Bang
a, I, :Nature, 178,310(1
956)].

The pharmacological effects of elastase have been studied using rats, rabbits, etc., and the following results have been revealed.

1) Inhibitory effect on lipid and calcium deposition on arterial walls.

2) Removes cholesterol and calcium from arterial walls.

3) Selective decomposition effect on denatured elastin.

4) Promotion of new generation of arterial wall elastic fibers.

5) Serum lipid lowering effect.

6) Lipoprotein metabolism improvement effect.

In a clinical study conducted based on the above research results, the following effects were found as a result of oral administration of porcine elastase.

1) Restoration of elasticity and extensibility of the arterial wall.

2) Improving effect on serum lipid abnormalities.

3) Improving effect on lipoprotein metabolism.

In the above study, elastase extracted from pig pancreas was used. 2. There are two types of elastase (elastase ■ and n) in the pancreas (
Bieth, J.: Front Matri.
x Blot m.

6.1 (1978)). The porcine elastase used here was a mixture of elastase (■) and (2), but most of it was elastase (■).

Previously, the present inventors used recombinant DNA technology to extract human elastase ffA and n, which are present in the human pancreas.
B (JP-A-62-130686 and JP-A-62-29
977) and human elastase II [A and I
[U (JP-A-62-29976 and JP-A-62-17
No. 5173) was obtained in large quantities.

Furthermore, human elastase I, which corresponds to porcine elastase I, is almost absent in the human pancreas (
Tan1, T, et, al, :J, Bloch
Em,.

101.591 (1987)) and Toyoshi obtained a large amount of human elastase I using a combination of DNA synthesis technology and recombinant technology (Japanese Patent Application Laid-Open No. 62-253381).
issue).

Problems to be Solved by the Invention The present inventors searched for an animal species that is evolutionarily closely related to humans and that produces elastase I in the pancreas. As a result, it was revealed that elastase I is produced in the pancreas of cynomolgus monkeys. Therefore, the present inventors used recombinant DNA technology to clone the monkey pancreatic elastase I cDNA, transferred the obtained recombinant DNA molecule into a suitable host, and expressed the monkey pancreatic elastase I cDNA. The present invention has been completed as a result of research and development of a technology that can obtain the desired elastase (2).

That is, the present invention provides monkey pancreatic elastase (1), a DNA encoding said monkey pancreatic elastase (2), a recombinant DNA expression vector containing said DNA, and said recombinant DNA.
This relates to a host transformed with the A expression vector.

The present invention is based on the general formula (■): (N)-Va 1-Va l -G 1 y-Gly
-Thr-Glu-Al a-Gly-Ar g-
As n-3ar-Trp-Pro-8ar-Gln-
11e-8er-Leu-Gin-Tyr-Leu-8
er-Gly-Gly-8er-Trp-Tyr-Hl
s-Thr-Cys-Gly-Gly-Thr-Leu
-11e-Arg-Gln-Asn-Trp-Val-
Met-Thr-Ala-Ala-Hia-Cys-V
al-Aap-8et-Pro-Lys-Thr-Ph
e-Arg-Val-Val-Val-Gly-Aap
-Hls-Asn-Leu-8er-Gin-Asn-
Asp-Gly-Thr-Glu-Gin-Tyr-V
al-8ar-Val-Gin-Lye-11a-Va
l-Val-Hls-Pro-Tyr-Trp-Aan
-8er-AIIn-Asn-Val-Ala-Ala
-Gly-Tyr-Asp-11e-Ala-Leu-
Leu-Arg-Leu-Ala-GIn-8er-V
al-Thr-Leu-Asn-8er-Tyr-Va
l-Gln-Leu-n-3ar-Trp-Pro-8
ar-Gln-11e-8er-Leu-Gin-Ty
r-Leu-8er-Gly-Gly-8er-Trp
-Tyr-Hls-Thr-Cys-Gly-Gly-
Thr-Leu-11e-Arn■-Gly-Gln-
Leu-Ala-Gin-Thr-Lau-Gin-G
ln-Ala-Tyr-Lau-Pro-8er-Va
l-Asp-Tyr-Ala-11e-Cys-8er
-8er-9er-8er-Tyr-Trp-Gly-
8er-Thr-Val-Lys-Asn-Thr-M
et-Val-Cys-Ala-Gly-Gly-As
p-Gly-Val-Hls-8er-Gly-Cys
-Gln-Gly-Asp-8er-Gly-Gly-
Pro-Leu-Hls-Cys-Leu-Val-A
in-Gly-Lys-Tyr-8er-Val-Hi
g-Gly-Vat-Thr-8er-Phe-Val
-8er-Lys-Gin-Gly-Cys-Asn-
Val-8er-Arg-Lys-Pro-Thr-V
al-Phe-Thr-Arg-Val-8er-Al
a-Tyr-11e-8er-Trp-mouthe-Asn-
Lys-Thr-11e-Ala-8sr-Asn-(
C) This relates to a DNA encoding monkey pancreatic elastase I or its equivalent, and the monkey pancreatic elastase I or its equivalent, which consists of the amino acid sequence represented by C).

In particular, in formula (I), the (N) terminal is a hydrogen atom, Me
t, or (N)-Th r -G 1 n-As p-Phe-
]'r o-Glu-Th r-Aa n-Ala
-Ar g-(C),(N)-Met-Thr-Gln
-Aap-Phs-Pro-Glu-Thr-Aan-
Ala-Arg-(C) or (N)-Me t-L
eu-Arg-Ph e -Leu -Va 1-P
he-Al a-Th r-Leu -Va 1-Le
u-Tyr-Gly-Hl a-8o r-Th r
-Gl n-As p-Phe-Pro-Glu-
Monkey pancreatic elastase I or an equivalent thereof having a part or all of a polypeptide represented by any one of Thr-As n-Ala-Arg-(C), and the monkey pancreatic elastase I or an equivalent thereof It concerns DNA that codes for things.

Particularly preferred (5')-GTA GTG TCA TGG CTG TCT GGA GGG ATG ACA AAG ACT AACCTG TACGTG CCA TAC GGCTAT CAG AGC CTG GGT CTG GCT GGCTGG CTG GC C CCCTCT TCCTCC ACCATG CACTCC is the general formula (■): GGA GGG ACT GAGCCCTCT
CAG ATT GGA GGT TCCTGG ACCCTCATCAGA GCT GCT CACTGC TTCCGCGTG GTG AGCCAG AACGAT AGT GTA CAG AAGTGG AA
CAGCAAT GACATCGCCCTG GTT ACCCTCAAT GTT CTG CCCCAG AACGACAGT CCC GGCAGG ACCAAG CAG ACCCTG CAG GTG GACTACGCC TACTGG GGCTCC GTG TGT GCT GGTGGA TG
CCAG GGT GCCGGG AGG TCT CTCCAG TAT CACACC CAG AACTGG GTG GAT TCC GTT GGA GAC GGCACCCAG ATCGTG GTG AACGTG GCT CTG CGCCTG A GCTAT GTC GAG GGA GCC TGCTACATC ACCAAT GGG CAG GCT TAT ATCTGCTCC ACT GTG AAG GGA GAT GGA GAT TCT GGG AAT AAC ACC CTC CCC AA T CAC AAT CCC CCC CAC ATC ACA CAC CTC ACC AAC GTT GT CCCCTCCAT TGCTTG GTG AAT
GGCAAG TATTCT GTCCACGGA G
TG ACCAGCTTT GTG TCCAAG C
AG GGT TGT AAT GTCTCCAGG
AAG CCTACA GTCTTCACCCGG
TCTCT GCT TACATTTOOTGG AT
A AAT AAA ACCATT GCCTCCAA
CX -(3') (wherein, X represents TAA, TGA or TAG)
It relates to DNA containing the base sequence represented by or a base sequence with the same effect as
At the end, ATG or (5')-ACCCAG GACTTT CCG G
AA ACCAACGCCCCGC-(3'), (5'
)-ATG ACCCAG GACTTT CCG
GAAACCAACGCCCGC-(3') or (5
)-ATG CTG CGCTTCCTG GTG
TTCGCA ACOCTG GTCCTT TAT
GGACACAGOACCCAG GACTTT C
CG GAA ACCAACGCCCG-(3')
Particularly preferred are polynucleotides containing part or all of the polynucleotide represented by any one of the following.

When the DNA of formula (It) has ATG at the 5' end, it encodes a polypeptide of formula (I) that has Met at the (N)-terminus.

Formula (II) (7) DNA has 5' end K(5')-A
ce CAG GACTTTCCG GAA ACCA
ACGCCCGC-(3'), (N)-Thr-Gln-As at the (N)-terminus of formula (1)
p-Phe-Pro-Glu-Thr-Asn-Ala
-Arg-(c) encodes a polye7'tide.

Formula (U) DNA has (5')-ATG at the 5' end
ACCCAG GACTTT CCG GAA ACC
When it has AACGCCCGC-(3'),
(N)-Met-Thr-G at the (N)-terminus of formula (1)
ln-Asp-Phe-Pro-Glu-Thr-As
Encodes a polypeptide having n-Ala-Arg-(C).

Formula (II) ODNA (5')-AT at the 5' end
G CTG CGCTTCCTG GTG TTCGC
A ACCCTG GTCCTT TAT GGA C
ACAGCACCCAG GACTTT CCG GA
A When having ACCAACGCCCCGC-(3'), (N)-Me t-La at the (N)-terminus of formula (I)
u-Ar g-Pha -Le u-Va 1-Phe
-Al a-Thr-Leu-Va 1-Leu-Ty
r-Gly y-Hl 5r-Bo r-Th r-Gl
n-As p-Phe-Pr o-Gl u-Thr
-Asn-Ala-Arg-(C).

The present invention also relates to an amine expression vector represented by general formula (1).

In particular, in formula (1), the (N) terminal is a hydrogen atom, Me
ts or (N)-Th r-G 1 n-Am p-Phe −
P r o-G l u-Th r-As n-Al
a-Ar g-(C), (N)-Me t-Thr-G
in-Aap-Phe-Pro-Glu-Thr-As
n-Ala-Arg-(C) or (N)-Met-
Leu-Arg-Ph e -Leu-Va 1-P
h e-Al a-Th r-Le u-Val-Le
u-Tyr-Gly-Hl a-8s r-Th
r-Gl n-As p-Ph e-Pro -G
1 u-Th r-As n-Ala-Arg-(
The present invention relates to a recombinant DNA expression vector containing DNA encoding monkey pancreatic elastase (2) or its equivalent, which has part or all of the polypeptide represented by any one of C).

Particularly preferred is a recombinant DNA expression vector containing a DNA containing the base sequence represented by the general formula 2Qr) or a base sequence equivalent thereto, and further comprising ATG or ATG at the 5' end of the DNA. , (5')-ACCCAG GACTTT CCG G
AA ACCAACGCCCCGC-(3'), (5'
)-ATG ACCCAG GACTTT CCG
GAAACCAACGCCCGC-(3') or (5
)-ATG CTG CGCTTCCTG GTG
TTCGCA ACCCTG GTCCTT TAT
GGACACAGCACCCAG GACTTT C
CG GAA Ace AACGCCCGC-(3#)
Particularly preferred are recombinant DNA expression vectors containing DNA having part or all of a polynucleotide represented by any one of the following.

Plasmids are used as recombinant DNA expression vectors, and in particular, pSV2-MFEI, pBsEX-MF
EI and pECEX-MFEI etc. are used.

The present invention also relates to a host transformed with each of the above recombinant DNA expression vectors.

As the host, Escherichia coli, Bacillus subtilis, yeast, animal cells, etc. are used.

As the animal cell, for example, CO81 cells are preferred.

As Bacillus subtilis, for example, 207-25 strain is preferred.

As E. coli, for example, YA21 strain is preferred.

Although the guanidine thiocyanate hot phenol method and the guanidine thiocyanate cesium chloride method can be used to extract pancreatic RNA, the guanidine thiocyanate-guanidine hydrochloric acid method is superior to the above two methods in the following points. Are better. That is, (1) It is easy to operate.

(2) High extraction and recovery rate.

(3) It is possible to extract from a relatively large amount of organs.

(4) DNA is not mixed in.

(5) RNA degradation is low.

Many of the mRNAs present in the cytoplasm of eukaryotic cells are
Since mRNA is known to have a po+JA sequence at its end, this feature is used to adsorb mRNA onto a (dT) cellulose column, and then elute and purify the residue.

Complementary double-stranded DNA is synthesized using reverse transcriptase using the obtained mRNA as a template.
Nuclease method (Efstratradis, A
aat al,: Ce1l, 7, 279 (1976)
), Land method (Land, H, et al.:
Nucleic Aefda Res, , 9.

2251 (1981)), Olcayama-Be
rg method (Okayama.

H, and P, Berg: Mol * Ce1l
, Biol, 2, 161 (1982): ],
Om Joon Yoo method [: O@ Joon Yo
o.

et al, :Proe, Natl *Acad
*Set *USA, 79.

1049 (1982) 3, etc., but the Okayama-Berg method was suitable for the purpose of the present invention.

Next, the obtained recombinant plasmid is transferred to E. coli, for example, DH
5 strains for transformation. Recombinants can be selected using tetracycline resistance or ampicillin resistance as an indicator.

In order to select clones containing elastase-encoding DNA from the obtained recombinants, P
Porcine pancreatic elastase IeDNA labeled with CTan
1 @ T a et al -: J * Bioc
A colony hybridization method using hem*g 1011591 (1987)) as a glove was used.

Determination of the nucleotide sequence contained in the selected clone was performed using the method of dideoxynucleotide synthesis chain termination using phage M13 (Messing, J., at al.
m: Nucleie Ac1ds Res, 9,3
09 (1981)) and Maxam-Gi 1be
rt method (Maxam, A, Me and G11b
@rt.

W, :Proc, Natl, Aead, Sci, US,
A, 74.560 (1977)], but in the present invention, the method of dideoxynucleotide synthetic chain termination is employed to obtain D, which completely encodes monkey pancreatic elastase 1.
Clones with NA were determined.

Next, elastase c DNA was extracted from the obtained clone.
can be cut out, ligated to a suitable expression vector, and introduced into a suitable host for expression.

In E. coli, promoters include tryptophan (trp) f promoter lactose (lac
) promoter tryptophan lactose (taa
) promoter lipoprotein (1pp) promoter lambda (λ) PL promoter derived from bacteriophage, poIJ-e peptide chain elongation factor Tu (tufB
) You can use a 7" motor.

Examples of the host include bacteria such as Escherichia coli and Bacillus subtilis. Examples of the method for transforming and introducing the DNA of the present invention into the Escherichia coli host include the Hanahan method.

D,:J,Mol,Biol,, 166,5
57 (1983)], simulated calcium method [: Dag
ert, M., and Ehrlich, S.
.

D, Gene, 6.23 (1979)), low method [Yasutaka Takagi: Gene Manipulation Manual, p. 49. Kodansha Scientific (1982)) etc. may be adopted.

In the present invention, Hanahan's method was preferred.

The host (recombinant) obtained in this way is cultured in a known medium, and the culture has a row represented by formula (I). Ripeptide (at its (N)-terminus, Met or (N)-Thr-Gl n-As p-Phe-Pr
o-Glu -Th r-As n-Al a-Arg
-(C),(N)-Met-Thr-Gln-Aap-
Phe-Pro-Glu-Thr-Asn-Ala-A
rg-(C) or (N)-Me t-Leu-Ar
g-Phe-Leu-Va 1-Phe-Ala-T
hr-Leu-Va 1-Leu-Tyr-G 1
y-Hl 5-8o r-Th r-Gl n-As
p-Ph e-Pr o-Glu-Th r-Ays
Polybeno represented by any one of n-Al a-Arg-(C) produces and accumulates the same effect, and by collecting it, the object of the present invention can be achieved.

Glucose □,
A medium consisting of kabuminic acid, for example, M9 medium (Mi
Ller, J.: Expert
lnMo1e-curarGaneties,
431-433 (Cold Spring Harb
or Lab #, Nevr York (1972
) ) E etc. are mentioned.

Culture of the recombinant is usually carried out at 15-43°C for 3-24 hours, and aeration and agitation can be added as necessary. In addition, when using animal cells as a host, 3-1
Requires 0 days of culture.

After culturing, the recombinant cells are collected by a known method such as centrifugation. When Bacillus subtilis, yeast, animal cells, etc. are used as a host, depending on the choice of vector, the produced elastase exits the cell and is contained in the supernatant.

On the other hand, when E. coli is used as a host, the elastase produced is an insoluble protein contained within the cell. It is often included in 1 nclusion body.

In this case, rather than destroying the bacterial cells and centrifuging,
The produced elastase is obtained as a precipitate.

As a method for destroying bacterial cells, ultrasonic treatment, lysozyme treatment, freeze-thaw treatment, etc. can be employed. The supernatant liquid or precipitate E, 1. Isolation of elastase from L-products can be carried out by commonly known protein purification methods.

Elastase produced according to the present invention exhibits biological activity equivalent to that of elastase extracted and purified from pig pancreas, and can be used for the same purpose and using the same method.

EXAMPLES The present invention will be described in more detail with reference to Examples below, but these are not intended to limit the scope of the present invention.

Example (1) Separation of mR and NA from monkey pancreas 3.8 g of cynomolgus monkey pancreas was soaked in guanidine thiocyanate solution (4
M guanidine thiocyanate, 1 H sarcosyl, 2
0 mM ethylenediaminetetraacetic acid (EDTA)% 2
5 mM sodium citrate (PH7,0), 100
After disruption and denaturation using a polytron in mM 2-mercaptoethanol and 0.1% anti-7ohm A), the mixture was centrifuged to obtain a supernatant.

To this was added 0.025 times the amount of 1M acetic acid and 0.75 times the amount of ethanol, and after cooling at -20°C for several hours, a precipitate was obtained by centrifugation. This precipitate was then mixed with a guanidine hydrochloride solution (7.5M guanidine hydrochloride, 25mM sodium citrate (pH 7).
, 0), 5mM dithiothreitol (DTT)), 0.025 times the amount of 1M acetic acid and 0.015 times the amount of ethanol were added, and after cooling at -20°C for several hours, centrifugation was performed. The precipitate obtained here was resuspended in a guanidine hydrochloride solution, acetic acid and ethanol were added,
After cooling to -20°C, the precipitate was collected by centrifugation. Next, this precipitate was washed several times with ethanol to remove the mixed guanidine hydrochloride, and after dissolving in distilled water, RNA was precipitated with ethanol. The precipitate was collected by centrifugation to obtain 157rv RNA.

Of the RNA thus obtained, 16.8 m9 was added to a high salt solution (
0.5 M NaCt, 20 rnM Tris-HC
(dT) in L (pH 7,5), lmM EDTA, 0.1% sodium dodecyl sulfate (SDS)).
After adsorption onto a cellulose column, the mRNA containing PoIJ (A) was added to the elution solution (lomM Tris-HCL).
(pH 7,5), 1mM EDTA.

62 ttt! mRNA was obtained.

(2) Production of cDNA puncture The production of cDNA puncture was carried out by Okayams-Berg
I did it according to the law. That is, 5μy of mRNA and 2
Add 4 units of reverse transcriptase to 20μt reaction solution [50mM
Tris-HCL (pH 8,3), 8mM Mg
Cl2.30mM KCl-, 0.3mM DTT, 2
mM dATP, 2 mM dGTP, 2 mM dCTP, 2 mM dTTP, 10 μci α-P-dCTP, 1.4 μy vector primer DNA (purchased from PL-Phar-macia)
] for 60 minutes at 42°C.

2μL of 0.25M EDTA and 1μt of 10chi SD
After stopping the reaction by adding S, protein was removed with 20 μt of phenol/chloroform. After centrifugation, 9.20 μt of 4M ammonium acetate and 80 μt of ethanol were added to the aqueous layer, and the mixture was cooled at −70° C. for 15 minutes. Then,
The precipitate was collected by centrifugation, washed with 75% ethanol, and then dried under reduced pressure.

The precipitate was mixed with 15 μt of terminal transferase reaction solution (140 mM potassium cacodylate, 30 mM Tr
is-HCt(pi(6,8), 1 mM cobalt chloride, 0.5 mM DTT, 0.21111
OpolyA, 100mM acTp).

After warming the reaction solution at 37°C for 3 minutes, 18 units of terminal deoxynucleotidyl transferase was added and reacted for 5 minutes.
After stopping the reaction by adding M EDTA and 0.5 µL of 10T SDS, centrifuging the 0 reaction solution, which was subjected to protein removal with phenol/chloroform, took the aqueous layer, and added 15 µt of 4M ammonium acetate and 60 µt of ethanol. Mix well. After incubating at -70°C for 15 minutes, the precipitate was collected by centrifugation and fc.

The precipitate was diluted with 10μt of restriction enzyme buffer (50mM NaC).
t, 10 mM Trim-HCL (pH 7,5
), 10mM MgCl2.1mM DTT), added 2.5 units of restriction enzyme H1nd3, and
Digestion was carried out for approximately 1 hour at ℃. Next, after protein removal with phenol/chloroform, ethanol precipitation was performed. -70℃
After cooling for 15 minutes, the precipitate was collected by centrifugation and
μt TE (10mM Tri8-HCL (pH 7,
5), dissolved in 1 mM EDTA).

Next, 1μt of the above sample was added to an oligo-dG linker (P
10 ng of L-Pharmacim was added to the reaction solution (10 mM Tris-HCL (pi47.
5), 1mM EDTA, 100mM NaCt)
In addition, the mixture was heated at 65°C for 5 minutes, and then kept at 42°C for 30 minutes.

After cooling the reaction in water, add 10μt of 10x ligase buffer (10mM ATP, 660mM Tri
s-HCt (pH 7,5), 66mM MgC22,
100 mM DTT), 78 μt of distilled water, and 8 units of T4 DNA I) gauze were added, and the mixture was kept at 12° C. overnight.

Next, KG/! of 10 μt IM! ,, 1 unit glue?
DNA polymerase with nuclease H133 unit■
, 4 units of T4 DNA ligase, 0.5 At of nucleotide solution (20mM dATP, 20mM dGT
P, 20 mM dCTP, 20 mM dT
TP), 0.1 μL of 50 μI/μ bovine serum albumin (BSA) was added, and the mixture was incubated at 12°C for 1 hour and then at 25°C for 1 hour.

After diluting this reaction solution 5 times, immediately
an's method (Hanahan, D*: J, M
Ol 6 Blol,.

166.557 (1983)), large positive picture I)
The H5 strain was transformed to produce a monkey pancreatic cDNA puncture.

(3) Isolation of recombinant bacteria containing monkey pancreatic elastase l cDNA DNA of 22,800 strains of recombinant bacteria from the monkey pancreatic elastase l cDNA bank mentioned above was obtained from Grunatein, M.
, andHognaaa, D*S* method (Proc6 Natl @ Aead.

Set, USA, 72, 3961 (1975)) on a nitrocellulose filter.

Among these DNAs, monkey pancreatic elastase-1
To select for those containing cDNA, a porcine pancreatic elastase I cDNA fragment was used as a probe.

Porcine pancreatic elastase l eDNA (JP-A-60-2
07583) using the nick translation method (Rl
gby, P, W, at al,: J, Mo1. Btol
,.

113.237 (1977)) and hybridized with the above cDNA puncture.

As a result, 35 positive clones were obtained, of which 2
The length of cDNA contained in the plasmid for the four strains was investigated. One clone with a cDNA length of about 1 kb was selected, and the plasmid it contained was called pMFE 1-1.
It was named 8.

A restriction enzyme map of the eDNA inserted into pMFEl-18 is shown in FIG. 1, and its entire base sequence and the amino acid sequence encoded by it are shown in Table 1.

Table 1 Base sequence of monkey pancreatic elastase I eDNA and amino acid sequence encoded by it GlnLysIlsVaIVaIHIaProTyrT
rpAmnserAsnAsnVaIAImA1mMe
tLeuArgPheLsuValPheA1mG1y
TyrAspIleA1aL@uL@uArgL@uA
1aG1nserVaIThrL@uThrLauVa
IL*uTyrG1yHlassrThrGlnAsp
PheProGluThrAmnAlaArgVaIV
aIG1yGlyThrG1uA1aG1yArgAs
nserTrpProSarG1nI1sserLeu
GlnTyrLeuserG1yG1ySerTrpT
yrHigThrC)ysG1yG1yThrLauI
1eArgG1nAsnTrp'VaIMetThrA
1aA1mSe rVa IAspTyrA1 aI
1 ecysse rsa rse rse rTyr
TrpGlyse rHiacyaVaIAspsar
ProLysThrPheArgVaIValvaIG
IyAspHlsAsnLeuserGlnA+nAs
pG1yTbrG1uG1nTyrVaISsrva1
GlyCysG1nGlyAsps@rGlyG1yP
roLauHiscysLeuVaIAsnpMFE1
-18 is a siG1yLysTyrSerVaIHisG1yValT consisting of 6 amino acids.
hrSerPheVal SerLyaGln region encoding activation peptide, Gly encoding activation 4 peptide consisting of 10 amino acids
cysAsnVaISerArgLyiProThrV
It can be seen that it has both the region encoding alPheThrArgValSerA1a and the region encoding the mature elastase protein consisting of 0 amino acids.

In addition, pMFE1-18 is 5' and TyrI1@SerTrpI1@AinLymThrI
It also contains 1*A1aSerAgn**+ and 3' untranslated regions.

As a result of comparing the amino acid sequences deduced from the cDNA sequences, we found that monkey pancreatic TTCCTGAGTCCAATGACCTTTCCCAA
AATGGTTCTTAGATCTGCAAC Visceral elastase I has 8% and 8% homology with pig and rat pancreatic elastase, respectively AGAACTTGCGATCATCAAGTAAAAAA
It became clear that there is ACATTCTAAAAGACTATTGAIC.

pMFE1-18 contains full-length eDNA synthesized from monkey pancreatic elastase l mRNA using reverse transcriptase, so by transferring this cDNA to an appropriate expression vector, it can be used to infect E. coli, Bacillus subtilis, yeast, animal cells, etc. Monkey pancreatic elastase I can be produced in large quantities using this as a host. Production of monkey pancreatic elastase I using animal cells In order to produce monkey pancreatic elastase I using animal cells as a host, the eDNA and the expression vector were linked together using the procedure shown in Figure 2 to create SV as an expression vector.
40 promoter, engineer/no1 promoter, 4S A signal, Small T antigen gene intervening sequence (
pSV2 grasmid containing the intron) was used.

An expression plasmid (PSV2-MFEI) in which the vector and cDNA are ligated in the order of the transcription direction,
From K on analyzing restriction enzyme cleavage patterns.

Selected.

The constructed expression plasmid pSV2-MFEI was introduced into animal cells (CO81 cells) by the calcium phosphate method (
transfection) by the calcium phosphate method, Graham and van
I followed the method of d@r Eb (Virology,
52.

456 (1973) L cos i cells used for transfection, I x 10' cells were sown in a petri dish with a diameter of 10 cIn.
The cells were cultured overnight in Dulbecco's modified Eagle's medium containing 10% tallow serum. Next, 300 μI of pSV2-MFE
1 plasmid was suspended in 12.55-mL sterile distilled water,
．． After adding 75- of 2.5 M CaCl and mixing well, add 1
．． 5 rnt of 10X HeBs solution (210mM HEPES, 1.37M NaC4, 7mM
KCl, 10.6mM N&2HPO4,55,5
(mM glucose, pH 7.05) was added dropwise to form a precipitate of DNA and calcium phosphate. After the precipitate was allowed to stand at room temperature for 30 minutes to ripen, one strain per Petri dish was added to CO81 cells that had been replaced with 10 g of fresh medium containing fetal bovine serum in advance. After culturing this for 12 hours at 37°C in the presence of 5% CO2, the culture medium was discarded and replaced with new Dulbecco's modified Eagle's medium that does not contain fetal bovine serum, and further cultured at 37°C in the presence of 5% CO2. and cultured for 48 hours. The transfected CO81 cells obtained here are monkey pancreatic elastase l rr + RN
A, and the culture supernatant was used to measure elastase activity.

Extraction of mRNA from CO81 cells Monkey pancreatic elastase I mRNA transcribed from expressed cilasmid in transfected CO81 cells.
To confirm that A exists.

mRNA was extracted from Co51 cells and subjected to Northern Pro hybridization as shown below. After 48 hours of culture, CO81 cells were placed in a Petri dish and 1 tnt of guanidine thiocyanate solution (4M guanidine Thiocyanate, 1% Sarcosyl, 20mM
of EDTA, 25 mM sodium citrate (p
H7,0), 100 mM 2-mercagutoethanol, and 0.1% antiform A) were added to lyse the cells. Next, this solution was passed through a 21ff-di injection needle several times to reduce the molecular weight of the high-molecular DNA, and then layered on top of a solution containing 5.7 M cesium chloride and 0.1 M EDTA.
Using Hitachi RPS40 swing rotor, 30.
Centrifugation was performed at 00 rpm and 20°C for 17 hours. The RNA precipitate obtained was washed with a small amount of ethanol and diluted with 300μ
It was dissolved in t of distilled water.

The extracted total RNA was extracted using the method of Aviv and Leder (Pr
oe.

Natl, Acad, Sci, USA 69.1408
(1972) ), several microliters of mRNA was purified by applying it to an (aT) cellulose column. purified m
Using half the amount of RNA, the method of Thomas [Pr.
oc, Natl, Aead, Scf.

Northern blot hybridization was performed according to USA, 77.5201 (1980)).

The glove has a nick translation method (Rlgb).
y, P, W, et al,, J, Mo1. Bi*1.1
13.

237 (1977)) was used. 32P-labeled monkey pancreatic elastase l cDNA was used. As a result, pSV
2-MFEI-transfected CO81 cells with a size of about 1.8 kb that hybridizes only to the mRNA of CO81 cells.
RNA was detected. When pSV2-MFEI is transcribed, it is estimated that a 1.8 kb mRNA will be generated when transcription is terminated by the polyA signal contained in the vector, and the results obtained by Northern blot hybridization are It matched the estimated value. From this, it was concluded that the introduced pSV2-MFEl was expressed in cos i cells using the SV40 promoter.

Monkey pancreatic i2 elastase IcD integrated into pSV2
Since NA retains a signal peptide region, the expressed elastase is expected to be secreted into the medium as heproelastase. Therefore, elastase activity in the culture solution after 48 hours of culture was measured. Elastase activity was measured using a method using a synthetic substrate by Bieth et al. (Front *Matrix, B1o1.6,1(1
978) ) was used.

200 μt of IM Trls-HCt in the culture supernatant of 1-
buffer (pH 8,5) and 50 μL of 10 m9/rnt
After activating gloelastase by adding trypsin and incubating at 25°C for 15 minutes,
Added trypsin was inactivated by adding 7jL of 101n9/rnt of soybean trypsin inhibitor. Next, N
- 10.4 μt of 125 dissolved in methylpyrrolidone
mM succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide (Sue-Ala-A
la-Ala-pNA) was added and kept at 25°C for 1 hour, and then the absorbance at 410 nm was measured.

As a result, no elastase activity was detected in the culture medium containing only CO81 cells, whereas pSV2-MF
Elastase activity was observed in the culture solution of cosi cells transfected with EI.

Next, α1-antitrypsin or elastatinal, which is a specific inhibitor of elastase, was added to the culture fluid that showed elastase activity, and whether or not it was inhibited by them was investigated. When elastatinal or α1-antitrypsin is added to a trypsin-activated sample, the decomposition activity of the synthetic substrate is strongly inhibited, and the expressed monkey pancreatic elastase I exhibits the same inhibition mode as natural pig pancreatic elastase I. It was shown that it has. Furthermore, it was shown that most of the secreted elastase was gloelastase, which was not activated by trypsin treatment.

In this practical example in which psV2-MFEI was introduced into COS IIB j@, the production of monkey pancreatic elastase in summer was short-term (transient expression
n), but the pSV2-MFEI plasmid is injected with an appropriate selection marker (e.g. neo gene, dihydr
rofolate reduetase gene, etc.) and introducing it into CHO cells etc., it is possible to obtain a cell line capable of producing simian elastase (2) over a long period of time.

(5) Production of monkey pancreatic elastase I using Bacillus subtilis The construction method of the expression vector is shown in FIG. First, plasmid p containing monkey pancreatic elastase l cDNA
MFE1-18 was digested with the restriction enzyme EcoR1, and a DNA fragment containing the C-terminal side from the 10th amino acid from the N-terminus of mature elastase I was isolated. In this DNA fragment,
A synthetic oligonucleotide (Fig. 4) encoding part of the signal peptide of Bacillus subtilis α-amylase, the activation peptide of elastase (10 amino acids), and 9 amino acids from the N-terminus of mature elastase (2) was added to T4.
They were linked using DNA ligase. Next, this DNA was cut with restriction enzyme Bgt2, and a DNA fragment containing e DNA was separated.

On the other hand, plasmid pTUB228 (Ohmura) in which the α-amylase gene of Bacillus subtilis is cloned.

K, at al,, J, Blochem, 95.87 (
(1984)) was cut with restriction enzyme H1nd3 to obtain α
- A 428 base pair DNA fragment containing the amylase promoter and part of the signal peptide, and the origin of replication5. Zoo base pair DNA fragments were isolated respectively.

The 428 base pair DNA fragment was further digested with the restriction enzyme Hpa 2
In addition, a 5,100 base pair DNA fragment was digested with restriction enzyme S.
After cutting with ma l, 385 base pairs from each,
A 4566 base pair DNA fragment was isolated. The above three types of D
After ligating the NA fragments with T4 DNA ligase, S
The restriction enzyme B gt2 site was blunt-ended using 1 nuclease. This is T4 DNA again! It was made m-shaped by treatment with J gauze.

This DNA was extracted using a conventional method to extract Bacillus subtilis strain 207-25 (mT6
3harM recE4amyEO7aro1906
1euA81ys21 (derived from Marburg strain) into protoplasts and regenerated, then 10 μy
A transformed strain capable of growing on this medium was obtained by culturing in a medium containing /- of kanamycin. In order to select the desired plasmid from several transformed strains, colony hybridization was performed using the synthetic oligonucleotide shown in FIG. 4 as a probe.

The positive clones obtained were isolated as ciblasmids, and their nucleotide sequences were determined to confirm that they were the desired clones. The expression plasmid thus obtained was p
It was named BSBX-MFEI.

The aforementioned monkey pancreatic elastase I expression plasmid pBSE
Bacillus subtilis 207-25 strain into which X-MFEI was introduced, 50
1 t of LG medium containing μg/- kanamycin (1% Bactodrypton (Difeo), 0.5% yeast extract (Difco), 0.5 t NaC2
, 0.2% glucose, p! -17,0) at 35℃,
Culture was performed with reciprocating shaking for 48 hours. 1 m/! of this culture supernatant! Add 200 μL of IM Tris-HCt buffer, (p
H8,5) and 50 μt of 10 m9/rnt trypsin and incubating at 25°C for 15 minutes to activate gloelastase.
/- soybean trypsin inhibitor was added to inactivate the added trypsin. Next, 125 of 10.4μt
Sue-Ala-Ala-Ala-Ala-p in mM
After adding NA and incubating at 25°C for 1 hour, 410n
The absorbance of m was measured.

As a result, no elastase activity was detected in the culture solution containing only the 207-25 strain, whereas pBSEX-
Elastase activity was observed in the culture solution of the 207-25 strain into which MFEI had been introduced.

Next, C1-antitrypsin or elastatinal, which is a specific inhibitor of elastase, was added to the culture solution showing elastase activity, and whether or not it was inhibited by them was investigated. When elastatinal or C1-antitrypsin was added to a sample activated by trypsin, the decomposition activity of the synthetic substrate was strongly inhibited, similar to elastase produced in animal cells. Furthermore, trypsin treatment was required for activation, indicating that the elastase produced was gloelastase.

(6) Production of monkey pancreatic elastase I using E. coli Expression vectors pF and cEX-MF using E. coli as host
The construction method of E1 is shown in FIG. The expression vector contains pUC8 (Pharmaei
(purchased by Company A) was used. Monkey elastase cDN
A cilasmid pMFE1-18 containing restriction enzyme B
After cutting with gtl to make it linear, it was partially cut with restriction enzyme Ace3 to separate a fragment containing elastase I cDNA. This was treated with 81 nuclease to make both ends blunt. The obtained DNA fragment was inserted into the Sma 1 cleavage site of the plasmid ptycs as T4 DN.
It was incorporated using A+7 gauze.

Using the plasmid thus constructed, E. coli J.
M103 strain was transformed. Plasmids were extracted from several transformed strains, and those whose cDNA transcription direction matched the orientation of the lactose operon promoter were selected by restriction enzyme mapping. This elastase I expression plasmid was named pECEX-MFE 1 (Figure 5).

5 of elastase c DNA in pECEX-MFEI
The nucleotide sequence and amino acid sequence near the 'terminus are as follows. Monkey pancreatic elastase expressed by pECEX-MFE1 has a bond with the β-galactosidase gene on the N-terminal side of its activation target. This results in a fusion protein with 7 amino acids added.

Next, several strains of E. coli were transformed with pECEX-MFE 1 to obtain strains capable of producing monkey pancreatic elastase I. The bacterial strain containing the obtained expression plasmid was
xTY-ampicillin medium (1.6% Bactodrypton, 1% yeast extract, 0.5% NaC
t, 50 μl/ml ampicillin) and 3
The cells were cultured at 7°C for 15 hours. After culturing, the culture solution was centrifuged to collect the bacterial cells, and the bacterial cells equivalent to 2.4 x 108 cells were collected in a 15 μt tube.
SDS solution (2 parts SDS, 5 parts 2-mercaptoethanol, 10% glycerin, 60 mM Trim
-HCt (pH 6,8)) and heated at 100°C for 3 minutes, followed by the method of Laemmli et al. [Natur
5DS according to e, 227.680 (1970):l
-Produced proteins were analyzed by polyacrylamide gel electrophoresis.

As a result, a large amount of elastase production was observed in the YA21 strain. The yield of elastase fusion protein is YA.
It corresponded to 20% of the total protein produced by 21 strains.

Although a relatively large amount of elastase fusion protein was produced in the Z984 strain as well.

The yield was less than half of that of the YA21 strain. The production amount of elastase fusion protein in the other two strains of E. coli (strain H8101 and strain MC4100) was significantly low.

Since the elastase fusion protein forms an inclusion body within the bacterial cell, it could be purified relatively easily. That is, Y transformed with pECEX-MFEI
A culture solution of A21 strain, 6.0 g of bacterial cells forming IL, inclusion, and ngodi, was obtained. Obtained bacterial cells 6
．． 50rnM Trim-H containing lysozyme 0.2 mcI/rnt and deoxycholic acid 1/-
Bacteria were lysed by treatment in Ct buffer 1 (pH 8,0). Undestroyed bacterial cells were removed by low-speed centrifugation (1,500xg, 10 minutes), followed by high-speed centrifugation (11,000x &
, 20 minutes), and the inclusion bodies were collected as a precipitate. Since this inclusion body still contained a large amount of bacterial cell fragments, it was suspended in 50 mM Tris-HC2 buffer containing 5 m97rd of Triton X-100, and then subjected to high-speed centrifugation (11,000x, 9. 2
0 minutes).

The cleaned incruge, Ndedi, is treated with a small amount of Tris-
It was suspended in HC1 buffer and stored at 4°C.

Purification in this manner yielded 270 m9 inclusion bodies, which contained approximately 50% of the monkey pancreatic elastase fusion protein. Also, p
It was confirmed by immunoblotting (immunoblotting) that the inclusion cod ζ day produced by strain YA21 transformed with ECEX-MFlml is an elastase fusion protein
This was confirmed using the no-blotting method.

As mentioned above, most of the elastase produced by E. coli exists in the insoluble fraction of the bacterial cell as an inclusion body, but some of it is soluble and exists in a state that retains enzyme activity. ing. Therefore, detection of this enzyme activity was performed as follows.

YA21 strain transformed with pECEX-MFEI was transformed into 2
The cells were cultured with shaking in 1 t of XTY-ampicillin medium at 37°C for 15 hours. After completion of the culture, the bacterial cells were collected by centrifugation at 3,000x for 9.5 minutes, and mixed with 20-buffer A (50mM Tris-HCt (pH 8,0), 1mM Fj
After suspending in DTA, 50 mM NaCl) and adding 10 mg of lysozyme, the mixture was incubated at 5°C for 20 minutes. Next, deoxycholic acid was added at a final concentration of 6°C to 1 m9/-, heated to 20°C, deoxylynuclease was added at a final concentration of 0.1 m9/-, and the mixture was crushed using a Polytron crusher. The bacterial cells were crushed. The obtained lysate was 80,0OOxJ? After centrifuging for 40 minutes to remove bacterial fragments, the mixture was subjected to Sephadex G-75 column chromatography. The elastase active fraction was further purified by antibody affinity chromatography and used as a monkey pancreatic elastase active fraction in the following assay.

The elastase activity of the sample was measured by the method described below. First, the sample solution was treated with trypsin to activate proelastase.

Synthetic substrate 5ue-Ala-Ala-Ala-p-nlt
roani 11da and released p-n1t.
0 reaction, quantified by measuring the absorbance of roanilide at 410 nm, is 0.2
25°C in Tris-HCti buffer (pH 8,0) of M.
This was done by keeping it warm. As a result, pECE
Obvious activity was detected in the sample of YA21 strain transformed with X-MFE 1, indicating the production of active monkey pancreatic elastase I in E. coli.

At the same time, the elastase inhibitor elastase or C1-antitrypsin was added to the sample solution at a final concentration of 0.
When added at a concentration of 1 m9/rnt, it was also confirmed that the activity was inhibited by these.

(7) Production of monkey pancreatic elastase I using yeast When using yeast as a host, as in the case of animal cells, Bacillus subtilis, and Escherichia coli, the DNA of monkey pancreatic elastase I can be prepared using conventional methods. It was possible to link the vector to an expression vector, transfer it into host 1 cells, express it, and confirm the presence of elastase activity in the culture solution.

The host strain is described in the "Recombinant DNA Experimental Guidelines," and specifically, the 8288C strain and the like were suitable.

Furthermore, as a promoter, the ADHI gene encoding the alcohol dehydrogenase gene was suitable.

[Brief explanation of drawings]

Ki1 section is the plasmid pMFE1-1 obtained in the example.
The restriction enzyme map of 8 is shown, and part 24 indicates a part encoding a peptide considered to be a signal peptide. The beginning part encodes a peptide considered to be an activation peptide, and the left part shows the part encoding the mature elastase protein. Figure 2 shows the procedure for constructing the pSV2-MFEI plasmid.
This is a grasmid in which β-globin cDNA is inserted into the vector. On the other hand, pMFE 1-18 is OK.
This is a plasmid in which elastase I cDNA is inserted into the ama-Berg vector. S
Reactions such as 1 nuclease can be performed using molecular cloning [Manlatls, T* et al.
(ed, )MolecularC1onlng'
Co1d Spring Harbor Lab,
(1982)). Thick arrow is SV4
The promoter derived from 0 is shown, and the thin arrow indicates the direction of transcription. In the figure, B, H, and P are restriction enzymes Bgt2゜Hl, respectively.
nd 3 represents Pst 1. FIG. 3 shows the procedure for constructing the pBSEX-MFEI plasmid. pTUB 228 is a plasmid in which the Bacillus subtilis α-amylase gene is cloned into a vector having a Bacillus subtilis origin of replication. The thick arrow indicates the promoter of α-amylase, and the thin arrow indicates the direction of transcription. In Figure 0, B, E, and H. p and s are restriction enzymes Bgt2 and EcoRl, respectively.
, Hlnd3°Hpa 2 , Sma 1. Figure 4 shows a portion of the signal peptide region of the Bacillus subtilis α-amylase gene and the activation of elastase I (a synthetic NA linker encoding the peptide followed by 9 amino acids. The steps for constructing the MFE1 plasmid are shown. The shaded region indicates the β-galactosidase gene. The thick arrow indicates the lactose promoter operator. The thin arrow indicates the direction of transcription. In the figure, A, B
, I(, S are restriction enzymes Ace 3 and Bgt, respectively.
2, Hind3, and Sma1.

Claims (1)

[Claims] 1. General formula (I): [There is a gene sequence] [There is a gene sequence] Monkey pancreatic elastase I or its equivalent, consisting of the amino acid sequence represented by: 2. In the general formula (I) according to claim 1, the (N) terminus of monkey pancreatic elastase I or its equivalent is a hydrogen atom, Met, or [there is a gene sequence] or [there is a gene sequence]. Monkey pancreatic elastase I or its equivalent having part or all of the polypeptide represented by any one of the above. 3. DNA encoding monkey pancreatic elastase I or its equivalent according to claim 1. 4. DNA encoding monkey pancreatic elastase I or its equivalent according to claim 2. 5. General formula (II): [There is a gene sequence] [There is a gene sequence] Contains the base sequence represented by (in the formula, X represents TAA, TGA, or TAG) or a base sequence with the same effect. The DNA according to claim 3 or 4, characterized in that: 6. In the general formula (II) according to claim 5, 5 of the DNA
DNA characterized by having part or all of a polynucleotide expressed by either ATG or [contains a gene sequence] or [contains a gene sequence] at its end. 7. A recombinant DNA expression vector containing the DNA according to claim 3. 8. A recombinant DNA expression vector containing the DNA according to claim 4. 9. A recombinant DNA expression vector containing the DNA according to claim 5. 10. A recombinant DNA expression vector containing the DNA according to claim 6. 11. The recombinant DNA expression vector is a plasmid.
The recombinant DNA expression vector according to claim 7, 8, 9 or 10. 12. The recombinant DNA expression vector according to claim 11, wherein the plasmid is pSV2-MFE1. 13. The recombinant DNA expression vector according to claim 11, wherein the plasmid is pBSEX-MFE1. 14. The recombinant DNA expression vector according to claim 11, wherein the plasmid is pECEX-MFE1. 15. A host transformed with the recombinant DNA expression vector according to claim 7. 16. A host transformed with the recombinant DNA expression vector according to claim 8. 17. A host transformed with the recombinant DNA expression vector according to claim 9. 18. A host transformed with the recombinant DNA expression vector according to claim 10. 19. A host transformed with the recombinant DNA expression vector according to claim 11. 20. A host transformed with the recombinant DNA expression vector according to claim 12, 13 or 14. 21. The host according to claim 15, 16, 17, 18, 19 or 20, wherein the host is any one of Escherichia coli, Bacillus subtilis, yeast or animal cells. 22. The host according to claim 21, which is a COS1 cell. 23. The host according to claim 21, which is Bacillus subtilis strain 207-25. 24. The host according to claim 21, which is Escherichia coli YA21 strain.