JPH0675509B2 - Human / Pancreas Elastase III B - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human pancreatic elastase IIIB (the novel human pancreatic elastase disclosed by the present invention
It is named pancreatic elastase IIIB. ), A DNA containing a nucleotide sequence encoding the same, a host transformed with the same, and a method for producing human pancreatic elastase IIIB using the host.

Elastase is a type of serine protease,
It is an enzyme that hydrolyzes elastin, a fibrous insoluble protein. Elastin is a type of hard protein that forms connective tissues, tendons, aortic epidermis, and cartilage of higher animals.
It is slightly decomposed by pepsin and trypsin.

In the course of research on animal sclerosis, Balo 'et al. Found the degradation of elastin fibers in the blood vessel wall, and in 1949 estimated the existence of its degrading enzyme [Balo', J. and Banga, I .: Schweiz Z. Pathol .B
acteriol., 12 , 350 (1949)]. Next, Banga is 195
2 years, specifically degrades enzyme elastin, discovered within the pancreas, taken up in crystal form was designated as elastase [Banga, I:.. Acta Physiol.Acad.Sci.Hung , 3, 317 (1
952)].

Elastase has been confirmed to be present in the pancreas of most animals such as humans, monkeys, cats, and rabbits, and its content is 3.1 mg / pancreas g in humans, 2.2 mg / g in cattle, 10.2 mg / g in rats. It is a degree. The relationship between human elastase activity and age was recognized, and the elastase activity in the pancreas and plasma was markedly reduced in males aged 40 and over and females aged 60 and over [Loev
en, WA, and Maureen M. Baldwin.:Gerontologia, 17 , 170
(1971)].

In patients with animal sclerosis, the activity of pancreatic elastase is significantly lower than that of healthy individuals, or
It disappeared [Balo ′, J., and Banga, I .: Nature, 178 , 31
0 (1956)]. Further studies have shown that elastase not only enzymatically decomposes elastin but also promotes its biosynthesis.

The pharmacological action of elastase has been studied using rats and rabbits, and the following effects have been clarified.

1) Inhibition of lipid and calcium deposition on arterial wall 2) Action of removing cholesterol and calcium from arterial wall 3) Selective degradation action on degenerated elastin 4) Action to promote new generation of arterial wall elastic fiber 5) Serum lipid lowering action 6 ) Lipoprotein metabolism-improving effect In the clinical studies based on the above studies, oral administration of elastase revealed the following effects.

1) Recovery of elasticity / extension of arterial wall 2) Improvement of abnormal serum lipids 3) Improvement of lipoprotein metabolism In the above studies, elastase extracted and purified from porcine pancreas was used. However, when porcine elastase is administered to humans, it is a heterologous protein for humans, and therefore antibody production occurs, and there is a risk that re-administration to patients will cause anaphylaxis (JP-A-59-1118).
0). Therefore, when administered to humans, human elastase is preferably used. However, it was extremely difficult to obtain a sufficient amount of human elastase. Therefore, the present inventors have cloned the human pancreatic elastase IIIB gene using recombinant DNA technology and obtained the resulting recombinant DN.
Human molecule and pancreatic elastase IIIB by transferring A molecule into the host
The present invention has been completed as a result of conducting research and development of a technique capable of expressing a gene and obtaining the desired elastase IIIB.

At present, the existence of two kinds of human pancreatic elastase is already known, but its characterization has not been performed sufficiently, and one of them has an amino acid sequence of 12 of its pro part. And only 4 of the body have been identified [C. Largman et al; Biochim. Biophys. Ac
ta, 623 , 208 (1980)], the structure of which was unknown until now. However, the present inventors have revealed all of these amino acid sequences and all of their DNA sequences (Japanese Patent Application No. 60-72308, 6).
0-91986, 60-163964). By the way, it is clear that the amino acid sequence clarified by the present invention is different from the amino acid sequence clarified as described above, and that the present invention relates to the novel substance of the present invention.

That is, the present invention provides a DNA containing a nucleotide sequence encoding human pancreatic elastase IIIB, a host transformed with the DNA, and a human pancreatic elastase obtained by culturing a host transformed with the DNA. A method for producing IIIB is provided.

The present invention is represented by the general formula (N) -Val Val Asn Gly Glu Asp Ala Val Pro Tyr Se.
r Trp Pro TrP Gln Val Ser Leu Gln Tyr Glu Lys Ser
Gly Ser Phe Tyr His Thr Cys Gly Gly Ser Leu Ile Al
a Pro Asp Trp Val Val Thr Ala Gly His Cys Ile Ser
Ser Ser Arg Thr Tyr Gln Val Val Leu Gly Glu Tyr As
p Arg Ala Val Lys Glu Gly Pro Glu Gln Val Ile Pro
Ile Asn Ser Gly Asp Leu Phe Val His Pro Leu Trp As
n Arg Ser Cys Val Ala Cys Gly Asn Asp Ile Ala Leu
Ile Lys Leu Ser Arg Ser Ala Gln Leu Gly Asp Ala Va
l Gln Leu Ala Ser Leu Pro Pro Ala Gly Asp Ile Leu
Pro Asn Glu Thr Pro Cys Tyr Ile Thr Gly Trp Gly Ar
g Leu Tyr Thr Asn Gly Pro Leu Pro Asp Lys Leu Gln
Glu Ala leu Leu Pro Val Val Asp Tyr Glu His Cys Se
r Arg Trp Asn Trp Trp Gly Ser Ser Val Lys Lys Thr
Met Val Cys Ala Gly Gly Asp Ile Arg Ser Gly Cys As
n Gly Asp Ser Gly Gly Pro Leu Asn Cys Pro Thr Glu
Asp Gly Gly Trp Gln Val His Gly Val Thr Ser Phe Va
l Ser Ala Phe Gly Cys Asn Thr Arg Arg Lys Pro Thr
Val Phe Thr Arg Val Ser Ala Phe Ile Asp Trp Ile Gl
u Glu Thr Ile Ala Ser His- (C) (I) The present invention relates to human pancreatic elastase IIIB represented by the amino acid sequence. In the formula, (N) -terminal has a hydrogen atom, Met, or (N) -Met Met Leu Arg Leu Leu Ser Ser Leu Leu Le as a pro and pre moiety.
u Val Ala Val Ala Ser Gly Tyr Gly Pro Pro Ser Ser
It may have a part or all of Arg Pro Ser Ser Arg- (C).

In particular, the general formula (5 ')-GTT GTC AAT GGT GAG GAT GCG GTC CCC TAC
AGC TGG CCC TGG CAG GTT TCC CTG CAG TAT GAG AAA AG
C GGA AGC TTC TAC CAC ACC TGT GGC GGT AGC CTC ATC
GCC CCC GAC TGG GTT GTG ACT GCC GGC CAC TGC ATC TC
G AGC TCC CGG ACC TAC CAG GTG GTG TTG GGC GAG TAC
GAC CGT GCT GTG AAG GAG GGC CCC GAG CAG GTG ATC CC
C ATC AAC TCT GGG GAC CTC TTT GTG CAT CCA CTC TGG
AAC CGC TCG TGT GTG GCC TGT GGC AAT GAC ATC GCC CT
C ATC AAG CTC TCA CGC AGC GCC CAG CTG GGA GAC GCC
GTC CAG CTC GCC TCA CTC CCT CCG GCT GGT GAC ATC CT
T CCC AAC GAG ACA CCC TGC TAC ATC ACC GGC TGG GGC
CGT CTC TAT ACC AAC GGG CCA CTC CCA GAC AAG CTG CA
G GAG GCC CTG CTG CCG GTG GTG GAC TAT GAA CAC TGC
TCC AGG TGG AAC TGG TGG GGT TCC TCC GTG AAG AAG AC
C ATG GTG TGT GCT GGA GGG GAC ATC CGC TCC GGC TGC
AAT GGT GAC TCT GGA GGA CCC CTC AAC TGC CCC ACA GA
G GAT GGT GGC TGG CAG GTC CAT GGC GTG ACC AGC TTT
GTT TCT GCC TTT GGC TGC AAC ACC CGC AGG AAG CCC AC
G GTG TTC ACT CGA GTC TCC GCC TTC ATT GAC TGG ATT
GAG GAG ACC ATA GCA AGC CAC-X (3 ') (II) (wherein X represents TAA, TGA or TAG) or a DNA containing a base sequence having the same effect.

The above DNA (II) has ATG at its 5'end, or (5 ')-ATG ATG CTC CGG CTG CTC AGT TCC CTC CTC
CTT GTG GCC GTT GCC TCA GGC TAT GGC CCA CCT TCC TC
It may have a part or all of T CGC CCT TCC AGC CGC- (3 ′).

When ATG is present at the 5'end of DNA (II), the following polypeptide (III) (N) -Val Val Asn Gly Glu Asp Ala Val Pro Tyr Se is used.
r Trp Pro Trp Gln Val Ser Leu Gln Tyr Glu Lys Ser
Gly Ser Phe Tyr His Thr Cys Gly Gly Ser Leu Ile Al
a Pro Asp Trp Val Val Thr Ala Gly His Cys Ile Ser
Ser Ser Arg Thr Tyr Gln Val Val Leu Gly Glu Tyr As
p Arg Ala Val Lys Glu Gly Pro Glu Gln Val Ile Pro
Ile Asn Ser Gly Asp Len Phe Val His Pro Leu Trp As
n Arg Ser Cys Val Ala Cys Gly Asn Asp Ile Ala Leu
Ile Lys Leu Ser Arg Ser Ala Gln Leu Gly Asp Ala Va
l Gln Leu Ala Ser Leu Pro Pro Ala Gly Asp Ile Leu
Pro Asn Glu Thr Pro Cys Tyr Ile Thr Gly Trp Gly Ar
g Leu Tyr Thr Asn Gly Pro Leu Pro Asp Lys Leu Gln
Glu Ala Leu Leu Pro Val Val Asp Tyr Glu His Cys Se
r Arg Trp Asn Trp Trp Gly Ser Ser Val Lys Lys Thr
Met Val Cys Ala Gly Gly Asp Ile Arg Ser Gly Cys As
n Gly Asp Ser Gly Gly Pro Leu Asn Cys Pro Thr Glu
Asp Gly Gly Trp Gln Val His Gly Val Thr Ser Phe Va
l Ser Ala Phe Gly Cys Asn Thr Arg Arg Lys Pro Thr
Val Phe Thr Arg Val Ser Ala Phe Ile Asp Trp Ile Gl
In addition to u Glu Thr Ile Ala Ser His- (C), it encodes a polypeptide having Met at the N-terminus of (III).

At the 5'end of DNA (II), (5 ')-ATG ATG CTC CGG CTG CTC AGT TCC CTC CTC
CTT GTG GCC GTT GCC TCA GGC TAT GGC CCA CCT TCC TC
When it has T CGC CCT TCC AGC CGC- (3 '), the polypeptide (III) is added (II
(N) -Met Met Leu Arg Leu Leu Ser Ser Leu Leu Le at the N-terminus of I)
u Val Ala Val Ala Ser Gly Tyr Gly Pro Pro Ser Ser
Arg Pro Ser encodes a polypeptide having Ser Arg- (C).

The DNA of the present invention containing a base sequence represented by the formula (II) or a base sequence having the same effect as the base sequence can be produced, for example, by the following steps (a) to (d).

(A) Isolate mRNA from human pancreas.

(B) A cDNA bank is prepared using this mRNA and reverse transcriptase.

(C) From the prepared cDNA bank, for example, a porcine elastase I cDNA is used as a probe to isolate a plasmid containing a cDNA encoding the desired human elastase IIIB.

(D) Cut out the desired cloned DNA from this plasmid.

For the RNA extraction of the pancreas, the guanidine-thiocyanate-hot-phenol method, the guanidine-thiocyanate-cesium chloride method, etc. may be adopted, but
The thiocyanate-guanidine / hydrochloric acid method is superior to the above two methods in the following points. (1) The operation is easy.
(2) High extraction / recovery rate. (3) Extraction from a relatively large amount of organs is possible. (4) DNA is not mixed in.
(5) Less RNA degradation.

Most of the mRNAs present in the cytoplasm of eukaryotic cells are known to have a poly A sequence at their 3'ends, and this feature is utilized to apply mRNA to oligo (dT) cellulose columns.
Is adsorbed, and this is then eluted and purified.

A complementary double-stranded DNA is synthesized using the obtained mRNA as a template and a reverse transcriptase. The method is the S1 nuclease method [Efstratiadis, A. et al .: Cell, 7 , 279 (197
6)], Land method [Land, H. et al .: Nucleic Acids Res.,
9 , 2251 (1981)], Okayama-Berg method [Okayama, H. and
P. Berg: Mol. Cell. Biol., 2 , 161 (1982)], O. Joon Yoo
Law (O. Joon Yoo, et al .: Proc. Natl. Acad. Sci. USA, 79 , 10
49 (1982)] and the like, but for the purpose of the present invention,
The Okayama-Berg method was preferred.

Next, the obtained recombinant plasmid was transformed into E. coli, for example,
It is introduced into RR1 strain and transformed. Recombinants can be selected using tetracycline resistance or ampicillin resistance as an index.

To select a clone containing DNA encoding elastase IIIB from the obtained recombinants, 32P was selected.
Pig Elastase I labeled with (Japanese Patent Laid-Open No. 60-207583)
A colony hybridization method using cDNA as a probe was used.

The nucleotide sequence contained in the selected clone was determined by the method of dideoxynucleotide synthetic chain termination using Phage M13 [Messing, J. et al .: Nucleic Acids Res., 9 , 309.
(1981)] and the Maxam-Gilbert method [Maxam, AM and Gi.
lbert, W.:Proc.Natl.Acad.Sci.USA, 74 , 560 (1977)], the present invention adopts the method of dideoxynucleotide synthetic chain termination to completely eliminate human pancreatic elastase IIIB. Clones with the encoding DNA were determined.

Next, the elastase IIIB gene can be excised from the obtained clone, ligated to an appropriate expression vector, and introduced into an appropriate host for expression.

As a promoter, in E. coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan-lactose (tac) promoter lipoprotein (lpp) promoter, the lambda-derived bacteriophage full Urge (λ) P _L promoter, polypeptide chain elongation A factor Tu (tufB) promoter or the like may be used.

As the host, bacteria such as Escherichia coli and Bacillus subtilis, microorganisms such as yeast, and animal cells can be adopted.

As a method for transforming and introducing the Escherichia coli DNA of the present invention into a host, the method of Hanahan [Hanahan, D .: J. Mol. Biol.,
166 , 557 (1983)], calcium chloride method [Dagert, M. and
SDEhrlich: Gene, 6 , 23 (1979)], low pH method [edited by Yasutaka Takagi, Gene manipulation manual, p.49, Kodansha Scientific (1982)] and the like. In the present invention,
The Hanahan method was preferred.

The host (recombinant) thus obtained is cultivated in a known medium, and in the culture, the compound of the general formula (N) -Val Val Asn Gly Glu Asp Ala Val Pro TyrSe is added.
r Trp Pro Trp Gln Val Ser Leu Gln Tyr Glu Lys Ser
Gly Ser Phe Tyr His Thr Cys Gly Gly Ser Leu Ile Al
a Pro Asp Trp Val Val Thr Ala Gly His Cys Ile Ser
Ser Ser Arg Thr Tyr Gln Val Val Leu Gly Glu Tyr As
p Arg Ala Val Lys Glu Gly Pro Glu Gln Val Ile Pro
Ile Asn Ser Gly Asp Leu Phe Val His Pro Leu Trp As
n Arg Ser Cys Val Ala Cys Gly Asn Asp Ile Ala Leu
Ile Lys Leu Ser Arg Ser Ala Gln Leu Gly Asp Ala Va
l Gln Leu Ala Ser Leu Pro Pro Ala Gly Asp Ile Leu
Pro Asn Glu Thr Pro Cys Tyr Ile Thr Gly Trp Gly Ar
g Leu Tyr Thr Asn Gly Pro Leu Pro Asp Lys Leu Gln
Glu Ala Leu Leu Pro Val Val Asp Tyr Glu His Cys Se
r Arg Trp Asn Trp Trp Gly Ser Ser Val Lys Lys Thr
Met Val Cys Ala Gly Gly Asp Ile Arg Ser Gly Cys As
n Gly Asp Ser Gly Gly Pro Leu Asn Cys Pro Thr Glu
Asp Gly Gly Trp Gln Val His Gly Val Thr Ser Phe Va
l Ser Ala Phe Gly Cys Asn Thr Arg Arg Lys Pro Thr
Val Phe Thr Arg Val Ser Ala Phe Ile Asp Trp Ile Gl
The object of the present invention can be achieved by producing and accumulating the peptide represented by u Glu Thr Ile Ala Ser His- (C) or its equivalent substance, and collecting the peptide.

As the medium, a medium consisting of glucose, casamino acid, etc., for example, M9 medium [Miller, J .: Experiments in Molec
ular Genetics, 431〜433 (Cold Spring Harbor Lab., Ne
w York (1972))].

Cultivation of the recombinant is usually carried out at 15 to 43 ° C. for 3 to 24 hours, and if necessary, aeration and stirring can be added.
When using animal cells as a host, culturing for 3 to 10 days is required.

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

On the other hand, when Escherichia coli is used as a host, the produced elastase IIIB is an insoluble protein and
Often included in n body. In this case, the elastase produced by destroying the cells and centrifuging
IIIB is obtained as a precipitate.

As a method of destroying the bacterial cells, ultrasonic treatment, lysozyme treatment, freeze-thaw treatment and the like can be adopted. Isolation of elastase from the supernatant or the precipitate can be carried out by a commonly known protein purification method.

Human pancreatic elastase IIIB produced by the present invention
Shows a biological activity equivalent to that of elastase extracted and purified from human pancreatic juice, and can be used for the same purpose and by the same usage.

Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the scope of the present invention.

Example (1) Isolation of mRNA from human pancreas 5.5 g of human pancreas (autopsy sample) was treated with a guanidine thiocyanate solution (4 M guanidine thiocyanate, 1% sarcosyl, 20 mM ethylenediaminetetraacetic acid (EDTA), 25 mM sodium citrate). After disruption and denaturation with a polytron in (pH 7.0), 100 mM 2-mercaptoethanol, 0.1% antiform A), centrifugation was performed to obtain a supernatant. to this
A 0.025-fold amount of 1 M acetic acid and a 0.75-fold amount of ethanol were added, the mixture was cooled at -20 ° C for several hours, and then a precipitate was obtained by centrifugation. Next, the precipitate was suspended in a guanidine hydrochloride solution (7.5 M guanidine hydrochloride, 25 mM sodium citrate (pH 7.0), 5 mM dithiothreitol (DTT), and added with 0.025 volume of 1 M acetic acid and 0.5 volume. After adding a certain amount of ethanol and 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, and the mixture was cooled to -20 ° C. After that, the precipitate was collected by centrifugation, washed with ethanol several times to remove the mixed guanidine hydrochloride, dissolved in distilled water and then precipitated with RNA to precipitate RNA. Were collected to obtain 53.9 mg of RNA.

The RNA thus obtained was treated with high salt solution (0.5M NaCl), 20mM Tris.
-After adsorbing to the oligo (dT) cellulose column in HCl (pH 7.5), 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), mRNA containing poly (A) was eluted (10 mM).
Elution was performed with Tris-HCl (pH 7.5), 1 mM EDTA, 0.05% SDS) to obtain 255 μg of mRNA.

(2) Preparation of cDNA bank The cDNA bank was prepared according to the Okayama-Berg method. That is, 5 μg of mRNA and 24 units of reverse transcriptase were added to 20 μl of the reaction solution [50 mM Tris-HCl (pH 8.3), 8 mM
MgCl ₂ , 30 mM KCl, 0.3 mM DTT, 2 mM dATP, 2 mM dGTP, 2 mM
DCTP, 2 mM dTTP, 10 μCi α-32P-dCTP, 1.4 μg vector primer DNA (purchased from PL-Phar macia)]
The reaction was carried out at 42 ° C for 60 minutes.

After stopping the reaction by adding 2 μl of 0.25 M EDTA and 1 μl of 10% SDS, the protein was deproteinized with 20 μl of phenol / chloroform. After centrifugation, remove the aqueous layer and add 20 μl of 4M ammonium acetate and 80 μl of ethanol,
Cooled for a minute. Then, the precipitate was collected by centrifugation, washed with 75% ethanol, and then dried under reduced pressure.

Precipitate 15 μl of terminal transferase solution (140 mM potassium cacodylate, 30 mM Tris-HCl (pH 6.
8), 1 mM cobalt chloride, 0.5 mM DTT, 0.2 μg polyA, 1
It was dissolved in 00 mM dCTP).

After warming the reaction solution at 37 ° C. for 3 minutes, 18 units of terminal deoxynucleotidyl transferase was added and reacted for 5 minutes. Then 1 μl of 0.25 M EDTA and
After stopping the reaction by adding 0.5 μl of 10% SDS,
Deproteinization was performed with chloroform. After centrifuging the reaction solution, the aqueous layer was taken, 15 μl of 4M ammonium acetate and 60 μl of ethanol were added and mixed well. After leaving at -70 ° C for 15 minutes,
The precipitate was collected by centrifugation.

Precipitate 10 μl of restriction enzyme buffer (50 mM NaCl, 10 mM T
ris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT),
2.5 units of restriction enzyme HindIII was added and digested at 37 ° C for about 1 hour. Next, after deproteinizing with phenol / chloroform, ethanol precipitation was performed. After cooling at −70 ° C. for 15 minutes, the precipitate was collected by centrifugation and 10 μl of TE (10 mM Tr
It was dissolved in is-HCl (pH 7.5), 1 mM EDTA).

Next, 1 μl of the above sample was added to a linker (PL-
Purchased from Pharmacia) Reaction solution containing 10 ng (10 mM Tris
-HCl (pH 7.5), 1mM EDTA, 100mM NaCl), add 65 ℃
It was heated for 5 minutes at 42 ° C. and then kept at 42 ° C. for 30 minutes.

After cooling the reaction solution on ice, 10 μl of 10-fold ligase buffer (10 mM ATP, 660 mM Tris-HCl (pH 7.5), 66 mM MgC)
l ₂ , 100 mM DTT), 78 μl distilled water, 8 units T4 DNA
Ligase was added and the mixture was kept warm at 12 ° C overnight.

Then 10 μl of 1 M KCl, 1 unit ribonuclease
H, 33 unit DNA polymerase I, 4 unit T4 DNA ligase, 0.5 μl of nucleotide solution (20 mM dATP, 20 mM
DGTP, 20 mM dCTP, 20 mM dTTP), 0.1 μl of 50 μg / 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.

The reaction solution was diluted 5-fold, immediately method of Hanahan [Hanahan.D.:J.Mol.Biol., 166, 557 (1983 ) ] Yotsute, E. coli RR1 strain was transformed, human pancreas A cDNA bank was created.

(3) Isolation of recombinant strain containing human pancreatic elastase IIIB cDNA DN of recombinant strain of 4700 strains of the above human pancreatic cDNA bank
A is the method of Grunstein.M. And Hogness, DS [Proc.Nat
L. Acad. Sci., USA, 72 , 3961 (1975)] on a nitrocellulose filter.

Meanwhile, the porcine pancreatic elastase I cDNA fragment was digested with the restriction enzyme Pst1.
By using the nickel translation method [Rigb
y, PW et al. J. Mol. Biol., 113 , 237 (1977)].
Labeled with 2P. Using this DNA fragment as a probe, 40% formamide, 5-fold concentration of 0.15M sodium chloride and
0.015M sodium citrate solution (SSC), 0.1% BSA 0.1
% FICOL, 0.1% polyvinylpyrrolidone, 0.5% SD
S, 10 mM sodium phosphate, 100 μg / ml salmon sperm DNA
DNA fixed on the filter at 35 ℃ in the solution of
I met him. Then, autoradiography was performed to obtain 10 strains that react with the above probe. these
When the nucleotide sequence of the cDNA region of the plasmid contained in one of the 10 strains was determined, the nucleotide sequence of the cDNA encoding human / pancreatic elastase that was previously identified (Japanese Patent Application No. 60-
72308, Japanese Patent Application No. 60-91986, Japanese Patent Application No. 60-163964). This cDNA was named CL1.

(4) The amino acid sequence encoded by CL1 and human pancreatic elastase IIIB CL1 had the only open reading frame capable of encoding 270 amino acids. The amino acid sequence determined from the nucleotide sequence showed only about 30% homology with trypsin and chymotrypsin, but showed high homology of about 50% with porcine pancreatic elastase 1. In addition, an amino acid sequence near the active center commonly found in serine proteases such as trypsin and chymotrypsin (Gly-A
sp-Ser-Gly-Gly-Pro) was present in the same region. In addition, the 45th histidine residue, 95th aspartic acid residue, and 189th serine residue involved in the charge relay characteristic of serine protease were also present.

In addition, the amino acid sequence near the carboxyl terminus that forms the substrate-binding pocket in elastase is CL
211 which is very similar between that encoded by 1 and porcine elastase I, and which is characteristic of its substrate-binding pocket.
The second valine residue and the 223rd threonine residue were also present in the amino acid sequence encoded by CL1. Table 1 shows the nucleotide sequence of CL1 and the amino acid sequence encoded thereby.

The amino acid composition of the protein encoded by CL1 is Lar
Human pancreatic elastase I reported by gman et al.
Amino acid composition of [Largman.C. Et al. Biochemistry 15 ,
2491 (1976)], but apparently different in detail and also different in molecular weight.

Human preproelastase I encoded by CL1
In IIB, the C-terminal side of the arginine residue, which is the 28th amino acid from the N-terminus, is cleaved by trypsin, and the
Elastase IIIB is produced.

Human preproelastase I encoded by CL1
The amino acid sequence homology between IIB and human preproelastase IIIA encoded by CL2 (Japanese Patent Application No. 60-72308) found above was 93.3%, and the nucleotide sequences in both coding frames were The homology is 95.3%, which is a closely related protein but is clearly different. MRN corresponding to CL1
In human pancreas, the expression level of A is the mR corresponding to CL2.
It is about 10% of the expression level of NA.

CL1 is a full-length cDNA synthesized from human elastase IIIB mRNA by reverse transcriptase, so by transferring this cDNA sequence to an appropriate expression vector, E. coli, Bacillus subtilis,
Human pancreatic elastase IIIB can be mass-produced using yeast or animal cells as hosts.

(5) Production of human pancreatic elastase IIIB using animal cells Construction of expression plasmid psv2-CL1 In order to express human elastase IIIB cDNA using animal cells as a host, the cDNA and expression vector were ligated by the procedure shown in FIG. did. Expression vectors include sv40 promoter, enhancer, poly A signal, small T anti
The psv2 plasmid containing the intervening sequence (intron) of the gen gene was used.

A clone in which the vector and cDNA were ligated in the forward direction with respect to the transcription direction was selected according to the cleavage patterns of various restriction enzymes, and introduced (transfection) by the animal cell (COS1 cell) calcium helate method.

Transfection of expression plasmid psv2-CL1 into COS1 cells
従Tsuta the method of am and Van Der Eb [Virology 52, 456 (1973)].

As COS1 cells used for the transfer, 1 × 10 ⁶ cells were seeded on a dish having a diameter of 10 cm and cultured overnight in a Dulbecco's modified Eagle medium containing 10% fetal bovine serum. Then 30
After suspending 0 μg of psv2-CL1 plasmid in 12.55 ml of sterilized distilled water and adding 0.75 ml of 2.5M CaCl ₂ and mixing well,
While foaming the solution with a pipette, add 1.5 ml of 10
× HeBS solution (210 mM HEPES, 1.37 M NaCl, 4.7 mM KC
1, 10.6 mM Na ₂ HPO ₄ , 55.5 mM glucose, pH 7.05) was added dropwise to form a precipitate of DNA and calcium phosphate. 30
After allowing to stand for a minute at room temperature to mature the precipitate, 1 ml of this is
COS1 replaced with fresh medium containing 10% fetal calf serum
Add it to the cells per sheet, and then add 37
After culturing for 12 hours at 5 ° C in the presence of 5% CO ₂ , the culture solution was discarded and replaced with a new Dulbecco's modified Eagle medium containing no fetal bovine serum, and at 37 ° C in the presence of 5% CO ₂ , It was cultured for 48 hours. The transfected COS1 cells obtained here are used for confirmation of expression of human elastase IIIB mRNA and measurement of elastase activity in the culture supernatant.

MRNA extraction from COS1 cells To confirm the presence of human elastase IIIB mRNA transcribed from the expression plasmid in the transfected COS1 cells, the mRNA from COS1 cells was analyzed as follows.
NA was extracted and subjected to Northern blot hybridization.

COS1 cells after culturing for 48 hours, 1 ml per sheet
Guanidine thiocyanate solution (4M guanidine thiocyanate, 1% sarcosyl, 20 mM ethylenediaminetetraacetic acid, 25 mM sodium citrate (pH 7.0), 100 mM
2-mercaptoethanol, 0.1% antiform A) was added to thaw the cells. Next, after passing this solution through a 21-gauge injection needle several times to reduce the molecular weight of high-molecular-weight DNA,
The solution was overlaid on a 5.7 M cesium chloride and 0.1 M ethylenediaminetetraacetic acid solution, and centrifuged using a Hitachi RPS40 swing rotor at 30,000 rpm, 20 ° C. for 17 hours. Got here
The RNA precipitate was washed with a small amount of ethanol and dissolved in 300 μl of distilled water.

Next, the separated total RNA and the method of Aviv and Leder [Proc. Natl.
Acad.Sci.USA 69 , 1408 (1972)] and applied to an oligo dT cellulose column to purify several μg of mRNA. The method of Thomas [Proc. Natl. Aca] using half of the purified mRNA.
d.Sci.USA 77 , 5201 (1980)] was used for Northern blot hybridization. The probe has
Nick translation method [Rigby, PW et al. J. Mol.
Biol. 113 , 237 (1977)], 32P-labeled human elastase IIIB cDNA was used. As a result of hybridization, the size of hybridizing with the probe only to the mRNA of COS1 cells transfected with psv2-CL1 was about 1.
A strong band of 8 kb and a weak band of about 1.0 kb in size were detected.

When psv2-CL1 is transcribed, when the transcription is terminated by the poly A signal contained in the vector, mRNA having a size of 1.8 kb is produced, and when the transcription is terminated by the poly A signal contained in cDNA, the mRNA of 1.0 kb is produced. Presumed. The results obtained by Northern blot hybridization were in agreement with their estimates. Therefore, psv2 in COS1 cells
-CL1 is expressed in large amounts using the SV40 promoter,
It was revealed that most of the transcribed mRNA was not terminated by the poly A signal contained in the cDNA but terminated by the poly A signal contained in the vector.

Elastase activity in medium supernatant Since human elastase IIIB cDNA incorporated into psv2 retains the signal peptide region, it is expected that the expressed elastase will be secreted into the medium as proelastase. Therefore, the elastase activity in the culture medium after 48 hours of culture was measured. The measurement of elastase activity is
A method using a synthetic substrate of Bieth et al. (Front.Matrix.Biol.
No. 6 , 1 (1978)].

To 1 ml of the culture supernatant, 200 μl of 1M Tris-HCl (pH 8.5) and 50
After activation treatment of proelastase by adding 10 μl of 10 mg / ml trypsin and incubating at 25 ° C. for 15 minutes, 50 μl of soybean trypsin inhibitor solution (10
mg / ml) and 125 mM succinyl-L-alanyl-L-alanyl- dissolved in 10.4 μl N-methylpyrrolidone
L-alanine-p-nitroanilide (Suc-Ala-Ala-A
la-pNA) was added. This reaction solution was kept at 37 ° C. for 1 hour and then the absorbance at 410 nm was measured.

As a result of measurement using a culture solution of COS1 cells transfected with psv2-CL1, elastase activity was observed in the culture solution only when activated with trypsin, and human elastase IIIB was detected in COS1 cells. It was shown that it was produced.

In the present example in which psv2-CL1 was introduced into COS1 cells, the production of human elastase IIIB was short-term (transient expres
selection marker suitable for the psv2-CL1 plasmid (eg, neo gene, dihydrofolate reductase).
(For example, a gene) and introduced into CHO cells or the like, a cell line capable of producing human elastase IIIB for a long period of time can be obtained.

(6) Production of human pancreatic elastase IIIB using Bacillus subtilis Construction of expression vector The construction method of the expression vector is shown in FIG. That is, the plasmid PCL1 in which the cDNA of human elastase IIIB has been cloned is cleaved with restriction enzymes HindIII and BglII, and a 712 base pair DNA fragment containing a part of human elastase IIIB cDNA is isolated by agarose gel electrophoresis. did. To this DNA fragment, a synthetic oligonucleotide consisting of 85 base pairs (Fig. 3) encoding a part of the signal peptide of Bacillus subtilis α-amylase and 24 amino acids on the amino terminal side of elastase IIIB was ligated with T4 DNA ligase After that, a DNA fragment of 797 base pairs was separated by agarose gel electrophoresis.

On the other hand, the plasmid PTUB228 (K. Ohmura, T. Shiroza, K. Na.
kamura, A.Nakayama, K.Yamane, K.Yoda, M.Yamasaki, and
G.Tamura: J.Biochem. 95 , 87-93 (1984) restriction enzyme Hind
Cleavage with III, a 428 base pair DNA fragment containing the α-amylase promoter and part of the signal peptide, and a 5100 base pair DNA fragment containing the replication origin were each subjected to 1.2% agarose gel electrophoresis. Isolated. The 428 base pair DNA fragment was further digested with the restriction enzyme HpaII and 5100
The base-paired DNA fragment is cleaved with the restriction enzyme BclI and then 1.2
% Agarose gel electrophoresis with 385 base pairs each,
And a 4173 base pair DNA fragment were isolated. 3 types of D above
After ligating the NA fragment with T4 DNA ligase, the Bacillus subtilis strain 207-25 (m ₁₆₃ hsrM recE4 amyE07 aroI906l was prepared by a conventional method.
euA8 lys21; derived from Marburg strain) was incorporated into protoplasts and regenerated, followed by culturing in a medium containing 10 μg / ml of kanamycin to obtain a transformant capable of growing on this medium. The target recombinant is the 85 base pair DNA shown in FIG.
Isolate the plasmid from the positive clone obtained by the colony hybridization method using as a probe,
It was separated by examining the nucleotide sequence of the DNA. One of the clones thus obtained was named pHAM001.

Confirmation of Product The Bacillus subtilis strain 207-25 into which the expression vector of elastase IIIB, pHAM001 was introduced, was used in LG medium 1 containing 50 μg / ml of kanamycin (per Bacto Tryptone (Difc
o) 10g, Bacto Yeast Extract (Difco) 5g, NaCl5g, Gluco
SE2g, pH7.0), and reciprocal shaking culture was performed at 35 ° C for 48 hours.

After culturing, the culture solution is cooled to 4 ℃ and then 3.000 x G / 5.
After removing the bacterial cells by centrifugation for minutes, ammonium sulfate was added so as to achieve 55% saturation, and the mixture was stirred at 4 ° C for 12 hours. The insoluble matter generated by this operation was precipitated by centrifugation at 8,000 × G / 20 minutes, the supernatant was removed, and the precipitate was collected in 20 ml of 50 mM.
It was dissolved in Tris-HCl (pH 8.0). Add this solution to 500 ml of 50
It was dialyzed against mM Tris-HCl (pH 8.0) for 16 hours, and insoluble substances were removed by centrifugation at 8,000 × G / 20 minutes. The dialyzed solution was used as a crude elastase IIIB solution in the following assay.

The elastase activity of the sample was measured by the following method.
That is, the synthetic substrate N-carbobenzoxy-L-alanine-
p-nitrophenyl ester (sigma) was added to 50 mM Tris-HCl (pH
8.0) to make a 0.1 mM solution. Add 0.25 ml of elastase sample solution to 0.25 ml of this substrate solution, and at 37 ℃
After reacting for 30 minutes, the absorbance at 410 nm was measured. At the same time, the elastase inhibitor elastatinal or α-1-antitribucin was added to the sample solution at a final concentration of 0.1 m.
In addition to g / ml, the degree of inhibition of the activity was measured. In the 207-25 strain into which the plasmid correctly bound to the promoter portion was introduced, the absorbance increased by 0.24 as compared with the control solution, and elastase activity could be detected. It was also confirmed at the same time that this elastase activity was suppressed by the above-mentioned two inhibitors.

The reaction of the restriction enzymes and other enzymes used in this example was carried out according to the buffer solution and reaction conditions described in the instructions added when the enzyme was purchased.

(7) Production of human pancreatic elastase IIIB using Escherichia coli The plasmid PCL1 in which the cDNA of human elastase IIIB was cloned was cleaved with restriction enzymes EcoRI and BglII to isolate a 1640 bp DNA fragment containing the cDNA of human elastase IIIB. . This was partially cleaved with the restriction enzyme Fnu4HI, and then
Treatment with S1 nuclease gave a 788 bp DNA fragment with the single-stranded portion as a blunt end. Separately, the plasmid puc8 was cleaved with a restriction enzyme SmaI and treated with phosphatase to separate a DNA fragment containing a promoter and an operator region of the lactose operon and a part of β-galactosidase.

The above-mentioned two kinds of DNA fragments were mixed with 30 μl of a solution containing T4 DNA ligase (66 mM Tris-HCl (pH 7.6), 6.6 mM Mgcl ₂ , 10 mM DT).
T, 1 mM ATP, 2.5 unit T4 DNA ligase) at 6 ° C, 7
Both DNA fragments were ligated by incubating for 2 hours (Fig. 4).

PH of the human elastase expression plasmid constructed in this way
It was named EX102. Various Escherichia coli was transformed with pHEX102 to obtain a strain capable of producing human elastase.

The human elastase expressed by this method becomes a fusion protein in which eight amino acids derived from β-galactosidase are linked to the N-terminus of mature human elastase as described below.

The DNA base sequence and amino acid sequence near the 5'end of the elastase gene in the human elastase expression plasmid pHEX102 are shown below.

The strain containing the elastase expression plasmid obtained as described above was treated with 2XTY-ampicillin medium (1.6% bactotryptone, 1% yeast extract, 0.5% NaC).
l, 50 μg / ml of ampicillin), and cultured at 37 ° C. for 15 hours. After culturing, centrifuge the culture solution to collect the cells and
X 10 ⁸ cells equivalent of 15 μl of SDS solution (2% SD
S, 5% 2-mercaptoethanol, 10% glycerin, 60 mM Tris-HCl (pH 6.8)) and suspended at 100 ° C for 3 minutes, and then the method of Laemmli et al. [Nature, 227 680 (19
70)] and subjected to SDS-polyacrylamide gel electrophoresis to analyze the produced protein.

The results showed that the YA21 strain produced a large amount of elastase fusion protein. The production of elastase fusion protein corresponded to 30% of the total protein produced by the YA21 strain. The X984 strain also produced a relatively large amount of elastase fusion protein, but the production was less than half that of the YA21 strain. The production amount of elastase fusion protein in the other two strains of Escherichia coli (HB101 strain and MC4100 strain) was extremely low.

Since the elastase fusion protein forms an inclusion body in the bacterial cells, it could be purified relatively easily. In other words, from the culture solution 1 of YA21 / pHEX102, the inclusion body
As a result, 6.6 g of the bacterial cells forming the bacterium were obtained. Obtained cells 6.
6 g, Lysozyme 0.2 mg / ml and deoxycholic acid 1 mg / ml
The cells were lysed by treatment in 50 mM Tris-HCl buffer (pH 8.0) containing Unbroken cells were centrifuged at low speed (1500Xg, 1
After removal by 0 min), inclusion bodies were obtained as a precipitate by high speed centrifugation (11000 × g, 20 min). This inclus
Since the ion body still contains a large amount of bacterial cell fragments,
The suspension was suspended in 50 mM Tris-HCl buffer containing 5 mg / ml of Triton X-100 and then washed by high speed centrifugation (11000 × g, 20 minutes). After washing, the inclusion body was suspended in a small amount of Tris-HCl buffer and stored at 4 ° C.

By refining in this way, 350 mg of inclusion bod
y was obtained, which contains about 50% elastase IIIB fusion protein. Also inclus produced by YA21 / pHEX102
The fact that the ion body is an elastase fusion protein means that
It confirmed by the blotting method.

As described above, most of the elastase produced in Escherichia coli becomes inclusion bodies and exists in the insoluble fraction of the cells, but some of them exist in a state that they are soluble and retain the enzyme activity. There is. The enzyme activity was detected as follows.

Escherichia coli X984 introduced with elastase IIIB expression plasmid
The strain was shake-cultured in 2XTY-ampicillin medium 1 at 37 ° C for 15 hours. After the culture was completed, the cells were collected by centrifugation at 3,000 XG / 5 minutes, and 20 ml of buffer solution A (50 mM Tris-HCl, 1 mM EDT was added.
A, 50 mM NaCl pH 8.0) was suspended, 10 mg of lysozyme was added, and the mixture was kept at 5 ° C. for 20 minutes. After that, deoxycholic acid was added to a final concentration of 1 mg / ml, heated to 20 ° C, and further deoxyribonuclease was added to a final concentration of 0.1 mg / ml, and cells were treated with a polytron homogenizer. Was crushed. The lysate thus obtained was added to 80,000XG / 40
After centrifugation for minutes to remove bacterial cell fragments, it was subjected to Sephadex G-75 column chromatography. The elastase active fraction was further purified by antibody affinity chromatography. After that, trypsin was added to 0.1 mg / ml and kept warm at 25 ℃ for 5 minutes.
Crude elastase was prepared by activating proelastase by adding soybean trypsin inhibitor to g / ml.
It was used as the IIIB sample solution in the following assay.

The elastase activity of the sample was measured by the following method.
That is, the synthetic substrate N-Carbobenzoxy-L-alanine-
Add 50 mM Tris-HCl (pH) to p-nitrophenyl ester (Sigma).
8.0) to make a 0.1 mM solution. Add 0.25 ml of elastase sample solution to 0.25 ml of this substrate solution, and at 37 ℃
After reacting for 30 minutes, the absorbance at 410 nm was measured. As a result, the absorbance increased by 0.36, and elastase activity could be detected. At the same time, elastatinal or α-1-antitrypsin, which is an inhibitor of elastase, was added to the sample solution to a final concentration of 0.1 mg / ml, and it was confirmed that the activity was inhibited by these. .

(8) Production of human pancreatic elastase IIIB using yeast As in the case of Escherichia coli, Bacillus subtilis and animal cells, the human pancreatic elastase IIIB cDNA is used as a host.
Was ligated to a suitable expression vector by a conventional method, transferred into a host cell and allowed to express, and the presence of elastase activity in the culture could be confirmed.

S. cerevisiae described in "Recombinant DNA Experimental Guideline"
Can be used as a host, but specifically, the S288C strain and the like were suitable. On the other hand, as a vector, YEp13
Etc. were suitable. As the promoter, the ADH1 gene encoding the alcohol dehydrogenase gene was suitable.

[Brief description of drawings]

FIG. 1 shows the procedure for constructing the psv2-CL1 plasmid. psv2-
β-globin is a plasmid in which the β-globin cDNA is inserted into the psv2 vector, and PCL1 is a plasmid in which the human elastase IIIB cDNA is inserted into the Okayama-Berg vector. Reactions such as S1 nuclease are described in “Molecular cloning” [Maniatis, T. et al. (Ed) “Molec
ular cloning "Cold Spring Harbor Lab (1982)]. Thick arrows indicate sv40-derived promoters, thin arrows indicate the direction of transcription. H indicates HindIII, B indicates B.
glII and P indicate PstI. FIG. 2 shows the procedure for constructing the pHAM001 plasmid. pTUB228
Is a plasmid in which the α-amylase gene of Bacillus subtilis has been cloned into a vector having a replication origin of Bacillus subtilis. The hatched region indicates human elastase IIIB cDNA, and the thick arrow indicates the α-amylase promoter. FIG. 3 shows an 85 base pair synthetic DNA containing a signal peptide region of the Bacillus subtilis α-amylase gene and a cDNA corresponding to the amino terminal side of mature human elastase IIIB. FIG. 4 shows the procedure for constructing the pHEX102 plasmid. The hatched region indicates human elastase IIIB cDNA, and the thick arrow indicates the lactose promoter operator.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication C12N 5/10 // A61K 37/547 ABX 8314-4C C12N 9/66 9359-4B (72) Invention Hidehiko Furukawa 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo stock company (72) Inventor Toshinori Omine 1-2-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo stock company

Claims (9)

[Claims] 1. A general formula (N) -Val Val Asn Gly Glu Asp Ala Val Pro Tyr Se.
r Trp Pro Trp Gln Val Ser Leu Gln Tyr Glu Lys Ser
Gly Ser Phe Tyr His Thr Cys Gly Gly Ser Leu Ile Al
a Pro Asp Trp Val Val Thr Ala Gly His Cys Ile Ser
Ser Ser Arg Thr Tyr Gln Val Val Leu Gly Glu Tyr As
p Arg Ala Val Lys Glu Gly Pro Glu Gln Val Ile Pro
Ile Asn Ser Gly Asp Leu Phe Val His Pro Leu Trp As
n Arg Ser Cys Val Ala Cys Gly Asn Asp Ile Ala Leu
Ile Lys Leu Ser Arg Ser Ala Gln Leu Gly Asp Ala Va
l Gln Leu Ala Ser Leu Pro Pro Ala Gly Asp Ile Leu
Pro Asn Glu Thr Pro Cys Tyr Ile Thr Gly Trp Gly Ar
g Leu Tyr Thr Asn Gly Pro Leu Pro Asp Lys Leu Gln
Glu Ala Leu Leu Pro Val Val Asp Tyr Glu His Cys Se
r Arg Trp Asn Trp Trp Gly Ser Ser Val Lys Lys Thr
Met Val Cys Ala Gly Gly Asp Ile Arg Ser Gly Cys As
n Gly Asp Ser Gly Gly Pro Leu Asn Cys Pro Thr Glu
Asp Gly Gly Trp Gln Val His Gly Val Thr Ser Phe Va
l Ser Ala Phe Gly Cys Asn Thr Arg Arg Lys Pro Thr
Val Phe Thr Arg Val Ser Ala Phe Ile Asp Trp Ile Gl
u Glu Thr Ile Ala Ser His- (C) DN encoding human pancreatic elastase IIIB
A. 2. A (N) -terminal has a hydrogen atom, Met, or (N) -Met Met Leu Arg Leu Leu Ser Ser Leu Leu Le
u Val Ala Val Ala Ser Gly Tyr Gly Pro Pro Ser Ser
Human pancreatic elastase IIIB having a part or all of Arg Pro Ser Ser Arg- (C)
The DNA according to claim 1, which encodes 3. A general formula (5 ')-GTT GTC AAT GGT GAG GAT GCG GTC CCC TAC.
AGC TGG CCC TGG CAG GTT TCC CTG CAG TAT GAG AAA AG
C GGA AGC TTC TAC CAC ACC TGT GGC GGT AGC CTC ATC
GCC CCC GAC TGG GTT GTG ACT GCC GGC CAC TGC ATC TC
G AGC TCC CGG ACC TAC CAG GTG GTG TTG GGC GAG TAC
GAC CGT GCT GTG AAG GAG GGC CCC GAG CAG GTG ATC CC
C ATC AAC TCT GGG GAC CTC TTT GTG CAT CCA CTC TGG
AAC CGC TCG TGT GTG GCC TGT GGC AAT GAC ATC GCC CT
C ATC AAG CTC TCA CGC AGC GCC CAG CTG GGA GAC GCC
GTC CAG CTC GCC TCA CTC CCT CCG GCT GGT GAC ATC CT
T CCC AAC GAG ACA CCC TGC TAC ATC ACC GGC TGG GGC
CGT CTC TAT ACC AAC GGG CCA CTC CCA GAC AAG CTG CA
G GAG GCC CTG CTG CCG GTG GTG GAC TAT GAA CAC TGC
TCC AGG TGG AAC TGG TGG GGT TCC TCC GTG AAG AAG AC
C ATG GTG TGT GCT GGA GGG GAC ATC CGC TCC GGC TGC
AAT GGT GAC TCT GGA GGA CCC CTC AAC TGC CCC ACA GA
G GAT GGT GGC TGG CAG GTC CAT GGC GTG ACC AGC TTT
GTT TCT GCC TTT GGC TGC AAC ACC CGC AGG AAG CCC AC
G GTG TTC ACT CGA GTC TCC GCC TTC ATT GAC TGG ATT
A base sequence represented by GAG GAG ACC ATA GCA AGC CAC-X (3 ') (wherein X represents TAA, TGA or TAG).
Item or the DNA according to Item 2. 4. ATG at the 5'end, or (5 ')-ATG ATG CTC CGG CTG CTC AGT TCC CTC CTC
CTT GTG GCC GTT GCC TCA GGC TAT GGC CCA CCT TCC TC
The DNA according to claim 3, which comprises a part or all of T CGC CCT TCC AGC CGC- (3 '). 5. A general formula (5 ′)-GTT GTC AAT GGT GAG GAT GCG GTC CCC TAC characterized by including the following (a) to (d):
AGC TGG CCC TGG CAG GTT TCC CTG CAG TAT GAG AAA AG
C GGA AGC TTC TAC CAC ACC TGT GGC GGT AGC CTC ATC
GCC CCC GAC TGG GTT GTG ACT GCC GGC CAC TGC ATC TC
G AGC TCC CGG ACC TAC CAG GTG GTG TTG GGC GAG TAC
GAC CGT GCT GTG AAG GAG GGC CCC GAG CAG GTG ATC CC
C ATC AAC TCT GGG GAC CTC TTT GTG CAT CCA CTC TGG
AAC CGC TCG TGT GTG GCC TGT GGC AAT GAC ATC GCC CT
C ATC AAG CTC TCA CGC AGC GCC CAG CTG GGA GAC GCC
GTC CAG CTC GCC TCA CTC CCT CCG GCT GGT GAC ATC CT
T CCC AAC GAG ACA CCC TGC TAC ATC ACC GGC TGG GGC
CGT CTC TAT ACC AAC GGG CCA CTC CCA GAC AAG CTG CA
G GAG GCC CTG CTG CCG GTG GTG GAC TAT GAA CAC TGC
TCC AGG TGG AAC TGG TGG GGT TCC TCC GTG AAG AAG AC
C ATG GTG TGT GCT GGA GGG GAC ATC CGC TCC GGC TGC
AAT GGT GAC TCT GGA GGA CCC CTC AAC TGC CCC ACA GA
G GAT GGT GGC TGG CAG GTC CAT GGC GTG ACC AGC TTT
GTT TCT GCC TTT GGC TGC AAC ACC CGC AGG AAG CCC AC
G GTG TTC ACT CGA GTC TCC GCC TTC ATT GAC TGG ATT
GAG GAG ACC ATA GCA AGC CAC-X (3 ') (wherein X represents TAA, TGA or TAG) A method for producing a DNA containing a nucleotide sequence. (A) Isolate mRNA from human pancreas. (B) A cDNA bank is prepared using this mRNA and reverse transcriptase. (C) A plasmid containing cDNA encoding human elastase IIIB is isolated from the prepared cDNA bank using a probe. (D) Cut out the desired cloned DNA from this plasmid. 6. ATG at the 5'end, or (5 ')-ATG ATG CTC CGG CTG CTC AGT TCC CTC CTC
CTT GTG GCC GTT GCC TCA GGC TAT GGC CCA CCT TCC TC
The method for producing DNA according to claim 5, which comprises a part or all of T CGC CCT TCC AGC CGC- (3 ′). 7. A general formula (5 ')-GTT GTC AAT GGT GAG GAT GCG GTC CCC TAC.
AGC TGG CCC TGG CAG GTT TCC CTG CAG TAT GAG AAA AG
C GGA AGC TTC TAC CAC ACC TGT GGC GGT AGC CTC ATC
GCC CCC GAC TGG GTT GTG ACT GCC GGC CAC TGC ATC TC
G AGC TCC CGG ACC TAC CAG GTG GTG TTG GGC GAG TAC
GAC CGT GCT GTG AAG GAG GGC CCC GAG CAG GTG ATC CC
C ATC AAC TCT GGG GAC CTC TTT GTG CAT CCA CTC TGG
AAC CGC TCG TGT GTG GCC TGT GGC AAT GAC ATC GCC CT
C ATC AAG CTC TCA CGC AGC GCC CAG CTG GGA GAC GCC
GTC CAG CTC GCC TCA CTC CCT CCG GCT GGT GAC ATC CT
T CCC AAC GAG ACA CCC TGC TAC ATC ACC GGC TGG GGC
CGT CTC TAT ACC AAC GGG CCA CTC CCA GAC AAG CTG CA
G GAG GCC CTG CTG CCG GTG GTG GAC TAT GAA CAC TGC
TCC AGG TGG AAC TGG TGG GGT TCC TCC GTG AAG AAG AC
C ATG GTG TGT GCT GGA GGG GAC ATC CGC TCC GGC TGC
AAT GGT GAC TCT GGA GGA CCC CTC AAC TGC CCC ACA GA
G GAT GGT GGC TGG CAG GTC CAT GGC GTG ACC AGC TTT
GTT TCT GCC TTT GGC TGC AAC ACC CGC AGG AAG CCC AC
G GTG TTC ACT CGA GTC TCC GCC TTC ATT GAC TGG ATT
A host transformed with a DNA containing a base sequence represented by GAG GAG ACC ATA GCA AGC CAC-X (3 ') (wherein X represents TAA, TGA or TAG). 8. ATG at the 5'end, or (5 ')-ATG ATG CTC CGG CTG CTC AGT TCC CTC CTC
CTT GTG GCC GTT GCC TCA GGC TAT GGC CCA CCT TCC TC
A host transformed with the DNA according to claim 7, which comprises a part or all of T CGC CCT TCC AGC CGC- (3 '). 9. The host according to claim 8, which is Escherichia coli, Bacillus subtilis, yeast or an animal cell.