JPS62130686A - Human pancreatic elastase iia - Google Patents

Abstract

PURPOSE:To obtain the aimed elastase, by cloning a human pancreatic elastase, ingressing the resultant recombinant DNA molecules into a host and expressing the pancreatic elastase gene. CONSTITUTION:An mRNA is separated from the human pancreas and used as a template to synthesize a complementary double stranded DNA. The resultant recombinant plasmid is then introduced into to transform Escherichia coli (RRI strain, etc.). Rat elastase IIcDNA is used as a probe in the resultant recombinant to determine a clone having DNA completely cloning a human pancreatic elastase expressed by the formula. Elastase gene is cut out of the resultant clone and linked to a suitable expression vector and introduced into a suitable host for expression. Thereby, the aimed elastase having the biological activity equal to that of the elastase extracted and purified from human pancreatic juice is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human pancreatic elastase-Uk, a DNA containing a base sequence encoding it, a host transformed with the same, and a method for producing human pancreatic elastase using the host.

Elastase is a type of serine protease,
It contains an enzyme that hydrolyzes elastin, a fibrous insoluble protein. Elastin is a type of hard protein that constitutes the connective tissue, key, aortic envelope, and cervical cord of higher animals.
Stones slightly degraded by pepsin and trypsin.

While researching arteriosclerosis, Balo et al. recognized the decomposition of elastin fibers in blood vessel walls, and in 1949 they deduced the existence of the degrading enzyme (Ba1o', J. and
B an gal engineer: Schweiz Z, Pat
hol, Bacteriol, 12°350 (19
49) ), followed by Banga in 1952,
An enzyme that specifically degrades elastin was discovered in the pancreas, isolated in crystal form, and named elastase.
Banga, Eng.: Acta Physiol, Ac.
ad.

Sci, Hung, 3, 317 (1952)]
.

Elastase has been confirmed to exist in the pancreas of most animals, including humans, monkeys, cats, and rabbits, and its content is 3.1 to 1/g of pancreas in humans and 2.2/g in cows. rat is 1o
, about 2my/9. Human elastase activity and
It is recognized that there is a relationship with age, men over 40 years old, women 60 years old
Elastase activity in the pancreas and plasma was significantly decreased in patients with 50% and above [Loeven, W, A, and M
aureen M, Baldwin: Gerontol
ogia, 17, 170 (197i)].

In patients with arteriosclerosis, pancreatic elastase activity was significantly lower than that in healthy subjects or absent (Balo', J., antiBan
ga, 1. : Nature, S'8,310
(1956)]. Further, subsequent research has revealed that elastase not only enzymatically degrades elastin, but also promotes its biosynthesis.

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

1) Effect of suppressing lipid and calcium deposition on the arterial wall 2) Effect of removing cholesterol and calcium from the arterial wall 3) Effect of selectively decomposing denatured elastin 4) Effect of promoting new generation of elastic fibers in the arterial wall 5) Effect of lowering serum lipids 6 ) Riboprotein metabolism improvement effect In a clinical study conducted based on the above research, the following effects were revealed as a result of oral administration of elastase.

1) Restoring elasticity and extensibility of arterial walls 2) Improving serum lipid abnormalities 3) Improving lipoprotein metabolism Elastase extracted and purified from porcine pancreas was used in the above research. However, when porcine elastase is administered to humans, since it is a foreign protein to humans, antibody production occurs, and repeated administration to the patient poses the risk of causing anaphylaxis (Japanese Patent Laid-Open No. 59-11
1so).

Therefore, when administering to humans, it is preferable to use human elastase. However, it has been extremely difficult to obtain a sufficient amount of human elastase. Therefore, the present inventors used recombinant DNA technology to clone the human pancreatic elastase gene, and transferred the obtained recombinant DNA molecule into a host to express the human pancreatic elastase gene. The present invention was completed as a result of research and development of a technology that would enable the production of elastase.

Currently, the existence of two types of human pancreatic elastase is already known, but their characterization has not yet been fully performed. The sequence includes the 12 activation peptide portions and the mature elastase (
N)-terminal four amino acid sequences have been revealed (
Largman, C, et al; Blochim, B
iophys, Acta, 623°208 (1980
) ), and its structure has remained completely unknown until now. However, the present inventors have now revealed all of its amino acid sequences and all of its DNA sequences.

The amino acid sequence revealed by the present invention is that (N
) - the terminal 16 amino acids are the 16 amino acid sequence of human pancreatic elastase-■ reported by Largman, C. et al. (Cys Gly Asp Pro 'rh = T
yrPro Pro Tyr Val Thr Arg
Val Val G], y Gly ), which clearly corresponds to the sequence of human pancreatic elastase-H.

That is, the present invention provides human pancreatic elastase+1A
The present invention provides a method for producing human pancreatic elastase-11A by culturing a DNA containing a base sequence encoding a nucleotide sequence, a host transformed with the DNA, and a host transformed with the DNA. .

The present invention relates to DNA encoding human pancreatic elastase-11A represented by the general formula %, human pancreatic elastase-11A represented by the amino acid sequence, and human pancreatic elastase-IIA represented by (1).

In the formula, at the (N)-terminus, there is a hydrogen atom, Met, or Met element Arg Thr Leu Leu Le
u, Ser Thr LeuVax Axa G17
It may have Ala Leu 5er.

In particular, the general formula %formula% ([) (In the formula, X represents TAA, TGA or TAG.

) or a DNA containing a base sequence equivalent to the base sequence.

The above DNA (II) has ATG or (5')-ATG4TA AGG AC! at its 5th end. GC! T.G.
(!TG C, TG TOOACT TTGGTG
GCT GGA Gcc CTCAGT-(3).

When DHA C+1> has ATG at the 5' end, it encodes a polypeptide having Met at the (N)-terminus of (1) in addition to polypeptide (1).

(5')-ATG ATA AGG ACG CTG at the 5-end of DNA (II)
CTG CTG Tcc ACT TTGGTG GC
! When having T GGA Goo'bTc AGT-(3), in addition to the polypeptide (+), Met engineering 1e ArgThr Leu Leu Leu is added to the (N)-terminus of (r).
Ser Thr Leu Van Ala Gly A
encodes a polypeptide with la Leu Ser.

The DNA of the present invention containing the base sequence represented by formula (II) or a base sequence equivalent thereto can be produced, for example, by the steps (a) to (a) shown below.

(b) Isolate mRNA from human pancreas.

(b) cDNA using this mRNA and reverse transcriptase
Create A bank.

(c) From the prepared cDNA bank, for example, a plasmid containing cDNA encoding the desired human elastase is isolated using rat elastase I [cDNA as a probe.

) Clone the desired DNA from this plasmid.
Cut out.

The guanidine thiocyanate hot phenol method, the guanidine thiocyanate cesium chloride method, etc. can also be used to extract pancreatic RNN oil, but the guanidine thiocyanate-guanidine hydrochloric acid method has the following advantages.
It is superior to the above two methods. (1) Easy to operate. (
2) High extraction and recovery rate. (3) It is possible to extract from a relatively large amount of organs. (4) No DNA is mixed in. (5) Less RNA degradation.

Many of the mRNAs present in the cytoplasm of eukaryotic cells are
Since mRNA is known to have a polyA sequence at its end, this feature is utilized to adsorb mRNA onto an oligo(aT) cellulose column, and then elute and purify it.

Using the obtained mRNA as a template, complementary double-stranded DNA is synthesized using reverse transcriptase, using the S1 nuclease method [Efstratiadis,
A.

et al: C! ell, 7.279 (1976
) ), Land method (La, nd.

H, et al.: Nucleic Ac1ds Re
e, 9.2251 (1981)], Okayama-
Berg method (Okayama, H, and P, Be
rg: Mol.

Ce11B1o1, 2, 16i (1982))
, 01Joon Yoo method CLIP, Joon Yo
o,et al,: Proc, Natl, Acad,
Sci.

USA, 79, 1049 (1982)), but for the purpose of the present invention, Okayama-Be
The rg method was preferred.

Next, the obtained recombinant plasmid is introduced into E. coli, for example, RR1 strain, and transformed. 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,
A colony hybridization method using P-labeled rat elastase l [cDNA as a probe was used.

The nucleotide sequence contained in the selected clone was determined using the method of dideoxynucleotide synthesis chain termination using phage M1'k (Messing, et al.: Nucle
ic Ac1cLs Res,, 9, 309 (1
981)] and the Maxam-Gilbert method (M
axam, A, M, and G11bert.

W, :Proc, NatlAcad, Sci,
USA, 74, 560 (1977)], but in the present invention, a dideoxynucleotide synthetic chain termination method was employed to determine a clone having DNA completely encoding human pancreatic elastase.

Next, the elastase gene is extracted from the obtained clone, ligated to an appropriate expression vector, and introduced into an appropriate host for expression.

Promoters include tryptophan (trp) promoter, lactose (1aC) promoter, tryptophan-lactose (1a) promoter, outer membrane major protein (lpp) promoter, bacteriophage-derived lambda (λ) PL promoter, and protein chain elongation factor T.
u(tufB) promoter, etc. can be used.

As hosts, in addition to bacteria such as Escherichia coli and Bacillus subtilis, and microorganisms such as yeast, animal cells can be used. In the present invention, Escherichia coli (IJ 392. YA21, etc.) is suitable as a host.

As a method for transforming and introducing the DNA of the present invention into a host, Hanahan's method (Hanahan, D.
:J, Mo1. Biol, υkon, 557 (1983
)], calcium chloride method (Dagert, M, an
d E3. D. Ehrlich: Gene, 6.2
3 (1979)), the low pH method [Yasutaka Takagi (ed.): Gene Manipulation Manual + p. 49 + Kodansha University Press (1qs2)), etc. may be employed. In the present invention,
The method of Hanaha H was preferred.

The host (recombinant) obtained in this way is cultured in a known medium, and the polypeptide represented by (1) (with Met or Met engineer A at its (N)-terminus) is added to the culture.
rg Thr Leu Leu Leu Ser Th
r Leu Van Ala Gly Al-a Leu
It may have Ser. ) or its equivalent effect can be produced and accumulated, and the object of the present invention can be achieved by collecting it.

As a medium, a medium consisting of glucose, casamino acids, etc., for example, M9 medium (Miller+J, 'Eixp
elements in Molecular Gen
etics, 431-433 (Oo'ld Sprin.
g Harbor Lab, New York (19
γ2))].

The recombinant is usually cultured at 15-43°C for 3-24°C.
Aeration and stirring can be added as necessary. In addition, when using animal cells as hosts, 3 to 10
Requires incubation for days.

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 Escherichia coli is used as a host, the produced elastase acts as an amnestic protein in 1 nclus within the cell.
It is often included in the ion body.

In this case, destroy the bacterial cells and centrifuge them.
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. Elastase can be isolated from the supernatant or precipitate using commonly known protein purification methods.

Elastase produced according to the present invention exhibits the same biological activity as elastase extracted and purified from human pancreatic juice, and can be used for the same purposes and in the same manner.

In addition, the human pancreatic elastase-+1A of the present invention is 12
It can also exist as a two-chains elastase in which the Arg-Va1 bonds at positions 1 and 13 are broken and Oys at position 1 is connected to day 01 in the molecule through a disulfide bond. Therefore, the present invention provides such two-c
It also includes hais type elastase.

EXAMPLES The present invention will be specifically explained below with reference to Examples, but these are not intended to 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 guanidine thiocyanate solution (
4M guanidine thiocyanate, 1% sarcosyl,
20mM ethylenediaminetetraacetic acid (EDTA), 2
5mM sodium citrate (CpH 7.0), 100m
After disruption and denaturation with a polytron in M 2-mercaptoethanol and 0.1% antiform A), the mixture was centrifuged to obtain a supernatant. KO, 025 times the amount of 1M acetic acid and 0.75 times the amount of ethanol were added, and after cooling at -20°0 for several hours, a precipitate was obtained by centrifugation. Next, this precipitate was mixed with a guanidine hydrochloride solution (7,5
Suspend M guanidine hydrochloride, 25 mM sodium citrate (pH 7,0), 5 mM dithiothreitol (DTT), add 0.025 times the amount of 1M acetic acid and 0.5 times the amount of ethanol, -20
After cooling at °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 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. Collect the precipitate by centrifugation, 53.977
1p RNA was obtained.

The RNA thus obtained was added to a high salt solution (0.5M Na07).
.

20mM Tris-Ho7 (pH 7,5), 1 m
M EDTA 10. After adsorption onto an oligo(aT) cellulose column in sodium dodecyl sulfate (SDS), the poly(A)-containing mRNA was absorbed into the elution fluid (SDS).
iomM TriS-HCI(1)Hy, 5), 1
Elution was performed with mM CI EDTA, 0.05% (5Ds) to obtain 255 μl of mRNA.

(Preparation of the 21c DNA bank The preparation of the cDNA bank was carried out by Okayama-Berg
I did it according to the law. That is, 5 μg of mRNA and 2
Add 4 units of reverse transcriptase to 20μl of reaction solution [50mM
Trls-act! (pH 8,3), 8mM MgC
42,30mM KCI! , 0.3 mM DTT,
-2mM aATP, 2mM (IG'Tp,
2mM da'rp, 2mM dTTP, 1
0 μOi of α-32P-40TP, 1.4 μI of vector primer DNA (PL-Pharmac
The mixture was reacted for 160 minutes at 42° C.

2 μl of 0.25 M KDTA and 1 μl of 10% SD
After stopping the reaction by adding S, protein was removed with 20 μl of phenol/chloroform. After centrifugation, 9.20 μl of 4M ammonium acetate and 80 μl of ethanol were added to the aqueous layer, and the mixture was cooled at −70° C. for 115 minutes. Next, the precipitate was collected by centrifugation, washed with 75% ethanol, and then dried under reduced pressure.

The precipitate was added to 15 μl of terminal transferase reaction solution (140 mM potassium cacodylate, 30 mM Tr.
is-HCI! (pH 5,3), 1mM cobalt chloride, 0,5mM DTT, 0.2μ9 polyA
, 10.0 mM 1iCTP).

After warming the reaction solution at 37°C for 3 minutes, 18 units of terminal deoxynucleotidyl transferase was added and allowed to react for 5 minutes.

Then 1 ttl of 0.25 M EDTA and 0.5
After terminating the reaction by adding μi of 10% SDS, protein was removed with phenol/chloroform. After centrifuging the reaction solution,
Discard the aqueous layer, add 15μl of 4M ammonium acetate, 60μl
1 of ethanol was added and mixed well. 15 to -70°C
After waiting for a minute, the mixture was centrifuged and the precipitate was collected.

Add the precipitate to 10 μl of restriction enzyme buffer (50 mM NaC
I, 10 mM Trit3-HCj' (pH 7,
5) , 10 mM MgC! j'2, j mM DT
Dissolve 25 units of restriction enzyme H1nd 3 in
was added and digested at 37°C for 1 hour.

After protein removal with phenol-chloroform, ethanol precipitation was performed. After cooling at -70°C for 15 minutes, the precipitate was collected by centrifugation and 10ttl of TI (10m
TriS-H (!f (pH 7,5), 1 mM ED
TA).

Next, 1 μl of the above sample was added to the linker with oligo cLG (
(Purchased from PL-Pharmacia) 10n, 9 was added to the reaction solution (10mM Tris-HC1! (pH 7,5
), 1mM 1iiDTA1100mM Na0J
), heated at 65°C for 5 minutes, then heated to 42°C.
It was kept warm for 30 minutes.

- After cooling the reaction in water, add 10 μl of 10x ligase buffer (10mM ATP, 660mM Tris-Hol! (pH 7,5), 66mM MgO
! ! 2.100mM DTT), 78μl distilled water, 8
Unit's T4 DNA ligase was added and the mixture was incubated at 12°C overnight.

Next, 10 μl of 1M
GTP, 20 mM acTP, 20 mM dTTP), 0.1 μl of 50 μν μl bovine serum albumin (BSA) were added and incubated at 12 °C for 1 h, then at 25 °C.
After diluting the reaction solution 5 times at °C for 1 hour, immediately
an's method (Hanahan, D.: I, Mo.
1. Biol, , 166°557 (1983)
], E. coli RRI strain was transformed into human pancreatic c.
A DNA bank was created.

(3) Isolation of recombinant bacteria containing human pancreatic elastase-11Ac cDNA DNA of 4700 recombinant bacteria from the human pancreatic cDNA bank mentioned above was collected from Grunstein, M. and
Hogness.

D, S, method (Proc, NatJ, Acad, Sc
i,, USA, 72.

3961 (1975)] was fixed on the nitrocellulose filter.

Among these DNAs, human pancreatic elastase
To select those containing 11Ac cDNA, rat autologous pancreatic elastase II C! DNA fragments were used as probes. Rat pancreatic elastase If c
DNA was isolated using the following method. First, a cDNA bank was created using mRNA extracted from rat pancreas. On the other hand, MacDonald, R.

J. et al. [Biochem, 21, 1453 (
Part of the base sequence of rat pancreatic elastase It mRNA (151-
1, which is complementary to (corresponding to the 156th amino acid sequence)
Oligodeoxynucleotide 5' consisting of 7 mar
d (GGOATAGTCCACAACCA)3 was synthesized. The 5-terminus of this oligodeoxynucleotide is Ma
xam, A, M, and G11bert.

W, method (Proc, NatlAcad, Sci
, USA, 74.560 (197γ)] was used as a probe, I'M elastase II was extracted from the rat pancreatic cDNA bank mentioned above.
cDNA clones were selected.

Next, the obtained rat pancreatic elastase c DNA
A 200 base long fragment was cut out using restriction enzymes Aat 2 and Hind 3, and then subjected to nick translation method (Rl
gby, P. W. et al.: J. Mo1. Biol
,, 113.

237 (1977)) with 32p. Using this DNA fragment as a probe, Alwine, J.
O. et al.'s method [Proc, Natl, Acad, ee
l, USA, 74, 5350 (1977))
The clones were allowed to associate with the DNA fixed on the nitrocellulose filter, and eight clones that reacted with the probe were obtained by autoradiography. The plasmid contained in one of these strains was designated pH2 to 2.

A restriction enzyme map of the DNA inserted into pH2E2 is shown in FIG. Table 1 shows the entire base sequence and the amino acid sequence encoded by it.

(Yo) bzy tte vat 5er Phe Gly
Ser Arg Leu Gly Cys AsnT
yr Tyr Hls Lys Pro Se
r Val , Phe Thr Arg Val
5erAAT TACATCGACTGG ATC
AAT TCG GTG ATT GCA AA
TAsn Tyr Ile Asp Trp Il
e Asn Ser Val Ile Ala
AsrAACT Enter A CCA AAA GAA GTC
CCT GGG ACT GTT TCA GACAs
n CHI TTG GAA AGG TCA CAG AAG
GAA AAT AAT ATA ATA
AAGTGA CAA CTA TGCpH2
It can be seen that K'l has both a region encoding a signal peptide consisting of 16 amino acids and a region encoding a mature elastase protein consisting of 253 amino acids. Also, as mentioned above, Largman
, C. et al. (Biochim, Biophys.

Acta, 623, 208 (1980)) reported that the sequence of 16 amino acids at the terminal end of human pancreatic elastase (2) completely matches that of pH 2.

Furthermore, the amino acid sequence encoded by pH2Fi2 and Ma
cDonald, R.J., et al.
21, 1453 (198'2)), there is a high degree of homology of 84 strands with that of rat pancreatic elastase (■).

Therefore, pH2E2 encodes human pancreatic elastase ■.

pH2E2 is a full-length cDNA synthesized from human elastase mRNA using reverse transcriptase, so by transferring this cDNA to an appropriate expression vector, it can be used as a host in Escherichia coli, Bacillus subtilis, yeast, animal cells, etc.・Pancreatic elastase-11A (3 pieces) can be mass-produced.

(4) Human pancreatic elastase using animal cells
Production of II'A Construction of 2A into the expression plasmid psV4- In order to produce human elastase-11A using animal cells as hosts, the cDNA and the expression vector were ligated according to the procedure shown in FIG. As an expression vector, SV40 promoter, enhancer, poly A signal, Small
A psV2 plasmid containing the intervening sequence (intron) of the lT antigen gene was used.

An expression plasmid (pSV2-]102A) in which the vector and cDNA were ligated in the correct direction with respect to the transcription direction was selected by analyzing the restriction enzyme cleavage pattern.

Expression plasmid pSV2-H into co11 cells
2A (7:) The constructed expression plasmid pSV2-R2A is
The cells were introduced (transfected) into animal cells (cos1 cells) by the calcium phosphate method. 7) Transfection by the calcium method is carried out using Grah
Following the method of am and van eLer Eb (Vir
ology.

■, 456 (1973)).

For the CO3i cells used for transfection, 1X106 cells were seeded in a Petri dish with a diameter of 106 m, and cultured overnight in Dulbecco's modified Eagle's medium containing 10 hours of tallow serum. Next, 300 μg of psV '1-12A plasmid was suspended in 12.55 ml of sterile distilled water,
2.5 MC of mj'! Add aoj'2 (after mixing, add 1.5 ml! of 10X HeBS solution (2iomM HF1PEiS, 1.37 M MaCl,
4.7mM KCJ, 10.6mM N+L2H
PO4, 55, 5mM glucose, pH 05) was added dropwise to form a precipitate of DNA and calcium phosphate. 3
After leaving at room temperature for 0 minutes to ripen the precipitate,
1 mt per plate was added to CO81 cells that had been previously replaced with fresh medium containing 10 g of tallow serum. This is 3
After culturing for 12 hours at 7°C in the presence of 15% CO2, the culture medium was collected and replaced with new Dulbecco's modified Eagle's medium without tallow serum, and further cultured at 37°C with 0.5% CO2.
The cells were incubated for 48 hours in the presence of 2. Note that the transfection L7', :C! O81 cells were used to confirm human elastase-A mRNA, and the culture supernatant was used to measure elastase activity.

Extraction of mRNA from cos 1 cells Human elastase-W A mR transcribed from the expression plasmid into transfected C081 cells.
To confirm the presence of NA, mRNA was extracted from C081 cells and subjected to Northern blot hybridization as follows.

For cos 1 cells after 48 hours of culture, 1 petri dish, 1 frll! of guanidine thiocyanate solution (4
M guanidine thiocyanate, 1 H sarcosyl, 2
0 mM E! DTA, 25mM sodium citrate (pH 7.0), 100mM 2-mercaptoethanol, 0.1% antiform A) were added to lyse the cells. Next, this solution was passed through a 21 gauge syringe needle several times to reduce the high molecular weight DNA, and then layered on top of a solution containing 5.7M cesium chloride and 0.1M KDTA. Using a swinging rotor, 3
Centrifugation was performed at 0,000 rpm and 20° C. for 17 hours. The RNA precipitate obtained here was washed with a small amount of ethanol, and
Dissolved in 0 μl of distilled water.

The extracted total RNA was extracted using the method of Aviv and Leder [:P
roc.

Natl, Acad, Sci, USlA 69
, 140B (1972)].
The mRNA was purified by applying it to a cellulose column for several microseconds. Half of the purified mRNA was used to perform the 'rhomae method (Proc, Natl, Acad.

Northern blot hybridization was performed according to 8ci, USA 77, 5201 (1980)). For probes, nick translation method (
Rigby, P. W. et al., J. Mo.
1. Biol.

P-labeled human elastase-II A cDNA was used. As a result, cos1a cells containing psV2-]i2A (7)
There is a strong band of approximately 1.8kl in size that hybridizes with the mRNA probe, and a strong band of approximately 1.8kl in size that hybridizes with the mRNA probe. Okb
A weak band was detected. When 2A is transcribed into psV2-, a 1.8 kb mRNA is generated when transcription is terminated by the polyA signal contained in the vector.
It is estimated that when transcription is terminated by the polyA signal contained in the cDNA, a 1.0 kb mRNA is generated. Results obtained by Northern plot hybridization were consistent with these estimates.

Therefore, 2A in pSV2- is C! S in O81 cells
Using the V40 promoter, human elastase
It was shown that a large amount of MA mRNA was synthesized.

Elastase activity in the medium supernatant p, human elastase-II incorporated into S V 2
Since AcDNA 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. 1m
Trie-HCI buffer (pH 8,0) to 1 culture supernatant
was added to a final concentration of 0.2M, and then 101n
9/frLI! Add 50 μl of trypsin and incubate at 25°C.
Ciproelastase was activated by incubating the mixture for 15 minutes. Next, 50 μl of soybean trypsin.
Glutaryl-Ala-Aha-Pro-L with inhibitor solution (1omy/mz) and synthetic substrate
Add eu-p-ni and troanilide and heat at 25°
The enzymatic reaction was carried out by incubating at C. After the reaction,
The released p-n1troanilide was quantified by measuring absorbance at 410 nm.

As a result, obvious elastase activity was detected when measured using the culture medium of O81 cells transfected with J-1 psV2-12A, indicating the production of active human elastase-11A in cos1 cells. At this time, proelastase activation treatment with trypsin was essential.

In this example, in which pSV2-2A was introduced into cos 1 cells, the production of human elastase-11A was short-term (one day of expression); however, an appropriate selection marker ( For example, neo gene, dihydro
folate redu, ctase gene, etc.) and introducing it into CHO cells, etc., it is possible to create a cell line that can produce human elastase-11A for a long period of time.

(5) Human pancreatic elastase-11A using Bacillus subtilis
Construction of expression vector for production The method for constructing the expression vector is shown in FIG.

First, plasmid pH2E'l containing human elastase-U A cDNA was cut into a linear chain with restriction enzyme H1nd 3, and then partially cut with restriction enzyme Pvu 2 to linearize plasmid pH2E'l containing human elastase-IA cDNA. 3k
The DNA fragment of 1) was isolated. This is added to the restriction enzyme Ava.
By partial cleavage with 2, a fragment containing a cDNA encoding the third amino acid downstream from the N-terminus of the activation peptide of elastase-[IA was obtained.

This DNA fragment contains part of the signal peptide of Bacillus subtilis α-amylase and two amino acids (Oys) on the N-terminal side of the activation heftide of elastase-11A.
.

Synthetic oligonucleotide (4th
) were combined using T4 DNA IJ gauze.

On the other hand, plasmid pTUB 228 (Oh, Mura
.

K. et al., J. Biochem, 95
．． 87 (1984)) with the restriction enzyme H1nd 3 to obtain a 428 base pair DNA fragment containing the α-amylase promoter and signal peptide, and a 5,100 base pair DNA fragment containing the replication origin. of,
Each was isolated. The 428 base pair DNA fragment was further digested with restriction enzyme Hpa 2 and 5,100 base pair DNA
After cutting the A fragment with the restriction enzyme Sma i, DNA fragments of 385 base pairs and 4566 base pairs were isolated from each fragment. After joining the above three types of DNA fragments with T4 DNA IJ gauze, Bacillus subtilis strain 207-25 (m〒63 hsrM recTj24 amyE○7
aro 9061eu'A31ys 21; Ma
rburg strain) into protoplasts,
After regeneration, the cells were cultured in a medium containing 10μ&/fnl of kanamycin to obtain a transformed strain capable of growing on this medium. 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 resulting positive clones were isolated as ciblasmids, and their nucleotide sequences were determined to confirm that they were the desired clones. The expression plasmid thus obtained was named pBI9KX-E2A.

Elastase activity in medium supernatant Human elastase-11A expression plasmid pBs as described above
Bacillus subtilis 207-25 strain into which KX-R12A was introduced, 5
LG medium II containing 0 μg/ml kanamycin! (
1% Bactodryptone (Difco), 0.5 T yeast extract (Difco), 0.5% Na
C! 3 at 7.2 g/l glucose, pH 7.0-)
Culture was performed at 5°C for 148 hours with reciprocal shaking. After culturing, the culture solution was cooled to 4°C, and the bacterial cells were removed by centrifugation at 3,000:xg for 5 minutes. Ammonium sulfate was added to the supernatant at 55°C. The mixture was added to saturation and stirred at 4°C for 12 hours. 8.00 (l) of insoluble matter produced by this operation
xg for 20 minutes, and after removing the supernatant, the precipitate was mixed with 20frLlt+0.2M Tri
It was dissolved in s-HCI buffer (pH 8,0).

This solution was dialyzed against 11 0.2 M Tris-HO7 buffer (pH 8,0) for 16 hours, and insoluble substances were removed by centrifugation in a, OoOXg, for 20 minutes. This dialyzed fluid was used as a crude Elas-Tase IIA sample solution in the following assay.

Elastase activity of the sample solution was measured by the following method.

First, gloelastase is activated by treating the sample solution with trypsin, and then the synthetic substrate Glutary
l -Ala-Ala-Pro -Leu-p -ni
p-nlt released by reacting with troanilide
Roanilide was quantified by measuring absorbance at 4 TOnm. The reaction temperature was 25°C.

As a result, 207-2 in which pBsEX-E2A was introduced
Clear activity was detected in the sample solutions of 5 strains, indicating the production of active human elastase-11A in Bacillus subtilis (6) Human pancreatic elastase + using Escherichia coli
The plasmid pH2F)2 containing the produced human elastase-IIA cDNA of 1A was cut into a linear chain with the restriction enzyme Bind 3, and then partially cut with the restriction enzyme Pvu 2 to form a linear chain containing the elastase-U A cDNA. 3 kb D
The NA fragment was isolated. By partially cleaving this with the restriction enzyme Dde i, a fragment containing the cDNA encoding the elastase-[IA activation peptide downstream was obtained. Phosphate ((LATP, acTp.

Both ends were made blunt by treating this DNA fragment with dGTP, dTTP). The obtained DNA fragment was integrated into the Sma 1 cleavage site of plasmid pUC8 using T4 DNA IJ gauze.

Using the plasmid thus constructed, E. coli J.
Mj03 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-+1A expression plasmid was named 1pH2EX-2 (Figure 5).

The base sequence and amino acid sequence near the 5th end of the elastase cDNA in 1H2HX-2 are as follows.

←β-galactosidase←1←Elastase□ThrM
ET Engineering ThrAsn Ser Leu Ser
Cys GlyAsp-・-ATG ACCATG A
TT ACG AAT TCOCTCAG TGT
GGG GAC! ... Therefore, human elastase-11A expressed by pH21X-2 has two amino acids (Leu, Ber) derived from the signal peptide and six amino acids derived from β-galactosidase on the N-terminal side of its activation peptide. It becomes a fusion protein in which the amino acids of

Next, several strains of E. coli were transformed with pH2EX-2 to obtain strains capable of producing human elastase-11A. The strain containing the obtained expression plasmid was transformed into 2xTY-
Ampicillin medium (1.6% bactodryptone, 1%
Yeast extract, 0.5 Na07.50
ambicillin (ttji/ml) and incubated at 37°C.
It was cultured for 115 hours.

After culturing, centrifuge the culture solution to collect the bacterial cells, 12.4XI
A quantity of bacterial cells equivalent to Q8 cells was added to a 15 μE SDS solution (2 parts SDS, 5% 2-mercaptoethanol, 10
% Glycerin, 60mM Trle-HCI! (pH
6,8)) After K suspension and heating for 100'0.3 minutes, the method of Laemmli et al. (Nature, 227.
680 (1970)) to analyze the produced proteins.

As a result, production of a large amount of elastase was observed in strain 1121. The yield of elastase fusion protein is 11
This was equivalent to 20% of the total protein produced by 21 strains.

Although the 1984 strain also produced a relatively large amount of elastase fusion protein, the production amount was less than half that of the 1121 strain. The production amount of elastase fusion protein in the other two E. coli strains (HBl (strain N and strain MO4100) was significantly low.

The elastase fusion protein is 1nclusion in the bacterial cell.
Since it forms ocly, it could be purified relatively easily. In other words, pH 21! Culture solution II of 1121 strain transformed with -2! From , 1nclusion
6.5 g of bacterial cells forming a body was obtained.

6.5 g of the obtained bacterial cells were mixed with 5Qm containing 0.2μ/ml of lysozyme and 11n9/fnl of deoxycholic acid.
Bacteria were lysed by treatment in Tris-H (!I! buffer (pH 8, 0) of M.
1,500xg, 10 minutes), then high-speed centrifugation (11,000xg, 20 minutes) to remove 1 nclus.
ion 1) O(17) was recovered as a precipitate.
Since the fusion body still contains a large amount of bacterial fragments, we added Triton X-100 to 5 m and 97 m.
50mM Tris-HC1 containing 1! After suspending in a buffer solution, the suspension was washed by high-speed centrifugation (11,000xg, 20 minutes). The washed 1nclusion'bod7 was suspended in a small amount of Tris'-HC1 buffer and stored at 4°C.

By purifying in this way, 1 nc of 30'O
A 1usion body was obtained, which contained about 50% of the elastase-+1A fusion protein. Furthermore, it was confirmed by immuno-blotting that the 1nclusion body produced by the YA21 strain transformed with pH2EX-2 was an elastase fusion protein.
Confirmed by law.

As mentioned above, most of the elastase produced in Escherichia coli exists in the insoluble fraction of the bacterial cell, such as the 1Nelusion Bocly, but some of it is soluble and retains its enzyme activity. Existing. Therefore, detection of this enzyme activity was performed as follows.

1984 strain transformed with pH2Ex-2 was transformed with 2xTY
-Ampicillin medium 11! Shaking culture was carried out at 37°C for 15 hours. After culturing, the bacterial cells were collected by centrifugation at 3,000xg for 5 minutes, and the cells were collected at 2om! ! Buffer A (50mM Tris-HC!l (pH 8,0), imM E
D.T.A.

Suspend lysozyme in 50mM NaCJ) and add 10mM
After adding g, the mixture was kept warm at 5'O for 20 minutes. Next, deoxycholic acid was added to a final concentration of 11 m9/f'rLI! It was heated to 20°C, and deoxyribonuclease was added to a final concentration of 0. After adding T mg/mt, crush the bacterial cells using a PoIJ) Ron crusher.
1) It was. The resulting lysate was centrifuged at 80,000xg for 40 minutes to remove bacterial fragments, and then separated using Sephadex G-7.
5 column chromatography.

The elastase active fraction was further purified by antibody affinity chromatography and purified by elastase-I
It was used as an IA sample solution in the following assay.

The elastase activity of the sample was measured by the method described below. First, proelastase was activated by treating the sample solution with trypsin.

Next, the synthetic substrate Glutaryl-A/1a-Ala-
p-n1troani'1ide released by reacting with Pro-Leu-p-nitroanilide
was quantified by measuring absorbance at 410 nm. Reactions were carried out in 0.2 M Trie-H containing synthetic substrates.
CI: It was carried out by incubating at 25°C in a buffer solution (pH 8,0). As a result, clear activity was detected in the sample solution of the 1984 strain transformed with I) H2KX-2, and active human elastase-1 in E. coli was detected.
Production of 1A was shown.

(7) Human pancreatic elastase-IA using yeast
Human pancreatic elastase = If A
The cDNA can be ligated to a suitable expression vector using a conventional method, transferred into a host cell, and expressed.
It was possible to confirm the presence of elastase activity in the culture.

S described in "Recombinant DNA Experimental Guidelines".

CereVi81ae can be used as a host, and specifically CereVi82880 strain was suitable. On the other hand, YKp13 and the like were suitable as vectors.

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

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme map of plasmid pH2Fi2 obtained in the example, and the mouth valve indicates a portion encoding a peptide considered to be a signal peptide. The oral valve shows the portion encoding the mature elastase protein. FIG. 2 shows the procedure for constructing the psV2-Fi2A plasmid. 1) SV2-β globin is a plasmid in which β-globin cDNA is inserted into a pSv2 vector. On the other hand, pa2z 2 is Oka7ama-Ber
This is a plasmid in which human elastase-IA cDNA is inserted into a g vector. Reactions such as S1 nuclease are described in "Molecular Cloning" (Mania
tis, T., et al. (ed.)”Mo1ec
ularC! 110n1n″Co1d SpringH
The method described in Arbor Lab, (?sao)) was followed. Thick arrows indicate the SV40-derived promoter, and thin arrows indicate the direction of transcription. In the figure, B, H, S, and V each represent the restriction enzyme Bg12. H1n43, Pst i, Pvu:? represents. Figure 3 shows the procedure for constructing the 111BSEX-2A plasmid. pTUB228 is a plasmid in which the Bacillus subtilis α-amylase gene is cloned into a vector having a Bacillus subtilis origin of replication. Thick arrow is α−
The amylase promoter is shown, and the thin arrow indicates the direction of transcription. In the figure, A, H. p, s, and v are restriction enzymes Ava 2 and Hl, respectively.
nd3. Hpa2. Represents Small and Pvu2. Figure 4 shows a part of the signal peptide region of the Bacillus subtilis α-amylase gene and elastase-If A cDNA.
A synthetic NAIJ linker containing the N-terminal side of the activation peptide of A is shown. Figure 5 shows the procedure for constructing the pH''1BX-2 plasmid. The shaded region indicates the β-galactosidase gene. The thick arrow indicates the lactose promoter operator, and the thin arrow indicates the direction of transcription. ,D,H,S,V
are restriction enzymes Dde1. H1n43. Sma1.
Represents Pvu2. Patent Attorney Sankyo Co., Ltd. Patent Attorney Osamu Kashi (385jz base '51) 1
One day ゛

Claims (1)

[Claims] 1. Human pancreatic elastase-IIA or its equivalent, which is represented by the amino acid sequence represented by the general formula [Amino acid sequence is available]. 2. Human pancreatic elastase-IIA or its equivalent, which is represented by the amino acid sequence set forth in claim 1, which has a hydrogen atom, Met, or [amino acid sequence is available] at the (N)-terminus. 3. DNA encoding human pancreatic elastase-IIA represented by the general formula [amino acid sequence included]. 4. The DNA according to claim 3, which encodes human pancreatic elastase-IIA having a hydrogen atom, Met, or [amino acid sequence] at the (N)-terminus. 5. Claims No. 1 characterized by containing a base sequence represented by the general formula [there is a gene sequence] (wherein, X represents TAA, TGA, or TAG) or a base sequence with the same effect. Section 3 or 4
DNA described in section. The DNA according to claim 5, which has ATG or [amino acid sequence] at the 6 and 5 ends. 7. A general formula [There is a gene sequence] [There is a gene sequence] characterized by including (a) to (d) described below (in the formula, X indicates TAA, TGA or TAG). A method for producing DNA containing the expressed base sequence or a base sequence equivalent thereto. (b) Isolate mRNA from human pancreas. (b) cDNA using this mRNA and reverse transcriptase
Create a bank. (c) Isolate a plasmid containing cDNA encoding human elastase from the prepared cDNA bank using a probe. (d) Target cloned DNA from this plasmid
Cut out. 8. The method for producing DNA according to claim 7, which has ATG or [gene sequence is present] at the 5th end. 9. A host transformed with a DNA containing the base sequence represented by the general formula [gene sequence is available] (wherein, X represents TAA, TGA, or TAG) or a base sequence with the same effect. 10. A host transformed with the DNA according to claim 9, which has ATG or [gene sequence] at the 5th end. 11. The host according to claim 9 or 10, which is E. coli, Bacillus subtilis, yeast, or animal cell.