JPS6229977A - Human pancreatic elastase iib - Google Patents

Abstract

PURPOSE:The titled elastase, consisting of a specific amino acid sequence, capa ble of exhibiting a biological activity equal to that of pancreatic juice, usable for the similar purpose and by the similar usage, obtained by the recombinant DNA technique and useful for arteriosclerosis, etc. CONSTITUTION:An mRNA is separated from human pancreas, and a cDNA bank is prepared by using the above-mentioned mRNA and a reverse transcriptase to isolate a plasmid containing a cDNA coding human elastase IIB from the resultant bank. A recombinant plasmid prepared therefrom is then introduced into Escherichia coli, etc., and a recombinant is collected from the resultant transformant. A clone containing a DNA coding the elastase IIB is selected from the above-mentioned recombinant and linked to a gene expres sion vector. The resultant gene expression vector is then introduced into host Escherichia coli and the resultant recombinant host is cultivated in a culture medium, e.g. glucose, at 15-43 deg.C for 3-24hr. The resultant culture is treated to afford the aimed human pancreatic elastase IIB consisting of an amino acid sequence, e.g. amino acids expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides human pancreatic elastase-IIB, a DNA containing the base sequence encoding it, a host transformed with Sotoriki 2, and a method for producing human pancreatic elastase using the host. Regarding. Elastase is a type of serine protease.
It is an enzyme that hydrolyzes elastin, a fibrous protein. Elastin is a type of hard protein that constitutes the connective tissues of higher animals, the outer skin of the aorta, and the cervical cord, and is slightly degraded by pepsin and trypsin. Ba1. While researching arteriosclerosis, a' 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, a
ndBanga, 1. : Schweiz Z, Pa
thol, Bacteriol, 12. 350 (1949)]. Next, Bangafl
, 1952, discovered an enzyme that specifically degrades elastin.
It was discovered in the pancreas, extracted in crystal form, and named elastase.ftl [Banga, 1. : Acta
Physjoko, Acad. Sci, Hung, 3, 317 (1952)
]. It is well known that elastase (':i, hi]...monkey) [n, rabbit, etc.] is present in the pancreas of most animals, and its content is 3.1 m9/liver J. , 22 cows
m9/ & , rat is about 102 渭9/, 9. A relationship between human elastase activity and age was observed, leading to vomiting, and elastase activity in the pancreas and plasma was significantly reduced in men over 40 years old and women over 60 years old.C
Loeven, w, A,, and Mau, re.
en M, Ba, ], dwj-n: Oeronto
logia, 17, 170 (1971)]. In patients with arteriosclerosis, the activity of pancreatic elastase was significantly lower than that in healthy subjects or disappeared [Ba1o', J., a, ncl.
Banga, T.: Nature, 178,
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 ) Improving effect on lipoprotein metabolism 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 prepared from porcine pancreas extract 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 may pose a risk of anaphylaxis (Japanese Patent Application Laid-open No. 111-1982).
80). 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. At present, the existence of two types of human pancreatic elastase is well known, but their characterization has not yet been fully performed, and the amino acid sequence of one of them, pancreatic elastase-■ The 161 chain has been clarified from the N-terminus [Largman, C, et al.
]ochim, Biophys, Acta, 623
. 208 (+980)], and its structure is currently 1
One thing was unclear. However, the inventor of the present invention
All of its amino acid sequences and all of its DNA sequences have been revealed (tau).The amino acid sequence revealed by the present invention differs from the slightly revealed amino acid sequences as described above,
It is clear that the present invention relates to a completely new substance C (that is, the present invention relates to a DNA containing a base sequence encoding human pancreatic elastase-IIB, a host transformed using the DNA, and human pancreatic elastase by culturing a host transformed with the DNA.
A method for producing IIB is provided. The present invention relates to DNA encoding human pancreatic elastase-IIB represented by the amino acid sequence represented by the general formula %, and human pancreatic elastase-IIB represented by (■). and in the formula, 04)-terminal hydrogen atom, 1vlet, or MetI], eArgThrLeuLeuLeu, 5e
rThrLeuVal Ala Gly Ala Le
It may have u Ser. In particular, it relates to a DNA containing a base sequence represented by the general formula % (in which X represents TAA, TGA, or TAG) or a base sequence equivalent thereto. The above DNA (ff) has ATG at its 5' end,
Or it may have (5') - [There is an amino acid sequence]. When n1*A(II) has all AT() at the 5' end, it encodes a polypeptide having Met at the (river-end) of (■) in addition to polypeptide (I). DNA (T) When the 5' end of the polypeptide has (5') - [There is an amino acid sequence] - (3'), in addition to polypeptide (I), (r)
It encodes a polypeptide having [amino acid sequence] at its (N)-terminus. Represented by formula (II) 1. The DNA of the present invention containing a base sequence or a base sequence having the same effect as that described above can be produced, for example, by the steps (a) to (b) 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, using rat elastase It c DNA as a probe, cD encoding the desired human elastase is extracted.
A plasmid containing IiA is isolated. ) From this plasmid, clone the desired DI-
Cut out IA. Can the guanidine thiocyanate hot phenol method or the guanidine thiocyanate cesium chloride method be used for pancreatic RNA extraction?The guanidine thiocyanate-guanidine hydrochloric acid method has the following points:
This method 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) DI=lA is not 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(dT) cellulose column, and then elute and purify it. Using the obtained mRNA as a template, complementary double M-strand DNA is synthesized using reverse transcriptase.
Nuclease method [Efstratiadis, A. et al.: Ce1l, 7, 279 (19
76) ], La, nd method [Land. H, et al.: Nucleic Ac1ds
Res,, 9, 2251 (1981)], Ok
ayama-Berg method [: Okayama, H,a
nd P, Berg: '#Ao],. Ce11. Biol, 2, 161 (1982
) ), O, Joon Yoo method [0, Joon Yo
o, et al, : Proc, Natl+Aca
d, Scj,. USA, Biol., 1049 (1982)], but for the purpose of the present invention, the
The g method was preferred. Next, the obtained recombinant plasmid is introduced into E. coli, for example, the 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, 32
Rat elastase se21 cDNA labeled with P
The colony hybridization method was used, using the probe as a probe. The nucleotide sequence contained in the selected clone was determined using the method of ditheoxynucleotide synthesis chain termination using phage IJ13 [Messing, J. et al.: Nuclei
c Ac1ds Res, 9, 309 (198
1)] and the Maxam-Gbert method [Ma
xam, A, M, and G11bert, W
, :Proc, Nat],, Acad, Sci,
tJsA, 74, 560 (1977)], but in the present invention, a ditheoxynucleotide 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 (1a) promoter, tryptophan-lactose (1ac) promoter, outer membrane major protein (lpp) promoter, bacteriophage-derived lambda (λ) Pl, promoter, protein button volatilization factor. Use the Tu (t+rrB) promoter or the like. In addition to microorganisms such as seawater, Escherichia coli, Bacillus subtilis, and yeast, animal cells can be used as hosts. In the present invention, the large intestine (LE392, YA 21, etc.) is suitable as the host. As a method for transforming and introducing the DsAt host of the present invention, Hanahan's method [Hana, han, D
, :J. Mo1. Biol,, 166, 557 (198
3) '3, Calcium chloride method CDagart, IV
I, and S, D, Ehrlich: Gene
, 6, 23 (1979)], the low pH method [edited by Zashiki Takagi: Gene Manipulation Manual, p. 49, Kodansha University Press (1982)], etc. In the present invention, the method of Hana and Han was suitable. The host (recombinant) obtained in J: is cultured in a known medium, and the culture contains a polypeptide (■) represented by (■) (Met or Met I at its (+4)-terminus).
], e Arg Thr Leu Leu L
eu Ser Thr LeuVal Ala
G], y A], a Leu 5er. ) or its equivalents are produced and accumulated, and the objects of the present invention can be achieved by collecting them. As a medium, a medium consisting of glucose, casamino acid, etc., for example, M9 medium [Mi1]-er, LIT,
:Experj, ments in 1. Aolecu
lar () enetics, 431-433 (Co
ld Spring I (arbor Lab,, N
ew York (1972)]. 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 is produced as an insoluble protein.
It is often included in 10n bOdy. In this case, by destroying the bacterial cells and centrifuging,
The elastase produced is obtained as a precipitate. As a method for destroying bacterial cells, ultrasonic treatment, lysozyme treatment, freeze-thaw treatment, etc. can be employed. Isolation of elastase from the supernatant or precipitate 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 human pancreatic juice, and for the same purpose,
The g-Met bond is broken and the first Cys becomes Cy in the molecule.
two-ch connected to s by a disulfide bond
It also exists as ains type elastase. Therefore, the present invention also includes such two-chais 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
N human pancreas (-f!I test sample) was treated with a guanidine thiocyanate solution (4M guanidine thiocyanate, 1% sarcosyl, 20rnM ethylenediaminetetraacetic acid (E
DTA), 25mM sodium citrate (pH 7,0
), 100 mM 2-mercaptoethanol, and 01 antiform A) were disrupted and denatured using a polytron, followed by centrifugation to obtain a supernatant. Add this to 0025 times the amount of l.
) Add acetic acid and 075 times the amount of ethanol, -2
After cooling at 0° C. for several hours, a precipitate was obtained by centrifugation. Next, this precipitate was added to a guanidine hydrochloride solution (7.5 ia of guanidine hydrochloride, 25
Suspended in mM sodium citrate (pH 7,0), 5 mM dithiothreitol (DTT), 0025
Add twice the amount of 1M acetic acid and 05 times the amount of ethanol, -
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, 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 bathing in distilled water, it was rinsed with ethanol.
NA was precipitated. 53. Collect the precipitate by centrifugation.
9719 RNAs were obtained. The thus obtained RNAf high salt bath solution, 5+t't
Jact. 20 mM Tris-I (cz (pH 7,5), i
After adsorbing onto an oligo(aT) cellulose column in 0.1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), mRNA containing poly(A) was dissolved in an elution solution (
10mM Tris-HC,/: (pH 7,5),
255 μg of nnRNA was obtained by elution with 1 mM EDTA, 0.05% SDS). (2) Preparation of cDNA bank The cDNA bank was prepared by Olcayama-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-Hcz (pH 8,3), 8mM ivfg
ct2.30 m1vl KCt, 0.3 mM D
'rT, 2 mM d, ATP, 2 ml, 4
dOTP, 2mM dCTP, 2m+, d' of 4
l'TP, α-32P-dCTP of 101tC1
, 1°4 μy vector brimer DIJA (P
(purchased from L-Pharmacia)] at 42°C, 6
It was allowed to react for 0 minutes. 2 μl of 0.25 M El) T8 and 1 μl of 10% S
After stopping the reaction by forcefully adding DFE, protein was removed with 20 μl of phenol/chloroform. After centrifugation, remove the aqueous layer and add 20μl of 4M vinegar & ammonium to 80%
μt of ethanol was added and cooled to −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 mixed with 15 μl of terminal transferase reaction solution (140 mM potassium cacodylate, 30 mM Tri
s-HCbep[(6,a), i mM cobalt chloride,
0.5 mM DTT, 0.211EI poly
A, 100 ml of dCTP). After warming the reaction solution at 37°C for 3 minutes, 18 units of terminal theoquine nucleotidyl transferase was added and allowed to react for 5 minutes. Next, 1 μl of 0.25 M EDTA and 0.5 μl of 10% SDS were added to stop the reaction, and protein was removed with phenol/chloroform. After centrifuging the reaction solution for 7 t, the aqueous layer was removed and 15 μl of 4 M acetic acid was added. Ammonium and 60 μt of ethanol were added and mixed well. After leaving it at -70°C for 15 minutes, it was centrifuged and the precipitate was collected. The precipitate was diluted with 10 μt of restriction enzyme buffer (50 mM NaC).
1,10mM Tris-Hcz (pH 7,5), 10
2mM MgCl2, imM DTT),
Add 5 units of restriction enzyme Elf, Hind 3;
Digestion was performed at 7'C for approximately 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 10 μt of Tε (10 mM Tris-HCt (pH 7,5), i mM EDTA
) dissolved in Next, 1μt of the above sample was added to an oligo-dG linker (P
(Purchased from L-Pharmacia) 10 n, 9 was added to the reaction solution (iomM Tris-HCt (pH 7,
5), i.m.M. EDTA. (100 mM Nact), heated at 65°C for 5 minutes, and then incubated at 42°C for 30 minutes. After cooling the reaction in water, add 10 μt of 10 medium ligase buffer (10 mM ATP, 660 mM Tris).
-HCf(p[(7,5), 66 mM MgCl2
, 100 mM DTT), 78 μl of distilled water, and 8 units of T4 DNA ligase were added, and the mixture was incubated at 12° C. overnight. Next K, 10μt (7) 1'; 6ct, 1 unit of bonuclease H133 units of DI-JA polymerase ■, 4 units of T4 DNA ligase,
05μl of nucleotide solution (20mM dATP,
d()TP of 20111M. 'lo mM dCTP, 20 mM dTTP),
50μg/μ of 01μt as bovine serum albumin (BSA)
was added and kept at 12°C for 1 hour and then at 25°C for 1 hour. After diluting this reaction solution 5 times, immediately [(ana
, 1ian's method [Hanahan, D.: J
, Mo1. Biol. 166, 557 (1983)], the E. coli RRI strain was transformed to produce human pancreatic cDNA.
The DNA of the recombinant strain of bacteria is
n + M, an d HOgn e sS r
I), S, method [Proc, Natt, Acad
, Sci., USA, 72. 3961 (1975)] was fixed on a nitrocellulose filter by FC. For this purpose, a rat pancreatic elastase I cDNA fragment was used as a probe. Rat pancreatic elastase II
CDNA was isolated by the following method. First, a cDNA bank was created using mRNA extracted from one slice of rat pancreas. On the other hand, MacDonald, R. J, et al. [:Bj,ocllem,, 21, 1453
(1982) ] Part of the base sequence of rat pancreatic elastase Tl mRNA (
Amino acid sequence of 151st to 156th i\(/ko)
An oligothioxynucleotide 5' d consisting of 17 mar, complementary to
ACCA, ) 3' was synthesized. The 5' end of this oligothioxynucleotide is Maxam, A, M, a
nd G11bert. W, method [Proc, Natt, Acad, S
ci, USA, 14, 560 (1977)] was used as a probe, and the elastase l c
DNA clones were selected. Next, the obtained rat pancreatic urine elastase ■c D
Restriction enzyme accumulation from NA to Aat 2 and Hind 3 to 20
A fragment with a length of 0 bases was cut out and subjected to the nick translation method [Rigby, P. W. et al.: J.
MomBio, /:,, 113. 237 (+977)] and labeled with 32P. Using this Di fragment as a probe, Alwine, J.
C. et al.'s method [Proc, Natt, Acad,
Sci,, USA, 74, 5350 (+977
)] was used to associate with the DNA fixed on the upper nitrocellulose filter, and eight clones that reacted with the probe were obtained by autoradiography. The plasmid contained in one of these strains was diluted to pH2E8.
It was named. A restriction enzyme map of the DNA inserted into pH2Fll is shown in FIG. Table 1 shows the entire base sequence of Ushida and the amino acid sequence it coats. [Amino acid sequence is available] [Amino acid sequence is available] pH2E8fd, has all of the region that coats the signal peptide consisting of 161 amino acids and the region that encodes the mature elastase protein consisting of 253 amino acids. I know that there is. Unfortunately, pH2E8 also contains 5' and 3' untranslated regions. p) (2E8 coated amino acid sequence and MacD
onald, R.J., et al. [Biochem, 21
, 1453 (1982)] reported that the rat
This is a cDNA encoding pancreatic elastase. For human pancreatic elastase, Larg
ma, n, C, et al. (: Biochj, m. Bj, ophys, Acta, 623, 208
(1980)] reported the (N)-terminal 16 amino acid sequence, which is clearly different from that of pH2E8. Therefore: 1) 82E8 encodes a novel elastase. T) 82F8 is a full-length cDNA synthesized from human elastase mRNA using reverse transcriptase, so by transferring the cDNAi to an appropriate expression vector,
Human pancreatic elastase-nBk can be produced in large quantities using E. coli, yeast, animal cells, etc. as hosts. (4) Human pancreatic elastase using animal cells
(2) Production of B In order to produce human elastase-11B using animal cells as hosts, the cDNA was fully ligated with the expression vector according to the procedure shown in FIG. S as an expression vector
V40 promoter, enhancer, poly A signal, small T antigen gene intervening sequence (
psV2 Shirasumi P containing the intron was used. Expression plasmid l' (pSV2-E2B) in which the vector and cDNA are ligated in order with respect to the direction of transcription.
The results were determined by cutting with restriction enzymes and fully analyzing the sequence. The constructed expression cilasmid psV2-E2B was introduced (transfected) into animal cells (C08I cells) using the calcium phosphate method. Transfection by the calcium phosphate method was performed by Graham and V.
fc (Virolo
gy. 52.456 (1973)]. C08I cells used for transfection were seeded at 1×10 cells in a Petri dish with a diameter of 10 cm, and cultured overnight in Dulbecco's modified Eagle's medium containing 10 μl of fetal bovine serum. Next, 300 μ7 of pSV2-E2B' plasmid was suspended in 12.55 ml of sterile distilled water and 0.0 μl of pSV2-E2B' plasmid was suspended in 12.55 ml of sterile distilled water.
After adding 75 me of 2.5 M CaCA22 and mixing well, 1. , 5 ml of 10 x HeB5 mW (
210mM HEPES, 1.37M NaC7
, 4,7mM KCA. 10,6mM (D Na2HPO4, 55,5mM
glucose, P147. (15) The entire solution was added dropwise to form a precipitate of DliA and calcium phosphate. After leaving it at room temperature for 30 minutes to fully ripen the sediment, 1 m per petri dish.
1f, was added to C08I cells which had been previously replaced with a fresh medium containing all of the 10 batches of tallow serum. This was heated to 37℃ and 5% 00
After culturing in the presence of 2 for 12 hours, the culture medium was discarded and replaced with a new Dulbecco's modified Eagle's medium that does not contain fetal bovine serum, and further cultured at 37°C under 15% CO2 concentration (l trowel 4
Cultured for 8 hours. The transfected C08I cells obtained here were engineered to contain human elastase-uB.
Human elastase-nB m transcribed from the transfected 7i (CO8I live cell plasmid)
To confirm the presence of RNA, mRNA was extracted from C08I cells as follows and Northern
A Plot Biblical Questionnaire was conducted. After culturing for 48 hours, the C08I cells were treated with 1 me of guaninonethiocyanate bath (4M of guaninonethioneane-1-, 1 part of the solution,
2 [1mM EDTA, 25mM sodium citrate (pt(7,+1), 1.fl (lmM 2
-Mercaptoethanol, 01% antiform A) was added to lyse the whole cells. Next, this solution was passed through a total of 21 ges injection needles several times to reduce the polymer DliA'i, and then mixed with 5.7 M cesium chloride and 0.5 M cesium chloride. E of I M
layered on top of the solution containing DTA and heated at 30,000 rpm for 20 min using a Hitachi RPS 40 swinging rotor.
Centrifugation was performed at ℃ for 17 hours. The RIiA precipitate thus obtained was washed with a small amount of ethanol and dissolved in 300 μt of distilled water. Extracted total RNA: Aviv Leder's method (PTO OC. Natl, Acad, Sci, USA 69.1
408 (1972)) to purify several micrograms of mRNA. Thomas' method (P
Doc, Natl, Acad, Sci. Northern blot hybridization was performed according to USA 77.520111980). To the probe (engineering, two-translation method [Rig
by, P. W. et al., J. Mo1. B
iol, 113, 237 (1977)) 3
2p-labeled human elastase-IB cDNA f was used. As a result, psV2-E2B fully introduced C03I
A strong band of approximately 1.8 kb in size that hybridizes with the probe only to cellular mFtNA, and a strong band of approximately 1.8 kb in size that hybridizes with the probe only to cellular mFtNA. O
A weak pan 1:' of kb was detected. psV2-E2
When B is transcribed, when the transcription is terminated by the poIJ A signal contained in the vector, a 1.8 kb mRNA is generated.
When A is generated and transcription is terminated by the yW IJ A signal contained in the cDNA, 1. It is presumed that Okb mRNA is generated. Results obtained by Northern blot hybridization were consistent with these estimates. Catch, psV2- E2B &X C0
7' o motor IT'lJ of 5V4o in 3I cells
It was shown that a large amount of human elastase-IIB mRNA k was synthesized using this method. T) Human elastase-r [BcD
Since NA retains a signal peptide region, the expressed elastase is expected to be secreted into the medium as heproelastase. Therefore, total elastase activity in the cultured cells after 48 hours of culture was measured. Tris-HC1 buffer (pH 8,0) was added to 1 ml of the culture supernatant to a final concentration of 02M, and then 50 μl of trypsin (10μ/ml) was added and incubated at 25°C for 15 minutes. Elastase activation treatment? I did it. Next, 50μt of soybean trypsin inhibitor bath solution (l
Omg7m13) and the synthetic substrate Glo, t
aryl-Ala-Ala-Pro-Leu-p-n
Enzyme reaction was carried out by adding 1troanilide f and keeping warm at 25°C. After the reaction, liberated yt p
-n1troanilide f was quantified by measuring the absorbance at 410 nm. As a result, obvious elastase activity was detected when measured using the culture medium of CO31 cells transfected with psV2-E2B, indicating the production of active human elastase-IB in CO81 cells. At this time, proelastase activation treatment with trypsin was essential. In this example, in which pSV2-E2B was introduced into CO3] cells, the production of human elastase-IIB was transient expression, but pSV2-E2B cilasmid was injected with an appropriate selection marker (e.g., neo gene, dihydrofolat).
e reduct, ase gene, etc.) and CH
By introducing it into O cells, etc., it is possible to construct all cell lines capable of producing human elastase-11Bi over a long period of time. (5) Human pancreatic elastase-n using Bacillus subtilis*
Construction of expression vector for production of B The method for constructing the expression vector is shown in FIG. 1. A plasmid containing human elastase-IB cDNA k was cut into a linear chain by cutting with pH2E8i restriction enzyme Hind3, and then partially cut with restriction enzyme Pvu 2 to create a plasmid containing human elastase-DB cDNA k. ．． A 3 kb DNA fragment was isolated. By partially cleaving this with the restriction enzyme Hha], a fragment containing cDNA 'j (containing the 8th amino acid sequence from the terminal side of the activation peptide of elastase-IB) was obtained. Synthetic oligonucleotides (Fig. 4) encoding part of the signal peptide of Bacillus subtilis α-amylase and the 7 amino acids at the 1q terminus of the activation peptide of elastase-IB were injected into T4D1iA.
It was bonded to ligase. On the other hand, plasmid pTUB 228 (Ohmura,
K, etal, J, Biochem, 95
．． 87 (1984): ] Digested with the restriction enzyme Hind 3 to obtain a 428 base pair DNA fragment containing all of the α-amylase promoter and part of the signal peptide, and a 5,100 base pair DNA fragment containing the replication origin. Well, it's isolated. The 428 base pair DliA fragment was further digested with restriction enzyme Hpa 2 and fc5,100 base pair DNA fragment G restriction enzyme Sma], and then the 385 base pair and 4566 base pair DliA fragment gold 1 were obtained, respectively. Female. Above: After ligating the three DIiA fragments using T4 DNA ligase, Bacillus subtilis 207-2
5 strains (m'i 63 hsrM recE4 a, my
EO7aro l9061euA81ys21: M
arburg strain) and allowed to regenerate, 10μ? /ml! Cultured on a medium containing kanamynon (-1) A transformed strain capable of growing on this medium was obtained. In order to select all of the desired plasmids from several transformed strains, the synthetic oligonucleotide shown in Colony hybridization was performed using the plasmid as a probe.A plasmid was isolated from the obtained clone of the positive 1 cow, and its nucleotide sequence was completely determined to confirm that it was the desired product.In this way, The resulting expression plasmid was named pBsEX-E2B. Elastase activity in the medium supernatant Human elastase-IB expression plasmid pBsE as described above
Bacillus subtilis 207-25 strain into which all X-E2B was introduced was
q/ml! LG medium containing kanamycin of 17!4]%
Bactodrypton (Difco), 05% Yeast Extract (Dirco), 05% NaCL
, 22μ glucose, pH 7.0) at 35°C, 48
Cultured with reciprocating shaking for hours. After the culture was completed, the culture solution was cooled to 4°C, centrifuged at 3,000xg for 5 minutes to remove all the bacterial cells, and ammonium sulfate was added to the supernatant to reach a total saturation of 55%, and the mixture was incubated at 4°C. Stirring was carried out for 12 hours.The insoluble matter generated by this operation was precipitated by centrifugation at 8,000×g for 20 minutes, and after removing the supernatant, the precipitate sound was 20
m1 02M Tris-HCt buffer U (p) (8,
o) dissolved in Main solution: 1.0.2 M Tr
Dialysis was performed against 1s-HCL buffer (pH 8,0) for 16 hours, and insoluble substances were removed by centrifugation at 48,000×g for 20 minutes. This intradialysis crude elastase JIB sample solution was used in the following assay. Elastase activity of the sample solution was measured by the following method. The sample solution was treated with +-lipsin without stirring to fully activate proelastase, and then treated with the synthetic substrate Glutar.
yl-Ala-Ala-Pro-Leu-p-nitr
Reacted with o-anilide and released 7tp-ni 廿oanj, 1ide f41, Onm absorbance -t
It was quantified by determining i++. The reaction temperature was 25°C. As a result, pBsEX-
Clear activity was detected in the sample solution of strain 207-25 into which E2Bi was introduced, indicating the production of active human elastase-IB in Bacillus subtilis. (6) Production of human pancreatic elastase-[B used in Escherichia coli] Plasmid pH2E8 containing human elastase-FIB cDNA f was cut into a straight button shape with the restriction enzyme Hind3, and then cut with the restriction enzyme Pvhi2. Digest approximately 1.3 kb of D containing elastase-IIB cDNA k.
The entire I-coA fragment was isolated. Restriction enzyme Dd, e 1
Partial cleavage of the cDNA encoding downstream of the activation target of elastase-IB
I got the NA fragment. Next, DNA polymerase K], en
ow fragment and four deoxyribonucleosides 5
'-Triphosphate (d, A, TP, d, cTP, d
OTP. This DNA fragment was treated with dTTP) to make both ends blunt. T4D was added to the Sma ] cleavage site of the entire plasmid pUC8 of the resulting DNA fragment.
It was assembled using NA IJ gauze. Using the thus constructed plasmid, all 103 Escherichia coli JM strains were transformed. Grasmi 1" was extracted from some of the transformed strains, and the direction of transcription of the cDNA matched the orientation of the promoter of the lactose operon. Restriction enzyme mapping VC,,l: was selected. This elastase-IIB expression plasmid w pH2EX
-8 (Fig. 5). The base sequence and amino acid sequence near the 5' end of the elastase cDNA in pH2EX-8 are as follows. (-β-galactosiguse→1←elastase=Thr
Met Engineering 1eTl〕l'ASI〕5erLeuSerC
ysG1yVa, 1”'AT'G ACCATG
ATT ACG AAT TCCCTCAG〒T
GT GGG GTC... Therefore, I) H2E
Human elastase expressed by χ-8 - [B is the 1-terminal side of its activation pezotide (C, two amino acids derived from the signal peptide (Leu, Ser
) and β- form a fusion protein in which six amino acids derived from lactosidase are linked. Next, some strains of E. coli at pH2EX -8? After transformation, a human elastase-■B gold-producing vine strain was obtained. All strains containing the obtained expression plasmid, 2xT
Y-Ampicillin medium (Bactodrypton of Section 16, 1
% yeast extract), 0.5% L]aC1,
5011ri/fnl Noah 7 bicillin),
The cells were cultured at 7°C for 15 hours. After culturing, centrifuge the culture solution to collect all the bacterial cells, and collect 2.4 x 1-08 cells equivalent of 1 bacterial cell.
After suspending in 5 μt of SDS solution (2% SDS, 5% 2-mercaptoethanol, 10% glyceriferous, 60 mM Tris-HCl (pH 68)) and heating at 100°C for 3 minutes, Laemmli
(Natqre, 227.680 (1970)
)) 5DS-polyacrylamide gel electrophoresis was performed to analyze all of the produced proteins. As a result, a large amount of elastase production was observed in the YA21 strain. Elastase fusion protein production Q engineering Y
This corresponded to 20 units of the total protein produced by the A21 strain. Although a relatively large amount of elastase fusion protein was produced in the X984 strain, the production amount was less than half that of the YA21 strain. The production amount of elastase fusion protein in two other strains of E. coli (IB 101 strain and MC4100 strain) was significantly low. The elastase fusion protein is 1nclusion in the bacterial cell.
Since it was formed as ody f, it was possible to purify it relatively easily. Transformed with pH2Eχ-8;i
YA, culture solution 1 of 21 strains! From 1nc1. usi
6.5 Li of fully formed on-body bacterial cells were obtained. 50 m containing 5.5 microbial cells obtained, 02 μg/ml of lysothyme and 61 mg/me f of deoxycol
M (7) was lysed by treatment in TTis-HCA buffer (pH 8,0). Undestructed bacterial cells, low speed dispersion.
After removing by separation (1,500Xg, 10 minutes), 1 nc was removed by high-speed centrifugation (11,000Xg, 20 minutes).
The fusion body was recovered as a precipitate. This 1nclusion body K still contains a large amount of bacterial body fragments, so Triton X-100f 5
50 mM Tris-HCL containing my/m/, f
After suspending in buffer, high-speed centrifugation (11,000X
g. 20 minutes). 1nclusion washed
Bodyl! , suspended in a small amount of Tris-HCA buffer and stored at 4°C. By purifying in this way, 300 μ inc]
, a usion body was obtained, which contained about 50% of the elastase=■B fusion protein. It is produced by the YA21 strain transformed with 1k, pH2EX-8, and the i1nclusion body is an elastase fusion protein.
This was confirmed using the ting method. As mentioned above, most of the elastase produced in Escherichia coli exists in the insoluble fraction of the 1-inclusion body bacterial cell, but some of it is soluble and exists in a state where the enzymatic activity is preserved. are doing. Therefore, the detection of the activity of this enzyme was carried out as follows. Freshly transformed 984 strain Ke2XTY- with pH2EX-8
The cells were cultured in ampicillin medium 1 at 37° C. for 15 hours with shaking. After the completion of the culture, the intestinal bodies were collected by centrifugation at 3,000Xg for 5 minutes, and diluted with 20ml of buffer AC30mM Tr.
is −HCt(p)! 8.0), ]mM EDT
A, 50 mM NaCt) was added, and lysozyme'i 10 Tn7 was added, followed by incubation at 5°C for 20 minutes. Next, deoxycholic acid was added to a final concentration of 1 m97 ml, heated to 20°C, deoxyribonuclease was added to a final concentration of 0.1 my/me, and the bacterial cells were disrupted using a Polytron crusher. . The resulting bacteriolysate was centrifuged at 80,000×g for 40 minutes to remove bacterial fragments, and then subjected to Sephadex G-75 column chromatography. The elastase active fraction was further purified by antibody affinity chromatography and used as an elastase-HB sample solution in the following assay. The elastase activity of the sample was measured by the following method. First, by treating the sample with 1kl-IJ Punun,
A friend that activates proelastase. Next, the synthetic substrate G111jary1-Ala-A, 1a
-PrO-Leu-p -nitroanilide
It was quantified by measuring the total absorbance of the released p-n1troanilide at 410 nm. The reaction was carried out by keeping the synthetic substrate Kanakura in a 0.2 M Tris-HCA buffer (pH 8,0) at 25°. As a result, clear activity was detected in the sample solution of strain 984 transformed with pI-12EX-8, indicating the production of active human elastase-■B in E. coli. (7) Production of human pancreatic elastase-[B using yeast] When using yeast as a whole host, human pancreatic elastase-[B c
The DNA f can be ligated to a suitable expression vector using a conventional method, transferred into a host cell, and fully expressed, and the presence of elastase activity in the culture can be fully confirmed. S described in "Recombinant DNA Experimental Guidelines". cerevisiae f host, but
Specifically, the 5288c strain was suitable. on the other hand,
As a vector, YEp 13 and the like were suitable. In addition, promoters include the ADH1 gene, which encodes the entire alcohol dehydrogenase gene.

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme map of plasmid pH2E8 obtained in the example, and the entire portion encoding the peptide considered to be signal peptide 1 is shown. The part indicates the entire coding part of the mature elastase protein. FIG. 2 shows the procedure for constructing psV2-E2B plasmid P. This is a plasmid engineered from psV2-β-globin, with β-globin cDNA inserted into the pSV2 vector. On the other hand, pH2E8 &X Okayama-Be
This is a plasmid in which human elastase-ITB cDNA is inserted into the rg vector. Reactions such as s1 nuclease are called "molecular cloning" (Man
iatis, T. et al. (ed.)” Molecular
Cloning”Col 1d Spring Harbor
Lab, (1,980)]. Thick arrow+X indicates all promoters derived from SV 40, thin arrow indicates direction of transcription. In the figure, B, H, S, v +・
Restriction enzymes Bg12, Hind3, Pst
I, Pvu 2 fully displayed. Figure 3 shows the procedure for constructing the pBsEX-E2B plasmid. pTUB 228 is a plasmid in which the α-amylase gene of Bacillus subtilis is cloned into a vector having a Bacillus subtilis origin of replication. The thick arrow indicates the α-amylase promoter, and the thin arrow indicates the direction of transcription. In the figure, restriction enzymes A, H, P, S,
Represents a2, Small, Pvu2i. FIG. 4 shows a synthetic peptide containing part of the signal peptide region of the Bacillus subtilis α-amylase gene and the activation peptide 11111k of the elastase-■B cDNA. Shows J car. Figure 5 shows the procedure for constructing the pH2Eχ-8 plasmid. Show. 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. In the figure, D)
], S, and Vl restriction enzymes IMe 1 and B, respectively.
ind 3, Sma 1, PVIJ, 2 k.

Claims (1)

[Claims] 1. Human pancreatic elastase-IIB 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-IIB 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] at the (N)-terminus. 3. DNA encoding human pancreatic elastase-IIB represented by the general formula [amino acid sequence included]. 4. The DNA according to claim 3, which encodes human pancreatic elastase-IIB 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 base sequence] (wherein X represents TAA, TGA, or TAG) or a base sequence with the same effect as the base sequence. Section 3 or 4
DNA described in section. The DNA according to claim 5, which has ATG or [there is a base sequence] at the 6 and 5' ends.
. 7. A base sequence represented by the general formula [there is a base sequence] characterized by including (a) to (d) described below (in the formula, X represents TAA, TGA, or TAG) or a base sequence thereof. A method for producing DNA containing base sequences with the same effect. (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. The DNA according to claim 7, which has ATG or [there is a base sequence] at the 8 and 5' ends.
manufacturing method. 9. A host transformed with a DNA containing the base sequence represented by the general formula [base sequence is available] (wherein, X represents TAA, TGA, or TAG) or a base sequence with the same effect. 10. The DNA according to claim 9, which has ATG or [base sequence] at the 5' end.
A host transformed with 11. The host according to claim 9 or 10, which is E. coli, Bacillus subtilis, yeast, or animal cell.