JPS61192288A - Bionic production of human elastase ii - Google Patents

Abstract

PURPOSE:To obtain human elastase II effectively, by subjecting a DNA chain having a base sequence to code polypeptide of a specific amino acid sequence to formation of plasmid, transformation of bacterium and its culture process. CONSTITUTION:A DNA chain having a base sequence to code polypeptide having an amino acid sequence selected from a group of sequences from B to D and from A to D of the formulas I and II is prepared, the DNA chain is subjected to a process consisting of (i) formation of a plasmid containing the DNA chain in a state capable of developing its genetic information, (ii) transformation of bacterium with the plasmid, and (iii) cultivation of the prepared transformant, and human elastase precursor is produced in the culture mixture.

Description

[Detailed description of the invention] Le Ming's back! Shellfish technology field This invention, human elastase ■! 1. Concerning physical engineering and manufacturing. Furthermore, the present invention provides (1) human elastane [! ■Improving the production performance of cows using cattle engineering 1) N
A chain (2) is transformed into a plasmid containing this DNA chain in a state in which the genetic information of the king can be expressed.
Elastase-producing microorganisms, i.e. DN
The present invention relates to a product that exclusively utilizes the properties of the Δ chain, and (3) a method for producing human elastase (2) by culturing this microorganism, that is, a method using the above-mentioned DNA chain.

In general, 1-lastase is a type of so-called serine protease produced by Qli mammalian pancreatic cells, etc., and is an extremely unique protease that degrades elastin, which is an insoluble hard protein. ]]Ra1 3 - Scouse is oral! The so-called porcine 1-lastase extracted from the pancreas of pigs has already been put into practical use as a drug (blood-metabolism improving agent). Similar medicinal effects are expected for human elastase.

In the pancreatic cells of mammalian animals, the 211i class of elastases are produced, which are physicochemically, enzymatically and antigenically different from each other and are called elastase Ⅰ and elastase I, respectively. Incidentally, porcine elastase, which has been put into practical use as a pharmaceutical, has the former as its active ingredient.

Elastase is produced in pancreatic cells as pre-proelastase, and when secreted outside the cell, it becomes proelastase (an inactive elastase precursor), and then undergoes the action of enzymes such as trypsin outside the cell. It is thought that upon receiving the enzyme, it is converted into elastase which has elastin-degrading activity.

= 4 - Prior technique * For Pig Elasturbi■, D, H, St+ott
The amino acid sequence of Mo is reported by onet at (Nature, 2251'eb 28.802 (
1970) ).

In addition, for rat elastase ■ and ■, Ra
ymond J, HacDonald et al.
Molecular biological analysis conducted by et al. revealed that related mRNA△
The nucleotide sequence and amino acid sequence of B
10ChOIIliSIry, 21.1453-14
63(InO2)).

Furthermore, for human elastase, C. largman
At al., 1 and ■ were purified from human pancreatic tissue, and their properties were investigated and reported (Bio
chemistry,' 1!1(11), 2491(
1976)). Furthermore, the terminal amino acid sequence (16) of human elastase has also been reported (C,
largman at: Bioc
himica at Biophysica 8ct
a 623 to 1980) 20B-212).

Regarding the amount of elastase present in the pancreas, it has been reported that pigs have a large amount of T-lastase ■, and humans have a large amount of elastase 11 (Biocb, 15).
2491 (1976)).

SUMMARY OF THE INVENTION The present invention provides a novel means for producing human elastase 11 using recombinant DNA technology.

However, according to the present invention, the biotechnological production ability of elastase 1) can be improved.
(3) It is characterized by having a base sequence overlapping a polypeptide having an amino acid sequence selected from the group consisting of (3).

Furthermore, the microorganism capable of producing human elastase according to the present invention has a DNA chain having a base sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) below. be transformed with a plasmid containing genetic information in a state capable of expressing genetic information;
It is characterized by

Furthermore, the biotechnological production method of human elastase ① according to the present invention includes iM which encodes a polypeptide having an amino acid sequence selected from the group consisting of
Prepare a DNA strand with a single base sequence, create a plasmid containing this DNA strand in a state where its genetic information can be expressed, transform this plasmid with There are two methods, b and e, which are characterized in that human elastase (2) is not produced in the culture when one culm is grown after culturing the transformant.

(1) Of the amino acid sequences shown in Figures 1 and 2, those from C to D; (2) the first
(3) Among the amino acid sequences shown in Figures 1 and 2, those from B to D are shown in Figures 1 and 2. ,
From △ to ]).

The present invention can also be said to relate to elastase produced by a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) above.

Effects According to the present invention, the shape of elastase is changed. Produced by microorganisms! can be done.

Therefore, compared to the method of extracting from pancreatic organs, there is a possibility that human elastase (2) can be produced and isolated more effectively.

Since the human elastase (1) produced by the present invention is of human type, it is expected to have a medicinal efficacy superior to that of porcine elastase (2) when used as a medicine.

Furthermore, according to a preferred embodiment of the present invention (details will be described later), it is possible to produce human elastase, which is directly active, as a DNA expression product (when elastase is extracted from the pancreas, an inactive Since it exists as proelastase, it was necessary to perform an activation treatment in addition to a purification operation).

Although recombinant DNA technology has made great progress today, it is still difficult to predict the gene sequence encoding each protein and its expression (production). Currently, human elastase ■ has not been achieved, so it is surprising that human elastase ■ could be produced by the present invention (
It should be said that there was no such thing.

= 8 constant The biological production ability of the human l-1-lastase according to the present invention is determined by Figure 1 and 2
Of the amino acid sequences shown throughout the diagram, C to D
1b, which contains a polypeptide which is from B to D, or from 8 to D. In addition, Figure 1 shows amino acid sequences 1 to 18 starting with △.
Needless to say, number 0 is shown, and Figure 2 shows the following numbers 181 to 269 (corresponding to the 269th amino acid in Figure 2) -ton followed by <DNA is also shown. ).

Here, the term "rDNAΔ strand" refers to a complementary double strand of polydeoxyribonucleic acid having a certain length. In the present invention, this rDNA chain is specified by the amino acid sequence of the polypeptide encoded by it, and since this polypeptide has a finite length as described above, this DNA chain also has a finite length. It is of goodness.

However, this DNA strand contains the gene for human elastase and is responsible for the biological production of this polypeptide;
This J: biotechnological production is not only possible on the A chain, but also on its 5'-upstream and/or 3
The biotechnological production of this polypeptide becomes possible when a DNA strand of an appropriate size is bound to the -side IZ stream. Therefore, in the present invention, when referring to ``rr)NA chain'', the term ``NA chain'' refers to a chain of this specific length (in terms of the corresponding amino acid sequences in Figures 1 and 2, the length of CD, BD, or Δ-D). In addition, it shall also include those in the form of linear or circular DNA chains whose members are DNA strands of this specific length (however, given the nature of the invention, this DNA strand may not be human).・This does not mean that it does not include cases where it exists in the pancreas.This is why the DNA strand according to the present invention is called "a DNA strand that has biotechnological production ability of 1- ■Raster 1■". ).

11 The typical state of existence of the DNA strand according to the present invention is as follows:
This is the form 4T of the buenosmid, which has this DNA strand as part of its members, and the form present in the plasmid, ``C9'', ``Vl'', Escherichia coli, and ``N1''.

According to the present invention, plasmids as preferable forms of existence other than one of the N∆ chains are present in a wide range of forms to foreign genes. The present invention D, such as a plasmid vector that exists stably and is capable of replication.
It is a combination of a promoter that directs the transcription of the NA strand to mRN△. As plasmid vectors and promoters, known vectors can be used in appropriate combinations, and the following can be exemplified.

ζ ~ polypeptide encoded by a blind gene As mentioned above, the I) NA chain according to the present invention is =
J: is specified in the amino acid sequence of the polypeptide that encodes 1-.

This polypeptide has C-, B-D, and B-D of the amino acid sequences shown in Figures 1 and 2.
Or those from A-D. Here, the polypeptides whose amino acid sequences are C-1'), B-D, A-D are Hi1~Elastase■, and HumanP[1
Elastase ■ and Hi 1 ~ Prealoelastu 12T
It is I.

Hill-elastor 1ffi II consists of 241 amino acids.

Human proelastase (B-D) corresponds to the above-mentioned Hi-1-elastase IF (0-D) with 12 amino acids bonded to the N-terminus.

Human preproelastase TI (A-D) has roughly 1 molecule at the N-terminus of this human proelastase TI (B-D).
It is a combination of six amino acids.

The amino acid sequences of these 1 raster compounds (including the 3 compounds listed below) have not been previously known.

Base Sequence of DNA Strand Human Elastor 1 Since there are three types of compounds, there are basically three types of DNA strands that determine the base sequence that causes the error.

Human] The DNA strand containing 21-domains of 2-lastase is
This is Shino having the 0-D base sequence shown in Figures 1 and 2 or its degenerate P1 form. Here, [degenerate f1 body 1] is defined as a DNA chain that differs only in the degenerate codon and can encode the same polypeptide. For example, Figs. For example, the codon (G1-T) corresponding to Val at the N-terminus corresponds to one of the amino acids for a DNA chain having the base sequence C-D. In the present invention, those which are in a heavy relationship and are changed to GTC, for example, are referred to as degenerate isomers.

A preferred embodiment includes at least one starting cordon AT G adjacent to the 5'-side upstream and a stopping cordon at the 3'-side end ifl L (one of which is e.g.
GA).

The DNA strand containing human proelastase is the first
It has the base sequence B-D (7) in Figure 2 or its degenerate isomer. The DNA chain containing human proelastase ■ also has a start codon ΔTG at its 5'-end and at least one stop codon at its 3'-end (
Preferably, one of them has, for example, TGA).

The DNA strand encoding human proelastase■ is the first
It has a partial base sequence as shown in Figure 2 or its degenerate form. This DNA strand has an initiation codon ATG at its 5'-end, but this DNA strand also has an initiation codon ATG at its 3'-end.
It is preferable to add at least one stop (one of which is, for example, TGA) at the end.

In any of the above-mentioned DNA strands encoding elastase compounds, the C-terminal A of the polypeptide
3' of the cordon (ΔΔC in the illustrated example) corresponding to sn
- Downstream there can be a long stretch of DNA strand as an untranslated region (the first part of which is usually a stop codon such as TAG),
Further, a poly(A) chain common to all eukaryotic genes may be added to the 3' downstream of A. If a poly(A) chain is added, its 5'-[stream-]
The untranslated region 1- usually contains a poly(Δ) addition signal called ΔΔTAAA. In addition,
The base sequence of the DNA strand (from ATG at the 5'-end to the poly(Δ) strand at the 3'-end) shown in Figures 1 and 2 is the 1q cDNA from mRNA of human 1 to preproelastase. was determined using the Maki→Nam-Gilbert method.

One way to obtain a DNA strand having a base sequence containing the amino acid sequence 11 of the human elastase compound described above is to chemically synthesize at least a portion of the chain length according to the method of nucleic acid synthesis. It is to be.

Considering that the number of bound amino acids is at least 241, it can be said that an enzymatic method using mRNA obtained from the pancreas is preferable to this chemical synthesis method.

That is, in order to produce the human elastase gene, mRNA is obtained from an organ containing human elastase mRNA, and this is used as a template to inject C into human elastase using reverse transcriptase.
A common method is to create a DNA bank and use an appropriate probe from the CDN bank to obtain a clone containing the human elastase 2 gene. As this probe, it is convenient to use a portion of the rat elastase Trill gene. It is generally known that proteins that control the same function in the living world have homology at the amino acid-foam structure level as well as at the DNA base sequence level, and in this case, the more closely related they are phylogenetically, the higher the homology. . As a result of comparing humans and rats after obtaining the genes, the present inventors found that both amino acid and DNA sequences are 8.
Approximately 0% homology was observed. Rat Elaster L'I
There has already been a report on the gene (cited above), D.
The NA base sequence is known.

Therefore, the method performed by the present inventors is as follows. In other words, Rat Elastor 1i If 3! A part of the DNA base sequence of Butsuko was chemically synthesized, and using it as a probe, screening was performed from cDNA△DNA created using reverse transcriptase from rough 1 to pancreatic 11mRN△, and the shoe 1 to elastase■ gene was screened. I got a clone. It was determined by examining the DNA base sequence that this gene was the rat elastase gene. This gene was cut with a restriction enzyme to approximately 770bp (tu
Which fragment was obtained for pair 321), and this fragment was labeled with 321) and used as a probe. A CI) NΔ bank created from human pancreatic mRNA using reverse transcriptase was screened using the above program. The distribution number 11 was determined using the Maki 4 Tsumu Gilbert method.

In this way, the DNA strand of the present invention was obtained from the J: Sea urchin 1 to pancreatic CI) N△ bank in the form of being integrated into the plasmid 1-, but of course this DNA strand was completely synthesized by chemical synthesis. Or, create it from the middle of 1 and take 1.
! l? As mentioned above, it is also possible to write

Since the DNA strand of the present invention obtained by the method described above contains the genetic information for producing human elastase protein, it can be introduced into microorganisms using biotechnological techniques for transformation. human elastase protein can be produced in this transformed microorganism.

As long as a suitable plasmid vector exists in the host microorganism, various types of microorganisms can be used.
The lZ product can be transformed with a plasmid containing the DNA strand of the present invention as a member.

One group of such microorganisms is [5cherichia c
oli, and the group of counts is 5accharoIIlyc
es cerevisiac.

The procedure for creating the transformed transformant (the production of human 1-1 lastase If) and the method itself are as follows:
Since it may be a technique commonly used in the fields of molecular biology, bioengineering, or genetic engineering, in the present invention, except for what is described below, (b) can be carried out according to these commonly used techniques.

Expressing the gene of the DNA strand of the present invention in a microorganism 'c5't
! In order to do so, it is necessary to relink this gene into a plasmid vector that stably exists in the microorganism. The plasmid vectors used in this case are 1) B R322 for E. coli, and -(Y) for yeast.
Rp7 etc. - all known ones are used.

On the other hand, in order to express the gene of the DNA strand of the present invention in the wII4: product, it is necessary to avoid transcription of the wII DNA into mRNAΔ. For this purpose, a promoter gene, which is a signal for transcription, can be incorporated into the 5'-upstream of the DNA strand of the present invention. About this promoter I, 1
Already 1: r p, l, <) c.

ompF (for E. coli), AI) H1, G A
L7, IIGK, TFgu [] 1 (for acid 01) -
Various types of S are known, and any of these can be used in the present invention.

In addition, at the stage of translating mRNA into protein, a sequence (called an SD sequence in E. coli) necessary for the liposome, which is the site of protein synthesis, to bind to the tip of the translation initiation site, is placed in front of ATG, which is the initiation signal for protein synthesis. It is necessary to take the next step. Note that the 3' untranslated region containing the poly(Δ) addition signal is not considered to be particularly necessary for protein synthesis, so it can be removed if necessary. However, when the host microorganism is Mm, these 3
'The presence or absence of untranslated regions is said to be involved in the amount of protein synthesis, so a large amount of human elastase
Care must be taken when trying to synthesize proteins.

Now, in general, if you want to make an elastase protein, the above-mentioned operations are necessary, but among the DNA strands of the present invention whose base sequences are BD and A-, the precursor peptide is Since it contains a sequence that encodes the 1-elastase protein, when the 1-elastase protein is made, the 1-elastase protein containing the precursor peptide (the 1-elastase 1ffi protein)
Ir precursor Nawa 15 proelastase ■ or prepro J
It is possible to create a raster, and the base sequence C-D
It is also possible to create proteins without this precursor pebbly. However, if it does not contain the ir1 cylindrical body bood (Δ-C), A, which is a signal for starting protein synthesis,
Since TG is required, it is advisable to add at least one methionine before the N-terminal amino acid. If so, one or more amino acids can be added before the N-terminal amino acid, or some amino acids can be deleted at the N-terminus or C-terminus. For adjustment and other purposes, it is also possible to combine a DNA strand of an appropriate length as RinnoJ-.Processing of this J:RinnoNA strand can be done using the current gene T
In terms of scientific technology, it can be easily carried out using restriction enzymes and rlNA chemical synthesis technology.

Transformation of microbes using the plasmid thus prepared can be carried out by any method commonly used in the fields of genetic engineering and biological sciences. Regarding the general matters, please write a book or review! Speaking of T, Haniatis.

In E., F., Fr1tsch, and J., Samhroo:
“Mo1ecular Cloning-＾ 1abo
raroy Manual J, Co1d S
pring 1larborLaboratory,
(1982).

The transformant has a new trait (that is, human elastase ■) based on the genetic information introduced into the DNA strand of the present invention with
production ability) and J:, except for trait 4 derived from the vector used and the lack of the corresponding trait due to lack of some genetic information from the vector used during genetic recombination, which may occur in some cases. , which is the same in its xenotype or phenotype or mycological properties as the host microorganism used.

Production of protein The transformant clone obtained in the above manner produces human proelastase, human proelastase 12m, or human proelastase in the culture when it is cultured. Produces preproelastase■. When Blaire loelascus I is secreted outside the pancreas, it becomes bu[1 elastase■, which is then broken down into trypsin etc. on the pad of the finger and becomes a mature protein. This invention is said to convert into elastase.
It is thought that when the precursor protein of I-lastase 2 is obtained, it can be treated with trypsin or the like to obtain a product identical to human puelastase 2.

In addition, when expressing the chymosin gene in yeast, it has been reported that the production efficiency was more than 10 times that of the production of chymosin by direct production of chymosin (Blow 1 = E-sin). ,H,,1,Dobson
.

N, ^, Robcrts, H, r, 1 suite, ,
1. S, Fmtagl, S, W old te.

P, A, l-owe, 1', Pal:e, A,, 1
．． Kingsman, S., 14. Kingsn+an
Gene, 24.1-14 (1983)), and in the present invention, it is also possible to easily overcome mass production by utilizing the sequence of this blep [1]. , it does not essentially change that for the host microorganism used. Alternatively, the recovery of produced proteins from a culture, that is, a bacterial cell or from a culture solution, can be carried out according to a conventional method.

Experiment example ■, Cloning of LAT1 ~ Elastase ■ Genetic Wistar L1 ~ (obtained from Charles River Japan Co., Ltd.)
After anesthesia with ether, the pancreas was immediately removed, and RNA was obtained by using guanidine diocyane-1 as a denaturing agent and purifying it by density gradient centrifugation (detailed experimental conditions can be found in Kazu1 (ν, Yoshiaki Fujizuki [see Cytology IL, 298-303 (1982)). To obtain this RNA AJ:su mRNA, it was purified by oligo dT cellulose column chromatography, and oligo dT cellulose column chromatography was used. mR with poly(Δ) adsorbed to
I only got NA. Approximately 10 pancreases were obtained from each rough animal, and 200 μq of mRNA was obtained. Next, this m
cDNA was prepared by converting RNA into starch μ-] and using the Okayama Bark method. The experimental procedure is as follows (detailed experimental conditions can be found in Katsuya Shigesada: [Cytology J2, 61 (1)
~626 (1983)).

′! Vector brimer DNA with 50 additions of 1 and 1 added to the end of mRNA d1 and d1.
Add reverse transcriptase, vector primer D
Use one part of NA as a brimer and add ff1 to mRNA.
=i TJ complementary strand DNA is synthesized. Then this DN
Add dCTP and a terminal transferer to the A fragment, and add 20 dCs to both ends. This vector primer DNA was extracted with the restriction enzyme t-1indm.
Cut a part of the part () to remove the dC(Nl addition part over a certain distance. On the other hand, one end is 1lind[[
Prepare a linker-1-]N△ fragment which has the same cleaved end and has about 20 dGs added to the other end, and add this to the above 11indll [digested product to link both DNAs in a circular form. Speak.

Next, RN as e l-(, DNA volumer L'
Ha'; and DNAΔNA-se remove the mRNA portion and turn it into DNA'A. In this way, using the rat pancreatic vector primer DNA1.511<h and the vector primer DNA1.511<h,
Add 1NΔ fragment Q and 0771O to a DNA (q
is.

Using this GDNA, E. coli RRT strain (Bol 1var,
F.: Gene, 2, 95 (1977)), approximately 50,000 clones were obtained as transformants.

Among these Escherichia coli, we searched for those with the gene known as rat elastase II.
W gene (Biochemistry S21. 14)
53-1463 (1982)) was chemically synthesized and used as a probe.

More specifically, AULJ GCA AAG AAG UAΔ3 corresponds to the C-terminal part of the rat elastase gene.
'TAA CGT TTCTTG ATT5 f
Arrange lXlX columns. The top is rat elastase mRNA
The lower part is a probe of the synthesized A nucleotide, which is the complementary strand to the mRNA. The synthesis method was based on the solid phase phosphotriester method (for details, see F.
, 0htsuka, Hjkehara.

D, 5oll: Nuclcic Ac1ds Re
5earch 10.655.3-6570 (19
82)). This synthetic oligonucleotide was labeled by transferring a phosphate group from γ[32P]ΔTP using T4 polynucleotide kinase. Using this as a probe, transformants containing cDNA were screened. The screening method was performed using the Ronnie hybridization method. In this case, the bacterial cells grown on the 21-cellulose filter were denatured with alkaline, and the l)NA in the bacterial cells was burned onto the filter. When the filter is placed in a hybridization solution containing probe DNA, I'j, which is homologous to the DNA sequence, binds under certain conditions. 1 (Kodansha))
. This is then subjected to autoradiography to determine whether it is positive or negative. Clones containing homologous sequences are colored black. In fact, about 2000 clones were screened and 12 positive clones were obtained. Hybridization conditions are 6XSSC15
X Denhardt solution/35°C/16 hours, 6x SS
Washed twice at C/35°C/30 minutes. For these clones, the plasmids were purified using the alkaline method (T, Haniat
is, c, r, "rth sch, , 1.5 anbro
ok knee 2 l - mouth (olecular Cloning-A I-o
When a restriction enzyme cleavage map was prepared and compared with that reported in the literature, it was found to be a complete match. In this way, rat elastase Imbutsu was obtained.

■ Cloning of elastase TEM gene from rat ■ mRNA was obtained in exactly the same manner as for the lastase III gene, and cDNA was roughly constructed. 200 μa of mRNA was obtained from about 20 human pancreases. Zarani, mRNA5.0/lQ.

Vector brimer DNA 1.5μQ and linker D
Approximately 30,000 transformants containing cDNA were obtained using NA 0.08Ilq. Among these, human elastase II il! Butsuko was screened,
A portion of rat elastase I [31f gene was used as a probe. The rat elastase ■ gene was digested with the restriction enzyme [)de■ and then separated by agarose gel electrophoresis to obtain a fragment of approximately 770 bp. α(3”P) was added to this fragment by the nick translation method.
) d CT P and insert the iso1 heave label I
For details on the Konitsuku 1-Lance 1 Nosion method, please refer to the above-mentioned "Gene Manipulation Manual") This was used as a probe and screened using the Ronnie hyperdivision method, which was previously adopted when cloning the Kutsu 1-Elastase ■ gene. did. Hybridization conditions are 6xSSC
in 15x Denhardt's solution/60°C/16 hours;
Washing was carried out at 60°C/30 minutes/twice. Approximately 10.00
0 J: 15 clones were obtained, among which the one with the most distinct cDNA portion (referred to as E I-I E 12 strain).

In addition, the cDNA-containing plasmid contained in this is 1)
The base sequence of the DNA portion other than the beta primer was determined for the HE I-2. jn base sequencing was performed using the Maxam Gilber 1-method, reading the strands in both directions (details of the method can be found in Mitsuru Takanami and Tatsuo Oi, eds.
DN△Sequence Analysis Manual □Al” (see Kodan 1)). The determined sequences are as shown in Figures 1 and 2. From this DNA base sequence, amino acids translated according to the -ton table are also shown.

This amino acid sequence was converted into the N-terminal amino acid sequence of the pro form of human elastane L4II (Cys-Gly-Δ5p-pr) reported by C,l-argman et al.
o-Thr-Tyr-pro-P r O-1-V r
-V a I -T h r -A r CJ -Va
1-Val-Gly-GIV), the N-terminal amino acid sequences of Brohi] to elastase Ir (B-D portion) were completely identical.

Deposit of Microorganisms The following microorganisms related to the present invention were refused to be deposited by the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(1) Human elastase ■ gene-containing plasmid p1
E. coli RR1 strain (El-IE12 strain) containing ~IE[2
.

[Brief explanation of drawings]

Figures 1 and 2 show the DNA base sequence 11 of the human elastase gene OJ: and its precursor gene and the amino acid sequence deduced from the base sequence. Figure 1 shows the 180th Figure 2 shows the amino acids from the 181st onwards. Applicant's Representative Immediately Ino Mata Seishi I Thr Tyr Pro Pro Tyr Vat T
hr Arg Val Val 1CCCTGG CA
G GTCTCCCTG CAG TACAGCTCC
. Pro Trp Gln Val Ser Leu G
in Tyr Ser Set TCCCTG ATA
GCCMCAGCTGG GTCCTG ACG・Se
r Leu Ile Ala Asn Se
r Trp Val Leu ThGACAT
T GCCCTG CTCAAA CTG GCT A
ACCCGly Arg Leu Gln Thr A
sn Gly Ala Val Pr figure; GCGGT GAA GAA GCG AGG CC
CMCAGCTGG;ly Gly Glu Glu△
la Arg Pro Asn Ser Trp\AT
GGCAAG TGG TACCACACCTGCG
GA GGG\sn Gly Lys Trp
Tyr His Thr Cys Gly
GlyECT GCCCACTGCATCAGCTCC
TCCAGG ACCrGG AACTCCAACCA
A ATCTCCAAA GGG AAC rabbit 2 GTG' GACTAT GCCACCTGCTCCA
GCTCT GCCVal Asp Tyr Ala
Thr Cys Ser Ser Ser AIOTT
G GAA AGG TCA CAG MG GAA
ATA ATA TAA diagram TGG TGG GGCAGCAGCGTG AAA
ACCAGT ATGTrp Trp Gly Ser
Ser Val Lys Thr Ser MetG
TCTTCCACG CGG GTCTCCAAT TC
CATCGACTAA AGT GACAACTAT
GCA AAT C Hand υcntii-IIE Book October 1985 3L+

Claims (1)

[Claims] 1. Biotechnological production ability of human elastase II, characterized by having a base sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) below. A DNA strand with (1) Of the amino acid sequences shown in Figures 1 and 2, those from C to D; (2) Among the amino acid sequences shown in Figures 1 and 2, those from B to D. (3) Among the amino acid sequences shown in FIGS. 1 and 2, those from A to D. 2. Among the base sequences that encode polypeptides shown in Figures 1 and 2, C to D
The DNA strand according to claim 1, which is from B to D, or from A to D. 3. Transformed with a plasmid containing a DNA strand having a base sequence encoding a polypeptide with an amino acid sequence selected from the group consisting of (1) to (3) below in a state in which the genetic information can be expressed. A microorganism capable of producing human elastase II, characterized in that: (1) Of the amino acid sequences shown in Figures 1 and 2, those from C to D; (2) Among the amino acid sequences shown in Figures 1 and 2, those from B to D. (3) Among the amino acid sequences shown in FIGS. 1 and 2, those from A to D. 4. The microorganism according to claim 3, wherein the microorganism is Escherichia coli. 5. The DNA strand has the base sequence from C to D among the base sequences shown in Figures 1 and 2,
The microorganism according to any one of claims 3 to 4, which is a microorganism from B to D or from A to D. 6. Prepare a DNA strand having a base sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) below, and transform this DNA strand into a state in which its genetic information can be expressed. A human elastase II organism characterized by producing human elastase II in a culture by subjecting it to the steps of creating a plasmid containing the plasmid, transforming a microorganism with this plasmid, and culturing the resulting transformant. Engineering manufacturing methods. (1) Of the amino acid sequences shown in Figures 1 and 2, those from C to D; (2) Among the amino acid sequences shown in Figures 1 and 2, those from B to D. (3) Among the amino acid sequences shown in FIGS. 1 and 2, those from A to D. 7. The method according to claim 6, wherein the microorganism is Escherichia coli. 8. The DNA strand has the base sequence from C to D of the base sequences shown in Figures 1 and 2,
8. A method according to any one of claims 6 to 7, wherein the method is from B to D or from A to D.