JPS61111688A - Bio-engineering preparation of human elastase i - Google Patents

Abstract

PURPOSE:To produce and separate human elastase I, in high efficiency, by carrying out specific transformation and cultivation of a DNA chain having a base sequence coding the polypeptide of amino acid sequences selected from specific three groups. CONSTITUTION:A DNA chain having a base sequence coding a polypeptide of amino acid sequences selected from specific three groups, is prepared beforehand, and a plasmid containing said DNA chain in a state to enable the exression of the genetic information is prepared. A microbial cell is transformed by the plasmid, and the obtained transformation is cultured to accumulate human elastase I in the culture product. Human elastase I can be produced by the transformed microorganism by this process. Accordingly, the production and separation of human elastase I can be carried out more effectively than the extraction from pancreas.

Description

BACKGROUND OF THE INVENTION TECHNICAL FIELD This invention relates to the biotechnological production of human elastase. More specifically, the present invention relates to (1) a DNA strand capable of producing human elastase in a biotechnological manner; (2) a plasmid transformed with a plasmid containing this DNA strand in a state in which its genetic information can be expressed. A human elastase producing microorganism, that is, one that exclusively utilizes the properties of the above DNA strand, and (3) a method for producing human elastase by culturing this microorganism, that is, a method that uses the above DNA strand.
Regarding.

Generally, elastase is a type of so-called serine protease produced by mammalian pancreatic cells, etc., and is a very specific protease that degrades elastin, which is an insoluble hard protein. Elastase has been confirmed to be effective in treating arteriosclerosis in humans when administered orally.
So-called porcine elastase, which has been conventionally extracted from pig pancreas, has already been put into practical use as a drug (vascular metabolism improving agent), and human elastase is expected to have similar medicinal effects.

In mammalian pancreatic cells, two types of elastase are produced, which are different from each other physicochemically, enzymologically, and antigenically, and are called elastase ① and elastase ②, respectively. Incidentally, porcine elastase, which has been put into practical use as a pharmaceutical, has the former as its active ingredient.

Elastase is the pancreatic QfRJ for both ■ and ■, respectively.
It is produced as pre-proelastase within the cells, becomes proelastase (an inactive elastase precursor) when secreted outside the cell, and then undergoes the action of enzymes such as trypsin outside the cell to transform into elastase with elastin-degrading activity. It is considered to be.

For the previous 5M crying pig elastase, D, H, 5hotto
The amino acid sequence has been reported by Net al. (Nature, 225 Feb 28.802 (1
970) ).

In addition, for rat elastase and ■, Ra
ymond J, MacDonald et al., the base and amino acid sequences of related mRNAs have been elucidated (Bio
chemistry, 21.1453-1463(1
982) ).

Furthermore, for human elastase, C.Largman
et al., ■ and ■ are I%u from human pancreatic tissue.
and their properties have been investigated and reported (Bio
chemistry, 15(11), 2491(19
71)). Furthermore, the N-terminal amino acid sequence (16 sequences) of the pro form of human elastase ■ has also been reported (C, Largman et al: B
iochimica et BiophysicaAc
ta 623 (1980) 208-212).

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

That is, the DNA chain capable of biotechnological production of human elastase according to the present invention has a base sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) below. This is a characteristic feature.

Furthermore, the microorganism capable of producing human elastase according to the present invention is capable of expressing DNAI having a base sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) below. It is characterized in that it has been transformed with a plasmid containing the same state.

Furthermore, the biotechnological production method of human elastase according to the present invention involves preparing 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. , this DNA strand is subjected to a process consisting of creating a plasmid containing the genetic information in a state capable of expressing it, transforming a microorganism with this plasmid, and culturing the obtained transformant. It is characterized by producing human elastase.

(1) Of the amino acid sequences shown in Figures 1 and 2, from C to D, (2) the first
Among the amino acid sequences shown in Figures and Figures 2, those from B to D, (3) Figures 1 and 2.
Among the amino acid sequences shown throughout the figures, those from A to D.

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

Effects According to the present invention, human elastase (2) can be produced by a transformed microorganism.

Therefore, compared to the method of extracting from pancreatic organs, it is possible to produce and isolate Toelas fusae more effectively.

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

Furthermore, according to a preferred IM embodiment of the present invention (details will be described later), it is possible to produce human elastase (2), which has direct activity 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 has not yet reached the point where it is possible to always predict the success of cloning and expression (production) of genes encoding individual proteins. Since this is the current situation, it should be said that it was unexpected that human elastase f could be produced by the present invention.

Definition The DNA chain capable of biotechnological production of human elastase according to the present invention, that is, the human elastase gene,
encodes a polypeptide whose amino acid sequence is from C to D, from B to D, or from A to D among the amino acid sequences shown in FIGS. 1 and 2; It is something to do. It goes without saying that Figure 1 shows numbers 1 to 180 of the amino acid sequence starting from 8, and Figure 2 shows the following numbers 181 to 269 (Figure 2 shows the 269th amino acid). Following the cordon corresponding to <DNA is also shown).

Here, the term "rDNA strand" means 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 it encodes, and since this polypeptide has a finite length as described above, this DNA chain also has a finite length. It is of length. However, this DNA strand contains the human elastase gene and enables the biotechnological production of this polypeptide, but such biotechnological production cannot be performed only with this finite length of DNA chain. Instead, this polypeptide can be produced biotechnologically with a DNA chain of an appropriate length attached to its 5'-upstream and/or 3'-downstream. Therefore, when referring to "rDNA chain" in the present invention, the corresponding amino acid sequence in Figures 1 and 2 of this specific length is C-D.
, B-D or A-D), it also includes DNA chains of this specific length that are in the form of linear or circular DNA chains (however, It goes without saying that the nature of the invention does not include the case where this DNA strand exists in the human pancreas.
This is why the NA chain is called ``DNA'a having biotechnological production ability of human elastase.'')

Representative states of the DNA strand according to the present invention are:
This DNA strand is in the form of a plasmid as a member, and as a plasmid it exists in microorganisms, especially Escherichia coli and yeast.

A plasmid as one and preferred form of existence of the DNA strand of the present invention includes a plasmid vector that stably exists and can replicate in microorganisms, such as a parasenger or a foreign gene, and a plasmid vector that can be used to mR the DNA strand of the present invention.
It is a combination of a promoter and a promoter for transcription into NΔ. As the plasmid vector and promoter, known vectors can be used in appropriate combination, and the following can be exemplified.

As mentioned above, a DNA chain according to the invention is specified by the amino acid sequence of the polypeptide it encodes.

This polypeptide has the amino acid sequence C-018-D or AD among the amino acid sequences shown in Figures 1 and 2. Here, polypeptides whose amino acid sequences are CD, BD, and AD are:
Human elastase■, human proelastase■
and human preproelastase.

Human elastase consists of 242 amino acids.

Human proelastase (B-D) corresponds to the above human elastase I (C-D) with 12 amino acids bonded to the N-terminus.

Human preproelastase I (A-D) contains roughly 15 nucleotides at the N-terminus of human proelastase I (B-D).
It is a combination of amino acids.

The amino acid sequences of these elastase compounds (b including the above three types) have not been previously known.

Base Sequence of DNA Strand Human Elastase ■ Since there are three types of compounds, there are basically three types of DNA strands that have the base sequences encoding them.

The DNAI encoding human elastase is
It has the base sequence CD in the figure or its degenerate isomer. As used herein, "degenerate isomers" shall mean DNA strands that differ only in degenerate codons and are capable of encoding the same polypeptide. For example, for a DNA chain with the base sequence C-D in Figures 1 and 2, the Neton corresponding to any of its amino acids, for example, the codon (GTT) corresponding to Val at the N-terminus, has a degenerate relationship with this. In the present invention, those which are changed to, for example, GTC, are referred to as degenerate isomers.

A preferred embodiment is one having a start codon ATG adjacent to the 5'-side upstream and at least one stop codon (one of which is, for example, TGA) adjacent to the 3'-end.

The DNA strand encoding human proelastase■ is the first
It has the base sequence B-D in Figure 2 or its degenerate isomer. D encoding human proelastase L'I
The NA chain also has a start codon ATG 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 the base sequence A to D in Figure 2 or its degenerate isomer. This DNA strand has an initiation codon ΔTG at its 5'-end, but this DNA strand also has an initiation codon ΔTG at its 3'-end.
It is preferable to have at least one stop codon (one of which is TGA, for example) at the end.

In any of the above-mentioned human elastase compound DNAa, the C-terminal tol of the polypeptide is
3' of the cordon (CAC in the illustrated example) corresponding to is
- Downstream, a DNA strand as an untranslated region (the first part of which is usually a stop codon, such as TAG) can continue for a certain length, and its rough A poly(A) chain common to eukaryotic genes may be added to the downstream of the 3'-side. When a poly(A) chain is added, the above-mentioned untranslated region upstream on the 5' side usually contains a poly(A> addition signal called AΔTAAA.J5, The DNA strand shown in Figures 1 and 2 (from the 5'-terminal ATG to the 3'-
The base sequence of the terminal poly(A>chain) was determined by the Magisam-Gilbert method for CDNA obtained from m1llA of human preproelastase.

Obtaining a DNA strand One means of obtaining a DNA strand having a base sequence encoding the amino acid sequence of the human elastase compound described above is to chemically synthesize at least a portion of the strand according to a nucleic acid synthesis method.

Considering that there are at least 242 bound amino acids, mR obtained from human pancreas is better than this chemical synthesis method.
It can be said that it is preferable to use an enzymatic method using NA.

That is, in order to produce human elastase lff1 gene, mRNA is extracted from an organ containing human elastase mRNA.
The general method is to obtain a human elastase II gene, use this as a template to create a cDNA bank using reverse transcriptase, and use an appropriate probe from the cDNA bank to obtain a clone containing the human elastase II gene. be. As this probe, it is convenient to use a portion of the rat elastase gene. In general, it is known that proteins that control the same function in the living world have homology not only at the amino acid-substructure level but also at the DNA base sequence level, and in this case, the more closely related they are phylogenetically, the higher the homology. . It can be said that humans and rats are quite similar, and the results of the comparison after gene acquisition by the present inventors show that amino acids,
Approximately 60% homology was observed in both DNA sequences. There has already been a report on the rat elastuse II gene (cited above), and the DNA base sequence is known.

Therefore, the method performed by the present inventors is as follows. That is, the DN of the rat elastase l1ll gene
A part of the A base sequence was chemically synthesized, and using it as a probe, a CDNA bank created from rat pancreatic mRNA using reverse transcriptase was screened.
A shoe 1-elastase ■ gene clone was obtained. It is D that this gene is the rat elastase■ gene.
It was estimated by examining the NA base sequence. This gene was cut with a restriction enzyme to obtain a fragment of about 700 bp (base pairs), which was labeled with 32P and used as a probe. A CDNA bank created from human pancreatic R mRNA using reverse transcriptase was screened using the above probe. The nucleotide sequence of the clone with the longest CDNA portion among the obtained clones was determined by the Mackinham-Gilbert method.

In this way, the DNA strand of the present invention is derived from human pancreatic cDNA as described above.
Although the DNA strand was obtained by being integrated into an Escherichia coli plasmid from a DNA bank, it is of course possible to obtain the DNA strand by chemically synthesizing it completely or partially.

Transformants Since the DNA strand of the present invention obtained as described above contains genetic information for producing human elastase I protein, it is introduced into a microorganism by biotechnological techniques to create a transformant. Human elastase I protein can be produced in this transformed microorganism.

Host microorganisms As long as a suitable plasmid vector exists, various microorganisms can be used as an invention DNA! It can be transformed with a plasmid containing Q as a member.

One group of such microorganisms is Escherichia c.
oli, and the other group is Saccharomyces
cerevisiae.

Transformation As mentioned above, it was confirmed for the first time by the present invention that the genetic information possessed by the DNA strand of the present invention was expressed in microorganisms, but the creation of transformants (and the production of human elastase) The procedures or methods themselves may be those commonly used in the fields of molecular biology, bioengineering, or genetic engineering, and therefore, in the present invention, procedures other than those described below may be carried out in accordance with these commonly used techniques. do it.

In order to express the gene of the DNA strand of the present invention in a microorganism, it is first necessary to connect the gene to a plasmid vector that stably exists in the microorganism. The plasmid vector used at this time is p
All known compounds such as BR322 and YRp7 for yeast can be used.

On the other hand, in order to express the gene of the DNA strand of the present invention in a microorganism, it is necessary to transcribe the DNA into rr+RNA. For this purpose, a promoter gene, which is a signal for transcription, may be incorporated into the 5'-upstream of the DNA strand of the present invention. This promoter has already been tr
p, lac, ompF (for E. coli), ADHI
, GAL7, PGK, TRPl (all for yeast), etc., and the present invention does not use any of these.
can do.

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

Now, generally speaking, if you want to make elastase protein, the above operations are necessary, but among the DNA strands of the present invention, those with base sequences B-D and A-D encode precursor peptides. When making human elastase I protein, it is possible to make human elastase I protein (referred to as human elastase precursor protein), that is, promoterase or preproelastase, when making the human elastase I protein. If the sequence C-D does not contain this precursor peptide (A-C), at least one methionine is added before the N-terminal amino acid because ATG, which is a signal for starting protein synthesis, is required. I have to let it happen. In addition, if elastase activity is maintained when producing Toelasfusae protein, it is possible to add one or more amino acids before the N-terminal amino acid, or add some amino acids to the N-terminal side or C-terminal amino acid. -Can also be scraped at the end.

Furthermore, a DNA strand of an appropriate length can be attached as a linker for adjusting the reading frame or for other purposes. With current genetic engineering technology, such processing of the NA strand can be easily carried out using restriction enzymes and DNA chemical synthesis technology.

Transformation of microorganisms with the plasmid thus prepared can be carried out by any method commonly used in the fields of genetic engineering and biotechnology. For general information, please refer to suitable etiology or reviews, such as T. Haniatis.

E, F, Frltsch, J, Sambroo K:
rHolecular C1onina-A Labo
raroy HanualJ, Co1d Sprin
g Harbor Laboratory, (1982
), and for specific examples of the present invention, reference may be made to Experimental Examples described later.

The transformant includes new traits (i.e., the ability to produce human elastase) due to the genetic information introduced by the DNA strand of the present invention, traits derived from the used vector, and, in some cases, new traits resulting from the genetic recombination that may have occurred. Except for the lack of corresponding traits due to the lack of some genetic information from the vector used, it is the same as the host microorganism used in terms of its xenotype or phenotype or mycological properties.

Expression of genetic information/Production of protein When the transformant clone obtained as described above is cultured, it produces human elastase t'I, human broelastus fusae, or human breproelastasee in the culture. arise. Prebroelas fuzee becomes proelastase■ when secreted outside the pancreas, and this is further degraded by 1~ribusin etc. in the duodenum. It is said that the human elastase T.
When the precursor protein is obtained, it is thought that if it is treated with trypsin or the like, a protein identical to human elastase can be obtained.

Furthermore, it has been reported that when the chymosin gene is expressed in yeast, the production efficiency is more than 10 times higher when prochymosin is produced than when chymosin is produced directly (J, Helor, H, J, Dobson.

N.A., Roberts; H.F., Tuitc;
J, S, Emtagl, S, W old te.

P, 8, Cowe, T, Pate, A, J, K
ingSman, s, +4. niingsmanGe
ne, 24.1-14 (1983)), and the present invention may also utilize the sequence of this prepro portion to facilitate mass production.

The culture or growth conditions for the transformant are essentially the same as those for the host microorganism used. In addition, the produced protein can be recovered from the culture, that is, the bacterial cells and/or the culture solution, according to conventional methods.

Experimental Example ■: A method of cloning Wistar rats (obtained from Charles River Japan) with the rat Elasfusae gene and immediately removing the pancreas after ether anesthesia, using guanidine thiocyanate as a denaturing agent, and purifying it by cesium chloride density gradient centrifugation. Detailed experimental conditions for obtaining RNA C31, Kazufu Juyo, Yoshiaki Fujii: "Cell Engineering" L, 298-303 (1982)). This RN
In order to obtain mRNA from A, it was purified by oligo dT cellulose column chromatography, and oligo dT
We obtained an mRNA sample with poly(A>) that adsorbs to . Approximately 1 g of pancreas was obtained from one rat, and 200 μg of mRNA was obtained. Next, using this mRNA as a template, cDNA was extracted by the Okayama-Burk method. It was created.

The experimental procedure is as follows (detailed experimental conditions can be found in Katsuya Shigesada: "Cell Engineering" Niji, 616-626 (1983)
).

That is, mRNA and vector primer DNA having 50 oligo dT added to the end are mixed, and reverse transcriptase is added to convert 67 portions of the vector primer DNA into a primer to synthesize complementary strand DNA to the mRNA. Next, dCTP and terminal transferase are added to this DNA fragment to add 20 dCs to both ends. A portion of this vector primer DNA portion is cut with restriction enzyme 1-1ind to remove a certain length of the dCC-added region. On the other hand, one end is l-1in
It has the same cleavage end as dnr and 2 dG at the other end.
A linker-DNA fragment with approximately 0 linkers added is prepared, and added to the 1-1indl [[[[[[[]] digested product, both DNAs are linked in a circular form.

Next, RNaseH, DNA polymerase and DN
A ligase removes the mRNA portion and replaces it with DNA. In this way, rat pancreatic mRNA2.
Using 5μq and vector primer DNA 1.5μq, add linker DNA fragment 0607μq to create C0NA.
I got it.

Using this cDNA, E. coli RRI strain (Bol 1var,
F.: Gene, 2, 95 (1977)')
About 50,000 clones were obtained as transformants.

In order to search for E. coli that has the rat elastase II gene, we investigated the known rat elastase II gene (
The Biochemistry, 21. 1453-
1463 (1982)) was chemically synthesized and used as a probe.

More specifically, it is a base sequence corresponding to the C-terminal portion of the rat elastase Im gene. The top is rat elastase mRNA
The lower part is a synthesized oligonucleotide probe, which is a complementary strand to mRNA. The synthesis method was based on the in-phase phosphotriester method (for details, see E.
, Ohtsuka, H, Ikehara.

D, 5oll: Nucoeic Ac1ds It
esearch 10.6553~6570 (19
82)). This synthetic oligonucleotide was labeled by transferring a phosphate group from γ[32P)ATP using T4 polynucleotide kinase. Using this as a probe, transformants containing cDNA were screened. The screening method was performed using colony hybridization method. That is, the bacterial cells grown on the nitrocellulose filter are denatured with an alkali, and the DNA inside the bacterial cells is burned onto the filter. When the filter is placed in a hybridization solution containing probe DNA, only DNA with homology to the base sequence binds under certain conditions (for details, see "Gene Manipulation Manual" edited by Yasutaka Takagi (Kodansha)) . This is subjected to autoradiography to determine whether it is positive or negative. Clones containing homologous sequences are colored black. Actually, we screened about 2000 clones.

Eight positive clones were obtained. Hybridization conditions were 6X PSC15x Denhardt's solution/3
5°C/16 hours and washed twice with 6XSSC/35°C/30 minutes. For these clones, the plasmids were purified using the alkaline method (T, Haniatis, E, F, F
r1tsch, J, 5anbrook:1N1o1
ecular Cloning-A Laborato
ry Manual J), created a restriction enzyme cleavage map, and compared it with that reported in the literature.
It was a perfect match. In this way, the rat elastase gene was obtained.

■ Cloning of the human elastase gene mRNA using exactly the same method as for the rat elastase ■ gene.
A was obtained and cDNA was roughly constructed. 200 μq of mRNA was obtained from 2 g of human pancreas. Zarani, mR
NA5.0μq1 Vector Brimer DNA1.5μq
and using linker-DN, Ao, 08μQ, CDN
Approximately 30,000 transformants containing A were obtained. Among these, we screened the human elastase Im gene, and used rat elastase I'+H as a probe.
I used part of the transmission. The rat elastase Im gene was digested with restriction enzymes Kpn and BclM, and then separated by agarose gel electrophoresis to obtain a fragment of approximately 700 bp. An isotope label was inserted into this fragment using αC32P (CJCTP) using the nick translation method. Screening was performed using the colony hybridization method used for cloning. Hybridization conditions are 6
XSSC15X in Denhardt's solution/60'C/16 hours, washed at 60°C/30 minutes/twice. Approximately 1
10 clones were obtained from 0,000 clones, and among them, the one with the longest CDNA portion (called El-IEL1 strain. Also, the CDNA-containing plasmid contained in this is called pHEL1) was The base sequence of the DNA portion was determined.

Base sequencing was performed using the Maxam-Gilbert method, reading strands in both directions (for details on the method, see Mitsuru Takanami,
(See Tatsuo Oi's rDNA Sequence Analysis Manual (Kodansha)). The determined sequence is as shown in Figures 1 and 2 (the actually obtained base sequence is the 5'
- side upstream CGT-CCT-ATC-ATC-ACA
-AAA-CTC-ATG). Also shown is the translation of this DNA base sequence into amino acids according to the codon table.

When this amino acid sequence was compared with that of porcine elastase reported by H. Shotton et al. (cited above), 58% homology was found as shown in FIG.

(2) Plasmid pUc8 containing the lac promoter of the human elassus Ii gene was obtained from Pharmacia Japan Co., Ltd. Use this plasmid with restriction enzyme H
It was cut with inclr.

On the other hand, for human elastase II gene, the above pH
After cutting ELI with the restriction enzyme AccI and partially cutting with NcoI, dATP, dC-P, dG-P, dT
Restriction enzyme cut points were blunted by running DNA polymerase Eklenow fragment in the presence of TP. Thereafter, a fragment of approximately 1 kb was obtained by agarose gel electrophoresis.

These two fragments are mixed, ligated with T4 ligase, and then introduced into E. coli (see Figure 4). Plasmids were obtained from the resulting transformants and their degradation patterns with various restriction enzymes were examined to confirm that the integration had been carried out as intended. The plasmid (1) KB4 obtained in this case is estimated from its DNA base sequence to correspond to human elastase (2) protein with Val at the N-terminus removed after one step and 10 amino acids added.

E. coli containing this plasmid pK84 was cultured for 100 d.
After culturing at 37°C overnight in the presence of ampicillin, the cells were collected by centrifugation. This was mixed with 20mM Tris-HCI (p
H8,0) was suspended in 1.5 d of buffer solution and disrupted by ultrasonication, then sodium hydroxide solution was added to this bacterial cell solution to a concentration of 0.0 IN, and after stirring at 5°C for 3 minutes, equimolar hydrochloric acid was added. Solution was added to neutralize. 10. at 5°C. 20 at OOOrpm
The supernatant was obtained by centrifugation for a minute. 100μ of this supernatant
The content of human elastase 12I in the supernatant was measured using an elastase 1 RIA kit (manufactured by Dynabot 2) using m. This kit was created for the purpose of quantifying human elastase (■) in blood using radioimmunoassay as its principle. As a result, 1.8 μ of human elastase was identified in the cultured cells. It was estimated that 102 molecules were expressed per bacterial cell.

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

(1) Human elastase gene-containing plasmid DH
E. coli RRI strain (EHEL1 strain) containing ELI.

[Brief explanation of the drawing]

Figures 1 and 2 show the DNA base sequence of the human elastase IEI gene and its precursor gene, and the amino acid sequence deduced from the base sequence.
Figure 2 shows the amino acids up to the 80th amino acid and from the 181st onwards. FIG. 3 shows the homology between the human elastase gene (upper row) and the porcine elastase gene (lower row). *A symbol indicates that the corresponding amino acids are identical. FIG. 4 is a flow sheet showing a method for constructing E. coli plasmid pKB4 using the Iac promoter. Applicant's representative Kiyoshi Inomata +10 Ala Ser Leu Pro Pro Ala G
ly Asp Ile Leu Pr. GCCTCA CTG CCT CCCGCT GG
T GACATCCTT CCC. ', CCCAG CrG GGA GAT GCCGT
CCAG CTC area m A CCCT GCT ATCA
CC mark C Figure 2 AACTGCCCCACA GAG GAT GGT
GGCTGG CAG GTC1m GGCTGCAA
CTTCATCTGG AAG CCT ACA GT
G 'TCCCACATCCTG AAT AAA G
AA TAA AGA TCT CTCTrp Trp
Gly Ser Thr Val Lys Lys
Thr Ding GG TGG GGT TCCACCG
TG AhaG AAA ACCSACGGT G
TG ACC, AGCTTT GTT TCT GGC
rTCACT CGA GTCTCCGCCTTCAT
CGAC:AA GGCCCA GCT GGCAGT
CCT GAT CGA procedural amendment form) March 28, 1985 1 Display of the case 1988 Patent Application No. 231832 2 Name of the invention Biotechnological production of human elastaseae 3 Person making the amendment Relationship to the case Patent Applicant 11M Beer Co., Ltd. Company 4 Agent February 6, 1985 (Delivery date February 26, 1985) 6 “Claims” and “Detailed description of the invention” in the specification to be amended J7
Contents of amendment 1) The scope of claims is amended as shown in the attached sheet. 2) The description shall be amended as follows. (1) Page 6, line 2, “D,” was corrected to [DM Shotton et al. < [), J. (2) Page 6, line 3, “al” was corrected to ral)J. (3) Correct page 6, line 4 r (Nature)J to [(Nach w-(, Nature))J. (4) Page 6, line 6, ``RaymondJ'' was corrected as ``Raymond Shea McDonald et al. (RaymondJ.) (5) Page 6, line 6, ``al'' was corrected as ra I) J. (6) Page 6, line 9 r (Biochemistry J is corrected as U (Biochemistry ([3iochemistry) J. River r) Condolences 0gucondolences 101 Ding force, Engineering, Biophysics force, Acta (Corrected as C, J. 8) Page 6, line 17, rActaJ is corrected to rACta)J. (9) At the top of the leftmost column of the table on page 12, "E, coli
oli) J and correction. (10) At the bottom of the leftmost column of the table on page 12, select rs, cerevis i aeJ, [S, cerevis i
ae)” and amended. (11) Page 19, line 13, between “A group is” and rEJ, [Escherichia coli (
” has been added. (12) Page 19, line 13 [coliJ, rcOIi) Corrected as J. (13) Page 19, line 14, between “Ichiwa” and “S”, add [Satsucharomyces cerevisiae (”).
The third line of the page, "King," is changed to [T. Maniatitis, E.F., Fritzsch, J. Samlczuk: Molecular Cloning Laboratory Annual, Cold Spring Harbor Laboratory (T.] Correction. (16) Page 23, line 6 [1laboratoryJ, rLaboratory
) J and correction. (17) Page 24, line 13 r (J, J,
G. N. M. Tagle, N. N. White, B. N. Law, T. Bate, N. G. Kingesman, N. M. Kingesman: Corrected as Gene (J,). (18) Page 24, Line 16 rGeneJ Corrected with rGene)J. (19) Correct “(B” in the second line of page 27 to “(F Poliver: Gene (BJ).” (20) Correct rGeneJ in the third line of page 27 to l”Gene)J. (21) Page 27, line 7, rBiochemistryJ, is corrected to [Biochemistry J. (22) Page 27, line 4 from the bottom, ``E,'' is changed to ``E.・Aids Research (E)
and correction. (23) Correct the third line rResearchJ from directly below the 27th line to rResearch)j. (24) Page 29, line 2 r (T, J, “T. Maniatis et al.: Molecular Cloning Laboratory Manual (
T,” he corrected. (25) On page 29, lines 3 and 4, "reference" was corrected to "reference in parentheses". (26) Page 31, line 7, “D,” is corrected to “DM Shotton (D,”). (27) Page 31, line 7, “n et al.” is corrected to “rn) et al.” . 3) Print the drawing as shown in the attached sheet (no changes to the content)
. Claim 1: Having the biotechnological production ability of human elastase (2), characterized by having a base sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of (1) to (3) below. DNA strand. (1) Of the amino acid sequences shown in Figures 1 and 2, those from C to D; (2) the first
Among the amino acid sequences shown in Figures and Figures 2, those from B to D, (3) Figures 1 and 2.
Among the amino M sequences shown throughout the figures, those from A to D. 2. The nucleotide sequence encoding the polypeptide is from C to D, from B to D, or from A to D among the nucleotide sequences shown in Figures 1 and 2. DNA according to claim 1
chain. 3. Transformed with a plasmid containing a DNA chain 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, characterized by the following. (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 is Escherichia coli.
The microorganism according to claim 3, which is Hia coli. 5. A DNA strand whose bases are from C to D, from B to D, or from 8 to D among the base sequences shown in Figures 1 and 2;
The microorganism according to any one of claims 3 to 4. 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. An organism of human elastase, characterized in that it produces human elastase in a culture by subjecting it to the steps of creating a plasmid containing the plasmid, transforming a microorganism with the 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) the first
Among the amino acid sequences shown in Figures and Figures 2, those from B to D, (3) Figures 1 and 2.
Among the amino acid sequences shown throughout the figures, those from A to D. 7. The microorganism is Escherichia coli >
A method according to claim 6. 8. A DNA strand whose bases are from C to D, from B to D, or from A to D among the base sequences shown in Figures 1 and 2;
The method according to any one of claims 6 to 7. 0 ”dl 7C77% rh 夛まProceedings Amendment May 20, 1985

Claims (1)

[Claims] 1. Biotechnological production ability of human elastase I, 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. Of the base sequences that code for 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 I, characterized by: (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. 1. A human elastase I organism characterized by producing human elastase I in a culture by subjecting it to a process consisting 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 among the bases 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.