JPH03254680A - Human acylamino acid-releasing enzyme-like polypeptide - Google Patents

Abstract

NEW MATERIAL:A human acylamino acid-releasing enzyme-like polypeptide having an amino acid sequence of the formula. USE:Production of human acylamino acid-releasing enzymes, etc. PREPARATION:A transformant is incubated and the objective polypeptide is collected from the cultured product. Said transformant is such as to be incorporated with recombinant plasmid containing the DNA coding the title polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a polypeptide having the amino acid sequence of human acyl amino acid releasing enzyme, its DNA sequence, and a method for producing the polypeptide.

[Conventional technology]

An acylamino acid releasing enzyme is a protein hydrolase that releases acylamino acids from proteins/peptides whose N-terminus is acylated. It is widely present in various animal tissues, and is thought to play an important role together with N''-acetyltransferase, which causes N-terminal acetylation, the most unique post-translational modification of proteins within cells. ing.

Protein N-terminal acetylation is a phenomenon widely observed in animals, plants, and their viruses.

Furthermore, it is known that in several types of eukaryotic cells, the N-terminus of more than half of intracellular soluble proteins is acetylated. For example, mouse L cell soluble protein 8
J, L., Brown (J, S.) found that 0% had undergone acetylation.

[The Journal of Biological Chemistry (The J
Our own of Biological Chem
istry) Volume 254, Page 1447 (1979))
. It is also known that the N-terminal amino acids of proteins undergoing acetylation have specificity, and acetylation is more likely to occur when glycine, alanine, serine, methionine, and aspartic acid are at the N-terminus.
Enzymology (Metbods in Bnzym)
106, p. 165 (1984)]
.

As described above, a relatively large number of proteins are acetylated at the N-terminus, and there are also many types of terminal amino acids. Several theories have been proposed regarding the biochemical significance of this N-terminal acetylation. One theory is that intracellular proteins are protected from protein hydrolases by post-translational modification called N-terminal acetylation, and this theory is thought to be related to the in vivo metabolism of proteins. There are also reports that N-terminal acetylation is involved in the passage of secreted proteins through biological membranes, but both theories still have many unknowns.

Acyl amino acid releasing enzymes are effective in studying the biochemical significance of these N-terminal acetylations.

That is, in order to study N-terminal acetylation, it is necessary to identify trace amounts of acetylated amino acid residues in proteins and peptides. For this, N
It is necessary to release an “-acetyl group or an N”-acetylamino acid, but no method has yet been found to release an acetyl group from an N”-acetylated protein. A method is known, and acylamino acid releasing enzymes are used to achieve this purpose.The method involves first cleaving a protein whose N-terminus is acylated with a highly specific protein hydrolase, and then releasing the acyl amino acid. An amino acid releasing enzyme decomposes it into short peptides that are easy to act on.Then, an acylamino acid releasing enzyme is used to release the acylamino acids, and the remaining peptide is analyzed by the Edman degradation method starting from the second amino acid, and the amino acid sequence is analyzed. is identified by mass spectrometry, reversed-phase HPLC analysis, etc.

As described above, acyl amino acid releasing enzyme is an extremely effective means for analyzing the primary structure of N-terminally acylated proteins.

Furthermore, DNA sequences encoding human acylamino acid releasing enzymes are useful for investigating the expression of acylamino acid releasing enzymes in the human body.

[Problem to be solved by the invention]

Acyl amino acid releasing enzymes are widely distributed in animal tissues, and purification from rat liver and brain, pig liver, bovine liver, sheep red blood cells, human red blood cells, and human placenta has been reported [Journal of Biochemistry]
of Biochemistry, Volume 77, Page 89 (1975) Journal of Neurochemistry
try) Volume 41, Page 201 (1983) Journal of Biochemistry Volume 93, Page 1217 (1983), The Journal of Biological Chemistry Volume 253, Page 5012 (1978)
), The Journal of Biological Chemistry Vol. 247, p. 2217 (1972) Biochemical and Biophysical Research Communications
Biophysical Re5earch Com
126, p. 933 (1)
985), European Journal of Biochemistry
Biochemistry) Volume 97, Page 205 (
1979)]. In addition, the genetic structure and the amino acid sequence deduced from it have been reported by the present inventors as an enzyme derived from pig liver [Journal of Biochemistry Vol.
Volume 06, page 548 (1989) and patent application No. 1-165
No. 216] and an enzyme derived from rat liver [The Journal of Biological Chemistry Vol. 264, No. 8
892 (1989): 1. However, the genetic structure and amino acid sequence of human-derived acylamino acid releasing enzymes are still unknown. Furthermore, there is no disclosure of an industrially advantageous method for producing a human acylamino acid releasing enzyme.

An object of the present invention is to provide a human acyl amino acid release enzyme-like polypeptide and a method for producing it using genetic engineering.

[Means to solve the problem]

To summarize the present invention, the first aspect of the present invention relates to a human acyl amino acid releasing enzyme-like polypeptide characterized by having the amino acid sequence shown in FIG.

Furthermore, the second invention of the present invention relates to a base sequence encoding the polypeptide of the first invention.

Furthermore, the third invention of the present invention relates to a method for producing the polypeptide of the first invention of the present invention, which comprises a transformation method in which a recombinant plasmid containing DNA encoding the polypeptide is introduced. The method is characterized by culturing the body and collecting the polypeptide of the first aspect of the present invention from the culture.

The present invention will be specifically explained below.

A known method can be used to clone the cDNA encoding human acyl amino acid releasing enzyme. for example,
From the liver and brain, where human acyl amino acid free enzyme is distributed, Bo! RNA containing J (A) is extracted and purified using a cellulose carrier to which oligo(dT) is bound. Using this as a template, reverse transcriptase is applied to generate cDNA, which is then connected to a plasmid or phage vector using methods such as the Okayama-Barb method or the Gubler-Hoffm-Ann method. and introduce it into a host to create a cDNA library.Such libraries are also commercially available, and can be purchased, for example, from Clontic.If a portion of the cDNA has already been obtained, A cDNA library is also constructed by the primer extension method. This method is particularly effective for cloning 5' cDNA.

In order to screen cDNA clones encoding the desired human acyl amino acid releasing enzyme from a cDNA library, it is effective to use as a probe a cDNA fragment of a pig or rat acyl amino acid releasing enzyme whose sequence has already been revealed. It is true. The DNA probe may be chemically synthesized or purified by cutting it out with a restriction enzyme from a vector containing cDNA.

To screen a library with a DNA probe, first the library is amplified on a plate, the grown colonies or plaques are transferred to a nitrocellulose or nylon filter, and the DNA is denatured by denaturation.
Fix A to the filter. This filter is incubated in a solution containing a DNA probe previously labeled with 32p, etc., and the DNA on the filter and probe D
A hybrid is formed with NA (hereinafter, this operation is referred to as hybridization). The incubation temperature is set based on the Tm (melting temperature) of the probe used. After hybridization, nonspecific adsorption is washed away and clones that have hybridized to the probe are identified by autoradiography. This operation is repeated to isolate clones for the next analysis.

If the recombinant is Escherichia coli, culture a small amount in a test tube, extract the plasmid using a conventional method, perform a cleavage reaction with restriction enzymes, and perform agarose or acrylamide gel electrophoresis to extract the cloned insert fragment. Examine the generation of. Furthermore, the electrophoretic pattern is transferred to a nitrocellulose or nylon membrane, and hybridization is performed by the method described above to examine whether the inserted fragment interferes with the hybridization of the DNA probe. Finally, the base sequence of the inserted fragment is determined by a known method. When the recombinant is a phage, the clones are analyzed using basically the same steps.

A previously cultured host E. coli is infected with the cloned phage, and phage DNA is prepared from the lysate.

A specific method for preparing phage DNA is described, for example, on page 100 of Sekisei Chemistry Experiment Course 1 "Gene Research Methods ■" (Tokyo Kagaku Doujin Publishing). The phage DNA is cut with a restriction enzyme and subjected to gel electrophoresis to confirm the inserted fragment and further examine whether it hybridizes with the probe DNA. Finally, the clone is confirmed by determining the base sequence.

By comparing the determined base sequence with known pig and rat cDNAs, the gene structure and amino acid sequence can be determined.

In this way, the amino acid sequence of human T-syl amino acid releasing enzyme and the corresponding DNA sequence can be determined. In addition, the structural gene of the obtained cDNA is connected to an expression vector so that it can be expressed in a suitable host cell, for example, yeast, and the human acyl amino acid releasing enzyme-like activity is obtained by introducing it into the host cell and culturing it. It is possible to produce a polypeptide having the following.

Confirmation of expression can be performed by measuring the hydrolysis activity of N-acyl peptide, for example, by measuring the amide of N-acetyl-L-methionine and 7-amino-4-methyl-coumarin (AMC). For example, a recombinant yeast cell extract is added to a measurement system using an acylamino acid releasing enzyme as a substrate, and 7-amino-4-methyl-coumarin produced by the release of N-acyl amino acids is measured using a fluorometer. This can be confirmed by measurement.

Conventional chromatography techniques are used to purify the human acyl amino acid releasing enzyme-like polypeptide from the culture of the transformant. That is, for example, cultured bacterial cells are vigorously stirred in the presence of glass beads to obtain a supernatant. This is fractionated by salting out using ammonium sulfate, and then the desired polypeptide can be obtained by chromatography such as ion exchange, gel filtration, or affinity chromatography using acetyl-L-methionylaminohexyl sepharose.

From the above, according to the present invention, the primary structure of human acyl amino acid releasing enzyme has been clarified, and it has become possible to provide a genetic engineering method for producing the enzyme.

〔Example〕

Examples of the present invention will be shown next, but these are not intended to limit the invention.

Example 1 Human acyl amino acid releasing enzyme cDNA cloning (1-1) Screening from cDNA library <Identification and isolation of positive clones> Human liver poly(A) RNA was obtained from Clontic (USA) (code: Number 6510) This poly (
A) cDNA was synthesized from 5 μg of RNA using an oligo(dT) primer using the cDNA Gakkai Kit (code number RPN, 1256) manufactured by Amarjam. Next is Amajam's ML c DNA cloning system/λgtl.

(Code number PRN, 1257), the cDNA was integrated into the EcoRI site of λgtlo and packaged into lambda phage in a cell-free system. However, EcoRI linker-[d (pGGAATTCC
) ) is made by Takara Shuzo ■, and the package is Giga Pack Gold (GIGA
PACK GOLD) was used.

<Identification and isolation of positive clones> Using the cDNA library as a host strain, C600Hf
Using the L strain, approximately 20,000 plaques were placed on 10 square petri dishes measuring 14 cm x 10 cm each.

That is, C6 in L medium containing 4 mg/- maltose.
00Hfl was cultured overnight at 37°C to a culture solution 0.2-
0.1-phage solution was mixed and kept at 37°C for 15 minutes. To this, add agarose to soft agar (L medium to a final concentration of 0.6%, autoclave it,
(Hereafter, this operation will be abbreviated as "blating").

Next, two hybridization filters were prepared from this plate. That is, a nylon film manufactured by Aljam Co., Ltd. (trade name: Hybon) was coated on the plate surface.
d-N)] for 30 seconds, and then 0.5 M
NaOH, 1,5M NaC] for 5 min on a paper soaked in a solution of 0.5M)
)-HCI buffer (pH 7,0), after treatment for 5 minutes (neutralization) on a paper soaked in a solution of 1.5 M NaCl, 2
117.53 g of SSCCNaC and 8.82 g of sodium citrate dissolved in water from Step 11].
The second sheet was dried on a paper (hereinafter referred to as filter treatment). The second filter treatment was performed with a contact time of 2 minutes between the plate and the nylon membrane.
The filter was irradiated for a minute to immobilize the DNA.

As a probe for hybridization, a 1782 bp fragment from 568 bp to 2349 bp was used, with the start codon of cDNA of acyl amino acid releasing enzyme derived from pig liver being set as 1.

That is, phage λAARI! containing the porcine acyl amino acid release enzyme cDNA! 419 DNA 40μg
After digestion with EC0RI, agarose gel electrophoresis was performed. After electrophoresis, the gel was diluted with 1 μg/- of ethidium bromide solution.
After staining for 0 minutes, the part containing the desired inserted fragment was cut out from the gel under ultraviolet irradiation. This was mixed with electroextraction buffer (0,004M Tris-acetic acid, 0.1mM EDTA).
Insert it into a dialysis tube filled with EEM solution (pH 8.0, hereinafter abbreviated as EEM solution), fill the external solution with the same solution,
Electricity was applied at V/cm for 20 minutes. After the gel was gently removed from the dialysis tube, the internal solution was thoroughly stirred and removed. To this DNA solution, an equal volume of phenol/chloroform mixture was added and stirred, and after centrifugation at 10,000 rpm for 5 minutes, the upper layer was collected (hereinafter abbreviated as phenol/chloroform extraction). To the upper layer, 1/lO volume of 3M sodium acetate solution and twice the volume of ethanol were added to precipitate the DNA (hereinafter abbreviated as ethanol precipitation). 15 to -70℃
After standing for 10 minutes, centrifugation was performed at 10.00 Orpm for 10 minutes, and the precipitate was rinsed with 80% ethanol and dried under reduced pressure.
-HCl pH 7.5, 0.1 mM EDTAI (hereinafter, this DNA fragment extraction and purification method will be abbreviated as electroextraction method). 50 μg of the obtained fragment was added to Takara Shuzo's Random Primer DNA Labeling Kit (code number 604).
5) with 32p and 1 x 10'cpm
A probe with a specific activity of /μg was obtained. 6X SSC using the total amount of this probe and the filter prepared above.

1% SDS, 100 μg/rn1 herring sperm DNA1
5x Denhardt [Bovine Serum 7J
L.

Bumin, polyvinylpyrrolidone, and Ficoll at a concentration of 0.1% each.
Hybridization was performed overnight at 5°C. The filters were then washed for 10 minutes in 6x SSC at room temperature. After further washing twice for 10 min in 2x SSC at room temperature,
Washed twice for 30 minutes in 0.2X SSC at 0.degree. After transferring the filter onto a paper and removing excess water,
Glued it to Whatman 3MMp paper and covered it with an intensifying screen overnight.
Autoradiographs were performed at 70°C.

As a result, one positive signal was obtained.

Plaques at positions corresponding to these signals were removed with agar and a 0.2-8M solution [NaCl 15.8g.

MgSO4・78202g, LM) Lith-HCl
Buffer (pH 7,5) 50-12% gelatin 5rnl
(dissolved in water to make a total volume of
(9 cm round Petri dish) The same operations and procedures as above were performed (
(hereinafter abbreviated as secondary screening).

As a result, a single plaque could be isolated. This clone was named λllA3.

Preparation of λllA3 DNA> Using the cloned phage as a host bacterium, use Escherichia coli L87.
The strain was used for liquid culture (Gene Research Methods ■, p. 100, Tokyo Kagaku Dojin Publishing).

In this way, approximately 40μ
λllA3 DNA of g was obtained.

Identification and extraction and purification of the inserted fragment> 20 μg of the prepared λ1A3 DNA was transferred to 100 μm 1X
37 with 120 units of EcoRI in EcoRI buffer (composition listed in Takara Shuzo Genetic Engineering Reagent Catalog)
The mixture was kept at a temperature of 1.5 hours, and RNA5eA was added at a final concentration of 50 μg/−, followed by keeping it warm for 10 minutes. Take out 5 μl from this reaction solution and add 1 μl of 6-fold concentrated electrophoresis buffer C0, 25% bromophenol blue, 0.25
% quijilene diγnol, 30% glycerol, below, 6
XEP buffer] was added, and the inserted fragment was identified by electrophoresis on a 1.0% agarose gel.

As a result, it was found that a 1.8 Kbp fragment was inserted into λ1IA3.

Next, add 20 μl of 6X to the remaining reaction mixture after EcoRI digestion.
EP buffer was added and electrophoresis was performed on a 1.0% agarose gel. After electrophoresis, the gel was stained with a 1 μg/− ethidium bromide solution for 10 minutes, and a portion containing the desired inserted fragment was cut out from the gel under ultraviolet irradiation. Then, the DNA fragments were extracted and purified by electroextraction and dissolved in 20 μATE buffer.

<Base sequencing of insert fragment> 4 μm of insert fragment solution of λBA3 and M 13mp18 (
Takara Shuzo) RF DNA was digested with EcoRI.
ng, ligation kit (Takara Shuzo code number 6
Ligation was performed using 021). Escherichia coli JM109 was transformed using 115 volumes of this ligation reaction solution, resulting in a transformation of 40 μg/ml! X-Ga1(5
-bromo-4-ri00-3-indolyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactoside) at a concentration of 20 μg/rnl. Late culture medium 0.
Mixed with 2- and spread on L-plate. This plate was incubated at 37° C. overnight and the white plaques formed were selected to obtain recombinants in which the fragment had been inserted. This clone was named M.3.

The phage of each 6 clones of the obtained recombinant were taken from the plaque, and the overnight culture of JM109 strain was diluted with 3/10 in L medium.
The cells were infected by adding 5 rnl of the diluted solution to 0.00, and cultured with shaking at 37°C for 6 hours. This is 9.00
Orpm centrifugation and alkaline lysis method from precipitated bacterial cells [Reference: 19g2, Cold Spring Harbor
Published by Laboratory, T, Maniastice (7,1 Bia
stis) et al., Molecular Cloning, A.
Laboratory Manual (Molecular Cl
oning＾Laboratory Manual),
RF DNA was prepared according to [Page 368] and dissolved in 30 μl of TE buffer. 3 μl of this DNA solution was enzymatically digested with 12 units of EcoRI at 37°C for 1 hour, and 50 μl
Add RNA5eA to give g/yd, and incubate at 37°C for 10
It was kept warm for minutes. 6 XEP buffer was added and electrophoresed on a 1% agarose gel, and it was confirmed from the cleavage pattern that each inserted fragment was present.

Next, to determine the orientation of the insert, each RF DNA was digested with a restriction enzyme whose recognition sequence was found in the insert. That is, acyl amino acid releasing enzyme cDNA derived from pig liver.
Presuming that the recognition sequence for the restriction enzyme PstI present in the base sequence also exists in human-derived cDNA fragments, Ps
Digestion with tI was performed. Analysis by 1% agarose gel electrophoresis revealed that Pst
It was found that there was a recognition sequence for I, and the orientation of the inserted fragment could also be determined. M for one direction, A3-1
, and the other one was named MHA3-2.

Next, in order to determine the entire nucleotide sequence of the inserted fragment of λllA3, MHA31, MllA3
-2. For clones with two different orientations, insert fragments were decomposed from the sea fence primer annealing position side to create derivatives with insert fragments of various sizes (hereinafter, this operation will be abbreviated as deletion). ).

Appropriate ones of these derivatives, MRA3-1, MHA3
-2 was subjected to DNA sequencing using the dideoxy method to determine the entire base sequence of the inserted fragment of λllA3. The region corresponds to base sequence numbers 568 to 2349 in Figure 2 and is shown in Figure 3.

(1-2) cDNA cloning using the primer extension method Next, in order to obtain a clone that covers a part of the structural gene that is not covered by the insert fragment of λ1IA3 described above, m
cDNA cloning was performed using the RNA primer extension method.

Identification and isolation of positive clones> As a primer, use the primer at position 128-147 from the 5' side of the inserted fragment of λ1IA3, that is, 695-7 in Fig. 2.
A 20bp sequence T complementary to the base sequence present at position 14
fLDNAP6 was used. The mRNA derived from normal human liver was obtained from the aforementioned Clontic Company. mRNA
5 μg and 1 μg of primer were added, and cDNA was synthesized using a CDNA platform kit manufactured by AmaJam.

This cDNA was converted into Ec of λgtlO as described above.

What was integrated into the RI site was packaged into lambda phage. This cDNA library was converted into C600H
Blating was performed using the f1 strain, and a square Petri dish 18
cells to form 20,000 phages per sheet,
This was transferred to two filters and filtered.

As a probe, N co I corresponding to positions -1 to 410 of the porcine acyl amino acid releasing enzyme cDNA base sequence
-E co47III fragment was used.

50 ng of this DNA was obtained from the above-mentioned Takara (TakaRa).
A probe with a specific activity of 9.6 x 10''cpm/μg (D) was obtained by labeling with 32P using a random primer labeling kit. Partial hybridization was carried out in 100 ml of hybridization buffer at 65°C. Filters were then washed for 1 minute in 6X SSC at room temperature and then in 2X SS at room temperature.
10 minutes at room temperature in 0.2x SSC, 0.1% SDS
After washing twice for 20 minutes in a vacuum cleaner, the filter was transferred onto a paper to remove excess water and mounted on Whatman 3MM paper. A portion of this was exposed to an intensifying screen and autoradiographed at -70°C. As a result, 19 positive signals were obtained. A secondary screening was performed on these 19 phages, and 12 single plaques were isolated (
λHA401, λHA402, λRA405, λHA4
10, λ□412, λRA421, λ□422, λ1I
A424, λ□A425, λllA426, λnA40
8, λ, named A409).

Preparation of λRA DNA and Identification of Insert Fragment Phage was prepared from a 40-ml culture medium by the liquid culture method as described above, and about 20 μg of DNA was obtained from it. This DN
When A0Jμg was enzymatically digested with 12 units of EcoRI at 37°C for 2 hours and analyzed by electrophoresis on a 1.0% agarose gel, insert fragments of about 700 bp to about 920 bp were confirmed.

Determination of the nucleotide sequence of the inserted fragment> For these clones, 15Pg
Extract and purify the insert fragment from the λ DNA of
Dissolved in buffer. 5 μl of the obtained DNA fragment and M1
3mp18 RF DNA EcoR1 digest 50μg
was ligated using a ligation kit (Takara Shuzo). Using 115 volumes of this reaction solution, JM1
The 09 strain was transformed and recombinant clones were obtained in the same manner as described above, and each clone containing an inserted fragment in the opposite direction was obtained.

For these clones, the M13 phage poIJ
It was deduced from the pig cDNA that S ma I, which has a recognition sequence within the IJ linker, also exists within the inserted fragment.

Therefore, when each RF DNA 1100n was digested with SmaI and half of it was analyzed by electrophoresis on a 1% agarose gel, it was found that each had a SmaI site near the center.

The remaining half was subjected to self-ligation using a ligation kit, and JM109 strain was transformed.

These Sma I fragment deleted clones and the original M13 recombinant clone were subjected to DNA sequencing by the dideoxy method to obtain recombinant λ DNA.
The entire nucleotide sequence of the inserted fragment was determined. Especially 5' of mRNA
The longest terminal clone, λ, A425, is shown in FIG. It was located at position 706 from the position corresponding to -27 of the base sequence number in Figure 2.

From the results of the base sequence analysis in Examples (1-1) and (1-2), the entire base sequence and amine sequence of the human acyl amino acid releasing enzyme constituent gene were determined.

The N-terminus was determined by comparison with pig and rat acyl amino acid releasing enzymes. The results are shown in FIG. That is, FIG. 2 is a diagram showing the base sequence of human acyl amino acid releasing enzyme and the corresponding amino acid sequence obtained from cDNA base sequence analysis.

Example 2 Construction of a plasmid expressing an acyl amino acid releasing enzyme-like polypeptide Insert fragments of λllA3, λ□425, and λHA401 were inserted into the S ma I site and the T ac I site in the region where the sea fence overlaps using the following procedure. Finally, pJM124, an E. coli-yeast shuttle vector with a yeast alcohol dehydrogenase promoter [Reference Niji Mbo Journal (The
, BMBO Journal) Volume 8, Page 2067 (1
989)] into an expression plasmid (pYHA201
) was constructed.

, 'l, A401 insert fragment has a length from the position corresponding to the 1st position of the base sequence number in Figure 2 to the position corresponding to the 606th position, and is
A coRI linker [d(pGGAATTCC)) was ligated to both ends. Therefore, on the 5' side, the start codon ATG became an N co I site (
...GGAATTCCATGG・). This λRA4
MuA401-I RF DNA in which the EcoRI insert fragment of 01 was incorporated into M13mp18 was Ncor,
After digestion with EcoRI, a DNA fragment of about 610 bp released was separated and purified. This is plasmid pTV119N
It was inserted into the Ncol-EcoRI site of [Takara Shuzo ■] and introduced into Escherichia coli JM109 to create plasmid pTHA1.
I got 01. Next, we created MHA4252 RF in which the EcoRI insert of λllA425 was incorporated into M13mp18.
The approximately 5oobp DNA fragment released by digesting the DNA with Tac I and the MHA3-I RF DNA mentioned above were combined with Tac I.
about 1672b released by digestion with ac I, EC0RI
The DNA fragment of p was isolated and purified. These two DNA fragments were ligated using a ligation kit, recovered by ethanol precipitation, digested with SmaI, 5EcoRI, separated on a 1% agarose gel, cut out a node of about 2090 bp, and separated and purified by electroextraction.

This was added to the SmaI-
It was integrated into ECORI and introduced into E. coli JM109 to obtain plasmid pTHA201.

Next, as mentioned above, NcoI- isolated and purified from MIIA401
An approximately 610 bp DNA fragment of EcoRI was made blunt-ended with the Flenow fragment. This is plasmid pJM1
24 was digested with BamHI to remove the BamHI insert fragment, which was made blunt-ended with the Flenow fragment and then introduced into E. coli HB 101 to create the plasmid pYHA.
I got 101. Additionally, pTHA201 was digested with EcoRI and blunt-ended with the Flenow fragment. Hind
After ligating the III linker [d(pCAAGCTTG)] using a ligation killer, HindII
Digestion was performed to generate 1indIII sites at both ends, and self-ligation was performed with diluted NAl 1 degree. This was introduced into E. coli JM109, and plasmid pTH
A201H was obtained. This plasmid pTHA201H was digested with S ma I and Hind III, and the released DNA fragment of about 2100 bp was separated and purified.

This was added to SmaIHi of the plasmid pYHA101 mentioned above.
ndIII and introduced into E. coli HB 101 to obtain plasmid pYHA201. This plasmid pYH
A201 encodes a 732 amino acid residue polypeptide of acyl amino acid releasing enzyme downstream of the yeast alcohol dehydrogenase promoter.

This plasmid pY)IA201 was transformed into yeast BJ 216
8 [MATa, ura3-52.1eu2. tr
pl, prc-407, prb11122, pep
4-3], or DKD-5D-H [MATa,
1eu2-3.1eu2-112. trpl, hi
s3] using an alkali metal treatment method [Reference: Journal
Journal of Bac
teriology) Volume 153, Page 163 (198
3):].

Yeast B J 2168 into which pYHA201 was introduced was
ccharo-myces cerevisiae B
J2168/pY) designated as IA201 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.
No. 0 (FBRM P-11570): ].

Example 3 Expression and purification of acyl amino acid releasing enzyme-like polypeptide in yeast Saccharomyces ce obtained in Example 2
revi-siae BJ2168/pYHA201 (
FBRM P-11570), 20 μg/- L-)liptophan, 30 μg/- L-leucine, 20 μg/-
- SD medium of 5rn1 containing uracil [0.17%
Bactoeast Nitrodiene Base W/○ Amino Acid (Difco), 0.5% ammonium sulfate, 2% dextrose] was inoculated, cultured in two parts at 30°C, and harvested at 5000 rpm. After discarding the supernatant and washing the bacterial cells with 2-ml water and centrifuging, 0.2M! 750 μl of the suspension was suspended in sodium chloride buffer (p)I 7.2). Add 400μl worth of glass beads (diameter 0) to this.
．． 35 to 0.5 mm) was added and vigorously stirred while cooling on ice to disrupt the bacterial cells. The glass beads and cell debris were removed by centrifugation, and the supernatant was collected and used as a yeast extract.

The acyl amino acid releasing enzyme activity of the extract thus obtained was measured as follows. 0.025mM N-acetyl-L-methionine and AMC amide.
5% dimethylformamide-0.2M sodium phosphate M solution (pH 7.2) 1.95- was heated to about 5% at 37°C.
After preheating for a minute, 50 μl of the above extract was added, mixed, and kept at 37° C. for 30 minutes. Next, add 250 μl of 90% trichloroacetic acid to stop the reaction, and turn the mixture at 3000 rpm.
The mixture was centrifuged for 5 minutes and the supernatant was collected. This supernatant 1-25
0 μl of 10% aqueous sodium hydroxide solution, 1-1.
5M! Potassium phosphate buffer (pH 7.2) was added, and the fluorescence intensity was measured using a fluorometer. That is, the excitation wavelength was 380 nm and the measurement wavelength was 440 nm, and the amount of liberated AMC was determined by preparing a standard curve in advance using AMC of known concentration and comparing it with that curve.

In addition, since AMC may be released by other hydrolases, t-butoxycarbonyl-L-
A control was performed using an amide of methionine and AMC as a substrate.

50 μl of the previously prepared extract had the activity of releasing about 2400 pmol of AMC in 30 minutes in the above reaction system. In addition, yeast BJ that does not carry a plasmid
2168 was treated in the same manner, but no acylamino acid releasing enzyme activity was observed.

Next, a human acyl amino acid releasing enzyme-like polypeptide was purified from 2Il of the BJ2168/pYHA201 culture solution. A cell extract was prepared in the same manner as described above.

However, as an extraction buffer, 5mM sodium phosphate buffer containing 1mM 2-mercaptoethanol (ptl
7.2) (hereinafter abbreviated as MP buffer) was used. Solid ammonium sulfate was dissolved in the extract to 60% saturation, and the resulting precipitate was collected by centrifugation at 1000 Orpm and dissolved in MP buffer containing a small amount of 0.2M NaCl. This was dialyzed against the same buffer, equilibrated with the same buffer, and applied to a DEAE-)Yopal (manufactured by Tosoh) column. After thorough washing with the same buffer, N
Linear gradient elution was performed with respect to aC1 concentration. Collect the acylamino acid free enzyme active fractions and add 0.2 M
Dialysis was performed against MP buffer containing NaCl. A portion of this was equilibrated with the same buffer and applied to an acetyl-L-methionyl-aminohexyl Sepharose column. After thorough washing with the same buffer, 0.6 M NaC
Elution was performed using a linear gradient gradient with respect to NaCl concentration using MP buffer containing 1, and acylamino acid free enzyme activity fractions were collected. Through the above operations, 100 μg of a human acylamino acid releasing enzyme-like polypeptide with a purity of 90% or more was obtained by SDS polyacrylamide gel electrophoresis.

Furthermore, a part of this was added to 10 m containing 0.2 M NaCl.
Purification was performed by HPLC using a TSK gel G4000PW (manufactured by Tosoh Corporation) column using M sodium phosphate buffer as an elution buffer. Approximately 2 μg of the obtained human acyl amino acid releasing enzyme-like polypeptide was analyzed using a peptide sequencer (manufactured by Applied Biosystems), and it was found that the N-terminus was protected. Considering that the N-terminus of natural porcine acylamino acid releasing enzyme is acetylated, the human acylamino acid releasing enzyme-like polypeptide expressed in yeast also has N-terminus.
It seems that the terminal is acetylated. Also, SDS
Since the molecular weight determined by polyacrylamide gel electrophoresis also agrees with the value calculated from cDNA,
The human acyl amino acid releasing enzyme-like polypeptide expressed in yeast was thought to have the same structure as the natural human acyl amino acid releasing enzyme.

〔Effect of the invention〕

From the above results, the present invention has revealed the amino acid sequence of human acyl amino acid releasing enzyme and its DNA sequence, and provided a genetic engineering method for producing a human acyl amino acid releasing enzyme-like polypeptide. Furthermore, by providing a target for investigating the expression of human acyl amino acid releasing enzyme in vivo, it is now possible to create probes and primers based on this DNA sequence, and to create antibodies based on the amino acid sequence. It has become possible.

[Brief explanation of drawings]

Figure 1 shows the amino acid sequence of the human acyl amino acid releasing enzyme of the present invention, and Figure 2 shows an example of the base sequence of the acyl amino acid releasing enzyme of the present invention obtained from cDNA base sequence analysis and the corresponding amino acid sequence. Figure 3 shows the restriction enzyme map of human acyl amino acid releasing enzyme cDNA and λ11A3 and λ obtained by screening. 425 is a diagram showing the position of the inserted fragment of 425.

Claims (1)

[Scope of Claims] 1. A human acyl amino acid releasing enzyme-like polypeptide characterized by having the amino acid sequence shown in FIG. 1 of the drawings. 2. A base sequence encoding the polypeptide according to claim 1. 3. Cultivating a transformant into which a recombinant plasmid containing DNA encoding the polypeptide according to claim 1 has been introduced, and collecting the polypeptide according to claim 1 from the culture. A method for producing the polypeptide according to claim 1.