JP3337178B2 - Clover yellow bain virus protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a nuclear inclusion body a (Nuclear Inclusio), which is a protease for maturation of clover yellow vein virus (hereinafter abbreviated as CYVV).
na: abbreviated as NIa), and a gene encoding the protease. By connecting the gene obtained in the present invention and a gene of an exogenous useful protein in the same reading frame via a specific cleavage sequence, it becomes possible to directly produce the useful protein in cells.

Furthermore, the gene can be expressed in host microbial cells or the like to purify NIa and used as a protease for cleaving a specific amino acid sequence.

[0003]

2. Description of the Related Art Potyvirus is a virus that has a single-stranded RNA consisting of about 10,000 bases as a genome and infects plants such as Solanaceae. In the virus genome, one very long protein is read. There is a frame [Dougherty.WG
and Hiebert, E. (1980) .Virology 101 : 466-474., Alli
son, R. et al . (1986). Virology 154 : 9-20.]. The viral functional protein is cleaved by the two types of proteases present in the reading frame, and becomes functional as each protein [Dougherty. WG and Carringto
n, JC (1988). Ann. Rev. Phytopath. 26 : 23-143.].
Tobacco etch virus (toba), a type of potyvirus
In cco etch virus (TEV), there are nuclear inclusions stained with trypan blue in infected cell nuclei. Furthermore, this inclusion body is composed of two types of proteins, one of which has been found to be a protease (nuclear inclusion-a: NIa) possessed by the virus (J. Virol., 61: 2540-2548). (1987)).

[0004] TEV and tobacco vein mottling virus (TVMV) were used to determine the entire nucleotide sequence of the virus genome, and as a result of homology search of amino acid sequences, the location of NIa in the genome was determined (Virology). , 154: 9-20 (1986): Nucleic Acid Re
s., 14: 5417-5430 (1986)). NIa recognizes and cleaves certain sequences of protein polymers translated from the viral genome. This specific sequence is Gln-Gly, Gln-Ser
, Gln-Ala, which protease cleaves peptide bonds between these amino acid sequences.

[0005] CYVV used in the present invention is a virus belonging to the potyvirus group. The molecular biological properties of the virus are based on the gene structure of a coat protein present at the 3 'end of the genome [Uyeda, I. et al. . (1991). Intervirol. 32 :
234-245.] Is known, and the genetic structure of the NIa region genome has not yet been elucidated. There are no reports that NIa itself was isolated.

On the other hand, when a foreign protein is produced by a genetic engineering technique, the gene encoding the foreign protein may not be expressed and the production efficiency may be extremely low. In such a case, it is difficult to express the target foreign protein in a large amount simply by linking the structural gene of the protein downstream of the control gene according to ordinary genetic engineering techniques.

In a method for expressing a fusion protein of a foreign protein of interest and a protein derived from Escherichia coli in a host such as Escherichia coli and finally producing the foreign protein of interest, the fusion protein is once purified after expression. , After the purification, a protease was added to cause a cleavage reaction,
Further, after the reaction, it is necessary to purify the desired target protein again, which requires a complicated operation as a whole.

[0008]

DISCLOSURE OF THE INVENTION The present inventors connect a DNA encoding the protease CYVVNIa with a DNA encoding a desired protein and transform a host cell using a transformation vector containing the DNA. Or D encoding the above protease CYVVNIa
By transforming host cells with a transformation vector containing NA and a transformation vector containing DNA encoding the desired protein, it was found that the desired protein was directly produced in the host cell. In addition, they have found that separately produced NIa cleaves a protein having the cleavage sequence in a test tube, and thus completed the present invention.

[0009]

The present invention provides: (1) a molecule comprising the amino acid sequence of amino acids 4 to 437 of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein Gln-Ala, A protein having a protease activity that specifically hydrolyzes a peptide bond between Gln-Gly or Gln-Ser, or one or more amino acid residues of the amino sequence are deleted, inserted, or substituted A protein comprising an amino acid sequence and having the protease activity; (2) a molecule comprising an amino acid sequence of amino acids 4 to 437 of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein Gln-Ala, Gln A protein having a protease activity of specifically hydrolyzing a peptide bond between -Gly or Gln-Ser; (3) a DNA encoding the protein of (1); (4) (2) (5) The DNA according to (3) or (4), wherein the molecule comprises the nucleotide sequence of nucleotide numbers 10 to 1311 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing in the molecule. 70% of the nucleotide sequence of nucleotide numbers 10 to 1311 of the nucleotide sequence shown in SEQ ID NO: 1 in the column list
DNA containing a sequence having the above nucleotide sequence homology and encoding a protein having a protease activity of specifically hydrolyzing a peptide bond between Gln-Ala, Gln-Gly or Gln-Ser in a substrate peptide; ) The nucleotide sequence of nucleotide numbers 10 to 1311 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing is 55%
DNA containing a sequence having the above nucleotide sequence homology and encoding a protein having a protease activity of specifically hydrolyzing a peptide bond between Gln-Ala, Gln-Gly or Gln-Ser in a substrate peptide, (8 (5) a DNA that hybridizes with the DNA of (5) and encodes a protein having a protease activity of specifically hydrolyzing a peptide bond between Gln-Ala, Gln-Gly or Gln-Ser in a substrate peptide; ) Hybridizing with the DNA described in (5) under the conditions of 60-70 ° C and 6 × SSC, Gln-Ala in the substrate peptide,
The DNA according to (8), which encodes a protein having a protease activity that specifically hydrolyzes a peptide bond between Gln-Gly or Gln-Ser; (10) (3), (4), (5), (6) ), (7),
(8) A recombinant DNA vector comprising the DNA of (9), (11) a host cell transformed with the recombinant DNA vector of (10), (12) a transformed host cell E. coli (1
(13) [I] A protein according to (1) or (2), wherein the following components are peptide-bonded from the N-terminal side to the C-terminal side: -Ala, a linker selected from any one of Gln-Gly or Gln-Ser, peptide component X, [II] Ala-X, Gl
(14) [I] a peptide bond comprising the following components in the direction from the N-terminal side to the C-terminal side: (i) a protein selected from the group consisting of y-X and Ser-X; A protein selected from the group consisting of: the protease according to (1) or (2), a linker selected from Gln-Ala, Gln-Gly or Gln-Ser, Pro-Y ₁ (where Y ₁ Represents an arbitrary peptide component.), [II] Ala-Pro-Y by autocatalysis of the component
_1, a protein that produces a peptide selected from any of the Gly-Pro-Y ₁ or Ser-Pro- Y _1, then and generating a Pro- Y ₁ by the catalytic action of aminopeptidase P, (15) [I] A protein in which the following components are peptide-bonded from the N-terminal side to the C-terminal side: the protease according to (1) or (2), Gln-Ala, Gln-Gly or A linker selected from any one of Gln-Ser, Pro-Y ₂ (where Y ₂ represents an arbitrary peptide component), [II] Ala-Pro-Y by autocatalysis of the component.
_2, generates a Gly-Pro-Y ₂ or Ser-Pro- peptide selected from one of Y _2, then aminopeptidase by the catalytic action of peptidases P generates Pro- Y _2, and then the proline imino peptidase catalytic protein and generates a Y ₂ by the action, (16) [I] following components from the N-terminal side in the direction of the C-terminal side is a protein are peptide bonds: (1) or ( 2) Protease described above, Gln-Ala, Z (where Z represents an arbitrary peptide component), [II] Ala-Z is generated by autocatalysis of the component, and then alanine aminopeptidase is produced. (17) (3), (4), (5), (6), (7), a protein characterized in that Z is produced by catalysis.
Gln-A is added to the 3 ′ end of the DNA according to (8) or (9).
la, DNA in which a DNA encoding a heterologous protein is connected via a DNA encoding Gln-Gly or Gln-Ser so as to be in the reading frame of the same protein, comprising DNA described in (18) or (17). (19) a host cell transformed with the recombinant DNA vector according to (18), (20) the transformed host cell is Escherichia coli (1)
(21) The following three steps: [I] The following components in the direction from the N-terminal to the C-terminal: Protease according to (1) or (2), Gln-Ala, A recombinant DN comprising a linker selected from any one of Gln-Gly and Gln-Ser, and a peptide component X comprising DNA encoding a protein to which a peptide bond is attached.
A step of transforming a host cell with the A expression vector, [II] culturing the host cell, and performing Ala-X, Gly-X or
A step of producing a peptide selected from any one of Ser-X, [III] from the host cell or a culture solution of the host cell,
isolating and purifying a peptide selected from any one of aX, Gly-X and Ser-X.
A method for producing a peptide selected from any one of X, Gly-X and Ser-X, (22) the following three steps: [I] the following components in the direction from the N-terminal to the C-terminal: : Protease according to (1) or (2), a linker selected from any one of Gln-Ala, Gln-Gly or Gln-Ser, Pro-Y ₁ (where Y ₁ is an arbitrary peptide component) A) transforming a host cell with a recombinant DNA expression vector comprising a DNA encoding a peptide-bound protein, [II] culturing the host cell, and constructing the host cell in the host cell. Ala-Pro-Y ₁ , Gly-Pr
o-Y ₁ or Ser-Pro- yielding peptide selected from any one of Y _1, further the Pro- Y ₁ by the catalytic action of aminopeptidase P expressed from a host cell-derived or vector of foreign [III] Pr from the host cell or a culture solution of the host cell.
isolating o-Y _1, purifying, more composed Pro-Y ₁ of the production method, (23) 3 subsequent steps: [I] N-terminal side of the following components in the direction of the C-terminal side: A protease selected from (1) or (2), a linker selected from any one of Gln-Ala, Gln-Gly or Gln-Ser, Pro-Y ₂ (where Y ₂ represents an arbitrary peptide component) ), Transforming a host cell with a recombinant DNA expression vector comprising DNA encoding a peptide-bound protein, [II] culturing the host cell, and constructing components in the host cell. Ala-Pro-Y ₂ , Gly-Pr
o-Y ₂ or Ser-Pro- Y yielding peptide selected from any one of _2, then by the catalytic action of aminopeptidase P expressed from vectors derived from the host cell or foreign to Pro- Y ₂ Generated, and
A step of producing Y ₂ by the catalysis of proline iminopeptidase derived from a host cell or expressed from a foreign vector; [III] From the host cell or a culture solution of the host cell, Y
_A method for producing Y ₂ , comprising the steps of isolating and purifying ₂ ; (24) the following three steps: [I] the following components from the N-terminal side to the C-terminal side: (1) or ( 2) A host cell comprising a recombinant DNA expression vector comprising a DNA encoding a peptide-bound protein, wherein Gln-Ala, Z (where Z represents an arbitrary peptide component), as described in 2) above. [II] culturing the host cell, and producing Ala-Z by autocatalysis of the components in the host cell;
A step of producing Z by the catalytic action of alanine aminopeptidase derived from a host cell or expressed from a foreign vector; [III] Z from the host cell or a culture solution of the host cell;
(25) a method for producing Z comprising the steps of:
(23) The production method according to (24) or (25), (26) In a test tube, Gln-Ala, Gln-Gly or Gln-Ala is catalyzed by the protease according to (1) or (2).
A peptide selected from any one of Ala-X, Gly-X or Ser-X is produced from a protein in which any one of Gln-Ser and peptide component X are peptide-bonded, and a reaction solution is used. Ala-X, Gly-X or Ser-X
(27) a method for producing a peptide selected from any one of Ala-X, Gly-X or Ser-X, comprising isolating and purifying a peptide selected from any one of: Gln-Ala, Gln-Gly or Gln-Ala by the catalytic action of the protease according to (1) or (2)
One of Gln-Ser and Pro- Y ₁ (however, Y ₁
Represents an arbitrary peptide component. ) Is from proteins that are peptide bonds, yielding _{Ala-Pro-Y 1, Gly} -Pro-Y 1 or Ser-Pro- peptide selected from any one of Y _1, further aminopeptidase P catalytically yielding Pro- Y _1, from the reaction solution, Pr
isolating o-Y _1, consists in purification, Pro-Y ₁ of the production method, (28) in vitro, (1) or (2) by the catalytic action described protease, Gln-Ala, Gln -Gly or
One of Gln-Ser and Pro- Y ₂ (however, Y ₂
Represents an arbitrary peptide component. ) Is from proteins that are peptide bonds, yielding _{Ala-Pro-Y 2, Gly} -Pro-Y 2 or Ser-Pro- peptide selected from one of Y _2, then the aminopeptidase P catalytically yielding Pro- Y _2, further proline imino peptidase by the catalytic action of peptidases yielding Y _2, from the reaction solution, isolating Y _2, preparation of Y ₂ consisting purification, (29) In a test tube, Gln-Ala and Z (where Z represents an arbitrary peptide component) are converted to Ala- by a catalytic action of the protease described in (1) or (2) above, in which the protein is peptide-bonded. To form Z, which is then catalyzed by alanine aminopeptidase.
And a method for producing Z by isolating and purifying Z from the reaction solution. First of the present invention
An object of the present invention is to provide the proteins described in (1) or (2), DNAs encoding these proteins and the like, DNA vectors containing the DNAs, and host cells transformed with the vectors. .

A second object of the present invention is to provide a specific cleavage amino acid sequence, ie, Gln-Ala, Gln-Gl, downstream of the above DNA.
Provided is a DNA in which a nucleotide sequence encoding a target protein is connected in the same reading frame via a nucleotide sequence encoding y or Gln-Ser, a protein encoded by the DNA, and a DNA vector comprising the DNA. Is to do. The vector is required to be capable of self-propagation in a host cell, that is, to contain a nucleotide sequence necessary for self-propagation.

Further, a third object of the present invention is to provide the above-mentioned DN.
An object of the present invention is to provide a host cell transformed with a recombinant vector containing A and capable of autonomous propagation in the host cell.

Furthermore, a fourth object of the present invention is to provide the above-mentioned DN
A. A host cell transformed with a recombinant vector capable of self-propagation in a host cell containing A, and cultured under conditions capable of expressing the DNA, wherein Ala, Gly, or Ser is added to the N-terminus. It is intended to provide a method for producing protein.

It is another object of the present invention to provide a method for directly producing a target protein having Pro at the N-terminus and a target protein having an arbitrary amino acid residue at the N-terminus.

The DNA of the present invention is preferably prepared by isolating an RNA genome from a potyvirus group virus, for example, which is known to propagate the virus by processing of the protein, and preparing it based on the RNA genome. Clover yellow bain virus [Uyed
a, I.et al. (1975) Ann.Phytopath.Soc.Japan 41: 92-20
3] is preferred.

A fifth object of the present invention is to produce NIa by culturing a host cell containing the above DNA and transformed with a recombinant vector capable of self-propagation in the host cell under conditions capable of expressing the DNA. And then recovering the protein, to provide a method for producing the protein.

The DNA of the present invention can be obtained, for example, by preparing genomic RNA from CYVV particles purified from an infected plant, and then converting it into double-stranded cDNA by a known method. In the present invention, the virus serving as a source of the genomic RNA is a potyvirus group clover yellow bain virus (for example, American Type Culture).
Collection No. PV 123; Clover yellow vein viru
s) is preferred.

[0017] In the present invention, the clover yellow bain virus refers to a virus that causes gangrene in broad beans.

In general, many types of potyvirus are known which differ in pathogenicity. Based on the serological relationship of the viral coat proteins and the homology of amino acid sequences, viruses with primary amino acid homology of 90% or more
ain) and less than 70% of the virus is heterogeneous (Distinct mem
bers) [Shukla, DD and Ward, CW (19
89). Arch.Virol. 106: 171-200.] The CYVV coat protein used in the present invention [Uyeda, I. et al. (1991).
Intervirol. 32: 234-245.]
CYVV is positioned as an independent virus within the potyvirus group. Therefore, the primary structure of the coat protein amino acid has 90% or more homology, or the anti-CYVV
Antiserum (eg, American Type Culture Collection
No.PV123: clover yellow vein virus antiserum)
A virus showing a positive result in the ELISA method using Clover yellow bain virus can be regarded as the same strain of clover yellow bain virus. As a virus-infected plant, common beans (Phaseo
lus vulgaris), broad bean (Vicia faba), pea (Pisum sativum) and the like can also be employed, but broad bean is preferable, and early broad bean is suitable.

As a method for purifying CYVV virus particles, the infected plant is crushed and squeezed with a suitable buffer, and purified by repeating the treatment with an organic solvent such as chloroform and the centrifugal separation method, followed by sucrose density gradient centrifugation. The method of separating as pure virus particles by using the method in combination is preferable.

The virus particles obtained as described above are
The presence of CYVV can be confirmed as the appearance of a symptom by confirming the virus particles with an electron microscope, or by inoculating the virus particles on broad bean.

As a method for extracting genomic RNA from the virus particles obtained as described above, a guanidine thiocyanate-phenol method, a guanidine thiocyanate
The trifluorocesium method, the phenol / SDS method, etc. can also be used.
y, WG and Hiebert, E. (1980) Viology 101: 466-474.
Is preferred.

The genomic RNA obtained as described above is
Whether a protease is encoded is determined by the genomic RN
A can be translated into protein in a cell-free translation system, and autolysis by the protease region present in all translation products can be detected by tracking changes in molecular weight with an anti-viral coat protein antibody.

For example, Xenopus la
evis) oocytes are translated by injection of genomic RNA [Gurdon, JB (1972). Nature 233: 177-182.], or rabbit reticulocyte lysate or wheat germ lysate [Schleif, RF and Wensink] , PC (1981). In "Pract
ical Methods in Molecular Biology "Springer-Verla
g, NY.], and the degradation of the translation product can be followed over time using an anti-enveloping protein antibody.

After using the genomic RNA obtained as described above as a template to synthesize a single-stranded cDNA using reverse transcriptase, a double-stranded cDNA can be synthesized from the single-stranded cDNA.
This method includes the S1 nuclease method [Efstratiadis, A.
et al. (1976). Cell 7: 279-288.], Okayama-Berg method [Okayama, H. and Berg, P. (1982). Mol. Cell. Biol. 2: 1.
61-170.] And the like, but in the present invention, the Gubler-Hoffm
An method [Gubler, U. and Hoffman, BJ (1983). Gene 25:26
3-269.] Is preferred.

The double-stranded cDNA thus obtained is inserted into a cloning vector, the resulting recombinant vector is introduced into a microorganism such as Escherichia coli, and transformed, and transformation is performed using tetracycline resistance or ampicillin resistance as an index. You can choose the body. Transformation of host cells was performed by the Hanahan method [Hanahan, D. (1983). J. Mol. Biol. 166: 5.
57-580.], Ie, by adding the recombinant DNA vector to competent cells prepared in the presence of CaCl ₂ , MgCl ₂ or RbCl. When a plasmid is used as a vector, it is necessary to have the above-mentioned drug resistance gene, and a lambda phage other than the plasmid can be used.

As a method for selecting a strain having the target NIa DNA from the transformants obtained as described above, for example, the following various methods can be adopted.

(1) Synthetic oligonucleotide probe
If the whole or a part of the amino acid sequence of the target protein is clarified (the sequence may be that of any region of the target protein as long as the sequence is a plurality of specific sequences), it corresponds to the amino acid sequence. (In this case, either a nucleotide sequence derived using codon usage or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences may be used. In the latter case, the type may be determined by including inosine. Using this as a probe (labeled with ³² P, ³⁵ S or biotin, etc.), the DNA of the transformant was hybridized with a denatured and fixed nitrocellulose filter, and the obtained positive strain was searched. , Select this.

(2) Prepared by polymerase chain reaction
Screening method using a probe When the whole or part of the amino acid sequence of the target protein has been elucidated, sense strand and antisense strand oligonucleotides corresponding to a part of the amino acid are synthesized, and these are combined to form a polymerase chain. Reaction (Saiki,
RK et al . (1988) Science 239 , 487-491) to amplify a DNA fragment encoding the desired NIa. As the template DNA used here, cDNA synthesized by reverse transcription from viral genomic RNA expressing NIa can be used. The DNA fragment thus prepared is labeled with ³² P, ³⁵ S, biotin, or the like, and colony hybridization or plaque hybridization is performed using this as a probe to select a desired clone.

(3) NIa is produced in other animal cells and
The transformant is cultured, the gene is amplified, and the gene is transfected into an animal cell (in this case, a plasmid capable of self-replication and containing a transcription promoter region or a plasmid capable of integrating into the chromosome of the animal cell). ), The protein encoded by the gene is produced, and the NIa activity of the cell extract is measured, or NI
A strain having a cDNA encoding the desired NIa is selected from the original transformant by detecting NIa using an antibody against a.

(4) Selection using an antibody against NIa
Methods Nuclear inclusions (mixture of NIa and NIb) from CYVV-infected plants
Or, in advance, cDNA is incorporated into an expression vector, and the protein is produced in the transformed strain.
A strain expressing the desired NIa is detected using an antibody against a and a secondary antibody against the antibody, and the target strain is selected.

(5) Selective Hybridization
The cDNA obtained from the transformant is blotted on a nitrocellulose filter or the like, and mRNA from cells expressing NIa is hybridized. Dissociate and collect. MR recovered
Inject NA into a protein translation system, for example, into Xenopus oocytes, or translate it into a protein in a cell-free system such as rabbit reticulocyte lysate or wheat germ, and examine the NIa activity of the protein, or use an antibody against NIa. Detect using
Select the target strain.

The DNA encoding the CYVV protein can be collected from the target transformant obtained by a known method [Maniatis, T. et al. (1982). In "Molecular Cloning".
A Laboratory Manual "Cold Spring Harbor Laborator
y, NY.]. For example, from cell D
The separation can be performed by separating a fraction corresponding to NA and cutting out a DNA region encoding the protein from the plasmid DNA.

The sequence of the DNA thus obtained can be determined, for example, by the Maxam-Gilbert chemical modification method [Maxa
m, AM and Gilbert, W. (1980). in "Methods in Enzymo
theory, 65: 499-276.] and dideoxynucleotide chain termination using M13 phage [Messing, J. and Vieira, J. (1
982). Gene 19: 269-276.].

By incorporating the DNA encoding the protein of the present invention thus obtained into an expression vector, prokaryotic or eukaryotic host cells can be transformed. Furthermore, the gene can be expressed in each host cell by introducing an appropriate promoter and a sequence related to expression into these vectors.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
) And the like. To express the gene of interest in these host cells, the host cells can be transformed with a replicon derived from a species compatible with the host, i.e., a plasmid vector containing an origin of replication and a promoter sequence such as lacUV5. good. Further, it is desirable that the vector has a sequence capable of imparting selectivity based on a phenotype (phenotype) to the transformed cell.

For example, as Escherichia coli, JM109 strain derived from E. coli K12 strain or the like is often used, and as a vector, pBR322 or pUC-type plasmid is generally used, but is not limited thereto. Both strains and vectors can be used. Examples of the promoter include, but are not limited to, a lactose promoter (lac) and a tryptophan / lactose promoter (trc) in Escherichia coli.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 [Ohmura, K. et al.
4). J. Biochem. 95: 87-93.], Etc., but is not limited thereto. As the promoter, a regulatory sequence of the Bacillus subtilis α-amylase gene is often used.If necessary, a DNA sequence encoding a signal peptide sequence of α-amylase may be ligated to allow extracellular secretion. Become.

Eukaryotic host cells include cells of vertebrates, insects, yeasts, plants, and the like. Examples of vertebrate cells include COS cells, which are monkey cells [Gluzman, Y. (198)
1). Cell 23: 175-182.] Is used.

As insect cells, caterpillars of the family Noctuidae (Spodop
tera frugiperda) -derived cells [Smith, GE et al. (198
3). Mol. Cell. Biol. 3: 2156-2165.
As yeast, baker's yeast (Saccharomycse cerevisiae)
And fission yeast (Schizosaccharomyces pombe). Plant cells include tobacco (Nicotiana tabacu
m) and rice (Oryza sativa). The cells exemplified here are generally used frequently as host cells, but are not limited thereto.

As an expression vector for vertebrate cells, those having a promoter, an RNA splicing site, a polyadenylation site, a transcription termination sequence and the like which are usually located upstream of the gene to be expressed can be used. If necessary, a replication origin may be provided. An example of the expression vector has an early promoter of SV40.
pSV2dhfr [Subramani, S. et al. (1981). Mol. Cell. Bio.
1: 854-864.] Etc., but is not limited thereto.

Examples of insect cell expression systems include noctuid caterpillar culture cells. Examples of the expression vector include a baculovirus (Polyhedrin) promoter, a polyadenylation site and an AcMNPV (Acutographa californica nuclear poly) necessary for performing homologous recombination upstream of the gene to be expressed.
hedrosis virus) having a part of the genome can be used, and pBacPAK8 [Matuura, Y. et al. (1987). J. Gen. Viro
l. 68: 1233-1250.], but is not limited thereto.

As eukaryotic microorganisms, yeasts are commonly used, and baker's yeast (S. cerevisiae) can be exemplified. Examples of yeast expression vectors include, for example, an alcohol dehydrogenase gene promoter [Bennetzen, JL and
Hall, BD (1982). J. Biol. Chem. 257: 3018-3025.]
And carboxypeptidase-Y [Ichikawa, K. et a
l. (1993). Biosci. Biotech. Biochem. 57: 1686-1690.]
Etc. are available. Further, by linking a DNA sequence encoding the signal peptide sequence of carboxypeptidase-Y, if necessary, extracellular secretion becomes possible, but is not limited thereto.

Examples of plant expression vectors include cauliflower mosaic virus early promoter 35
s promoter and Agrobacterium tumefa
pB which has a polyadenylation sequence of the nopaline synthesis gene of C. ciens and further has a gene transduction sequence by apical carcinoma
I121 [Jefferson, RA et al. (1987). EMBO J. 6: 3901-
3907.] can be used, but is not limited thereto. Plant cells can be introduced by root carcinoma infection or electroporation.

As an example, when Escherichia coli is used as a host cell, pKK388-1 has a trc promoter as an expression vector and is capable of autonomous growth in a strain derived from Escherichia coli K12, such as JM109. (Manufactured by Clontech) can be used. The expression vector can be easily introduced into Escherichia coli based on a commonly used transformation method of Escherichia coli such as the rubidium method. The strain thus obtained is inoculated into a commonly used LB medium or the like, and after a certain culture, isopropyl-β
-Activate the trc promoter by adding galactopyranoside (IPTG). Thereafter, after culturing for a certain period of time, the expressed protein can be extracted from the cells by crushing the cells with an ultrasonic crusher or the like. In the present invention, when producing a target protein having Pro at the N-terminus, Escherichia coli must be used as a host.

As described above, the fraction containing the protein of the present invention produced in the cells of the transformant can be separated and separated by various known separation procedures utilizing the physical and chemical properties of the protein. It can be purified. Specific examples of such a method include treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC). ), Various types of chromatography, dialysis methods, combinations thereof, and the like.

As a method for measuring the protease activity of the NIa protein purified as described above,
a A method of causing a protein to act on a substrate protein containing a cleavage sequence for the protein can be exemplified. That is, a gene encoding a fusion protein of a viral coat protein that is a natural substrate of the NIa protein and a nuclear inclusion body b (NIb) (Do
ugherty, WG et al. (1988). EMBO J. 7: 1281-1287)
Is isolated from the viral genomic cDNA and inserted into an expression vector. The resulting expression vector is introduced into a suitable host cell to produce a fusion protein. Produced protein,
The fusion protein is separated and purified by combining the treatment with a normal protein precipitant and various types of chromatography. The substrate protein thus separated and purified is reacted with the protease in a suitable buffer at a suitable temperature to collect a reaction product. After electrophoresis of the collected reaction product, Western blotting using an anti-enveloped protein antibody can be performed to confirm the protease activity as a difference in band mobility. In this method, a synthetic oligopeptide containing a protease cleavage sequence may be used as the substrate protein.

Further, the activity of the protein of the present invention can be confirmed without purifying the protein. That is, as described above
A gene encoding the protein is connected downstream of the trc promoter, and a cleavage sequence of the protein, for example, Gln-A
La, Gln-Ser or Gln-Gly can be measured by connecting a CYVV coat protein or the like in the same protein reading frame with the nucleotide sequence encoding the amino acid of Gln-Gly interposed therebetween. That is, Escherichia coli protein expressed in the same manner as described above can be detected as a difference in band mobility by Western blotting using an anti-CYVV coat protein antibody or the like. If there is protease activity, it can be detected as a band showing higher mobility than the fusion.

The band whose cleavage has been confirmed in this way is recovered from the gel, and the amino terminal sequence is analyzed by a conventional method, so that it can be analyzed whether or not cleavage has occurred at the desired peptide binding site.

As a method for producing a desired protein using the protease, the desired protein is GS
Once produced as a fusion with a protein such as T, the protease is allowed to act in a test tube and cleaved to produce the desired protein. The desired protein is directly fused with the protease as a host. After expression in a cell, a method for producing a desired protein by self-cleavage in the same host cell can be mentioned.

According to the above method, the desired NIa protein can be easily produced in high yield and high purity, and the thus obtained recombinant NIa of the present invention can be used as a protease.

At one or more sites in the amino acid sequence of the NIa protein consisting of amino acids 4 to 437 of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, one or more Proteins in which amino acid residues are deleted, inserted, or substituted may have the same protease activity as NIa. These proteins are also included in the present invention.

For example, it has already been known that a protein obtained by converting a nucleotide sequence corresponding to cysteine of the interleukin 2 (IL-2) gene to a nucleotide sequence corresponding to serine retains IL-2 activity. [Wa
ng, A. et al. (1984). Science 224: 1431-1433.]. Therefore, as long as these naturally occurring or artificially synthesized proteins have NIa activity, all DNAs consisting of these proteins and the same nucleotide sequences encoding the proteins are also included in the present invention.

Examples of the DNA comprising such an equivalent nucleotide sequence include the DNAs described in (3), (6), (7), (8) and (9).

The various DNAs of the present invention can be obtained from the NI
a Based on protein information, for example,
Triester method [Hunkapiller, M. et al. (1984).
e 310: 105-111.] and the like, and can also be produced by chemical synthesis of nucleic acids. [Grantham, R. et al. (198
1). Nucleic Acids Res. 9: r43-r74.]. Furthermore, these nucleotide sequence codons can be partially modified by a site-specific mutant synthesis method using a primer consisting of a synthetic oligonucleotide encoding the desired modification [(site sp
ecific mutagenesis), Mark, DF et al. (1984).
l.Acad.Sci.USA.81: 5662-5666.] and the like.

The codon for the desired amino acid is known per se and may be selected arbitrarily. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used. Whether a certain DNA hybridizes with the DNA described in (5) can be determined, for example, by subjecting the target DNA to a random primer method [Feinberg, AP and Vogelstein,
B. (1983) Anal. Biochem. 132: 6-13.] And the nick translation method [Maniatis, T. et al. (1982). In "M
olecular Cloning A Laboratory Manual "Cold Spring
Harbor Laboratory, NY.] [Α- ³² P] dCTP
Hybridization can be carried out using a probe labeled with the above. The DNA used for hybridization is adsorbed on a known method, for example, a nitrocellulose membrane or a nylon membrane, and solidified by heating or ultraviolet rays. Then, immerse in a prehybridization solution containing 6 × SSC, 5% Denhardt's solution, and 0.1% SDS, and keep at 55 ° C for 4 hours or more. Thereafter, the previously prepared probe is added to the same prehybridization solution so as to have a final specific activity of 1 × 10 ⁶ cpm / ml, and the mixture is incubated at 60 ° C. overnight. Then, 6 × SSC at room temperature
5 times for 5 minutes, then 20 minutes at 57 ° C
After washing for 1 minute and performing autoradiography, whether or not hybridization has occurred can be determined.

The method for producing a desired protein using the thus obtained NIa or its equivalent (hereinafter referred to as "NIa") includes an intracellular direct cleavage method and an extracellular cleavage method. Can be

[Direct Intracellular Cleavage Method] The DNA encoding NIa's is replaced with the cleavage sequence Gln-GlyGln-Ser or Gln-A.
With la interposed, they are connected in the same reading frame as the DNA encoding the desired protein. The obtained DNA encoding the fusion protein is inserted into a vector having a promoter and a terminator that suitably brings about expression in a host cell to be produced. The obtained vector is introduced into a target cell by a suitable method to obtain a transformant. The fusion protein expressed in the transformant is self-cleaved by the protease activity of NIa species, and Gly, Ser is added to the N-terminal side of the target protein.
Alternatively, a peptide in which Ala is bound to a peptide can be expressed in a cell.

In this case, any target protein can be used, but IL-11 is preferred.

Further, in this cleavage method, the above-mentioned N-terminal Gly, Ser or Ala is further cleaved by the catalysis of aminopeptidase P derived from a host cell or expressed from a foreign vector. Can be expressed.

That is, the DNA encoding the target protein starting from Pro is replaced with the cleavage sequences Gln-Ala and Gln-Gly.
Or connect with Gln-Ser in between. The obtained fusion protein-encoding DNA is inserted into a vector having a promoter and a terminator for suitably expressing the DNA. The expression vector thus prepared is introduced into a target host cell to obtain a transformant. The obtained transformant is
Induce with a suitable inducer for the promoter used. For example, when using an IPTG promoter, it is preferable to use IPTG as an inducing agent.

In the induced cells, a fusion of NIa and the target protein is first expressed. After this, the fusion
Self-cleavage occurs due to the protease activity of NIa present in the N-terminal region, and Ala, Se derived from the cleavage sequence
Proteins with additional r or Gly residues are produced.

Furthermore, the N-terminal amino acid residues of these proteins accumulated in the cell can be removed by the aminopeptidase P when the host cell has the aminopeptidase P (3.4.11.9). By this reaction, a protein whose N-terminus starts with Pro can be produced in a host cell.

When the host cell does not have aminopeptidase P, an expression vector containing a gene encoding aminopeptidase P is introduced into the host cell, and the aminopeptidase P is expressed. Proteins whose N-terminus begins with Pro can be produced in host cells. Aminopeptidase is a protein known per se, and a gene encoding this enzyme protein can be obtained by a method well known to those skilled in the art.

When a protein whose N-terminus starts with Pro in a host cell as described above, it is preferable to extend the culture time if necessary.

Furthermore, the Pro residue of the protein whose N-terminus starts with Pro is a proline iminopeptidase (3.4.) Expressed from a host cell or expressed by a foreign expression vector.
It can be removed by the catalysis of 11.5), and by this reaction, a protein whose N-terminus starts with any amino acid residue can be produced in a host cell. Proline iminopeptidase is a protein known per se, and a gene encoding this enzyme protein can be obtained by a method well known to those skilled in the art. Are well known to those skilled in the art.

Further, by the method described above, the N
A peptide having a terminally bound Ala peptide can be expressed in cells, but this Ala residue is removed by the catalysis of alanine aminopeptidase (3.4.11.14) derived from a host cell or expressed by a foreign expression vector. By this reaction, a protein whose N-terminus starts with any amino acid residue can be expressed in a host cell. Alanine aminopeptidase is a protein known per se, and the gene encoding this enzyme protein is:
It can be obtained by a method well known to those skilled in the art, and a method for constructing an expression vector encoding the gene and a method for introducing the vector into a host cell are also well known to those skilled in the art.

The target protein thus produced in the cell can be isolated and purified by the above-mentioned method.

[Extracellular Cleavage Method] DN Encoding NIa
A is stably grown in a suitable host cell, incorporated into a vector capable of expressing a heterologous protein, and introduced into a host cell to obtain a transformant. Expressed in the resulting transformant
NIa is purified by combining an ion exchange column, gel filtration column or reverse phase column. Or glutathione
It can also be expressed as a fusion with -S-transferase or maltose-binding protein, separated and purified on a glutathione column or maltose column, then cut with enterokinase or factor Xa, and then purified and used as NIa.

On the other hand, the protein intended for cleavage contains Gln-Gly, Gln-Ser or Gln-Gly in its amino acid sequence.
-If it has an Ala peptide bond, the protein itself can be used as a substrate, or glutathione-S-transferase protein or maltose binding protein, etc.
It is also possible to prepare a fusion sandwiching Gly, Gln-Ser or Gln-Ala and use this as a substrate. By reacting this substrate protein with the purified NIa in a suitable buffer, the protein can be cleaved in a test tube, and as a result, a target protein having Gly, Ser or Ala at the N-terminus can be obtained. Can be. Furthermore, by using the aforementioned aminopeptidase P, proline iminopeptidase, and alanine aminopeptidase, a target amino acid having Pro at the N-terminus and an arbitrary amino acid residue can be obtained. These obtained target proteins can be isolated and purified by the methods described above.

[0070]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Virus Purification Material The virus was propagated by adding the diseased leaves (CYVV-No. 30 isolate: Uy) to broad beans (early soybeans) in which the second leaves were spread.
eda, I. et al. (1975) .Ann.Phytopath.Soc.Japan 41:19
2-203) was triturated with 10 volumes of inoculation buffer (0.1 M Tris-HCl buffer, pH 7.0, 0.05 M EDTA, 1% 2-mercaptoethanol), and the resulting juice was carborundum (400 mL). (Mesh) Sprayed leaves were inoculated with a glass spatula. 8 after inoculation
On the 1010th day, the top leaf on which a reticulated mosaic symptom appeared was cut off and used as virus-purified material.

Purification of Virus Particles The virus-infected leaves were crushed with a mincing machine, and three times the volume of an extraction buffer (0.1 M Tris-HCl buffer, 0.05 M EDTA, 1% 2
-Mercaptoethanol, pH 7.0) was added and ground in a mortar. The ground solution was slowly stirred at room temperature for 1 hour with a motor equipped with a stainless steel stirring blade, and the juice was squeezed out with a double gauze. All subsequent operations were performed at 4 ° C. A 1/2 volume of chloroform was added to the crude juice, and the mixture was vigorously stirred with a warning blender, followed by centrifugation at 6,000 × g for 15 minutes. The aqueous layer after centrifugation is collected, and polyethylene glycol (PEG) is adjusted to a final concentration of 4%.
# 6,000), stir on ice for 1 hour, and add 1 hour on ice.
Let stand for hours. This juice was subjected to centrifugation at 6,000 × g for 15 minutes, and the precipitate was collected as a virus fraction. The obtained precipitate was washed with 50 ml of 0.5 mM urea in 10 mM phosphate buffer (pH 7.4).
), An equal amount of carbon tetrachloride was added, and the mixture was vigorously stirred for 5 minutes. After stirring, the mixture was centrifuged at 3,000 × g for 10 minutes to obtain an aqueous layer. This fraction was subjected to ultracentrifugation at 30,000 rpm for 90 minutes using a Hitachi RP-30 rotor at 4, and the precipitate was collected as a virus fraction. This precipitate was suspended in 10 mM phosphate buffer (pH 7.4) containing 1% Triton X100, and centrifuged at 8,000 × g for 1 minute at 4 ° C. to collect a supernatant fraction. The obtained supernatant is
10-40% sucrose density gradient column tube (40%: 10ml, 30%: 10ml, 20%: 10m) prepared with 10mM phosphate buffer (pH7.4)
l, 10%: 10 ml. (Leave at 4 ° C overnight).
Using a PRS25 rotor at 43,000C, 23,000 rpm, 120
Ultracentrifugation was performed. The sucrose density gradient column tube after centrifugation was fractionated with a fractionator (Model UA-2: manufactured by ISCO) equipped with an OD _{260 nm} detector. OD
A sucrose density of about 20 to 30% having an absorbance at _{260 nm} was used as a virus fraction. The recovered virus fraction was diluted 2-fold with 10 mM phosphate buffer (pH 7.4), and centrifuged at 40,000 rpm for 90 minutes at 4 ° C. using a Hitachi RP-65 rotor. The resulting precipitate was suspended in a 10 mM phosphate buffer (pH 7.4) to obtain a purified virus solution.

Isolation of viral RNA Since genomic RNA of CYVV is composed of about 10,000 bases and is 3'-terminally polyadenylated, the full-length RNA can be completely used by the usual phenol SDS method or guanidine thiocyanate method. Difficult to extract. Thus, viral genomic RNA was prepared by alkaline sucrose density gradient centrifugation. That is, 500 mg of virus solution containing 2 mg of virus
An equal volume of 500 μl of the digestion solution [200 mM ammonium carbonate, 2% SDS, 2 mM EDTA, 400 μg / ml bentonite, 20 μg / ml protease K (pH 9.0)] was added to the μl, and the mixture was allowed to stand at room temperature for 20 minutes. Then 1xSSC (0.15M NaCl, 0.015M
0% to 33.4% sucrose gradient column tubes (33.4%: 1.3) buffered with trisodium citrate, pH 7.0
4ml, 30.4%: 7.6ml, 27%: 7.0ml, 23%: 6.3ml, 18.7%:
5ml, 12%: 3.2ml, 0%: 2.7ml. After layering, let stand overnight at 4 ° C), and use Hitachi RPS-27 rotor at 15 ° C for 2 hours.
Ultracentrifugation was performed at 4,000 rpm for 9 hours. A hole was made in the lower end of the tube after centrifugation to fractionate a sucrose density gradient. The viral genomic RNA fraction was identified by measuring the absorbance at 260 nm of each fraction. The virus genomic RNA was thereby precipitated at a sucrose concentration of about 20 to 30%. Thus about
25 μg of genomic RNA was obtained.

CDNA Synthesis cDNA was synthesized using genomic RNA purified by the method described above as a template. cDNA was performed using cDNA Synthesis Sistem Plus (Amersham). Terminal deoxynucleotide transferase (BRL) was added to the cDNA purified by Sephadex G50 (registered trademark, manufactured by Pharmacia) column.
And a dCTP to add a poly C chain to the 5 'end. Opened by the restriction enzyme PstI and pre-
The mixture was mixed with a plasmid pBR322 (manufactured by BRL) to which a chain had been added, kept at 65 ° C for 5 minutes, and then kept at 57 ° C for 2 hours.
Thereafter, the mixture is cooled slowly, and the poly-G chain on the plasmid
The C chain was annealed.

The cDNA annealed with the transforming plasmid pBR322 was introduced into Escherichia coli HB101 by the calcium chloride method. LB liquid medium (10 g Bacto tryptone, 5 g Bacto yeast extract, 5 g sodium chloride, 1 liter with distilled water)
E. coli HB101 strain shaken overnight in 50 ml
Inoculate 0.5 ml into LB liquid medium, and until OD _550nm reaches 0.5
The cells were shake-cultured at 7 ° C. The cells were collected by centrifugation at 5,000 xg for 5 minutes at 4 ° C, and the precipitate was gently suspended in 25 ml of Tris-calcium buffer (10 mM Tris, 50 mM calcium chloride, adjusted to pH 7.4 with hydrochloric acid). And left on ice for 5 minutes. Then centrifuge again at 4,000xg for 4 minutes,
The precipitate was suspended in 5 ml of Tris-calcium buffer and left on ice for 2 hours. This was used as a competent cell. 100 μl of annealed DNA and 200 competent cells
μl and left on ice for 30 minutes. After standing, the mixture was incubated at 42 ° C. for 2 minutes, added with 1 ml of LB liquid medium, and cultured with shaking at 37 ° C. for 1 hour. Thereafter, the mixture was spread on an LB solid medium containing 12.5 μg / ml of tetracycline hydrochloride and 1.5% agar. 37 ℃
The culture was continued overnight to obtain a cDNA clone library.

Analysis of the selected cDNA recombinant plasmid of pNS51 was performed by the alkaline SDS method. That is, 2 ml of tetracycline-resistant, ampicillin-sensitive bacterial colonies containing the cDNA recombinant plasmid
Was inoculated into an LB liquid medium, and cultured with shaking at 37 ° C. overnight. 1
The cells were collected by centrifugation at 0,000 × g, and 70 μl of lysis buffer (0.17 g sucrose, 250 μl 1 M Tris-HCl buffer (pH 8.0), 200 μl 0.5 MEDTA (pH 8.0) was added in 20 ml of double distilled water. ), And add 16 μl of lysozyme solution (10
(mg / ml), suspended for 5 seconds, and allowed to stand at room temperature for 5 minutes. 160 μl of an alkaline SDS solution (0.2 M sodium hydroxide, 1% SDS) was added, the tube was inverted several times, stirred, and left on ice for 5 minutes. 5M potassium acetate 120
After adding μl, the mixture was again left still on ice for 5 minutes, and centrifuged at 10,000 × g for 5 minutes. The supernatant after centrifugation was transferred to a new tube, 250 μl of isopropanol was added, and the mixture was allowed to stand on ice for 30 minutes. After centrifugation at 10,000 xg for 5 minutes, precipitate
0 μl of TE buffer [10 mM Tris, 5 mM EDTA (pH 8.0
)] And dissolve in equal amounts of phenol, chloroform,
Isoamyl alcohol (25: 24: 1) was added, and the mixture was vigorously stirred and centrifuged (this operation is hereinafter referred to as phenol extraction). To the obtained aqueous layer, 3 M sodium acetate (pH 5.2) and 250 μl of ethanol were added, left on dry ice for 10 minutes, and then subjected to centrifugation at 10,000 × g for 5 minutes to collect a nucleic acid fraction (hereinafter, this operation was performed). Ethanol precipitation). This precipitate was dissolved in 50 μl of RNase A solution (10 μg / ml TE buffer) and incubated at 37 ° C. for 1 hour. After incubation, 30 μl of PEG solution (2.5 M sodium chloride, 20% polyethylene glycol #
8,000) and left on ice for 1 hour. The DNA fraction was recovered by centrifugation at 10,000 × g, and then twice ethanol precipitation was repeated to obtain purified plasmid DNA.

The purified plasmid DNA was digested with the restriction enzyme PstI, and 1% agarose gel electrophoresis was performed with TBE (100 mM Tris, 100 mM boric acid, 1 mM EDTA). The gel after the electrophoresis was subjected to a conventional method [Southern, EM (1975). J. Mol. Biol. 89:
503-517.], And Southern blot hybridization was performed. That is, the gel after electrophoresis was shaken for 40 minutes in a denaturing solution (1.5 M sodium chloride, 0.5 M sodium hydroxide), and then transferred to a neutralization buffer [0.5 M Tris, 3 M sodium chloride (pH 7.4)]. Shake time. Gel after shaking
Transfer onto a sponge containing 20xSSC (3M sodium chloride, 0.3M trisodium citrate, pH 7.0) and apply Hybond-N
Transferred to a membrane (registered trademark: manufactured by Amersham). Shake the transferred High Bond-N film with 1xSSC for 10 minutes, and
After drying for a while, the DNA was immobilized. Then, a prehybridization solution [5 ml formamide, 1 ml 50x Denhardt's solution (1% polyvinylpyrrolidone, 1% bovine serum albumin, 1% Ficoll 400), 2.5 ml 20xSSC, 100 μl yeast tRNA (50 mg / ml), 100 μl 10% SDS, 1.3ml
Double distilled water] and kept at 50 ° C for 3 hours.

The probe was used by cutting viral genomic RNA with Mg ²⁺ . That is, 1 μg virus RNA solution to 16 μl
4 μl 5x digestion buffer (125 μl 1M glycine pH 9.0)
, 25μl 1M magnesium chloride, 850μl double distilled water)
The mixture was incubated at 37 ° C for 3 hours, and denatured by heating and quenching 5 µl (100 ng) of the degraded RNA solution recovered by ethanol precipitation at 70 ° C for 5 minutes. Add 4 μl of 5x
Labeling buffer [25 μl 1 M Tris-HCl buffer (pH 7.9), 5
[mu] l 1M magnesium chloride, 2.5 [mu] l 1M dithiothreitol (DTT), [γ over ³² P of 9.2 [mu] l distilled water] and 1 [mu] l]
Add ATP (0.37 MBq), 10 μl double distilled water, and add 20 U T4
Polynucleotide Kinase [Takara Shuzo Co., Ltd.]
It was kept at 37 ° C for 30 minutes. Unreacted [gamma over ³² P] ATP is 5
It was removed by repeating ethanol precipitation twice.

The obtained probe was used at a final concentration of 5 × 10 ⁵ cpm / ml.
Hybridization solution [5 ml formamide, 2 ml 50% dextran sulfate 200 μl 50x Denhardt's solution, 2.5 ml 20x SSC, 50 μl yeast tRNA (5
0 mg / ml), 100 μl 10% SDS, 1.3 ml double-distilled water], and the Hybond-N membrane after prehybridization was transferred to the solution. The Hybond-N membrane that was incubated at 50 ° C overnight and hybridized was 0.1% at 60 ° C.
The plate was shaken with 2xSSC containing SDS for 30 minutes and washed. This operation
Repeat 3 times, and further in 0.1xSSC containing 0.1% SDS at room temperature.
Shake for 1 hour and dry. As a result of autoradiography, the number of bases hybridized with viral genomic RNA was approximately
PNS51 with an insert of 6,500 base pairs (bp) was obtained.

Determination of Nucleotide Sequence To prepare a restriction map of the cDNA cloned into pNS51, the plasmid pNS51 was replaced with the restriction enzymes BamHI, Ec
oRI, Hind, KpnI, NcoI, PstI, SalI, SphI, SacI,
SmaI, XhoI, and XbaI were cut with each or a combination of the two to prepare restriction enzyme maps. FIG. 1 shows the generated restriction enzyme map.

Each fragment obtained by cutting the restriction enzymes SalI and PstI was inserted into M13mp19 RF DNA. That is, pNS51 was cleaved twice with restriction enzymes PstI and SalI, and then subjected to 5% polyacrylamide gel electrophoresis under 1 × TBE conditions. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and a band was confirmed under ultraviolet light. The gel containing each DNA band was cut out with a razor, and was eluted electrically with an electrorelator (manufactured by Amicon) equipped with Centricon-30 (manufactured by Amicon). After that, use SalI alone or DNA with DNA ligation kit [Takara Shuzo Co., Ltd.]
Each fragment was inserted into M13mp19 that had been digested with PstI and SalI and then treated with alkaline phosphatase.

The recombinant M13mp19 RF-DNA into which each restriction enzyme fragment of pNS51 was inserted by T4 DNA ligase was introduced into Escherichia coli JM109 strain by the rubidium chloride method. M9 minimal agar medium [10
xM9 salt solution (0.145M disodium hydrogen phosphate, 0.172M
Potassium dihydrogen phosphate, 0.187M ammonium chloride, 0.
137M sodium chloride, pH 7.0) 10ml, 1M magnesium sulfate 100μl, 20% glucose 1ml, 1% thiamine hydrochloride 50μl, 0.01M calcium chloride 1ml, double distilled water 13ml
, And sterilized by filtration, and 50 ml of 3% Bacto agar is added to a petri dish. After adding 125 μl of potassium, make up to 500 ml with distilled water, and after autoclaving, add 5 ml of 1M magnesium chloride and 5 ml of 1M magnesium sulfate) and shake culture overnight. Overnight cultivation of E. coli JM109 strain into 50 ml of new SOB liquid medium.
6 ml was inoculated, and shake-cultured at 37 ° C. until the OD _{600 nm} reached 0.5. The cells were collected by centrifugation at 5,000 xg for 10 minutes at 4 ° C, and the precipitate was washed with 25 ml of TFB1 buffer [1M MES (pH 6.2) 5
ml, rubidium chloride 6.045g, calcium chloride dihydrate
0.735 g, manganese chloride tetrahydrate 4.94 g, pH 5.8 with acetic acid
And sterilized by filtration with distilled water, and allowed to stand on ice for 20 minutes. Then again at 3,000xg
After centrifugation for 5 minutes, the precipitate was washed with 2 ml of TFB2 buffer [1M MOPS
1 ml, calcium chloride dihydrate 1.102 g, rubidium chloride 0.12 g, glycerol 15 ml, adjusted to pH 6.5 with acetic acid, adjusted to 100 ml with double-distilled water, sterilized by filtration] and suspended on ice.
It was left still for 20 minutes and used as a competent cell. 1-10 μl of ligated DNA and 100 μl of competent cells
l and left on ice for 1 hour. After standing at 42 ℃
Incubate for 1.5 minutes, and add 500 μl of SOC liquid medium (5 ml of SOC,
20% glucose (90 μl) was added, and the cells were shake-cultured at 37 ° C. for 1 hour. 2xYT containing 0.8% agar in bacterial suspension after shake culture
(Make up to 1 liter with 16 g bactotryptone, 5 g bacto yeast extract, 5 g sodium chloride, double distilled water) Medium, 30 mM 100 mM IPTG, 10% 5-bromo-4-chloro-3
-Add 10 μl of indolyl-β-galactoside (X-gal) and 100 μl of indicator bacteria (culture solution of JM109 strain shaking overnight at 37 ° C. in 2xYT medium) and add the previously solidified 1.2% agar. The mixture was poured into a 2xYT solid medium containing the mixture, solidified, and cultured overnight at 37 ° C. As a result, recombinant phages were obtained as white plaques.

Each white plaque contains 1/100 of indicator bacteria
2xYT liquid medium was inoculated and shake-cultured at 37 ° C overnight. 10,0
After centrifugation at 00xg for 1 minute, the supernatant was frozen or stored at 4 ° C as a phage solution. On the other hand, the cells, which are the precipitate, are prepared in the same manner as the usual plasmid preparation method, and the RF double-stranded DNA (hereinafter referred to as RF-DNA), which is a phage replication intermediate, is recovered, and the presence or absence of the inserted fragment is determined. Confirmed by restriction enzyme digestion.

Thus, SalI which is an intermediate part of the 51PS5 '/ M13mp19 genome containing the PstI-SalI fragment on the 5' side of the CYVV genome is obtained.
A 51SS / M13mp19 containing the / SalI fragment was obtained. From these cloned phages, RF-DNA was purified by an alkaline SDS method and a cesium chloride density gradient centrifugation according to a conventional method. 5 μg of the purified DNA was cut with BamHI, which is a 5 ′ protruding end, and further cut with KpnI, which is a 3 ′ protruding end.
Then erase-a-base system (promega)
Exonuclease was added according to the attached protocol of, and the reaction time was separated stepwise from 1 to 10 minutes.
Each of the cDNA fragments inserted in was cut off stepwise from the 5 'side. In this case, since the vector side has a 3 'protruding end, it is not attacked by exonuclease. afterwards,
S1 nuclease treatment to blunt the DNA ends and T4 DNA
Ring closure with ligase.

Recombinant M13mp19 closed with T4 DNA ligase
RF-DNA was obtained from E. coli by the rubidium chloride method as described above.
Introduced into JM109 strain. As a result, recombinant phages having cDNA inserts of various lengths were obtained as white plaques.

From the obtained recombinant phage subclone, RF-DNA was extracted according to a conventional method, and clones having different insert fragment lengths were selected. Select clones, 2x containing indicator bacteria
YT liquid medium was inoculated and shake cultured at 37 ° C. overnight. 1.5 ml of the shake culture was centrifuged at 10,000 xg for 5 minutes at 4 ° C. Transfer 1 ml of supernatant to a new tube and add 250 μl
A 20% PEG aqueous solution (20% PEG # 8,000, 2.5 M sodium chloride) was added, stirred, and allowed to stand on ice for 30 minutes. At 4 ° C,
After centrifugation at 10,000 xg for 5 minutes, precipitate was
And the phage genome single-stranded DNA (hereinafter referred to as ssDNA) was recovered by phenol extraction and ethanol precipitation.

The nucleotide sequence of the nucleic acid was determined by the dideoxynucleotide chain termination method using phage ssDNA obtained from each subcloned phage as a template. That is,
The phage ssDNA obtained as described above was
-Sequenace Version 2.0 Kit for dCTP
Using (USB ^{Corp.), [α- 32 P] dCTP} ( specific activity: ～220TBq
/ Mmol, Amersham). Reaction product is 1x
5% polyacrylamide gel electrophoresis containing 8 M urea was performed under TBE buffer. After the gel was dried, autoradiography was performed to decode the nucleotide sequence.

The 5′-Pst-Sal fragment of pNS51 and M-Sal-Sa
The 3,839 bases were determined by nucleotide sequence analysis of the l fragment.

When the reading frame of the polypeptide (hereinafter referred to as ORF) was searched from the nucleotide sequence of the obtained nucleic acid, one ORF was detected on the + sense side of the virus genome. The sequence of the polypeptide was determined based on the detected ORF. When this and the known amino acid sequence were subjected to homology search using Genebank, TEV-HAT
And homology were observed. Viral proteases that mature using proteases such as TEV, plum pox virus, or the animal virus poliovirus are generally Gln-Ala, Gln-Ser, or Gln-Gly.
It is known to cleave peptide bonds between (Wi
llink, J. and van Kammen, A. (1988). Arch.Virol. 98: 1-2.
6.) Analysis of the cleavage site of the coat protein of CYVV-No. 30 (Uyeda, I. et al. (1991). Intervirology 32: 234-24
From 5.), a truncated sequence at three positions was observed in the obtained predicted polypeptide sequence. Of these, the polypeptide starting from the 4th Gly on the amino acid sequence and extending to the 437th Gln was determined as CYVV NIa.

Preparation of Fusion of CYVV-NIa and IL-11 In order to prove that the DNA provided in the present invention satisfies the following, the DNA and human interleukin 11 were
(Hereinafter referred to as "IL-11").
The same reading frame was connected via the gene encoding Ala.

What must be proved is, first, whether the DNA is a gene encoding NIa having protease activity, and second, whether the protease cleaves a desired amino acid sequence. Third, whether the protease activity of NIa acts in the same manner as in a virus-infected cell in the form of a fusion with the desired heterologous protein, and fourthly, the cleaved and produced heterologous protein has That is, whether the activity is maintained.

The NIa cistron has no initiation codon, since NIa itself is cleaved from the viral precursor protein. Therefore, it is necessary to add an initiation codon, ATG, to the 5 'end of the NIa gene. In addition, the 3 'end of the NIa gene must be connected with the same ORF as IL-11. In order to solve these problems, NIa was modified by polymerase chain reaction (PCR) using a restriction enzyme XhoI cleavage site present in the central part of the NIa gene.

An ATG corresponding to the initiation codon and a restriction enzyme Nc for cloning are provided at the 5 'end of the 5'-side (NIa5') fragment of NIa.
PCR primers were made to add the oI cleavage sequence. The prepared sequence is 5 'GTCCATGGGGAAAAGTAAGAGAACA 3'
(NSATG: SEQ ID NO: 3). 5'ACTCTGAGACCGTGCTCG
AG 3 ′ (referred to as NSX1: SEQ ID NO: 4). Plasmid
For 1 μg of pNS51 DNA, 1 μg of each of these synthesized primers, dNTP mix (25 mM dATP, 25 mM dCTP,
0.8 μl of 25 mM dGTP, 25 mM dTTP) and 10 × Taq buffer (buffer previously attached to the enzyme: manufactured by Promega) were added, and the mixture was adjusted to 100 μl with double-distilled water, followed by addition of 5 U of Taq DNA polymerase and PCR. . PCR program at 92 ° C
One minute, 37 ° C. for 1 minute, and 72 ° C. for 2 minutes were repeated 20 times, and a further 92 ° C. for 1 minute, 37 ° C. for 1 minute, and 72 ° C. for 30 minutes were performed once.
The amplified DNA product after PCR was subjected to 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation, and the DNA band was cut out together with the gel. The cut gel was subjected to electroelution using a centrilutor (manufactured by Amicon) previously equipped with Centricon-30 (manufactured by Amicon). After elution, centrifuge at 7,500 xg at 4 ° C
The eluted DNA fragments were concentrated and recovered by centrifugation at for 45 minutes. The obtained DNA concentrate was further purified by phenol extraction and ethanol precipitation.

1 μg of the plasmid obtained by replacing the SacI cleavage site of the plasmid pKK388-1 (manufactured by Clontech) with the XhoI cleavage site was digested with restriction enzymes NcoI and XhoI in advance, and alkaline phosphatase / cauffintestin [Alkali
ne Phosphatase Calf intestine: CIAP: manufactured by Takara Shuzo Co., Ltd.]. Recover the recovered DNA with Nc
After ligation was performed using 100 ng of pKK388-1 digested with oI and XhoI and dephosphorylated with a ligation kit (manufactured by Takara Shuzo Co., Ltd.), it was cloned into E. coli JM109. This gives pK
The plasmid pKNI5 'inserted in the forward direction downstream of the trc promoter of K388-1 was obtained (FIG. 2).

A plasmid pCD20-2 containing the cDNA of the precursor of IL-11 (Pre-IL11) containing a secretory signal sequence [Kawash
ima, I. et al. (1991). FEBS L. 283: 199-202.] is cleaved with restriction enzymes BamHI and ApaI to produce an IL-11 precursor (Pre-IL11).
And the region containing the SV40 promoter. This fragment was once cloned into the BamHI and ApaI sites of pBLUESCRIPT II SK + and cut again with restriction enzymes XhoI and KpnI. As a result, a gene encoding a protein lacking the N-terminus of mature IL-11 (Mat-IL11) can be isolated. The obtained N-terminal-deficient Mat-IL11 DNA fragment was previously subjected to restriction enzymes XhoI and KpnI.
Was reincorporated into pKNI5 'which had been cleaved with and subjected to CIAP treatment by T4 ligase to prepare pKNI5IL (FIG. 3). Four PCR primers were synthesized to connect the specific sequence on the C-terminal side of NIa and Mat-IL11 in the same reading frame across Ala. That is, 5'A
GGAAAAGAGTTCCTCGAGC 3 '(referred to as NSX2: SEQ ID NO: 5), 5' AATTGTTCATTCCAAGCACCTGGGCCACCACCTGGC 3 '
(Referred to as NSJ001P: SEQ ID NO: 6), 5′GCCAAGTGGTGGCCCAG
GTGCTTGGAATGAACAATT 3 '(NSJ002N: SEQ ID NO: 7) and 5'TTGTCAGCACACCTGGGAGCTGTAGAGCTC3' (ILSAC: SEQ ID NO: 8).

The first PCR is a reaction performed using pNS51 DNA as a template and a combination of primer NSX2 and primer NSJ002N (referred to as CNI3 section). Apart from this, pC
Using D20-2 DNA as a template, a combination reaction of the primers NSJ001P and ILSAC was also performed (referred to as 5'IL section). The PCR program consists of 92 ° C for 1 minute, 37 ° C for 1 minute, and 72 ° C for 2 minutes.
This was repeated once, and further performed once at 92 ° C for 1 minute, 37 ° C for 1 minute, and 72 ° C for 30 minutes. The product after each PCR was subjected to 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation, and the amplified DNA band was cut out together with the gel. DNA was electrically recovered from the cut gel by the method described above (FIG. 4).

The two obtained DNA fragments were subjected to the first PCR
The part of the primer NSJ001P NSJ002N used in
The 3 'end of the DNA fragment and the 5' end of the 5 'IL DNA fragment,
There is partial homology in the nucleotide sequence. Therefore, both
When PCR is performed again using the DNA fragment, the primer NSX2 and the primer ILSAC, a homologous portion hybridizes, and a fusion DNA of both DNAs is obtained by joining the homologous portion (FIG. 5). The collected CIN3 DNA fragment and 5'IL DNA fragment were mixed, and NSX2 and ILSAC were
R made. The PCR program is 1 minute at 92 ° C, 1 minute at 37 ° C.
10 minutes at 72 ° C for 2 minutes, then at 92 ° C for 1 minute.
45 ° C for 1 minute and 72 ° C for 2 minutes were repeated 20 times. Then 92 ° C
After performing phenol treatment and ethanol precipitation for 1 minute, 55 ° C. minutes and 72 ° C. for 30 minutes, 5% polyacrylamide gel electrophoresis was performed to obtain a CNI3IL DNA fragment in which CNI3 and 5′IL were fused. The above method is shown in FIG.

CNI3IL DNA fragment was cut with restriction enzyme XhoI,
A PCR fragment was inserted using T4 DNA ligase into pKNI5 'which had been digested with the restriction enzyme XhoI and dephosphorylated with CIAP in advance, and cloned into Escherichia coli JM109 strain. CNI3IL
To select a clone in which the fragment was inserted in the forward direction, a plasmid was extracted from the obtained clone, and the primer NS
PCR was performed using X2 and primer ILSAC. As a result, only the DN of the plasmid of the clone inserted in the forward direction is
A band is detected. By this method, NIa and Mat-IL1
A plasmid pKSUN9 was obtained in which 1 was connected by the same reading frame with the cleavage sequence Gln-Ala interposed (FIG. 6).

Production of Ala- IL11 in Microorganisms pKSUN9 functions in Escherichia coli, and the constructed recombinant gene is
The expression was confirmed by Western blotting. That is, Escherichia coli carrying the plasmid pKSUN9 (hereinafter referred to as KSUN9 strain) was shake-cultured with 42 μg / ml ampicillin in 5 ml of LB medium at 37 ° C. overnight. Then the new LB
2.5 ml of KS in 250 ml of medium (containing the same amount of ampicillin)
UN9 shake culture solution was added, and shake culture was performed at 37 ° C. until the OD _{600 nm} reached 1.0. In the culture solution when OD _600nm reaches 1.0,
Add IPTG to a final concentration of 0.1 mM and add 12 mM at 28 ° C.
The cells were cultured with shaking for a time. However, mature body IL-11 starting with Pro
When IPTG is produced, add IPTG to a culture solution with an OD _{600 nm} of 1.0 to a final concentration of 1 mM, and add
Shaking culture was performed for 36 hours or more.

By western blotting of the product
From the culture solution shake-cultured for 12 hours after adding the confirmation inducer (IPTG) or the culture solution shake-cultured for 36 hours, 4 ° C
The cells were collected by centrifugation at 3,000xg for 5 minutes.
The cells were suspended in 100 μl of a 20 mM borate buffer (pH 9.0). The cells were treated with an ultrasonic crusher [Handy Sonic UR-20P: manufactured by Tommy Seiko Co., Ltd.] for 2 minutes in a range 8 and crushed. The homogenate was centrifuged at 10,000 xg for 10 minutes at 4 ° C, and the supernatant was used as a soluble protein fraction. 5 μl of the obtained soluble fraction was subjected to the Laemmli method [Laemmli, UK (1970). Nature 2
27: 680-685.] And 12% polyacrylamide gel electrophoresis containing SDS. The gel after electrophoresis was transferred to transfer buffer (25 mM Tris, 192 mM glycine, 20 mM
% Methanol) for 5 minutes, and
Using an SD semi-dry transfer cell (manufactured by Bio-Rad Laboratories), the cells were transferred to a PVDF membrane (Trans-Blot Transfer Medium: manufactured by Bio-Rad Laboratories) by treatment at 15 V for 1 hour. Transcribed PVDF
The membrane was washed with PBS-Tw (80 mM disodium hydrogen phosphate, 20 mM sodium dihydrogen phosphate, 100 mM sodium chloride, 0.1% Tween 20) for 10 minutes. The PVDF membrane was transferred to PBS-Tw containing 5% skim milk [manufactured by Snow Brand Co., Ltd.], and blocking was performed at 37 ° C. for 1 hour. After blocking, the PVDF membrane was transferred to anti-IL-11 rabbit serum diluted 10,000-fold with PBS-Tw after washing once for 10 minutes and twice for 5 minutes in PBS-Tw. Incubated for a minute. Wash for 10 minutes in PBS-Tw
Once and two 5 minute washes, a horseradish peroxidase-labeled anti-rabbit Ig diluted 5,000-fold in PBS-Tw
G ・ Transfer into goat antibody (Amersham) and
Incubated for a minute. Then, wash again for 10 minutes in PBS-Tw 1
Washing was performed 4 times for 5 minutes. The washed PVDF membrane was transferred to ECL Detection Regent (Amersham). The band that reacted with the anti-IL-11 antibody was PVDF.
Detection was performed by contacting the membrane with X-ray film for 30 seconds to 5 minutes.

As a result of Western blotting, signal bands having a molecular weight of about 50 KDa and a molecular weight of about 23 KDa were detected from both the cells cultured with shaking for 12 hours and the cells cultured with shaking for 36 hours. Of these, the low molecular side 23KDa band
It showed almost the same mobility as the mature IL-11 used as a control. This signal band of 23KDa was considered to be IL-11 cleaved by the specific sequence Gln-Ala by the protease activity of NIa from the fusion of NIa and IL-11. In addition, of the two 23KDa proteins detected at this time, the protein obtained from the cells cultured with shaking for 12 hours was converted to the following 23KDa.
Set to a-ON, and the protein obtained from the cells cultured with shaking for 36 hours is set to 23 KDa-36 hr.

Purification of 23KDa-ON and 23KDa-36hr Proteins In order to determine the amino acid sequence of the N-terminal of 23KDa-ON and 23KDa-36hr proteins, these 23KDa proteins were purified. In the case of 23KDa-ON, induction with 0.1 mM IPTG
From the 250 ml culture solution that was shake-cultured for 23 hours, 23KDa-36
In hr, cells were recovered from a 250 ml culture obtained by inducing IPTG at 1 mM and shaking at 28 ° C. for 36 hours at 5,000 × g for 15 minutes at 4 ° C. The collected cells were suspended in 10 ml of 20 mM borate buffer (pH 9.0), and the cells were disrupted by a French press and an ultrasonic disrupter. 15,000xg at 4 ℃
The soluble protein fraction (supernatant fraction) was collected by centrifugation for 30 minutes.

The bacterial cell soluble protein fraction obtained as described above was purified using the following conditions under the following conditions:
For weak ion exchange column chromatography using

Column: CM Toyopearl Pack 650M [2.2 x
20 cm, manufactured by Tosoh Corporation] Elution buffer: A = 10 mM boric acid-sodium hydroxide (pH 9.
0), 13mM potassium chloride B = 10mM boric acid-sodium hydroxide (pH 9.0), 13mM potassium chloride, 400mM sodium chloride Flow rate: 2.5ml / 1min Fraction volume: 5ml / 1 tube Concentration gradient: eluent A → Eluent B (linear concentration gradient), eluted for 120 minutes, and the fraction containing IL-11 was identified by enzyme-linked antibody double sandwich method (ELISA).

The ELISA was performed as follows. That is, 1
50 μg / ml anti-IL-11 mouse monoclonal antibody
mM sodium carbonate buffer (pH 9.6) was added to each well of a 96-well immunoplate (Maxisorp: Nunc).
Aliquots of 100 μl were aliquoted and incubated at 37 ° C for 1 hour. Then, each well was treated with PBS-T (4 g sodium chloride, 0.1 g potassium dihydrogen phosphate, 1.45 g disodium hydrogen phosphate.
Wash 12 times with water salt, 0.1 g potassium chloride, 0.1 g sodium azide, make up to 1 liter with double distilled water, pH 7.4), dilute each fraction 100-fold with PBS-T, and add To 100
Dispensed in μl. After incubating at 37 ° C for 1 hour,
After washing with T, 100 μl of anti-IL-11 rabbit IgG diluted to 1 μg / ml with PBS-T was dispensed to each well. 37 ° C again
After incubation for 1 hour and washing with PBS-T, alkaline phosphatase-labeled anti-rabbit IgG / goat antibody (manufactured by Gibco BRL) diluted 3,000 with PBS-T
0 μl was dispensed into each well. Incubate again at 37 ° C for 1 hour, wash with PBS-T, and then use the substrate solution (dissolve paranitrophenyl phosphate to 0.01% in a 10% aqueous solution of diethanolamine adjusted to pH 9.8 with hydrochloric acid) Was dispensed into each well in an amount of 100 μl. At room temperature
After keeping the temperature for 30 minutes to 1 hour, the color development of each well was measured at an absorbance of 405 nm, and the desired fraction was identified.

The specified target fractions (fractions Nos. 19 to 25) were collected, concentrated 100-fold with Centprep-10 (manufactured by Amicon), and electrophoresed on a 12% polyacrylamide gel in the presence of SDS based on the Laemmli method. Was done. Then, it was electrically transferred to the PVDF membrane in the same manner as in Western blotting.
However, at this time, a pro-blot membrane (manufactured by Applied Biosystems) was used as the PVDF membrane. The membrane after the transfer was sufficiently washed with double distilled water. The transferred PVDF membrane is stained with Coomassie Brilliant Blue R250, and after decolorization with 50% methanol, the band corresponding to the protein that reacts with the anti-IL-11 antibody is cut out by Western blotting, and a protein sequencer (manufactured by Applied Biosystems) is used. In N
The terminal amino acid sequence was analyzed. As a result, the N-terminal sequence of the 23KDa-ON band reacted with the anti-IL-11 antibody was as follows.

Ala-Pro-Gly-Pro-Pro-Pro-Gly- (SEQ ID NO: 9) This sequence corresponds to -1 of the amino acid sequence of mature IL-11 protein.
It corresponds to the amino acid sequence at positions +6 to +6. From this 12
The 23KDa protein that reacts with the anti-IL-11 antibody obtained from the time section is formed by cleavage of the peptide bond of Gln-Ala, a specific cleavage sequence of NIa, from NIa and IL-11 fusion by the protease activity of NIa. Ala-IL-11 was thought to be.

On the other hand, anti-IL-1 similarly obtained from 36 hours
1 The N-terminal amino acid sequence of 23KDa-36hr that reacts with the antibody
It was as follows.

Pro-Gly-Pro-Pro-Pro-Gly-Pro- (SEQ ID NO: 10) This sequence corresponds to the amino acid sequence from position +1 to position +7 of mature IL-11. This result and the result previously obtained with 23KDa-ON are combined as follows.

The fusion of NIa and IL-11 was expressed in E. coli by induction with IPTG.

In the culture 12 hours after the induction of the expression, the expression of NIa
The peptide bond between the specific cleavage sequence Gln-Ala was cleaved by the protease activity of NIa and IL-11 fusion.

Ala-IL11 with extra Ala added to the N-terminus of mature IL-11 by cleavage of the peptide bond by NIa
Was expressed in E. coli.

After the expression of Ala-IL11 was confirmed, the cells were further cultured for 24 hours to mature Ala-IL11 into IL-11 in which the Ala residue present at the N-terminal was removed.

From the above, 23 hours obtained after 36 hours of culture were obtained.
It was concluded that the KDa protein was mature IL-11 beginning with Pro at the N-terminus.

From these results, it was found that IL which was a fusion with NIa
-11 is cleaved once between Gln-Ala at the junction of both proteins by the specific protease activity of NIa, and then N-terminal Ala is removed by a factor in E. coli by continuing culturing. It was confirmed that the protein started from Pro was matured.

The biological activity of the expressed Ala-IL11 or mature IL-11 is determined by the inhibitory factor for adipogenesis inhibition (Adipogenesis inhibitor).
ory factor: AGIF) activity was used as an index.
It is generally known that IL-11 has an activity of inhibiting adipogenesis [Kawashima, I. et al. (1991). FEBS L. 28
3: 199-202.].

The method of measuring AGIF activity used in the present invention is as follows. The cells used for the measurement were a mouse embryonic fibroblast cell line 3T3-L1 [Green, H. and Kehinde, O. (1974).
Cell 1: 113-116.] And purchased from ATCC. All cell cultures were performed at 37 ° C. in a humidified mixture of 10% CO ₂ -90% air. Culturing the cells for passage is performed using medium A [10% inactivated FBS
(Hycron) and 10 mM Hepes (HEPES, pH 7.2)
) Containing DMEM (Dulbecco's modified Eagle's medium: 4.5)
g / liter of glucose)]. Three
The differentiation induction of T3-L1 cells into adipocytes can be performed by the method of Rubin et al. [Rubin, CS et al. (1978). J. Biol. Chem. 253: 7570-
7578.].

Inhibition of morphological change of 3T3-L1 cells into adipocytes
Method of assay 3T3-L1 cells were suspended in medium A at a density of 1.0 × 10 ⁴ cells / 1 ml, and the suspension was dispensed into a 48-well multi-cluster dish (Coaster) at a volume of 0.5 ml / well. went. After 3 days in culture, the cells reach confluency. Then, replace the medium with fresh medium A,
After further culturing for 2 days, the medium was changed to medium B (10 mM HEPES (pH 7.2), 3% inactivated FBS,
μg / ml bovine insulin (Sigma), 8 μg / ml
DMEM (4.5 g / L) containing d-biotin (Sigma), 4 μg / ml pantothenic acid (Sigma), 1.0 μM texamethasone (Sigma) and 0.5 mM isobutylmethylxanthine (Alditch) Containing glucose)]. At this time, the sample was added at the same time. Thereafter, the medium was replaced with fresh medium B every two days, and the sample was added again. On the 4th to 7th day after starting to add the medium B and the sample, replace the medium with the medium
C [5% inactivated FBS, 10 mM HEPES (pH 7.2) and 100 ng /
ml of DMEM containing bovine insulin (containing 4.5 g / l glucose)].

After culturing in medium C for 2 days, the cells were fixed with 5% formaldehyde, and oil globules and cell nuclei accumulated in the cells were stained using oil red O and hematoxylin. After taking a micrograph, the number of cells accumulating fat granules stained red from the photograph and the number of nucleated cells stained with hematoxylin were counted, and the adipogenesis rate was calculated from the following formula.

Adipocyte conversion rate (%) = 100 × the number of fat-grain-accumulating cells / the number of nucleated cells The cell fixing method, oil red O staining and hematoxylin staining were described in the experimental manual [Yoshio Mitomo, Shojiro Takayama "Clinical" Laboratory Course “Vol. 12,“ Pathology ”(1982), Medical and Dental Medicine Publishing].

Method for measuring LPL inhibitory effect [Beutler, BA et al. (1985). J. Imm
unol. 135: 3972-3977.] as follows. 3T3- transformed into adipocytes using the method described in the section "Method for measuring the inhibitory action of 3T3-L1 cells on morphological changes to adipocytes"
L1 cells were prepared. However, no sample is added during the induction of differentiation into adipocytes. The medium was then replaced with fresh medium C and the sample was added. After culturing for 18 hours, remove the medium,
The cells were washed twice with PBS (-) [phosophate buffer saline, manufactured by Nissui Pharmaceutical Co., Ltd.]. Next, medium D [5% inactivated FBS
, 10 mM HEPES (pH 7.2), DMEM (containing 4.5 g / liter glucose) containing 100 ng / ml bovine insulin and 10 U / ml sodium heparin (manufactured by Novo Inductry)] in 300 μl of each well. After further culturing for 1 hour, 100 μl of the culture supernatant was taken and used for measuring LPL activity. The measurement of the LPL inhibitory effect was performed in triplicate for each sample, and the average value was determined.

The measurement of LPL activity was performed by Nilsson-Ehle and Schotz.
[Nilsson-Ehle, P. and Schotz, MC (1976).
Lipid Res. 17: 536-541.]. 100 μl of the culture supernatant prepared as described above was mixed with an equal volume of substrate solution: 13 mM
Glycerol tri ^{[9,10 (n) - 3 H} ] oleic acid (51.8KB
q / μmol, Amersham), 1.3 mg / ml L-α-phosphatidylcholine distearoyl (Sigma), 20 mg / ml bovine serum albumin (Sigma),
135mM Tris-HCl [Tris-HCl (pH8.1), Sigma], 16.5% (v / v) glycerol, 16.5% (v / v) inactivated
The mixture was mixed with FBS: and reacted at 37 ° C. for 120 minutes. 1.05ml
0.1M potassium carbonate-borate buffer (pH10.5) and 3.25ml
Methanol: chloroform: heptane = 141: 125: 10
The reaction was stopped by adding 0 (v / v) and the mixture was stirred vigorously.
Centrifuged at 0xg for 15 minutes. The ³ H count of the water-methanol layer is measured with a liquid scintillation counter.
One unit of LPL activity was defined as the activity to produce 1 μmol of fatty acid per minute. Incidentally, 13 mM glycerol-tri of substrate solution ^{[9,10 (n) - 3 H} ] oleic acid, Amersham Corp. Kuriseroru-tri ^{[9,10 (n) - 3 H} ] oleic acid (37.0GBq / mol ) Was diluted with triolein (Sigma) and purified by silica gel column chromatography.

[0123]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1320 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA antisense: No Origin Organism name: Clover yellow bain virus Sequence characteristics 1 -1320 E CDS 10-1311 E mat-peptide sequence AAG TTC CAA GGG AAA AGT AAG AGA ACA AGA CAA AAG TTG AAG TTC AGA 48 Lys Phe Gln Gly Lys Ser Lys Arg Thr Arg Gln Lys Leu Lys Phe Arg 1 5 10 15 GCG GCA AGA GAC ATG AAG GAT CGT TAT GAA GTG CAT GCC GAT GAG GGG 96 Ala Ala Arg Asp Met Lys Asp Arg Tyr Glu Val His Ala Asp Glu Gly 20 25 30 ACT TTA GTG GAA AAT TTT GGA ACT CGT TAT TCA AAG AAA GGC AAG ACA 144 Thr Leu Val Glu Asn Phe Gly Thr Arg Tyr Ser Lys Lys Gly Lys Thr 35 40 45 AAA GGT ACT GTT GTG GGT TTG GGT GCA AAA ACA AGA CGG TTC ACT AAC 192 Lys Gly Thr Val Val Gly Leu Gly Ala Lys Thr Arg Arg Phe Thr Asn 50 55 60 ATG TAT GGT TTT GAC CCC ACG GAG TAT TCA TTT GCT AGG TAT CTT GAT 240 Met Tyr Gly Phe Asp Pro Thr Glu Tyr Ser Phe Ala Arg Tyr Leu Asp 65 70 75 80 CCA ATC ACG GGT GCA ACA TTG GAT GAA ACC CCA ATT CAC AAT GTA AAT 288 Pro Ile Thr Gly Ala Thr Leu Asp Glu Thr Pro Ile His Asn Val Asn 85 90 95 TTG GTT GCT GAG CAT TTT GGC GAC ATA AGG CTT GAT ATG GTT GAC AAG 336 Leu Val Ala Glu His Phe Gly Asp Ile Arg Leu Asp Met Val Asp Lys 100 105 110 GAG TTA CTT GAC AAA CAG CAC TTA TAC CTC AAG AGA CCA ATA GAA TGT 384 Glu Leu Leu Asp Lys Gln His Leu Tyr Leu Lys Arg Pro Ile Glu Cys 115 120 125 TAC TTT GTA AAG GAT GCT GGT CAG AAG GTG ATG AGG ATT GAT CTA ACA 432 Tyr Phe Val Lys Asp Ala Gly Gln Lys Val Met Arg Ile Asp Leu Thr 130 135 140 CCC CAC AAC CCA TTG TTG GCA AGC GAT GTT AGC ACA ACC ATA ATG GGT 480 Pro His Asn Pro Leu Leu Ala Ser Asp Val Ser Thr Thr Ile Met Gly 145 150 155 160 TAT CCT GAG AGA GAA GGT GAA CTC CGT CAA ACT GGA AAG GCA AGG TTA 528 Tyr Pro Glu Arg Glu Gly Glu Leu Arg Gln Thr Gly Lys Ala Arg Leu 165 170 175 GTC GAC CCA TCA GAG TTG CCC GCG CGG AAT GAG GAT ATT GAT GCA GAG 576 Val Asp Pro Ser Glu Leu Pro Ala Arg Asn Glu Asp Ile Asp Ala G lu 180 185 190 TTT GAG AGT CTA AAT CGC ATA AGT GGT TTG CGC GAC TAT AAT CCC ATT 624 Phe Glu Ser Leu Asn Arg Ile Ser Gly Leu Arg Asp Tyr Asn Pro Ile 195 200 205 TCA CAA AAT GTT TGC TTG CTA ACA AAT GAG TCA GAA GGC CAT AGA GAG 672 Ser Gln Asn Val Cys Leu Leu Thr Asn Glu Ser Glu Gly His Arg Glu 210 215 220 AAG ATG TTT GGA ATT GGA TAT GGT TCA GTG ATC ATT ACA AAT CAA CAT 720 Lys Met Phe Gly Ile Gly Tyr Gly Ser Val Ile Ile Thr Asn Gln His 225 230 235 240 CTG TTC AGA AGG AAT AAT GGG GAG TTA TCA ATT CAA TCC AAG CAT GGC 768 Leu Phe Arg Arg Asn Asn Gly Glu Leu Ser Ile Gln Ser Lys His Gly 245 250 255 TAC TTC AGA TGC CGC AAC ACC ACA AGC TTG AAG ATG CTG CCT TTG GAG 816 Tyr Phe Arg Cys Arg Asn Thr Thr Ser Leu Lys Met Leu Pro Leu Glu 260 265 270 GGA CAT GAC ATT TTG TTG ATT CAG TTA CCA AGG GAC TTT CCA GTG TTT 864 Gly His Asp Ile Leu Leu Ile Gln Leu Pro Arg Asp Phe Pro Val Phe 275 280 285 CCA CAA AAG ATT CGC TTT AGG GAG CCA AGA GTG GAT GAC AAA ATT GTT 912 Pro Gln Lys Ile Arg Phe Arg Glu Pro Arg Val Asp A sp Lys Ile Val 290 295 300 TTG GTC AGC ACA AAT TTC CAG GAA AAG AGT TCC TCG AGC ACG GTC TCA 960 Leu Val Ser Thr Asn Phe Gln Glu Lys Ser Ser Ser Ser Ser Thr Val Ser 305 310 315 320 GAG TCC AGT AAC ATT TCA AGA GTG CAG TCA GCC AAT TTC TAC AAG CAT 1008 Glu Ser Ser Asn Ile Ser Arg Val Gln Ser Ala Asn Phe Tyr Lys His 325 330 335 TGG ATC TCA ACA GTA GCA GGA CAC TGT GGA AAC CCT ATG GTT TCG ACT 1056 Trp Ile Ser Thr Val Ala Gly His Cys Gly Asn Pro Met Val Ser Thr 340 345 350 AAA GAT GGA TTT ATT GTA GGT ATC CAC AGT CTT GCT TCA TTG ACA GGC 1104 Lys Asp Gly Phe Ile Val Gly Ile His Ser Leu Ala Ser Leu Thr Gly 355 360 365 GAC GTT AAC ATC TTC ACA AGC TTT CCG CCG CAG TTT GAG AAC AAA TAT 1152 Asp Val Asn Ile Phe Thr Ser Phe Pro Pro Gln Phe Glu Asn Lys Tyr 370 375 380 CTA CAG AAG CTC AGT GAA CAC ACA TGG TGT AGT GGA TGG AAA CTA AAT 1200 Leu Gln Lys Leu Ser Glu His Thr Trp Cys Ser Gly Trp Lys Leu Asn 385 390 395 400 CTT GGA AAG ATT AGT TGG GGT GGA ATC AAC ATT GTG GAG GAT GCA CCT 1248 Leu Gly Lys Ile Ser Trp Gl y Gly Ile Asn Ile Val Glu Asp Ala Pro 405 410 415 GAA GAG CCC TTT ATA ACA TCC AAG ATG GCA AGC CTT CTT AGT GAT TTG 1296 Glu Glu Pro Phe Ile Thr Ser Lys Met Ala Ser Leu Leu Ser Asp Leu 420 425 430 AAT TGT TCA TTC CAA GCA AGT GCG 1320 Asn Cys Ser Phe Gln Ala Ser Ala 435 440 SEQ ID NO: 2 Sequence length: 440 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Biological name: Clover yellow bain Virus sequence characteristics 4-437 E mat-peptide sequence Lys Phe Gln Gly Lys Ser Lys Arg Thr Arg Gln Lys Leu Lys Phe Arg 1 5 10 15 Ala Ala Arg Asp Met Lys Asp Arg Tyr Glu Val His Ala Asp Glu Gly 20 25 30 Thr Leu Val Glu Asn Phe Gly Thr Arg Tyr Ser Lys Lys Gly Lys Thr 35 40 45 Lys Gly Thr Val Val Gly Leu Gly Ala Lys Thr Arg Arg Phe Thr Asn 50 55 60 Met Tyr Gly Phe Asp Pro Thr Glu Tyr Ser Phe Ala Arg Tyr Leu Asp 65 70 75 80 Pro Ile Thr Gly Ala Thr Leu Asp Glu Thr Pro Ile His Asn Val Asn 85 90 95 Leu Val Ala Glu His Phe Gly Asp Ile Arg L eu Asp Met Val Asp Lys 100 105 110 Glu Leu Leu Asp Lys Gln His Leu Tyr Leu Lys Arg Pro Ile Glu Cys 115 120 125 Tyr Phe Val Lys Asp Ala Gly Gln Lys Val Met Arg Ile Asp Leu Thr 130 135 140 Pro His Asn Pro Leu Leu Ala Ser Asp Val Ser Thr Thr Ile Met Gly 145 150 155 160 Tyr Pro Glu Arg Glu Gly Glu Leu Arg Gln Thr Gly Lys Ala Arg Leu 165 170 175 Val Asp Pro Ser Glu Leu Pro Ala Arg Asn Glu Asp Ile Asp Ala Glu 180 185 190 Phe Glu Ser Leu Asn Arg Ile Ser Gly Leu Arg Asp Tyr Asn Pro Ile 195 200 205 Ser Gln Asn Val Cys Leu Leu Thr Asn Glu Ser Glu Gly His Arg Glu 210 215 220 Lys Met Phe Gly Ile Gly Tyr Gly Ser Val Ile Ile Thr Asn Gln His 225 230 235 240 Leu Phe Arg Arg Asn Asn Gly Glu Leu Ser Ile Gln Ser Lys His Gly 245 250 255 Tyr Phe Arg Cys Arg Asn Thr Thr Ser Leu Lys Met Leu Pro Leu Glu 260 265 270 Gly His Asp Ile Leu Leu Ile Gln Leu Pro Arg Asp Phe Pro Val Phe 275 280 285 Pro Gln Lys Ile Arg Phe Arg Glu Pro Arg Val Asp Asp Lys Ile Val 290 295 300 Leu Val Ser Thr Asn Phe Gln Glu Lys Ser SerSer Ser Thr Val Ser 305 310 315 320 Glu Ser Ser Asn Ile Ser Arg Val Gln Ser Ala Asn Phe Tyr Lys His 325 330 335 Trp Ile Ser Thr Val Ala Gly His Cys Gly Asn Pro Met Val Ser Thr 340 345 350 Lys Asp Gly Phe Ile Val Gly Ile His Ser Leu Ala Ser Leu Thr Gly 355 360 365 Asp Val Asn Ile Phe Thr Ser Phe Pro Pro Gln Phe Glu Asn Lys Tyr 370 375 380 Leu Gln Lys Leu Ser Glu His Thr Trp Cys Ser Gly Trp Lys Leu Asn 385 390 395 400 Leu Gly Lys Ile Ser Trp Gly Gly Ile Asn Ile Val Glu Asp Ala Pro 405 410 415 Glu Glu Pro Phe Ile Thr Ser Lys Met Ala Ser Leu Leu Ser Asp Leu 420 425 430 Asn Cys Ser Phe Gln Ala Ser Ala 435 440 SEQ ID NO: 3 Sequence length: 25 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTCCATGGGG AAAAGTAAGA GAACA 25 SEQ ID NO: 4 Sequence length: 20 Sequence type : Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACTCTGAGAC CGTGCTCGAG 20 SEQ ID NO: 5 Sequence length: 20 : Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGGAAAAGAG TTCCTCGAGC 17 SEQ ID NO: 6 Sequence length: 36 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AATTGTTCAT TCCAAGCACC TGGGCCACCA CCTGGC 36 SEQ ID NO: 7 Sequence length: 36 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCCAAGTGGT GGCCCAGGTG CTTGGAATGA ACAATT 36 SEQ ID NO: 8 Sequence length: 30 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTGTCAGCAC ACCTGGGAGC TGTAGAGCTC 25 SEQ ID NO: 9 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Protein Ala Pro Gly Pro Pro Gly 7 15 SEQ ID NO: 10 Sequence length: 7 Sequence type: amino acid Topology: linear Sequence type: protein Pro Gly Pro Pro Gly Pro 7 15

[0124]

Industrial Applicability The nuclear inclusion a gene derived from clover yellow bain virus of the present invention can be stably maintained and expressed in Escherichia coli. Furthermore, the expressed nuclear inclusion body a protein retains its activity as a specific protease,
Its activity can also work in the form of a fusion of a protease and a foreign protein. Therefore, when the N-terminal amino acid is Ala or Ser
Alternatively, it has become possible to easily and easily express a large amount of a polypeptide beginning with Gly, a polypeptide beginning with Pro at the N-terminal amino acid, or a target protein having an arbitrary amino acid residue at the N-terminus.

[0125]

[Brief description of the drawings]

FIG. 1 is a restriction map of the cDNA of the NIa region isolated from CYVV-cDNA.

FIG. 2 is a construction diagram of a plasmid pKNI5 ′ containing a 5 ′ region of NIa.

FIG. 3 is a construction diagram of a plasmid pKNI5IL containing a part of the IL-11 gene and the 5 ′ region of NIa.

FIG. 4 is a diagram of primers used for preparing a CIN3 DNA fragment and a 5 ′ IL DNA fragment in which the 3 ′ end of the NIa gene is fused to the 5 ′ end of the IL-11 gene.

FIG. 5 is a fusion diagram of a CIN3 DNA fragment and an IL5 ′ DNA fragment obtained by PCR.

FIG. 6 is a diagram showing the construction of plasmid pKSUN9.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // (C12N 1/21 C12N 15/00 ZNAA C12R 1:19) (C12N 9/50 C12R 1:19) (58) Field (Int. Cl. ⁷ , DB name) C12N 15/00-15/09 C07K 14/08 C12P 21/06 BIOSIS (DIALOG) WPI (DIALOG) SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq

Claims (25)

(57) [Claims] (1) a molecule containing the amino acid sequence of amino acid numbers 4 to 437 of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and Gln-Ala, Gln-Gly or Gln-Ser in the substrate peptide;
A protein having a protease activity of specifically hydrolyzing a peptide bond between them, or an amino acid sequence in which one or more amino acid residues of the amino acid sequence are deleted, inserted or substituted, and the protease A protein having activity. 2. The molecule contains the amino acid sequence of amino acids 4 to 437 of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and Gln-Ala, Gln-Gly or Gln-Ser in the substrate peptide.
A protein having a protease activity of specifically hydrolyzing a peptide bond between the two. [3] a D encoding the protein of [1];
NA. (4) D encoding the protein of (2)
NA. 5. The DNA according to claim 3, wherein the molecule contains the nucleotide sequence of nucleotide numbers 10 to 1311 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. 6. The D according to claim 3, wherein
A recombinant DNA vector comprising NA. 7. A host cell transformed with the recombinant DNA vector according to claim 6. 8. The host cell according to claim 7, wherein the transformed host cell is Escherichia coli. 9. [I] A protein in which the following components are peptide-bonded from the N-terminal side to the C-terminal side: the protease according to claim 1 or 2, Gln-Ala, Gln-Gly or The linker selected from any one of Gln-Ser, peptide component X, [II] The autocatalysis of the component, Ala-X, Gly-
A protein which produces a peptide selected from any one of X and Ser-X. 10. [I] A protein in which the following components are peptide-bonded from the N-terminal side to the C-terminal side: the protease according to claim 1 or 2, Gln-Ala, Gln-Gly or A linker selected from any one of Gln-Ser, Pro-Y ₁ (where Y ₁ represents an arbitrary peptide component), [II] Ala-Pro-Y by autocatalysis of the component.
_1, it generates a Gly-Pro-Y ₁ or Ser-Pro-Y ₁ of the peptide selected from any one, then, and generates a Pro-Y ₁ by the catalytic action of aminopeptidase P protein. 11. [I] A protein in which the following components are peptide-bonded from the N-terminal side to the C-terminal side: the protease according to claim 1 or 2, Gln-Ala, Gln-Gly or A linker selected from any one of Gln-Ser, Pro-Y ₂ (where Y ₂ represents an arbitrary peptide component), [II] Ala-Pro-Y by autocatalysis of the component.
_2, generates a Gly-Pro-Y ₂ or Ser-Pro-Y ₂ peptide selected from any one, then, generates a Pro-Y ₂ by the catalytic action of aminopeptidase P, then proline iminopeptidase A protein which produces Y ₂ by the catalytic action of 12. [I] A protein in which the following components are peptide-bonded from the N-terminal side to the C-terminal side: the protease according to claim 1 or 2, Gln-Ala, Z (provided that: Z represents an arbitrary peptide component.), [II] A protein characterized in that Ala-Z is generated by autocatalysis of the component, and then Z is generated by catalysis of alanine aminopeptidase. 13. Gln-Ala, Gln-Gly and Gln-Ser at the 3 ′ end of the DNA according to any one of claims 3 to 5.
A DNA in which a DNA encoding a heterologous protein is connected via a DNA encoding an amino acid sequence selected from the group consisting of: 14. A recombinant DNA vector comprising the DNA according to claim 13. 15. A host cell transformed with the recombinant DNA vector according to claim 14. 16. The host cell according to claim 15, wherein the transformed host cell is Escherichia coli. 17. The following three steps: [I] The following components from the N-terminal side to the C-terminal side: The protease according to claim 1 or 2, Gln-Ala, Gln-Gly or Gln-Ser A DNA selected from the group consisting of a linker selected from the group consisting of:
Transforming a host cell with a recombinant DNA expression vector comprising: [II] culturing the host cell, and in the host cell, Ala-X, Gly-X or
A step of producing a peptide selected from any one of Ser-X, [III] from the host cell or a culture solution of the host cell,
isolating and purifying a peptide selected from any one of a-X, Gly-X or Ser-X; and selecting from any one of Ala-X, Gly-X or Ser-X. Method for producing a peptide. 18. The following three steps: [I] the following components from the N-terminal side to the C-terminal side: the protease according to claim 1 or 2, Gln-Ala, Gln-Gly or Gln-Ser A DNA selected from the group consisting of: a linker selected from any one of the following: Pro-Y ₁ (where Y ₁ represents an arbitrary peptide component);
Transforming a host cell with a recombinant DNA expression vector comprising the following steps: [II] culturing the host cell, and in the host cell, Ala-Pro-Y ₁ , Gly-Pro
Yielding peptide selected from any one of -Y ₁ or Ser-Pro-Y _1, further generates a Pro-Y ₁ by the catalytic action of aminopeptidase P expressed from vectors derived from the host cell or foreign [III] Pr from the host cell or a culture solution of the host cell.
isolating o-Y _1, purifying, preparation of Pro-Y ₁ comprising more. 19. The following three steps: [I] The following components from the N-terminal side to the C-terminal side: The protease according to claim 1 or 2, Gln-Ala, Gln-Gly or Gln-Ser A linker selected from any one of the following: Pro-Y ₂ (where Y ₂ represents an arbitrary peptide component);
Transforming a host cell with a recombinant DNA expression vector comprising: [II] culturing the host cell, and in the host cell, Ala-Pro-Y ₂ , Gly -Pro
-Y ₂ or Ser-Pro-Y ₂ to produce a peptide, and then Pro-Y ₂ is catalyzed by aminopeptidase P derived from a host cell or expressed from a foreign vector. And further producing Y ₂ by the catalysis of proline iminopeptidase derived from the host cell or expressed from a foreign vector. [III] From the host cell or a culture solution of the host cell, Y
Isolating _2, purifying, more composed preparation of Y _2. 20. The following three steps: [I] The following components in the direction from the N-terminal to the C-terminal: Protease according to claim 1 or 2, Gln-Ala, Z (where Z is optional) Represents a peptide component of.), DNA encoding a protein having a peptide bond
Transforming a host cell with a recombinant DNA expression vector comprising: [II] culturing the host cell, and generating Ala-Z by autocatalysis of a component in the host cell;
A step of producing Z by the catalytic action of alanine aminopeptidase derived from a host cell or expressed from a foreign vector; [III] Z from the host cell or a culture solution of the host cell;
A process for isolating and purifying Z. 21. The method according to claim 17, wherein the host cell is Escherichia coli.
The production method according to any one of 8, 19 and 20. 22. In a test tube, any one of Gln-Ala, Gln-Gly or Gln-Ser and a peptide component X are peptide-bonded by the catalytic action of the protease according to claim 1 or 2. A peptide selected from any one of Ala-X, Gly-X or Ser-X is produced from the protein, and selected from any one of Ala-X, Gly-X or Ser-X from the reaction solution. A method for producing a peptide selected from any one of Ala-X, Gly-X and Ser-X, comprising isolating and purifying a peptide. 23. In a test tube, any one of Gln-Ala, Gln-Gly or Gln-Ser and Pro-Y ₁ (where Y ₁ is the same) by the catalytic action of the protease according to claim 1 or 2. It represents any peptide component.) than protein is a peptide _{bond, Ala-Pro-Y 1,} Gly-Pro-Y 1 or
Yielding peptide selected from any one of Ser-Pro-Y _1, further yielding Pro-Y ₁ by the catalytic action of aminopeptidase P, from the reaction solution, isolate Pro-Y _1, purified It consists in, Pro-Y ₁ of the production method. 24. In a test tube, one of Gln-Ala, Gln-Gly or Gln-Ser and Pro-Y ₂ (where Y ₂ is Ala-Pro-Y ₂ , Gly-Pro-Y ₂ or Gly-Pro-Y ₂
Yielding peptide selected from any one of Ser-Pro-Y _2, then yielding Pro-Y ₂ by the catalytic action of aminopeptidase P, further generates a Y ₂ by the catalytic action of proline iminopeptidase A method for producing Y ₂ comprising isolating and purifying Y ₂ from a reaction solution. 25. A protein in which Gln-Ala and Z (where Z represents an arbitrary peptide component) is peptide-bonded in a test tube by the catalytic action of the protease according to claim 1 or 2. , Ala-Z, and then catalyzed by alanine aminopeptidase, Z
And then isolating and purifying Z from the reaction solution.