JP2844870B2 - Method for producing polypeptide by recombinant DNA method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a polypeptide of interest by culturing a microorganism transformed by a recombinant DNA method. Correct, more uniform N
The present invention relates to a method for producing a polypeptide having a terminal.

By establishing a technique for obtaining a polypeptide having a uniform N-terminus, for example, in the application of a target polypeptide to medical treatment, it is thought that it can be used to overcome problems with antigenicity.

[Conventional technology]

When a gene encoding a target polypeptide derived from a eukaryote or the like is simply expressed by Escherichia coli using recombinant DNA technology, the gene is often simply expressed at a high level by a widely used gene expression system. May be a mixture in which a methionine residue derived from a translation initiation codon is added, or a mixture in which a methionine residue is added or not, and a mixture in which up to several amino acids in a target polypeptide are deleted.

Such an N-terminal heterogeneous polypeptide may not only change its stability and physiological functions, but also may have an antigenicity in the body when applied to pharmaceuticals. It is also conceivable to have inappropriate effects.

Therefore, conventionally, in order to align the N-terminus of the target polypeptide, a purified and extracted target polypeptide precursor is
Separately purified various Escherichia coli aminopeptidases were allowed to act in a test tube, and the N-terminal was homogenized through such a process.

Recently, A. Ben-bassat et al. (J. Bacteriology, 169, 751-7
57, 1987) clones methionine aminopeptidase (MAP) from Escherichia coli and uses it for the purpose of deleting the N-terminal methionine when expressing and producing interleukin 2 and ricin A in Escherichia coli.

[Problems to be solved by the invention]

However, the addition of purified aminopeptidase from E. coli to the purified target polypeptide results in increased impurities in the target polypeptide fraction. On the other hand, aminopeptidase itself requires cultivation of a bacterium that produces the aminopeptidase and purification of the enzyme, costs and time are required, and this method is considered to be improved in terms of economy.

In addition, in the method of removing the N-terminal methionine using methionine aminopeptidase, as a result of the action in vivo, 60% of the interleukin 2 has its excision from the N-terminus to the second alanine residue. In addition, N-terminal control of the produced polypeptide in the producing cells has not been established yet.

An object of the present invention is to solve the above problems and to provide a technique for preparing the N-terminus more uniformly in a target polypeptide-producing cell.

[Means for solving the problem]

The present inventors have noted that aminopeptidase P of Escherichia coli has extremely high specificity. By introducing the present aminopeptidase into the target polypeptide-producing cells, a highly efficient and economical method for producing a target polypeptide having a more uniform N-terminus was successfully developed, and the present invention was completed. I came to.

That is, the present invention provides a method for culturing an organism having a vector containing an aminopeptidase P gene and a vector containing a gene encoding a target polypeptide, and collecting the target polypeptide accumulated in the organism and / or in the culture solution. And a method for producing a polypeptide.

Aminopeptidase P (Aminopeptidase P [EC 3.
4.11.5], hereinafter referred to as AP-P. ) When the second amino acid from the N-terminal is bromoline, the first amino acid is cleaved and released. Therefore, X is cleaved and released at the amino acid sequence of X-Pro-Y- (X is an arbitrary amino acid, Y is an amino acid other than bromoline). The aminopeptidase P gene can be removed from any organism that produces aminopeptidase P, but can be isolated, for example, from E. coli.

Although the kind of the target polypeptide is not limited, it includes various physiologically active substances, and includes, for example, various interleukins, interferons, colony forming factors, lymphokines such as lymphotoxin, and various growth hormones. As the polypeptide in which the N-terminus of the target polypeptide is a brolin residue, human IL-6, basic fibroblast growth factor and the like are preferable. Human interleukin 6 (IL-6) is BSF-
2 (B-cell Stimulatory Factor-2)
Initially, factors that differentiate B cells into antibody-producing cells (BCDF: B-
It is a lymphokine cloned as a cell differentiation factor). (T.Hirano et.al.Nature
324, 73 (1986)] However, IL-6 is not merely responsible for B cell differentiation ability, but is produced by various cells, and in addition to antibody production enhancement ability, hematopoietic function enhancement ability and differentiation inducing ability. And so on. The first half of the gene encoding the target polypeptide (N of the target polypeptide
The DNA portion encoding the terminal region) is adenine (A) as long as the amino acid sequence of the polypeptide to be produced is not changed.
Alternatively, it is more preferable to use a DNA having a base sequence rich in thymine (T).

The vector containing the aminopeptidase P gene and the vector containing the gene encoding the target polypeptide may be contained in one vector, or may be separate vectors.

The method for obtaining the vector containing the aminopeptidase P gene and the gene encoding the target polypeptide is described below.For example, the chromosome of Escherichia coli is determined by a restriction enzyme, cloned into an appropriate vector, and transformed into Escherichia coli. In general, a vector containing the AP-P gene can be obtained by measuring the aminopeptidase P activity of each clone and preparing a plasmid DNA from the clone having the increased AP-P activity.

Also, generally, the target gene is prepared by preparing mRNA from cells producing the target polypeptide, preparing its cDNA, and then using a DNA probe prepared from information on a partial amino acid sequence of the target polypeptide to convert the target gene. Can be obtained. Alternatively, when the amino acid sequence of the target polypeptide is known,
It is also possible to directly produce the target gene with synthetic DNA.

When the vector containing the aminopeptidase P gene or the vector containing the gene encoding the target polypeptide does not contain a promoter or translation initiation codon, it is necessary to incorporate them.

Derived promoters not at questioning can be used, for example, trp promoter of E. coli, lac promoter, tac promoter synthetic promoter, trc promoter, P _l promoters of λ phage, etc. P _R promoter.

The ATG is added to the translation initiation codon immediately before the region encoding the target polypeptide on the DNA base sequence.

The organism into which the above-described gene expression vector incorporating these vectors is incorporated may be any of prokaryotes and eukaryotes. Examples of prokaryotes include Escherichia coli and Bacillus subtilis. Eukaryotes are, for example, yeast. Known methods may also be used to incorporate the gene expression vector into these organisms.For example, in Escherichia coli, the cells in the logarithmic growth phase are treated with 50 mM calcium chloride in ice for about 30 minutes, so that the cell wall of Escherichia coli can be treated. The structure changes, DNA is subsequently injected, and after about 10 minutes, a heat treatment is performed at 30 ° C. to 42 ° C. for 2 minutes, and then a culture medium is added, and the cells are cultured at 30 ° C. to 37 ° C. for about 60 minutes. Can be incorporated into living things.

The aminopeptidase P gene of Escherichia coli is already pUC19
Cloned on the plasmid of
105, 412-416 (1989)] This pUC-based plasmid cannot stably coexist in a single cell with a plasmid pBR-based plasmid used for large-scale expression of a target polypeptide. Therefore, the AP-P gene was re-transferred to pACYC177, which is a plasmid that can coexist with an expression vector having a pBR or pUC replication origin.
By producing a high-producing strain of P, a host for producing the target polypeptide was constructed.

On the other hand, the AP-P activity was increased by transferring the AP-P gene onto the target polypeptide expression plasmid, and conversely, by mounting the gene expression system of the target polypeptide on the AP-P expression plasmid. It has become possible to produce the target polypeptide in the production cells and obtain a more uniform N-terminal polypeptide. Furthermore, the aminopeptidase P activity of the host that expresses the target polypeptide can be increased by enhancing the expression of the aminopeptidase P gene in the chromosome, thereby aligning the N-terminals of the target polypeptide.

The target polypeptide and aminopeptidase P can be accumulated in the organism or in the culture solution by culturing organisms holding these vectors. The culture medium may be a known medium capable of culturing each organism, and the culture conditions may be known conditions. After the culture, the target polypeptide may be obtained by a known method.

[Action]

When an organism carrying the vector containing the aminopeptidase P gene and the vector containing the gene encoding the target polypeptide is cultured, first, a precursor polypeptide containing the target polypeptide and aminopeptidase P are produced in the organism or in the culture solution. Stored. Thus, aminopeptidase P acts on the precursor polypeptide to generate a N-terminally uniform target polypeptide.

〔Example〕

Example 1 Production method of human interleukin 6 The N-terminal amino acid of mature human interleukin 6 (hereinafter abbreviated as IL-6) is broline (Pro), but recombinant IL-6 produced by the recombinant DNA method is Generally, methionyl IL- with methionine added to its N-terminus
6, or a mixture with methionine excision.

Therefore, the present inventors have maintained a plasmid in which aminopeptidase P was cloned, and used Escherichia coli having enhanced aminopeptidase P activity as a host for producing IL-6.
By cloning the plasmid into a 6-production plasmid and mass-producing aminopeptidase P, IL-6 having N-terminals aligned with brolin was successfully produced.

(1) Example of producing IL-6 in a host with increased AP-P activity Construction of direct expression plasmid DNA for producing IL-6 pBSF-2D (E. coli pTBCDF-0 containing this plasmid)
2 / HB101 has been deposited as FERM P-9061. )
Is the IL described in J. Biochem. 104, 30-34 (1988).
-6 is a direct expression plasmid. It has a pBR origin of replication, and gene expression systems include the trp promoter and t
The SD sequence of rpL is deployed, and a trpA terminator is located downstream of the gene. However, pBSF-2D
After induction of the trp promoter by the addition of indoleacrylic acid (hereinafter abbreviated as IAA), the expression level of IL-6 was low, and no IL-6 granules could be confirmed in the cells.

Further, the translation termination codon on the IL-6 cDNA is amber (TAG), and Escherichia coli HB101 has an amber suppressor, so that the synthesis of the IL-6 polypeptide can be completely terminated at the TAG codon. Added IL-
6 may be produced.

Therefore, first, the conversion of pBSF-2D origin of replication to pUC and IL
Plasmid pBSF2C-DUC was constructed as shown in FIG. 1 with the aim of converting the -6 translation stop codon into an ocher (TAA). In the figure, the black band indicates the trp promoter / operator, the black triangle indicates the trpA terminator, and the white band indicates the IL-6 coding region.

As shown in FIG. 1, first, pBSF-2D was replaced with a restriction enzyme EcoR.
I, PvuII, and DNA fragment containing IL-6 cDNA and pUC19
(Messing, R. (1983) Methods in Enzymology, 101, 20-
78) was ligated with a large DNA fragment obtained by digestion with restriction enzymes Hinc II and EcoRI with T4 DNA ligase to obtain pBSF2-DUC having a replication origin as a pUC system.

PT13SNco (E. coli AJ-1 containing this plasmid)
2447 has been deposited as FERM P-10757. ) [J. Bio
chem. 104, 30-34 (1988)].
pT13SNco-UC was constructed by ligating the small DNA fragment obtained by digestion with vu II with the above pUC19 fragment.

Next, a large DNA fragment obtained by cleaving pBSF2-DUC with restriction enzymes HindIII and BanII, a synthetic DNA fragment BSCT (for IL-6 translation termination codon conversion) prepared by an ordinary method,
Furthermore, pT13SNco-UC was digested with restriction enzymes BamHI and HindIII, and the resulting DNA fragment having a trpA terminator and three DNA fragments was ligated with T4 DNA ligase to give pB13.
SF2C-DUC was constructed. The synthetic DNA fragment BSCT has the following sequence.

On the other hand, in order to arrange two SD sequences before the IL-6 coding region, a part of the IL-6 cDNA was modified with chemically synthesized DNA as shown in FIG. 2 (synthetic DNA fragment BSFN). The broken line in the figure indicates the SD array.

This synthetic DNA is a 39-mer to 45-mer chemically synthesized DN.
It was prepared as follows from six A fragments (BSFN1F, 1R, 2F, 2R, 3F, 3R). That is, after each DNA fragment chemically synthesized by a DNA synthesizer (model 380B manufactured by Applied Biosystems) is phosphorylated (BSFN1F and BSFN3R are not phosphorylated), BSFN1F and BSFN1 are not phosphorylated.
R, BSFN2F and BSFN2R, BSFN3F and BSFN3R were annealed, then they were mixed and ligated with T4 DNA ligase. Next, the reaction solution was subjected to 5% polyacrylamide gel electrophoresis, and after electrophoresis and ethidium bromide staining, a DNA band (approximately
130 bp) and purified to obtain a synthetic partial IL-6DN
A got. As shown in FIG. 3, this synthetic DNA fragment BSFN was ligated to a large pUC19 DNA fragment digested with restriction enzymes EcoR I and Acc I using T4 DNA ligase to obtain pUC-SD6N. The lattice band part in the figure shows the synthetic DAN part. The DNA base sequence of the synthetic DNA fragment BSFN was confirmed by using the pUC-SD6N from both directions by the dideoxy method.

Subsequently, as shown in FIG. 4, pUC-SD6N was replaced with restriction enzyme C.
After cutting with laI, blunt-ending with Klenow fragment, IL-6 obtained by digestion with TthHB81
DNA fragment (about 120 bp) having a part of pBSF2C-DUC
A DNA fragment (about 470 bp) having the latter half of IL-6 obtained by digestion with vu II, BamHI and TthHB81, and pBR322 as Eco RV,
PBR-SD7 was constructed by digesting with BamHI and ligating the resulting large DNA fragment with T4 DNA ligase. In the figure, the black band indicates the trp promoter / operator, the black triangle indicates the trpA terminator, the white band indicates the IL-6 coding region, and the lattice band indicates the synthetic DN.
Part A is shown.

Then the same fourth, as shown in the figure, the pBR-SD7 dam ^- After transformation into E. coli strains by the usual method, pBR-SD7 plasmids were prepared (dam ^-) give, which Cla I, BamH By cutting with I, a DNA fragment having semi-synthetic IL-6 DNA was obtained. Then, to this DNA fragment, a DNA fragment having a trp promoter and a trpA terminator obtained by cleaving pBSF2C-DUC again with restriction enzymes Cla I and BamHI was used as T4 DNA.
By ligating with ligase, a plasmid pBSF2-SD7 for high expression of IL-6 and direct expression was constructed and obtained.

Subsequently, E. coli HB101 was transformed with pBSF2-SD7, and IL
-6 high producing bacteria (E. coli pBSF2-SD7 / HB101 AJ-12448, F
ERM P-10758) was obtained.

Construction of plasmid DNA for mass production of aminopeptidase P. Plasmid pAPP4 for high production of aminopeptidase P is JB
105, 412-416 (1989), the aminopeptidase P derived from Escherichia coli HB101
It was cloned into UC19.

Since this pAAP4 cannot coexist with the above pBSF2-SD7 in one E. coli (indicating incompatibility), AP-P on pAPP4
Plasmid vector pA capable of coexisting gene with pBSF2-SD7
Transferred to CYC177.

As shown in FIG. 5, pACYC177 was first converted to the restriction enzyme Hinc.
II, followed by calf intestine alkaline phosphatase from calf intestine
Acted. On the other hand, a restriction enzyme Pvu II was allowed to act on pAPP4 to obtain a DNA fragment containing the AP-P gene. Next, both DNA fragments were ligated with T4 DNA ligase, and the origin of replication was derived from p15A, and AP-P
PAPP4-Km carrying the gene was constructed.

In the figure, Plac is the lactose promoter and the white band is AP
-P gene portion, Km ^r is kanamycin resistance and Ap ^r
Indicates ampicillin resistance, respectively. Note that this
The AP-P gene is under the control of the lac promoter. Next, this plasmid was transformed into E. coli HB101, and AP
-P high production department was obtained.

Similarly, an AP-P production plasmid pAPP4-KmΔB in which the Iac promoter on the present pAPP4-Km was deleted was constructed. As shown in FIG. 6, this transforms pAPP4-Km into restriction enzyme B.
By cutting with amHI and performing self-ligation, pAPP4-KmΔ
B was prepared. E. coli HB10
One strain was transformed to obtain an AP-P-producing strain.

Subsequently, Escherichia coli HB101 carrying PAAP4-Km or pAPP4-KmΔB was transformed with pBSF2-SD7 and selected with ampicillin and kanamycin, so that Escherichia coli pBSF2-SD7 and pAPP4-Km / HB101 (AJ
-12456) and E. coli pBSF2-SD7, pAPP4-KmΔB / HB101
(AJ-12457) was obtained. The strains are respectively No. 10766 and No. 10767.

Culturing and obtaining the product The selected transformant strain E. coli pBSF2-SD7, pAPP4-Km / H
B101 (FERM P-10766) or E. coli pBSF2-SD7, pAPP
4-KmΔB / HB101 (FERM P-10767) was added to a 2 × TY medium containing 50 μg / ml ampicillin and 50 μg / ml kanamycin (1.
6% bactotripton, 1% yeast extract, 0.5% NaCl, pH
7.0) Grow overnight in 5 ml at 30 ° C. Then, 5 ml of each culture suspension was added to 100 ml of M9-casamino acid medium (0.6% Na ₂ HPO ₄
・ 12H ₂ O, 0.3% KH ₂ PO ₄ , 0.05% NaCl, 0.1% NH ₄ Cl, 0.05% Mg
_{SO 4 · 7H 2 O, 0.00147} % CaCl 2, 0.2% glucose, 0.2% casamino acid, 0.02% L-leucine, 0.02% L-Burorin, 0.000
2% thiamine hydrochloride, 100 μg / ml ambicilin, 50 μg / ml
Kanamycin, pH 6.9) and cultured at 37 ° C. for 3 hours. Thereafter, 3-indoleacrylic acid (IAA) was added to a concentration of 25 μg / ml, and induction culture was performed at 37 ° C. for 21 hours.

Some of these cell suspensions were analyzed by phase contrast microscopy.
When observed at a magnification of 1500, the shape of granules was observed in both the producing cells.

Subsequently, each of the cell suspensions cultured as described above was centrifuged, and the cells were collected and concentrated at 30 mM NaC so that the cells were concentrated twice.
After adding 20 mM Tris-HCl buffer (pH 7.5) containing l, 37.5 ml of a lysozyme solution dissolved at 1 mg / ml with 0.5 M EDTA (pH 8.0) was added thereto. After stirring, the mixture was left on ice for 1 hour. Then, the cells were disrupted by sonication, and each granule was recovered by centrifugation at 6000 rpm for 5 minutes.

The granules were solubilized with 6 M guanidine hydrochloride, and the concentration of the target polypeptide IL-6 was adjusted to 100 μg / ml, and a 2 M guanidine hydrochloride solution was prepared, and oxidized glutathione 1 mM and reduced glutathione 10 mM were added thereto. And left at room temperature for 10-16 hours at pH 8.0. Next, Sepha
Guanidine hydrochloride was removed by gel filtration using dex G-25,
IL-6 fractions from both producers were obtained.

On the other hand, pBSF2-SD7 / HB101 was similarly cultured to prepare an IL-6 fraction. However, antibiotics depended on the addition and selection of ampicillin alone.

By SDS polyacrylamide gel electrophoresis, the molecular weight of the substances was almost the same as the value calculated from the amino acid composition.However, as a result of testing the N-terminal amino acid sequence with a protein sequencer, pBSF2-SD7 / HB101-derived
About 40% of the N-terminus of IL-6 had methionine residues, but pBSF2-SD7, pA
PP4-Km / HB101, pBSF2-SD7, and pAPP4-KmΔB / HB101 all have N-terminal methionine residues below the detection limit, and about 95% or more of the polypeptide having the sequence of mature IL-6, which is the target product. It was confirmed that there was.

In addition, as a physiological activity, a human B cell line CL4 producing IgM (T. Hirano et al., Proc. Natl. Acad. Sci., 82, 5490.198)
Using 5), the IgM production activity was measured. In other words, a test solution for measuring antibody production activity and 6 × 10 ³ CL4
FRMI 1640 medium containing CS (100 units of penicillin per ml,
Containing 100 μg of streptomycin, 10 μg of gentamicin and 16 mM of NaHCO ₃ ), and culturing the mixture in a 96-well microplate for 3 days at 37 ° C. in the presence of 5% CO _2. It was measured by immunoassay. Further, under these conditions, the antibody producing activity showing 50% of the maximum IgM production amount (the highest CL4 reaction) was 1 u / ml.

As a result, the three recombinant IL-6 obtained as described above
Showed a specific activity of about 6.0 u / ng, which was found to have the same physiological activity as natural IL-6 obtained from animal cells.

As described above, by producing aminopeptidase P in a large amount in IL-6 producing cells as in the present invention,
A more uniform N-6 terminus of IL-6 polypeptide could be produced and obtained.

(2) Cloning of the AP-P gene on an IL-6-producing plasmid, from which expression of IL-6 was produced in a host with enhanced AP-P activity Construction of plasmid DNA As shown in FIG. pBSF2-SD7 with restriction enzyme Hi
After digestion with nd III, followed by blunting the end with Klenow fragment, the DNA fragment having IL-6 gene obtained by digestion with EcoRI and pAPP4 were digested with restriction enzyme SacI, and T4 DNA 3 'by polymerase
After removing the protruding end to make it a blunt end, a large DNA fragment obtained by digestion with EcoR was ligated with T4 DNA ligase to give a plasmid pBSF encoding both IL-6 and AP-P.
2-SD7P1 was constructed and obtained. In the figure, the black band indicates the trp promoter, the lattice band indicates the IL-6 coding region, and the white band indicates the AP-P coding region.

Subsequently, by transforming this plasmid into Escherichia coli HB101, a production host vector system having high AP-P activity and simultaneously expressing IL-6 in large amounts could be constructed. In addition,
This bacterium (pBSF2-SD7P1 / HB101 AJ-12458) is
No. 10768.

Culture and acquisition of product E. coli pBSF2-SD7P1 / HB101 in the same manner as in Example 1 (1).
(FERM P-10768) was cultured to prepare and obtain IL-6.

IL-6 obtained by this method also has a physiological activity, and as in Example (1), the N-terminal amino acid sequence naturally has a mature form.
It should be considered IL-6.

As described above, a human IL-6 polypeptide having a more uniform N-terminus can be produced and obtained by using the method of the present invention.

(3) The AP-P gene on the E. coli chromosome
Expression and production of IL-6 in a host with enhanced -P activity Integration of the AP-P gene into the chromosome using homologous recombination ability of Escherichia coli As shown in FIG. A DNA fragment containing the AP-P gene obtained by digestion with
(Takeshita.S et al. (1987) Gene, 61, 63-74) and inserted into the Kpn I site to construct pHAP4. PMAN031 (Matsuyama.S), a plasmid whose replication function has become temperature-sensitive
(1985) J. Bacteriol. Voll62, 1196 to 1202) is digested with the restriction enzymes PstI and HindIII.
PTS1 was constructed by cloning the NA fragment into the PstI-HindIII fragment of pBR322. Next, this pTS1
A large DNA fragment cut with EcoR I and Hind III, and p
Similarly, HAP4 is digested with EcoR I and Hind III, and the resulting AP
By linking the DNA fragment containing the -P gene, pTSAP1 in which the AP-P gene was mounted on a plasmid having a temperature-sensitive replication origin was constructed.

Subsequently, the constructed pTSAP1 was transformed into Escherichia coli RR1 strain, and a strain which became ampicillin resistant was selected. This strain is grown in 5 ml of L medium (containing 50 μg / ml of ampicillin) for 30 minutes.
After culturing for 6 hours and diluting, the resultant was spread on an L-plate (containing 50 μg / ml of ampicillin), cultured overnight at 42 ° C, and a colony grown at 42 ° C was selected.

Next, in order to delete the RecA function of this strain, the transduction method using P1 phage was used. E. coli JC 10240 strain recA ^- a is, the recA ^- a strain which Tn10 in the vicinity are present. According to a conventional method, a P1 phage was prepared, which was then infected with the JC10240 strain to obtain a lysogen, and a P1 phage was prepared again from this bacterium, and two copies of the AP-P gene obtained earlier were obtained. were infected to RR1 strain became, strains became tetracycline resistance ^{(RR1: pTSAP1 (RecA -)} ) was selected. This strain was assayed RecA ability to ultraviolet radiation at ordinary method, recA ^- was found to be changed.

In addition, the resulting RR1: pTSAP1 ^- AP on the chromosome of (RecA)
The presence of two copies of the -P gene was confirmed by Southern hybridization. First, RR cultured at 37 degrees
1 strain and RR1: pTSAP1 (RecA ^-) respectively higher molecular DNA is prepared and after digestion of these DNA with restriction enzyme BamH I, or Sal I, subjected to agarose gel electrophoresis, GeneScr the DNA
Transcribed to eenPlus, the AP-P gene (EcoRI, Hi of pAPP4, Hi
Southern blot hybridization was performed using the ndIII fragment) as a probe. As a result, in the RR1 strain, Bam
In both HI digestion and Sal I digestion, only one band indicating the AP-P gene was present (FIG. 9, lane 1,
3), RR1: pTSAP1 (RecA -) to both restriction enzymes both in strain 2
This band was detected (lanes 2 and 4 in FIG. 9) and AP
It was confirmed that the -P gene was increased to two copies on the chromosome. When the AP-P activity was measured, RR1: pT
It was confirmed that the AP-P activity of the SAP1 (RecA ⁻ ) strain was about twice that of the RR1 strain.

RR1: pTSAP1 (RecA ^-) of IL-6 with production IL-6 limits the direct expression plasmid pBSF2-SD7 enzyme Ec
The DNA fragment containing the IL-6 gene and pHSG399 (Takeshita. S et al. (1987) Gene, 61, 63-74) are similarly digested with EcoRI and HindIII. Large DN
By ligating the fragment A, pBSF2-SD7-Cm was constructed (FIG. 10). Next, this plasmid was used for E. coli HB101 strain and R
R1: pTSAP1 (RecA ^-) respectively to strain, was transformed, HB101 / PBSF
2-SD7-Cm (AJ 12540 ), RR1: pTSAP1 (RecA -) / pBSF2
-SD7-Cm (AJ 12541) was obtained. The strains are respectively No. 11607 and No. 11608. These strains were cultured and cultured for IL-6 in the same manner as in Example 1.
Was prepared and obtained. When the amino terminus of IL-6 obtained from each strain was assayed using a protein sequencer, about 15% of the former was IL-6 having a methionine residue. In the latter strain, about 96% of which was the mature IL-
It was confirmed to be a polypeptide having the sequence of 6. In addition, it was also found that it has the same physiological activity as natural IL-6.

As described above, by producing IL-6 in an E. coli host in which the AP-P gene is made into two copies as in the present invention,
A human IL-6 polypeptide having a more uniform N-terminus was produced and obtained.

〔The invention's effect〕

According to the present invention, in the case of producing a target polypeptide by a recombinant DNA method, a target polypeptide having a more uniform N-terminus can be produced in a large amount at low cost.

[Brief description of the drawings]

FIG. 1 is a drawing showing the construction steps of plasmid pBSF2C-DUC. FIG. 2 is a drawing showing the base sequence of the synthetic DNA fragment BSFN. FIG. 3 is a drawing showing the construction steps of plasmid pUC-SD6N. FIG. 4 shows a plasmid for directly expressing IL-6, pBSF2-SD7.
4 is a drawing showing a construction process of FIG. FIG. 5 shows an aminopeptidase P expression plasmid.
1 is a drawing showing a construction step of pAPP4-Km. FIG. 6 shows an aminopeptidase P expression plasmid.
It is a drawing showing a construction step of pAPP4-KmΔB. FIG. 7 is a drawing showing the construction steps of pBSF2-SD7P1, which is an expression plasmid for both IL-6 and aminopeptidase P. FIG. 8 is a drawing showing the construction steps of plasmid pTSAP1. FIG. 9 is a drawing showing an autoradiogram of Southern blot hybridization. FIG. 10 is a drawing showing the construction steps of plasmid pBSF2-SD7-Cm.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Yoshimi Kikuchi 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Inc. Central Research Laboratory (56) References Bacteriology, 169, p. 751-757, 1987 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 21/00 C12N 15/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (8)

(57) [Claims] 1. An organism carrying a vector containing an aminopeptidase P gene and a vector containing a gene encoding a polypeptide of interest is cultured, and
Or a method for producing a polypeptide, comprising collecting a target polypeptide accumulated in a culture solution 2. The method for producing a polypeptide according to claim 1, wherein the gene encoding aminopeptidase P and the gene encoding the polypeptide of interest are present on the same vector. 3. The polypeptide according to (1), which is an organism having a chromosome with enhanced expression of the gene encoding aminopeptidase P and carrying a vector containing the gene encoding the polypeptide of interest. Manufacturing method 4. The method according to claim 1, wherein the organism is a prokaryote.
The method for producing the polypeptide according to (2) or (3) 5. The method for producing a polypeptide according to claim 4, wherein the prokaryote is Escherichia coli. 6. The method according to claim 1, wherein the organism is a eukaryote.
The method for producing the polypeptide according to (2) or (3) 7. The method for producing a polypeptide according to claim 1, wherein the N-terminus of the target polypeptide is a proline residue. 8. The method for producing a polypeptide according to (1), (2) or (3), wherein the target polypeptide is interleukin 6.