JPH01165385A - Secretory expression of protein - Google Patents

Abstract

PURPOSE:To efficiently produce a high-purity and highly active protein, by transforming a host cell with a vector containing a DNA, etc., capable of coding a secretory signal sequence expressed by a specific formula and cultivating the resultant transformant. CONSTITUTION:A vector, having DNAs significantly bound to a replicable vector and successively containing a promoter controlling transcription of the DNA, Shine-Dalgarno sequence, the DNA capable of coding a secretory signal sequence expressed by formula I (alpha represents KK, K or R; beta represents I or S; alpha represents N or S), the DNA, directly bound to the 3'-terminal thereof and capable of coding a protein and a terminater region containing a stop codon in the order mentioned from the 5'-terminal to the 3''-terminal sides is used to transform a host cell. This host cell is then cultivated to accumulate the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the secretion and production of proteins by recombinant DNA technology, and more particularly to the production of novel DNA proteins encoding improved signal sequences for bacterial toxins. The present invention relates to a method for secreting and expressing a protein having a complex higher-order structure using the method to easily obtain a highly pure active protein.

Problems that Japan is trying to solve Currently, methods for producing useful proteins using recombinant DNA technology are common, and most of them are produced using Escherichia coli (hereinafter referred to as E. (sometimes abbreviated as coli). Although this method has the advantage of requiring a short culturing time, there is a risk that the proteins produced within the microorganism may be degraded by host proteases, and the process of obtaining the target product in pure form is costly. Not only does this require time and effort, but there is also a risk that the process may involve deactivation of the protein.

To solve this problem, we secrete proteins produced within the host microbial cell into the periplasm (between the cytoplasmic membrane and the outer membrane) or outside the microbial cell using signal sequences involved in membrane permeation of proteins. Methods for accumulating these have been attempted (for example, see Japanese Patent Application Laid-Open No. 61-280292 and Japanese Patent Application Laid-open No. 61-92575).
JP-A-61-280292) and ampicillin resistance factor signal sequences are commonly used. However, alkaline phosphatase and ampicillin resistance factors are characterized by having almost no disulfide bonds in their molecules. In general, E. coli secreted proteins are poor in disulfide bonds (P, A, Maher and S, J, Sin
ger: Proceedings of the National Academy of Sciences (Proc, Nat.
1, Acad, Sci, USA), 8
3.

9001 (1986)), and the signal sequences of these proteins are combined with proteins rich in disulfide bonds, such as human superficial growth factor (hEGF), human basic fibroblast growth factor (hbFaF), and human acid fibroblast. Cell growth factor (haFGF), human angiogenin.

Human proinsulin, human insulin-like growth factor I
and ■, it is considered unreasonable to apply this method to the secretory expression of basic bovine pancreatic trypsin inhibitor.

It is known that these disulfide bond-rich proteins either have no physiological activity or become extremely weak due to incomplete disulfide bond formation or the wrong combination of disulfide bonds during the expression process. In order to return these inactive forms to active forms, an oxidation process following protein denaturation is required. Thus, in order to secrete a protein while maintaining its complex higher-order structure, it has been desired to develop an appropriate signal sequence.

Furthermore, JP-A No. 61-92575 describes a method for secreting a specific mature eukaryotic protein to the periplasm or outside the bacterial cell using DNA encoding a bacterial secretion signal sequence. . Specifically, in this method,
The DNA encoding the signal sequence is extracted from a bacterial secretory protein, a cell membrane protein, or a mutant thereof, that is, a natural one, and the eukaryotic protein to be secreted is a complement protein, especially a mammalian protein. You're getting growth hormones like human growth hormone.

Furthermore, Journal of Biological Chemistry (J, Biol, Chem,), 262, 10
189 (1987), an attempt was made to modify the signal sequence of the heat-labile enterotoxin B subunit protein produced by toxigenic Escherichia coli. However, there is no mention of increased protein secretion.

W. Means for Solving the Problem The present inventors have developed a method of secreting and accumulating proteins produced within the host microbial cell into the periplasm or outside the microbial cell using a signal sequence involved in protein permeation. In order to make improvements, we first used a chemically synthesized NA that corresponds to the naturally occurring bacterial secretion signal sequence to synthesize proteins, especially hEGF.
proposed a method to secrete and express the
As a result of further research, it was discovered that chemically synthesized NA, which corresponds to a partially modified natural bacterial secretion signal sequence, could also achieve the desired purpose. The present invention was achieved by advancing the universalization of DNA.

That is, 1 the present invention is (1) general formula % formula % [wherein α is KK, K or R, β is engineering or S,
γ indicates N or S]; (2) the DNA is meaningfully linked to a replicable vector; A promoter that controls DNA transcription, a Shine-Dalgarno sequence, a general formula % [where α is KK, K or R, β is ■ or S,
A DNA encoding a secretory signal sequence represented by γ represents N or S], a DNA encoding a protein directly linked to its 3' end, and a terminator region containing a stop codon are contained in this sequence. Above (1)
and (3) the DNA is meaningfully linked to a replicable vector, and from the 5' side to the 3' side of the vector, I) a promoter controlling transcription of NA, a Shine Dalgarno sequence; , general formula % formula % [wherein α is KK, K or R, β is engineering or S,
A DNA encoding a secretory signal sequence represented by γ represents N or S], a DNA encoding a protein directly linked to its 3' end, and a terminator region containing a stop codon are contained in this sequence. The present invention relates to a method for producing the protein, which comprises transforming a host cell using a vector, culturing the host cell, and then accumulating the protein in the periplasm or extracellular space of the host. The present invention has made it possible to secrete and express proteins with complex higher-order structures, especially proteins containing a high density of disulfide bonds, thereby making it possible to obtain highly pure active proteins through simple operations. 6. As mentioned above, the DNA encoding the secretory signal sequence represented by the general formula % (in which α, β, and γ are as described above) used in the present invention is a It originated from focusing on substances that encode modified signal sequences, and in particular, the starting point was synthetic RNA that encodes the modified signal sequence of the heat-labile enterotoxin A subunit produced by toxigenic Escherichia coli. I will explain these first.

Enterotoxins produced by toxigenic Escherichia coli exhibit toxicity such as diarrhea to humans and other animals [Yoshifumi Takeda, Yasushi Shimonishi, Tatsuo Yamamoto, Nagahisa Takeda, "Proteins, Nucleic Acids, Enzymes", 324 (1986)).

It is broadly classified into heat-resistant (ST) and heat-labile (LT) types, and subtypes are known for each.

The production of enterotoxins is controlled by E. coli plasmids, and like drug resistance factors, plasmids are advantageous for producing useful substances.
When using a chemically synthesized NA encoding the enterotoxin signal sequence, the hEGF gene was connected to the signal sequence of the ampicillin resistance factor of the E. coli plasmid pBR322, and this was secreted and expressed in E. coli under the control of the tryptophan promoter. (mentioned above, special application in 1973)
No. 2-61207) further found and confirmed that the secretion amount of hEGF was more clearly improved by using chemically synthesized NA encoding the signal sequence of the improved engineerotoxin A subunit.

As mentioned above, there are two types of enterotoxin signals: heat-resistant (ST) and heat-labile (LT). Among ST, there is ST Ia.

STI b, 5TII, etc., and LT include LTh, LTp (
There are A and B subunit proteins, respectively), and the one used in the present invention is the DNA corresponding to the modification signal of LTA.

The LTA signal sequence consists of 18 amino acids, and if this sequence is used as it is, it exhibits significantly lower protein secretion ability than when the 5TII signal sequence is used.

However, LTA is one of the shortest signal sequences, and it was thought that its secretion ability could be enhanced by slight modification. By mutually comparing various known enterotoxin signal sequences, an attempt was made to add elements effective as secretion signals to the LTA signal sequence. The effectiveness of the modified signal is determined by chemically synthesizing the DNA corresponding to the modified signal sequence, connecting it to hEGF DNA, inserting it into an expression vector, and then producing hE secreted and expressed in E. coli.
This was achieved by quantifying GF.

As a result of testing the effects of various signal sequences, it was discovered that the amino acid sequence shown below has excellent effects. In addition, amino acids are expressed by the following amino acid one-letter symbol (IUPAC-IUB Biochemical Nomenclature Committee confirmation rules).

A: Alanine B: Aspartic acid or asparagine C Nidistine D = Aspartic acid E: Glutamic acid F: Phenylalanine G Nigericin H: Histidine: Isoleucine: Lysine L: Leucine M: Methionine N: Asparagine P Nibroline Q: Glutamine R: Arginine S : Serine T: Threonine ■: Valine W Nitributophane Y: Tyrosine Z: Glutamic acid or Glutamine
TFIFFILLASNAYA.

MKNITFIFILLASSAYA.

MRNITFIFILLASNAYA.

MKKNITFIFILLASNAYA.

MKNITFI FF5LLASNAYA.

MRNITFIFFSLLASNAYA.

MKKNITFIFFSLLASNAYA etc. are mentioned as preferable ones, especially MKNITFI FF
ILLASNAYA.

MKNITFIFILLASSAYA.

Particularly preferred examples include MKKNITFIFILLASNAYA.

The proteins secreted and expressed in the present invention include human epidermal growth factor (hEGF), human basic fibroblast growth factor (
hbFGF), human acidic fibroblast growth factor (haFG)
F), human angiogenin, human proinsulin,
Human insulin-like growth factor [and ■.

Proteins rich in disulfide bonds, such as basic bovine pancreatic trypsin inhibitor, are cited as more effective, but it is of course effective for secretory expression of any other protein.

The DNA of the present invention is a DNA encoding a secretion signal sequence.
A itself or a DNA encoding a protein can be directly linked to its 3' end, and they can also be incorporated into a replicable vector.
At this time, DN
A promoter that controls the transcription of A, the general formula % formula % [wherein α is KK, K or R, β is Engineering or S,
γ represents N or S], a structural gene, and a terminator region containing a stop codon are included in this vector.

As a host to be transformed with the above vector, E. coli HB
IOI, E. coli DHI, E. coli C600, E. coli MM
294, E. coli RRI, and the like.

Specifically, the present invention relates to a recombinant product obtained by introducing a gene encoding a desired foreign protein into a vector equipped with a gene encoding an improved signal sequence of enterotoxin of toxigenic E. coli under the control of a promoter. DNA
By transforming a host using the method and culturing the obtained transformant under appropriate conditions, the target protein with the higher-order structure necessary for expressing physiological activity can be transferred to the outside of the E. coli cell or to the periplasm. The present invention relates to a method for producing proteins that involves efficient secretion into proteins.

Hereinafter, using the DNA of the present invention encoding amino acids corresponding to an improved signal sequence of enterotoxin, hEG
A detailed explanation will be given by taking as an example the case where F is secreted and expressed, but the present invention is not limited thereto.

Hereinafter, the invention will be explained in detail with reference to Examples.

In addition, the plasmid pTB described in Example 2 (2) below
413 was used to transform E. coli DHI strain into T. Maniat
is et al. “Molecular Cloning J”
cular cloning), pp. 250-251 (1
A transformant of Escherichia coli obtained by transformation by the method described in Col. Spring Harbor, 1982)
) D H1/p T B413 was sent to the Fermentation Research Institute (IFO) as IFO-14576, and from March 18, 1985 to the Microbial Research Institute (FRI) of the Ministry of International Trade and Industry as FERM P-9293. It has been deposited as.

Note that FIG. 1 shows the amino acid sequences of the signal peptide of enterotoxin LTA and specific examples of the signal peptide of the present invention, and FIG. 2 shows the chemically synthesized NA sequences corresponding to each signal peptide in FIG. 1. FIG. 3 shows gene fragments for chemically synthesizing the enterotoxin LTA signal peptide and the signal peptide of the present invention. FIG. 4 shows the amino acid and synthetic gene sequence of the signal peptide-hEGF junction, and FIG. 5 is a construction diagram of the plasmid used in the present invention.

Example 1, Synthesis of DNA fragments Each DNA fragment (LUI, LU2. LU
3゜M33U3. M3101J3. M312U1.
LLI, LL2. L12. M33L2゜M31
0L2. M312L1) was synthesized using β-cyanoethylphosphoramidite as a raw material using an Applied Biosystems model 380A-DNA automatic synthesizer. For deprotection purification, 0.2 μmole of the resin was heated with 2 mR of concentrated ammonia water at 55°C for 5 hours, and purified by reverse phase high performance liquid chromatography (hereinafter abbreviated as HPLC).
The dimethoxytrityl group at the 5' end was removed in 2 mQ of 0% acetic acid for 20 minutes, followed by reverse phase HPLC and ion exchange HPLC.
Purified with C.

(1) Preparation of signal-containing fragments [see Figures 2, 3 and 5 (A)] DNA fragments LU2, LU3, LLI, LL2 each 75Q
pmon e with 66mM Tris-HCl (pH 7,6)
, dissolved in 19.5μQ of a solution of 6.6mM magnesium chloride, 10mM β-mercaptoethanol, and 1mM adenosine triphosphate (hereinafter abbreviated as ATP),
3.5 units of T4 polynucleotide kinase (Takara Shuzo)
was added to make the total amount 20μQ, and treated at 37℃ for 1 hour.
’ end was phosphorylated. After treating these at 90°C for 3 minutes, the final concentration of the reaction was 66mM Tris-HCl (pH 7, 6).
), 6.6 m M magnesium chloride, 500 μM ATP
750 pm each of LUI and LL3 in the solution.

The mixture was added together with Qe, heated at 90°C for 3 minutes, and then quickly cooled on ice. Further, this was slowly cooled from 75°C to 20°C over 2 hours, and then treated at 15°C for 10 minutes. β-mercaptoethanol was added to this so that the final concentration was 10 mM, and 400 units of T4-DNA ligase (New England Biolab) was added to make the total volume 200 μQ.
It was treated at 15°C for 20 hours.

After the reaction, 10% polyacrylamide gel electrophoresis (hereinafter P
69 base pairs (bp) of DNA was recovered from the gel piece using AGE (abbreviated as AGE) [Journal of Molecular Biology J (J, Mo1. Biol, )
.

υtongue, 119 (1977)). This DNA was treated with 6 units of T4 polynucleotide kinase (Takara Shuzo) in the same manner as above to phosphorylate the 5' end.

(2) Preparation of hEGF fragment (hEGF/Hinf-Pst)C Figure 5 (
B)) Japanese Patent Application Laid-open No. 62-40290 (Patent Application No. 176-1982)
No. 976) Plasmid pT8 described in Publication Example 7
．． 413 (4,3k bp) and a standard buffer (hereinafter abbreviated as S, buffer) was prepared so that the final reaction concentration was 20mM Tris-HCl (pH 7°6), 7mM magnesium chloride, and 10mM β-mercaptoethanol. Sodium chloride was added to the mixture to give a final concentration of 50 mM, and 80 units of Hinf I (Takara Shuzo) and 105 units of Pstl (Nippon Gene) were added to make a total volume of 400 μQ, and the mixture was treated at 37° C. for 2 hours.

The reaction solution was subjected to 5% PAGE, and a 145 bp hEGF fragment was recovered from the gel.

(3) Preparation of vector fragment (pT RP771/C1a=Pst) (Figure 5 (C
)] Plasmid p'r RP771 (4,3 kbP) (
Tsutomu Kuroyo et al. “Nukreitsuku Acid Research J (Nu
cleic Ac1dsResearch), 11,3
077 (1983)) to 10 pg, add S, buffer and sodium chloride in the same manner as in 2(2), and add C1a
New England Biolabs, Inc.) 10 credits.

Add 15 units of Pstl to make the total amount 100μQ, and make 37
It was treated at ℃ for 2 hours. The reaction solution was subjected to 0.7% agarose gel electrophoresis (AGE), and a 3.6 kbp fragment was recovered from the gel.

(4) Preparation of plasmid pLAE4 [Figure 5 (D)] Signal fragment (2 (1)) 0.5 pmole, h E
GF fragment (2(2)) 0.5 pmole, vector fragment (2(3)) 0.05 pmole (0,1,
ttg) in 66mM Tris-HCl, 6.6mM Magnesium Chloride, 500mM ATP, 10mM
The mixture was placed in a β-mercaptoethanol solution, and 400 units of T4 DNA ligase was added to make a total volume of 20 μQ, and the mixture was treated at 15° C. for 20 hours.

(See Figure 3 and Figure 5 (A)) Among the DNA fragments used in the previous section 2 (1), LU3 was
In 3U3, LL2 was converted to M33L2 and treated in the same manner as above to prepare a fragment containing the modified signal M3-3.

This, hEGF fragment C2 (2)), vector fragment (2
(3) ) was processed in the same manner as in the previous section 2 (4).

4. Preparation of hEGF/secretion plasmid pMLA3-10 containing the modified signal of the present invention (see Figures 2, 3, and 5 (A)) Among the DNA fragments used in the previous section 2 (1) , LU3 to M3
10U3, LL2 was converted to M310L2 and treated in the same manner as above to prepare a fragment containing the modified signal M3-10. This, hEGF fragment (2(2)), and vector fragment (2(3)) were treated in the same manner as in the previous section 2(4).

(See Figure 3, Figure 3, and Figure 5 (A)) Among the DNA fragments used in the previous section 3, the LUI was M312U.
1, LLI was converted to M312L1 and treated in the same manner as above to prepare a fragment containing the modified signal M3-12.

This, hEGF fragment [2 (2)), vector fragment (2
(3) :] was processed in the same manner as in the previous section 2 (4).

(T. Maniatis et al. “Molecular Cloning”, 2
pp. 50-251 (1982) Cold Sping H
arbour) 5mQ glue, broth, inoculated with Escherichia coli I (BIOI),
Cultured overnight at 37°C. Glue this 200μQ to 20mQ.

The cells were planted in broth, cultured at 37°C until the number of Klett units reached 80, and cooled in water for 10 minutes. Incubate the culture at 4°C for 30
Centrifuge at 0 rpm for 5 minutes and collect 1 isolated bacterial cell into 10 mQ
After turbidity in physiological saline, 4℃, 2400 rpm
Centrifuged for 5 minutes. The cells were suspended in 10 mQ of a 50 mM calcium chloride solution and cooled on ice for 30 minutes. The centrifuged bacterial cells were suspended in 2 mΩ of 50 mM calcium chloride solution. To each 200 μQ of this l@ suspension was added 10 μα of each of the reaction solutions of 2(4), 3°4 and 5, and after cooling with water for 1 hour, the mixture was treated at 42° C. for 75 seconds. Add 0.8mu glue to this,
After adding broth and incubating at 37°C for 1 hour, centrifuge and supernatant 0.
．． After removing 8mQ, remove the bacterial cells! @2 turbid 100μQ glue, broth agar plates (tetracycline 6μ
g/mQ) and cultured overnight at 37°C.

The plasmid DNA of the transformant was extracted using the alkaline method (T, Ma
Niatis et al. “Molecular Cloning” (M
olecular Cloning), 368-36
9 pages (1982) Cold Sping t(arbo
ur), and after performing a cleavage reaction with C1aI and PstI, transformants E and coli in which the signal and hEGF gene were inserted correctly were obtained by 5% PAGE.
HB 101/ p L A E 4 , E.

coli HBIOI/pMLA3-3. E,col
i HBIOI/pMLA3-10. E. coli H
BIOI/pMLA3-12 was selected.

7. Secretion and expression of hEGF BH containing tetracycline at a concentration of 12.5 μg/mQ
Transformant E, co
li HBIOI/ p LAE 4, E, col
iHBIOI/pMLA3-3. E. coli HB
IOI/pMLA3-10, E. coli HBIOI
/pMLA3-12 was inoculated and cultured overnight at 37°C.

This 1 mQ was subcultured into 19 mQ of M9 medium (containing 1% casamino acids, 1% glucose, and 6°5 μg/mQ tetracycline) at 37°C for 8 hours. This culture solution 5n+Q was centrifuged at 800° rpm and 4° C. for 5 minutes to separate the culture supernatant and bacterial cells. Osmotic shock method (S.

J. Chan et al. Proceedings of the National Academy of Sciences of the United States of America.
J (Proceedings of Nation
nal Academy of Sciences of
the United 5tates of Ame
rica),? 8, 5401 (1981)].

8. Quantification of hEGF Culture supernatant fraction and periplasm fraction were subjected to CL8Sep
- Adsorbed with Pak (Waters Inc.), water 5II
Q.

20% acetonitrile [0.1% trifluoroacetic acid (T
After washing with 2 ml of 50% acetonitrile (containing 0.1% TFA), it was eluted with 2 ml of 50% acetonitrile (containing 0.1% TFA).

The solvent was distilled off, the residue was dissolved in water and subjected to reverse phase HPLC (TSK
-GEL, 0DS-120T; Toyo Soda). Elution was performed at an acetonitrile concentration of 25% to 40% (0
, 1% TFA) over 15 minutes.

Quantification was performed by determining the size of a peak showing the same retention time as standard hEGF (Yusui Pharmaceutical Co., Ltd.) using the height half-width method and comparing it with a standard product.

Secretory expression level of hEGF Compared to that using the natural signal peptide of enterotoxin LTA, the secretory expression efficiency of the protein using the signal peptide of the present invention is three times or more higher.

(1) Secretion expression of hEGF using modified signal M3-3 Transformant E obtained in Example 6 and Co11 HB were added to 5 mu of BHI broth (Difco) containing tetracycline at 12.5 μg/mQ'JM. 101/ p M
LA 3-3 was inoculated and cultured overnight at 37°C. This 4 mQ was added to 76 m fl of M9 medium (1% casamino acids, 1
% glucose, containing 6.5 μg/mQ tetracycline) and cultured at 37° C. for 8 hours.

(2) Purification of secreted and expressed hEGF 80 mQ of the culture solution obtained in (1) above was heated to 800 rpm.
, and centrifuged at 4°C for 5 minutes to obtain about 80 mQ of culture supernatant. Of these, 20mff was adsorbed onto C1g 5ep-Pak (Waters) and 10m of water was used! , 20% acetonitrile [containing 0.1% trifluoroacetic acid (TFA)] 2
After washing with IlIQ, 50% acetonitrile [0.1% T
It was eluted with 4mQ (containing FA). The remaining culture supernatant was also treated in the same manner. Distill the solvent of the eluate,
The residue was dissolved in 500 μQ of distilled water. This is 100μΩ
Reverse phase HPLC (TSK-GEL, 0
A peak containing hEGF was fractionated using a DS-120T (Dolly).

The elution conditions were an acetonitrile concentration of 25% to 40% (
0.1% TFA) with a 15 min linear gradient of 12
．． The eluate from 5 minutes to 12.8 minutes was fractionated. The solvent of this eluate was distilled off, and the residue was dissolved in 100 μQ of distilled water.
Re-fractionation was carried out twice under the same conditions. The solvent of the eluate was distilled off, and the residue was dissolved in 80 μm of distilled water. 6 of these
5μQ was subjected to ion exchange HPLC (TSK-GEL, DEA
A peak containing hEGF was fractionated using E-5PW (Dolly). The elution conditions were ammonium acetate concentration of 0.
．． 12.7 with a 15 minute linear gradient from 1M to 0.3M
The eluate from 13.4 minutes was collected. The solvent of this eluate was distilled off, the residue was dissolved in 100 μΩ distilled water, and reverse phase HPLC was performed under the same conditions as described above to obtain about 12 μg of purified hEGF.

(3) N-terminal sequence analysis of seminal hEGF
mole) Applied Biosystems Model 47
N-terminal sequence analysis was performed using a 7A gas phase sequencer. The reaction was carried out for two cycles starting from the N-terminus, and Asn-8
The N-terminal sequence of er was identified.

10. Amino acid analysis of hEGF 30 μg of purified hEGF obtained in the same manner as in Example 9 was
200μ of 5.7N constant boiling hydrochloric acid containing % thioglycolic acid
The solution was dissolved in Q and the container was sealed. After processing this at 110°C for 24 hours, Hitachi Model 835 Am1no
Amino acid analysis was performed using Ac1d Analyzer.

The results are shown below.

Asp+Asn 6.63(7), Ser 2.83
(3), Glu+Gln 5.12(5).

Pro 1.27(1), Gly 4.10(4),
Ala 2.00 (2), Cys (6).

Val 2.68 (3), Net 0.87 (1),
Ile 1.64 (2), Leu 4.91 (5)
.

Tyr 4.99 (5), Lys 1.76 (2),
1list, 97(2), Arg 3.24(3
).

Trp 1.76 (2) 11, Measurement of biological activity of hEGF Growth inhibition of human squamous cell carcinoma H5C-1 cells [Barnes, Journal of Cellular Biology (J, Ce11. Biol, ), 9
3.1 (1982)), the hEGF culture supernatant showed significant biological activity (Table 1).

In addition, proliferation promotion for BALB/c 3T3-A31 cells (Rose et al., Journal of Cellular Physiology (J, Ca11. Physiol
, ), 86°593 (1975)) and found that
hEGF culture supernatant showed significant biological activity (Table 2)
.

Table 1 Growth inhibition on H8C-1 cells 1) As a control, the plasmid pTRP described in Example 2 (3)
E. coli It in the same manner as in Example 6 using 771.
E. coli t (BI
A culture supernatant obtained by culturing OI/pTRP 771 in the same manner as in Example 9 (1) (a culture supernatant that does not express hEGF) was used.

Table 2 BALB/c 3T3-A 311jJJJ
Promoting proliferation against retention

[Brief explanation of the drawing]

FIG. 1 shows the amino acid sequences of the natural signal peptide of enterotoxin LTA and specific examples of the signal peptide of the present invention, and FIG. 2 shows the chemically synthesized NA sequences corresponding to each signal peptide in FIG. FIG. 3 shows a gene fragment for chemically synthesizing the natural enterotoxin LTA signal peptide and the signal peptide of the present invention. FIG. 4 shows the amino acid and synthetic gene sequence of the signal peptide-hEGF junction. FIG. 5 is a construction diagram of a plasmid for protein secretion and expression used in the present invention.

Claims (7)

[Claims] (1) General formula MαNITFIFβLLASγAYA [wherein α is KK, K or R, β is I or S,
γ represents N or S] A DNA containing a DNA encoding a secretion signal sequence represented by: (2) The DNA according to claim 1, wherein a DNA encoding a protein is directly linked to the 3' end of the DNA encoding a secretory signal sequence. (3) The DNA according to claim 1 or 2, wherein the signal sequence is represented by any one of MKNITFIFILLASNAYA, MKNITFIFILLASSAYA, or MKKNITFIFILLASNAYA. (4) The D according to claim 1, 2, or 3, wherein the DNA encoding the secretion signal sequence is chemically synthesized DNA.
N.A. (5) Claim 2, wherein the protein is human epidermal growth factor;
DNA according to 3 or 4. (6) The DNA is meaningfully linked to a replicable vector, and the DNA is linked from the 5' side to the 3' side of the vector.
A promoter that controls transcription of A, Shine-Dalgarno sequence, general formula MαNITFIFFFβLASγAYA [wherein α is KK, K or R, β is I or S,
γ indicates N or S]; a DNA encoding a protein directly linked to its 3'end; and a terminator region containing a stop codon. The DNA according to claim 1. (7) The DNA is meaningfully linked to a replicable vector, and the DNA is linked from the 5' side to the 3' side of the vector.
A promoter that controls transcription of A, Shine-Dalgarno sequence, general formula MαNITFIFFFβLLASγAYA [wherein α is KK, K or R, β is I or S,
γ indicates N or S]; a DNA encoding a protein directly linked to its 3'end; and a terminator region containing a stop codon. A method for producing the protein, which comprises transforming a host cell using a vector, culturing the host cell, and then accumulating the protein in the periplasm or extracellular space of the host.