JP2560214B2 - DNA sequence encoding a secretory signal peptide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a DNA sequence encoding a secretory signal peptide.

(Conventional technology and problems) Yeast has the characteristics of higher organisms in terms of cell structure and function,
It is also a useful microorganism as a raw material for foods, pharmaceuticals, feeds, etc., which is closely related to human daily life, and is expected to be developed as a host in genetic engineering.

However, since the cell surface of yeast has a strong cell wall outside the cell membrane, it is often difficult to purify useful substances (heterologous proteins) produced by gene recombination technology. It is desired to develop a system for secreting the bacterium outside the cells.

Further, since secretory production enables continuous culture, a large increase in the production amount is expected, and further the decomposition of the product by the intracellular protease is prevented, which is a great advantage.

(Means for solving problems) Kluyveromyces lact
The yeast having the linear plasmid pGKL1 derived from is kills certain yeasts that do not have pGKL1 by the killer toxin protein that it produces and secretes. This killer toxin protein is
It consists of three subunits of 97kd, 31kd and 28kd and is believed to be released into the medium by the N-terminal signal peptide of the killer toxin precursor. In addition, a killer gene and a killer resistance gene are present on pGKL1 and show resistance to the killer toxin produced by itself. pGKL
As a result of the determination of the entire nucleotide sequence of 1, this plasmid was 5
It has been shown that ORF3 is a 97kd and 31kd gene, ORF4 is a 28kd gene, and ORF5 is a killer resistance gene [Nucleic Acids]. Research, 12
Vol., 7581 to 7597 (1984), 15: 1031 to 1046 (1987) and JP-A-60-12983].

The present inventors previously coded a signal peptide capable of efficiently secreting a protein produced in yeast cells from the above-mentioned 97 kd and 31 kd subunit regions.
A DNA (hereinafter referred to as DNA) sequence was found (Japanese Patent Application No. 61-2807).
No. 47).

The present inventors, based on the above findings, as a result of further examination, also from the 28 kd subunit region in the ORF4, encode a signal peptide capable of efficiently secreting a protein produced in yeast cells. The present invention has been accomplished by finding a DNA sequence.

 The present invention will be described in detail below.

The DNA sequence encoding the secretory signal peptide of the present invention has 57 base pairs (bp) encoding the following 19 amino acids.
Consists of

MET LYS ILE TYR HIS ILE ATG AAG ATA TAT CAT ATA PHE SER VAL CYS TYR LEU TTT AGT GTT TGT TAT CTA ILE THR LEU CYS ALA ALA ATA ACA TTA TGT GCT GCT ALA GCA The DNA sequence encoding the secretory signal peptide of the present invention is , 28k of the killer toxin proteins in pGKL1 above
It can be isolated from the region encoding the signal peptide of d. Alternatively, it can be prepared by chemical synthesis.

When the DNA sequence of the present invention is inserted into a suitable plasmid vector between a promoter sequence and a foreign gene encoding a desired protein and expressed in a yeast host,
The target protein can be secreted efficiently.

The construction method and usefulness of the secretion vector containing the DNA sequence of the present invention using the mouse α-amylase gene as a marker will be described in detail below.

[Construction of Secretion Vector] (1) Construction of plasmid pKEN516 (11.1 kb) Insert the entire region of pGKL1 DNA into a circular plasmid.

Since the protein is covalently bound to less than 5'of (8.9 kb) of pGKL1 DNA, pGKL1 is first treated with 0.1N NaOH at 30 ° C. for 30 minutes to decompose the bound protein. Then, after neutralization, it is extracted with phenol and purified using Sephaacrylic S-1000 (Pharmacia).

This DNA was cleaved with BamHI and the two left and right fragments obtained were used as a cloning vector for E. coli and B. subtilis, plasmid pLS354 [EMBO Journal, 3, Vol. 761-766 (1984)], Sma. It is cloned into a fragment cut with I and BamHI to make pGKF106 (9.8 kb) containing the right fragment and pGKF107 (9.8 kb) containing the left fragment.

Then, a fragment (4.4 kb) obtained by cutting pGKF106 with BamHI and EcoRI and a fragment (5.5 kb) obtained by cutting pGKF107 with BamHI and PvuI were prepared. Cleaved with II, ligated with EcoR I linker (pGGAATTCC), then cleaved with EcoR I
The plasmid obtained by ligation and lacking the EcoR I-Pvu II fragment (2.1 kb) containing the tetracycline gene of pBR322 was ligated with the EcoR I-Pvu I fragment (1.2 kb including ori), and the entire region of pGKL1 was ligated. Plasmid containing pKEN516 (11.1kb)
Is obtained (FIG. 1).

(2) Construction of plasmid pNW002 (3.8 kb) The plasmid pKEN516 was cleaved with Xmn I and EcoR I, and the resulting 1.2 kb fragment containing the 28 kd killer toxin gene on pGKL1 was transformed into plasmid pUC13 (Pharmacia Catalog P-L).
Biochemicals, 1986 edition, p. 60, 27-4954-01)
The plasmid pNW002 (3.8
kb) (Fig. 1).

(3) Construction of plasmid pNW043 (2.8 kb) The 113 bp region encoding the signal peptide from 59 bp upstream of the translation initiation codon of the 28 kd killer toxin gene on pNW002 was added to plasmid pUC12 (Pharmacia Catalog P-L).
Biochemicals, 1986 edition, p. 60, 27-4952-01) to construct plasmid pNW043 (2.8 kb).

That is, pNW005 (3.8 kb) was cleaved with Rsa I and Pst I to obtain an Rsa I-Pst I fragment containing a DNA sequence encoding the secretory signal peptide of the present invention, which was used for Hin of pUC12.
Plasmid pNW043 (2.8kb) inserted into the cII-PstI site
(Fig. 2).

(4) Construction of plasmid pNW048 (2.8 kb) pNW043 was cleaved with BamHI, the DNA was treated with the nuclease BAL31 from the cleavage site, and then the bacterial alkaline phosphatase (BAP) was treated with Bal II linker (pCAGATCTG). ) Is ligated to form a plasmid pNNW048 (2.8 kb) containing the signal sequence of pGKL1 (second)
Figure).

(5) Preparation of plasmid pNW051 (4.3 kb) A plasmid pNW051 (4.3 kb) containing the signal sequence of pGKL1 and the promoter sequence of yeast phosphoglycerate kinase gene ( PGK ) is prepared.

Since the promoter of pGKL1 is said not to work in a circular vector, a plasmid pMA91 (9.5 kb) containing the promoter sequence of the phosphoglycerate kinase gene ( PGK ) of yeast (S. cerevisiae) [Gene, 24, 1 , Pp. 14 (1983)], the region containing the PGK promoter sequence is ligated upstream (5 'side) of the signal sequence of the above plasmid pNW048, and the plasmid containing this sequence is linked.
Create pNW051 (4.3kb).

That, EcoR I-Bgl II (1.4 which includes a promoter sequence PGK obtained by cutting pMA91 a (9.5 kb) with EcoR I and Bgl II
kb) fragment and the fragment containing the signal sequence of pGKL1 obtained by cleaving pNW048 with Pst I and Bgl II.
Plasmid pNW051 inserted into the EcoR I-Pst I site of UC12
Create (4.3 kb) (Fig. 2).

(6) Construction of plasmid pNW050 (4.1 kb) Pst plasmid pMSa104 [Cell, 179, 21 (1980)] containing amylase gene derived from mouse salivary glands
Pst I to Ps containing the amylase gene obtained by cutting with I
The plasmid pRE1075 (4.3 kb) prepared by inserting the t I fragment into the Pst I site of pUC13 (JP-A-62-96).
086) was digested with Apa I, the Apa I cleavage site was treated with S1 nuclease to make it blunt-ended, and then digested with Dra I to encode an amylase protein lacking the first 15 amino acids of the amylase precursor. The Apa I to Dra I fragment is removed (this gene lacks the amylase secretion signal). A BamHI linker (for example, 12mer) is ligated to this and cleaved with BamHI to obtain a DNA fragment having BamHI sites at both ends. After blunting this fragment, the plasmid pU
The plasmid pNW050 is prepared by inserting it into the Hinc II site of C9 [Gene, 19: 259-268 (1982)].

(7) Construction of plasmid pMT57 (5.2 kb) By the route shown in Fig. 3, the plasmid YRp7 Natur was constructed.
e, 282, pp. 39-43 (1979)] and plasmid pREl032 containing TRP1 and ARS1 (JP-A-61-271988) and 2 μm.
From the plasmid and the plasmid pBR322, TRP1 , Ap ^r and p
Yeast and E. coli shuttle vector pREl052 containing the replication origins of both BR322 and 2 μm plasmid (JP-A-61-271).
988) was prepared, and then pREl052 was digested with Aat II, and then treated with Mung bean nuclease to give a blunt end.
A Bgl II linker is ligated to this terminus, cut with Bgl II, and then ligated to construct a plasmid pMT57.

(8) Mouse amylase secretion vector pNW052 (8.1k
b) Construction pNW051 was digested with EcoR I and Pst I, and EcoR I-Pst I containing the promoter sequence of PGK and the signal sequence of pGKL1.
On the other hand, pNW050 was digested with Pst I and BamH I to obtain a Pst I-BamHI fragment containing the mouse amylase gene, and these two DNA fragments were isolated from EcoR I-Bg of pMT57.
l Inserted at the II site and secreted for mouse amylase
Create pNW052 (8.1kb) (Fig. 2).

The thus produced Saccharomyces cerevisiae introduced with pNW052 having the secretory signal sequence of the present invention can efficiently secrete mouse amylase into a culture solution injection.

In addition, there is a Pst I cleavage site downstream (3 ′ side) of the secretory signal sequence of pNW052, and by inserting an arbitrary structural gene in place of the mouse amylase gene, the desired proteins corresponding to each are inserted. It can be expressed and secreted. In addition, a multiple cloning site can be introduced by inserting multiple restriction enzyme cleavage sites into the Pst I cleavage site.

(Example) Hereinafter, the present invention will be described in more detail with reference to Examples.

The operations in the following examples were carried out by the following methods I to XI, unless otherwise specified. I [Cleavage and Recovery of DNA by Restriction Enzymes] The following three types of buffers for digestion by restriction enzymes are used (1) to (3)
etics (1981) (Cold spring Harbor, New York). As for the cutting conditions, a restriction enzyme of 2 units / μg DNA is used and treated at 37 ° C or 65 ° C for 30 minutes.

Then TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM
It is extracted once with phenol saturated with EDTA), phenol is removed with ether, 2 volumes of ethanol are added, and the mixture is left at -20 ° C for 30 minutes and then centrifuged to recover DNA.

(1) Low salt concentration buffer solution Consists of 10 mM Tris-HCl (pH 7.4), 10 mM magnesium sulfate and 1 mM dithiothreitol.

(2) Medium salt concentration buffer solution 50 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10 mM magnesium sulfate and 1 mM dithiothreitol.

(3) High salt concentration buffer solution Consists of 100 mM NaCl, 50 mM Tris-HCl (pH 7.4) and 10 mM magnesium sulfate.

II [Preparation of plasmid DNA from E. coli] (1) Mini preparation method (mini prep method) Nucleic Acids Res.7
Vol. 1513-1523 (1979) E. coli containing plasmid DNA was treated with 500 μl of L-broth (10 g peptone, 5 g yeast extract, 1 g glucose, 5
g of NaCl / l (pH 7.2) was used for overnight culture, and the cells collected by centrifugation were 100 μl of solution A [50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8.0). ) And lysozyme 2 mg / ml] and leave in ice water for 30 minutes.

Next, 200 μl of solution B [0.2% NaOH containing 1% SDS (sodium dodecyl sulfate)] is added in ice water and shaken to simultaneously denature the DNA.

After adding 150 μl of 3M sodium acetate solution (pH 4.8) and cooling with ice, centrifuge, adding 2 volumes of cold ethanol to the supernatant, and adding -70 ° C.
Cool for 10 minutes, then centrifuge and collect the precipitate. The precipitate was added to 400 μl of solution C [50 mM Tris-HCl (pH 8.0) and 0.
It consists of 1M sodium acetate], and after removing insoluble matter,
Cold ethanol is added to wash the precipitated DNA, dried under reduced pressure and stored at -20 ° C.

(2) Large-scale preparation method Escherichia coli containing plasmid DNA was cultured using 100 ml of L-broth (containing a drug in the case of a drug resistant plasmid), and after collecting and washing, 3 ml of solution A (50 mM glucose, Suspend in 10 mM EDTA, 25 mM Tris-HCl (pH 8.0) and lysozyme 2 mg / ml) and leave in ice water for 30 minutes.

Then, 4 ml of solution B [0.2% NaOH containing 1% SDS (sodium dodecyl sulfate)] is added in ice water and shaken to simultaneously denature the DNA.

Add 3 ml of 3M sodium acetate solution (pH 4.8), cool with ice, centrifuge, and add 0.6 volume of isopropanol to the supernatant.
Let stand for 20 minutes.

Centrifuge to collect the precipitate, wash with ethanol and wash with 2 ml of T.
Suspend in E (pH 7.5) buffer. Then 10 μl RNase A
Add (10mg / ml) and let stand at 37 ℃ for 30 minutes, then perform phenol extraction. 0.2 volume of 5M NaCl and 1/3 volume of 30% polyethylene glycol 6000 are added to the aqueous layer and the mixture is allowed to stand at -20 ° C for 40 minutes and then at 4 ° C for 40 minutes. Centrifuge to collect the precipitate, 400m
Dissolve in l of water, precipitate the DNA with ethanol, wash and
Dissolve in 0 μl TE buffer.

III [ligation by T4 DNA ligase] The two DNA fragments to be ligated were adjusted to 1 μg / 10 μl so that the ligation buffer [66 mM Tris-HCl (pH 7.5), 6.6 mM]
Of magnesium chloride, 10 mM dithiothreitol] and treated at 65 ° C for 10 minutes, then at 4 ° C
Add ATP (adenosine triphosphate) and then add T4
Add ligase to 0.1 unit / μg DNA for sticky ends and 1 unit / μg DNA for blunt ends 4
After reacting at ℃ for 18 hours, treat at 65 ℃ for 10 minutes.

IV [Transformation of E. coli] [Advanced Bacterial Genetic
s (1981) (Cold Spring Havor, New York)] 5 ml of L-broth was inoculated with E. coli and cultured overnight. 0.2 ml of this is seeded in 20 ml of L-broth and shake-cultured at 37 ° C. until the Kret unit reaches 60. Cells were collected and ice-cooled with 50 mM calcium chloride and 10 mM Tris-HCl (pH
8.0) and 10 ml of buffer solution consisting of and and chill on ice for 30 minutes. The centrifuged bacterial cells were suspended in 1 ml of calcium chloride solution, 0.1 ml of this was mixed with 10 μl of DNA solution and incubated at 0 ° C. for 30 minutes and 42 ° C. for 2 minutes, and then 1.5 ml of L-
Broth is added and incubated at 37 ° C for 30 minutes, and 0.1 ml of this is seeded on an agar medium.

V [Removal of DNA sticky end by Mung bean nuclease] DNA having sticky end is treated with 30 mM sodium acetate (pH 4.
6), 100 μl buffer consisting of 50 mM NaCl and 1 ml ZnCl ₂
1 μl (2 units / μl) of Mung bean nuclease was added to the solution and treated at 37 ° C. for 10 minutes, then 2 μl of
25% SDS solution, 10 μl of 0.5 M Tris-HCl buffer (pH 9.5)
And add 10 μl of 8M LiCl.

Next, 150 μl of phenol / chloroform mixed solution (1: 1) was added to this, and the mixture was extracted.
Add μl of 3M sodium acetate and 200 μl of ethanol, cool to −20 ° C., and centrifuge to recover the precipitated DNA.

VI [BAL31 treatment] Perform BAL31 treatment to digest both strands from both ends of the DNA. That is, 20 μl of 15 μg of ethanol-precipitated DNA
5x BAL31 buffer [3M sodium chloride, 60 mM calcium chloride, 60 mM magnesium chloride, 5 mM EDTA], add 79 μl of water and 1 μl (2 units) of BAL31 enzyme, and incubate at 20 ° C for 30 minutes To do. After reaction 3 times with phenol,
After extraction with ether three times, ethanol precipitation is performed.

VII [filling of sticky ends] 1 μg of DNA was added to 30 μl of 50 mM Tris-HCl (pH 7.2),
10 mM magnesium sulfate, 0.1 mM dithiothreitol, 50 μg / ml bovine serum albumin (BSA) and 0.2 mM dN
Dissolve in TP, add 1.25 units of klenow fragment (large fragment obtained by treating E. coli DNA polymerase I with trypsin) and allow to react at room temperature for 30 minutes. Then 1 μl of 0.
The reaction is stopped by adding 5M EDTA (pH8.0), phenol and ether are sequentially extracted to remove phenol, and DNA is recovered by ethanol precipitation.

VIII [Phosphorylation of Bgl II and EcoR I linkers] Bgl II linker (5'CAGATCTG3 ') and EcoR I linker (5'GGAATTCC3') (only one of the double chains is shown) Since there is no acid group, phosphorylation was performed with T4 kinase.

3 nmol of linker DNA was added to 41 μl of 80 mM Tris-HCl (pH
7.5) and dissolved in 12 mM magnesium chloride, 10 at 60 ℃
After incubation for 30 minutes, 10 mM 2-mercaptoethanol and 20 mM ATP are added at 37 ° C., 10 units of T4 kinase is further added, and the mixture is treated at 37 ° C. for 30 minutes and then stored at −20 ° C.

IX [Bgl II linker and ligation of EcoR I to blunt end] A ligation buffer [66 mM Tris-HCl (pH 7) was added to the blunt end DNA fragment (1.2 pmol end) and the linker DNA phosphorylated in VIII above (100 pmol end). .5), consisting of 6.6.mM magnesium chloride, 10 mM dithiothreitol]],
After adding 1 unit of T4 ligase and reacting at 4 ℃ for 18 hours, BamHI
Alternatively, by treating with Bgl II to remove the excess linker, the DNA is extracted with phenol saturated with TE buffer, the phenol is removed with ether, and the DNA is recovered by ethanol precipitation.

X [Treatment of DNA fragment with bacterial alkaline phosphatase (BAP)] In order to prevent self-ligation of plasmid DNA by the ligase reaction, the plasmid DNA is treated prior to the ligase reaction.
The digested fragment with the restriction enzyme is treated with BAP.

Plasmid DNA cleaved with restriction enzyme (10 pmol 5'end)
10 μl of 10 × BAP buffer [100 mM Tris-HCl (pH 8.
0), 1mM EDTA] and dissolve in 88.5μl water and 1.5μl
(0.75 units) of BAP was added and reacted at 60 ° C for 1 hour.
Add 0.25M EDTA. After that, phenol-chloroform extraction treatment is carried out twice and phenol extraction treatment is carried out twice, and then phenol is removed with ether and the plasmid DNA is recovered by ethanol precipitation.

XI [Yeast transformation (KU method) Saccharomyces cerevisiae in 10 ml of YEPD medium
After culturing at 30 ° C. overnight, the cells are collected, washed once with TE buffer, and then suspended in the same buffer so that the cell number becomes 2 × 10 ⁷ cells / ml.

An equal volume of 0.2 M lithium acetate (pH 7.
After adding 5) and holding at 30 ° C for 1 hour, dispense into 100 μl test tubes, add DNA to this at 0 ° C, and mix at 0 ° C for 30 minutes. T containing 100 μl of 70% polyethylene glycol 4000
Add E buffer, mix well and mix at 30 ° C for 1 hour, then 4
Hold at 2 ° C for 5 minutes, centrifuge to collect cells, and wash with sterile water.

This is suspended in 500 μl of sterilized water, 100 μl of which is inoculated on a selective medium, and cultured at 30 ° C. for 3 to 4 days to obtain a transformant.

Example (1) Preparation of pKKN516 (11.1 kb) pGKL1 DNA (8.9 kb) was treated with 0.1 N NaOH at 30 ° C. for 30 minutes to decompose the bound protein, and then neutralized, and then the DNA was extracted with phenol. , Sephaacrylic S-1000 (Pharmacia).

This DNA was digested with BamHI and the two left and right fragments obtained were ligated with the fragments obtained by digesting pLS354 with SmaI and BamHI, respectively, and pGKF106 (9.8 kb) containing the right fragment and the left fragment. We obtained pGKF107 (9.8 kb) containing the fragment.

Then pGKF106 was digested with BamHI and EcoRI to obtain D
The NA fragment (4.4 kb) and the DNA fragment (5.5 kb) obtained by cleaving pGKF107 with BamHI and PvuI were used to transform EcoR I-Pvu of pKKN405.
Ligation with the I fragment (1.2 kb containing ori) gave a plasmid pKKN516 (11.1 kb) containing the entire region of pGKL1 (Fig. 1).

(2) Construction of pNW002 (3.8 kb) pGKL1 obtained by cleaving pKKN516 with Xmn I and EcoR I
A 1.2 kb fragment containing the above 28 kd killer toxin gene was added to pUC
Plasmid pNW002 was inserted into the EcoR I-Sma I site of 13
(3.8kb) was created. (Fig. 1).

(3) Construction of pNW043 (2.8 kb) pNW002 was cleaved with Rsa I and Pst I to collect an Rsa I-Pst I fragment (113 bp) containing the secretory signal sequence of pGKL1. A plasmid pNW043 (2.8 kb) was constructed by ligating to the Pst I site.

(4) Construction of pNW048 (2.8 kb) pNW043 was cleaved with BamHI, DNA was treated with the nuclease BAL31 from the cleavage site, and then bacterial alkaline phosphatase (BAP) was treated with Bgl II linker (pCAGATCTG). Was ligated to prepare a plasmid pNW048 (2.8 kb) containing a signal sequence (Fig. 2).

(5) Construction of pNW051 (4.3 kb) EcoR I-Bgl II (1.4k containing the promoter sequence of PGK obtained by cutting pMA91 (9.5 kb) with EcoR I and Bgl II
b) The fragment and the fragment containing the signal sequence of pGKL1 obtained by cleaving pNW048 with Pst I and Bgl II were designated as Ec of pUC12.
Plasmid pNW051 (4.3kb) inserted at oR I-Pst I site
Was prepared (Fig. 2).

(6) Construction of plasmid pNW050 (4.1 kb) pMSa104 was digested with Pst I and the resulting Pst I to Pst I fragment containing the amylase gene was inserted into the Pst I digestion site of pUC13 to obtain pREl075 (4.3 kb). Obtained. It is then cleaved with Apa I, the Apa I cleavage site treated with S1 nuclease to make it blunt-ended, and then cleaved with Dra I to encode an amylase lacking the first 15 amino acids of the amylase precursor. The Apa I to Dra I fragment was collected. A BamH I linker (12mer) was ligated to this and cut with BamH I to obtain a DNA fragment having BamH I sites at both ends. This fragment was made blunt-ended and then inserted into the Hinc II cleavage site of pUC9 to prepare pNW050.

(7) Construction of plasmid pMT57 (5.2 kb) EcoR I-Pst I obtained by cutting pREl032 with EcoR I and Pst I.
Pst I obtained by cutting the fragment (0.8 kb) and 2 μm plasmid with EcoR I, filling in sticky ends, and then cutting with Pst I
The -EcoR I fragment (2 kb) was ligated with the Pvu II-EcoR I fragment (2.3 kb) obtained by cutting pBR322 with EcoR I and Pvu II to prepare pREl051 (5.1 kb), which was used as EcoR I. After partial cleavage with, the cohesive ends were filled in and ligated to create pREl052 (5.2 kb) lacking one EcoR I cleavage site (3rd
Figure).

After digesting pREl052 with Aat II, blunt ends and Bgl I
Insert I linker (5'CAGATCTG3 ') and pMT57 (5.2k
b) was created.

(8) Construction of pNW052 (8.1 kb) pNW051 was cut with EcoR I and Pst I, and EcoR I-Pst I containing the promoter sequence of PGK and the signal sequence of pGKL1.
On the other hand, pNW050 was digested with Pst I and BamH I to obtain a Pst I-BamHI fragment containing the mouse amylase gene, and these two DNA fragments were isolated from EcoR I-Bg of pMT57.
l Inserted at the II site and secreted for mouse amylase
pNW052 (8.1kb) was constructed (Fig. 2).

(9) Confirmation of amylase activity (secretion) Saccharomyces cerevisiae 20B12 strain was transformed by the KU method using the plasmid pNW052 prepared by the above method.

The obtained transformant was cultured at 30 ° C. for 20 hours in a medium containing 2% casamino acid, 1% yeast extract and 2% glucose, and the supernatant obtained by centrifugation was subjected to Methods in Enzymolog.
y The amount of amylase in the supernatant was 1000 μg / l as a result of measurement according to the method described in Vol. 1, page 499 (1955).

(Effects of the Invention) Saccharomyces cerevisiae introduced with a plasmid containing a DNA sequence encoding the secretory signal peptide of the present invention can efficiently secrete a heterologous protein into a culture solution, as shown in the above Examples. .

[Brief description of drawings]

1 to 3 show plasmids pNW002, pNW052 and pNW052, respectively.
It is a schematic diagram which shows the structural route of pREl052.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 865) (56) References Nucleci Acids Reseach. 12 [15] (1984) P. 6011-6030 Biochemical and Biophysical Research Communications 144 [2] (1987) P. 613-619

Claims (1)

(57) [Claims] 1. A DNA sequence encoding a secretory signal peptide represented by the following formula: MET LYS ILE TYR HIS ILE PHE SER VAL CYS TYR LEU ILE THR LEU CYS ALA ALA ALA.