JPH0256076B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to plasmids. In particular, it relates to plasmids useful as secretive vectors.

(Structure of the Invention) Yeast is a useful microorganism that has cell structures and functions that are characteristic of higher organisms, and is closely related to human daily life as a raw material for foods, medicines, feeds, etc. It is expected to be developed as a host in engineering.

However, because the yeast cell surface layer has a strong cell wall outside the cell membrane, it is often difficult to purify useful substances (heterologous proteins) produced by genetic recombination technology. Therefore, it is desired to develop a system that secretes products outside the bacterial cells. In addition, continuous culture is possible for batch production, which is expected to greatly increase the production amount, and furthermore, it has great merits such as preventing the degradation of the product by intracellular proteases.

The α-factor gene of the yeast Saccharomyces cerevisiae has been studied and its sequence has been elucidated [Cell, Vol. 30, pp. 933-943 (1982)]. Also yeast α
Studies have been conducted to secrete proteins using the leader sequences of factor genes (Japanese Patent Application Laid-Open No. 132892-1989).
(Japanese Patent Application Laid-Open No. 1960-196093).

The present inventors have searched for a signal peptide that can efficiently secrete useful substances produced within the bacterial cells using yeast as a host, and found that the signal peptide in the leader sequence of the yeast alpha factor gene A specific peptide consisting of a 22-residue amino acid sequence exhibits protein secretion activity, and a plasmid is prepared in which a polynucleotide encoding this peptide is linked upstream of a foreign gene (structural gene) encoding the desired protein. The present invention was accomplished by discovering that a target protein can be effectively secreted when expressed in an appropriate host.

That is, the gist of the present invention is to link the promoter sequence of the S. cerevisiae α factor gene and a polynucleotide encoding a peptide consisting of the following amino acid sequence upstream of the structural gene, and to connect the S. cerevisiae α factor gene downstream of the structural gene.
A region connecting the terminator sequence of the factor gene and containing the ampicillin resistance gene ( ^Apr ) and origin of replication (ori) of plasmid pBR322, 2 μm
A plasmid containing a region containing the plasmid replication origin (ori) and a region containing the tryptophan synthase gene (TRP1) of Satucharomyces cerevisiae.

Met Arg Phe Pro Ser lle Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser Ala Leu Ala
Located in Ala Pro Val.

To explain the present invention in detail, the unique feature of the plasmid of the present invention is that a polynucleotide encoding a peptide consisting of the promoter sequence of the S. cerevisiae α-factor gene and the above amino acid sequence is linked upstream of the structural gene. That's what happens.

A polynucleotide (hereinafter referred to as DNA) encoding a peptide consisting of the above amino acid sequence is
It consists of DNA represented by a 66 base pair (bp) base sequence that encodes a peptide consisting of the 22 amino acid sequence shown in FIG. This DNA can be synthesized or, for example, yeast chromosomal DNA.
can be isolated from the α-factor gene.

The plasmid of the present invention can be easily prepared by the method described below, and the desired protein can be produced by selecting a foreign gene encoding a desired protein as a structural gene, introducing it into an appropriate host, and expressing it. It can be secreted effectively.

Below, the usefulness of the plasmid of the present invention as a secretion vector will be explained in detail using the human β-endorphin gene (β E DNA) as a structural gene (marker).

[Construction of secretion vector] (1) α-factor of Satucharomyces cerevisiae
Preparation of DNA-containing plasmid pLS01 (4.4kb) The details will be described in the Examples below, but chromosomal DNA of Satucharomyces cerevisiae was cut with EcoR and a DNA fragment of around 2kb was added to the plasmid.
pUC13 (Pharmacia catalog, P-L
Biochemicals, p. 27, 27-4973) EcoR
Transform E. coli and insert into
Collect white colonies containing DNA and add the following 16 base nucleotides homologous to the yeast α factor DNA MFα1 and MFα2 5′GGCCAACCAATGTACT3′ (-Gly-Gln-Pro-Met on the C-terminal side of α factor).
- Corresponding to Tyr) as a probe, perform colony hybridization and select positive colonies.
Next, plasmid DNA is separated, and plasmid pLS01 (4.4 kb) containing yeast α-factor DNA is selectively collected.

(2) Preparation of plasmid pREl032 (5.8kb) Plasmid YPp7 (5.7kb) [DNA fragment containing TRP1 of Satucharomyces cerevisiae and
Plasmid containing pBR322, Nature,
282, pp. 39-43 (1979)] was partially cut with EcoR, filled with sticky ends, and then ligated.
One EcoR site of YRp7 was removed
Prepare pREl032 (5.8kb).

(3) Preparation of plasmid pUC8-βE (2.9 kb) Plasmid pYT3-24 (described in JP-A-58-92696, p. 2) was cut with Hae to generate human β
- Endorphin gene (93bp, hereinafter β E
160bp of Hae~Hae containing DNA)
Collect fragments.

On the other hand, Fuage M13mp7 (Nucleic Acids
RF of Research Vol. 9, pp. 309-321)
Hinc-Hinc fragment obtained by cutting DNA (double-stranded DNA) with Hinc and dissolving it in ethanol.
The 160 bp DNA fragment containing the above βE DNA was ligated to the Hinc site excluding 5'GACCTGCAGGTC3', and E. coli was transformed. Prepare RF DNA from the transformed strain and cut it with BamH to obtain 172bp BamH ~ BamH
The fragment was transferred to plasmid pCU8 [Bethesda
pUC8-β by inserting it into the BamH site of the BRL catalog (August 1, 1983), Cat/No. 5359SA published by Research Laboratories, InC.
Get E (2.9kb).

(4) Preparation of plasmid pREl046 (0.4kb) EcoR~Hind fragment (1.4kb) containing the promoter sequence and leader sequence (including the nucleotide sequence of the present invention shown in Figure 1) of pLS01 (4.4kb)
and the Hind~EcoR fragment (0.2 kb, containing βE DNA) of pUC8-βE (2.9 kb), pREl032
(5.8kb) into the EcoR cleavage site.
Obtain pREl046 (7.4kb). (See Figure 2) (5) Preparation of plasmid pREl052 (5.2kb) EcoR containing TRP1 of pREl032 (5.8kb)
Pst fragment (0.8kb), Pst~EcoR fragment (2kb) containing the replication origin of the 2μm plasmid,
Pvu~EcoR fragment (2.3kb) of pBR322,
EcoR after ligation to create pREl051 (5.1kb)
Cut it partially, fill it with sticky ends, and connect it.
pREl051 lacks one EcoR cleavage site
pREl052 (5.2kb) was obtained. (See Figure 3) (6) Preparation of plasmid pREl059 (6.8kb) The promoter sequence of pREl046 obtained in (4) above,
Contains leader sequence and β E DNA sequence
EcoR~Sma fragment (1.6kb) and pLS01
(4.4kb) Hinc including terminator sequence
-Collect EcoR fragment (0.3kb).

Meanwhile, pREl052 (5.2kb) was cut with EcoR and
Next, bacterial alkaline phosphatase (BAP)
After removing the terminal phosphate group by adding
Connect the above two DNA fragments and create a plasmid
Obtain pREl059 (6.8kb). (See Figure 4) (7) Preparation of plasmid pREl066 (6.6kb) Cut pREl059 with Hinc, Ban and EcoR to create a Ban~Hinc fragment (1.5kb), Hinc
~EcoR fragment (0.5kb) and Bam~EcoR
Prepare fragments (4.6kb) and ligate them together.
Plasmid PREl066 (6.6 kb) in which the Hinc to Hinc site of pREl059 is deleted is obtained. (5th
(See figure) (Effect of the invention) Plasmid pREl066 has only the short signal sequence of the present invention consisting of only 66 bases as shown in Fig. 1 upstream of the β E DNA sequence, but this plasmid can be used, for example, in yeast. Satucharomyces
When introduced into S. cerevisiae YNN27 strain and culturing the transformed strain, β-endorphin can be secreted into the culture solution with high efficiency, as shown in Examples below.

(Example) The present invention will be explained in more detail by giving examples below.

In addition, the operations in the following examples were performed in accordance with the following methods, unless otherwise specified.

[Cutting and recovery of DNA with restriction enzymes] Use the following four types of buffers for cutting with restriction enzymes.
Bacterial Genetics (1981) (Cold spring
Harbor, New York). The cutting conditions were as follows: 2 units/μg DNA of restriction enzyme, 37
℃ or 65℃ for 30 minutes.

Then, TE buffer (10mM Tris-HCl PH
Extract once with phenol saturated with 8.0 and 1 mM EDTA, remove the phenol with ether, add 2 volumes of ethanol, and incubate at -20°C.
After leaving for 30 minutes, centrifuge to collect DNA.

(1) Low salt concentration buffer consisting of 10mM Tris-HCl (PH7.4), 10mM magnesium sulfate and 1mM dithiothreitol.

(2) Medium salt concentration buffer 50mM NaCl, 10mM Tris-HCl (PH
7.4) 10mM Magnesium Sulfate and 1mM
dithiothreitol.

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

(4) Restriction enzyme Sma buffer 20mM KCl, 10mM Tris-HCl (PH
8.0), 10mM magnesium sulfate and 1mM
dithiothreitol.

[Plasmid from E. coli HB101
Preparation of DNA] (1) Mini preparation method (mini prep method) Nucleic
Acids Res. Vol. 7, pp. 1513-1523 (1979)] Escherichia coli HB101 was used as a host, and 0.5 ml of L-broth (10 g of peptone, 5 g of yeast extract, 1 g of glucose, 5 g of NaCl/1
The cells were cultured overnight using a solution containing PH7.2) and then centrifuged to collect the bacteria.
The suspension was suspended in 25mM Tris-HCl (PH8.0) and 2mg/ml of lysozyme and left at room temperature for 30 minutes.

Then add 200μ of solution B [1%
0.2N with SDS (sodium dodecyl sulfate)
of NaOH] and shake, and at the same time
Performs DNA denaturation. Add 150μ of 3M sodium acetate solution, cool on ice, centrifuge, add cold ethanol to the supernatant, cool to -20°C, centrifuge, and collect the precipitate.

The precipitate was dissolved in solution C (50mM Tris-HCl and
After removing insoluble matter, add cold ethanol to wash the precipitated DNA, dry under reduced pressure, and store at -20°C.

(2) Large-scale preparation method Inoculate E. coli HB101 into 200 ml of L-broth (containing the drug in the case of drug-resistant plasmids), culture overnight, collect the bacteria, and inoculate 15 ml of STES.
Buffer [TES buffer (10mM Tris-HCl (PH7.4), 1mM EDTA and 50mM NaCl
EDTA, lysozyme and ribonuclease A (manufactured by Sigma) were suspended in 25% sutucarose and
Add 600 μg/ml and 50 μg/ml of mM, and further add 500 μg/ml of pronase E in ice water. Next, add SDS to 1%,
Shake at 37°C, return to ice water, add NaCl to a final concentration of 1M, and centrifuge.
Add 2 volumes of cold ethanol to the supernatant and incubate at -20
The DNA is collected as a precipitate by centrifugation at a temperature of 0.degree. C., dried under reduced pressure, and stored at -20.degree.

[Legation using T4 DNA ligase] The two DNA pieces to be ligated are prepared in a ligation buffer [66mM Tris-HCl (PH7.5), 6.6mM magnesium chloride,
consisting of 10mM dithiothreitol] and treated at 65°C for 10 minutes, followed by 66μM dithiothreitol at 4°C.
After adding ATP (adenosine triphosphate) and T4 ligase at 0.1 unit/μg DNA for sticky ends or 1 unit/μg DNA for blunt ends, react at 4°C for 18 hours. , and process for 10 min at 65 °C.

[Advanced Bacterial Transformation
Genetics (1981) (Cold Spring Havor, New,
York)] E. coli HB101 is inoculated into 5 ml of L-broth and cultured overnight. Transfer this 0.2ml to 20ml L-
Plant in broth and incubate with shaking at 37°C until the number of cretunate units reaches 60. The bacterial cells were collected and cooled on ice.
Suspend in 10 ml of a buffer consisting of 50 mM calcium chloride and 10 mM Tris-HCl (PH 8.0) and cool on ice for 30 minutes. The centrifuged cells were suspended in 1 ml of calcium chloride solution, and 0.1 ml of this was added to a 10μ
After mixing with the DNA solution and incubating at 0°C for 30 minutes and at 42°C for 2 minutes, 1.5ml of L-broth was added and incubated at 37°C for 30 minutes, and 0.1ml of this was planted on an agar medium.

V [Removal of DNA sticky ends using S1 nuclease] DNA with sticky ends was removed in 200μ high salt concentration buffer [30mM sodium acetate (PH4.25), 0.3M
20 units/μg DNA of S1 nuclease was added, treated at 22°C for 40 minutes, extracted with phenol saturated with TE buffer, and the phenol was removed with ether. , add ethanol, cool to -20°C, and collect the precipitated DNA by centrifugation.

[Phosphorylation of Bam H linker] Bam H linker (manufactured by BRL) 5′CCGGATCCGG3′ GGCCTAGGCC does not have a phosphate group at the end, so T4
Phosphorylation was performed with kinase.

3 nmol of BamH linker, 41μ of 80
After dissolving in mM Tris-HCl (PH7.5) and 12mM magnesium chloride and incubating at 60℃ for 10 minutes, 10mM 2-mercaptoethanol and 20mM ATP were added at 37℃, and 10 units of T4 kinase was added. After adding and treating at 37°C for 30 minutes, store at -20°C.

[Linking Bam H linker to blunt end] Connect the blunt end DNA fragment (1.2 p mol end) and the Bam H linker phosphorylated above (100 p mol end).
mol end) in ligation buffer [66mM Tris-HCl (PH7.5), 6.6mM magnesium chloride, 10
1 unit of T4 ligase and react at 4°C for 18 hours, then treated with BamH to remove excess BamH.
Remove the linker, extract the DNA with phenol saturated with TE buffer, remove the phenol with ether, and recover the DNA by ethanol precipitation.

[Bacterial alkaline phosphatase (BAP) treatment of plasmid DNA] To prevent self-ligation of plasmid DNA,
Prior to ligating the restriction enzyme site of the plasmid DNA with the DNA fragment, the plasmid DNA is
Process with BAP.

Plasmid DNA (10p) cut with restriction enzymes
mol 5′ end) in 200μ BAP buffer [10mM
of Tris-HCl (PH8.0) and 0.1mM EDTA], 100 units of BAP was added, and 65
After reacting at ℃ for 1 hour, extraction treatment was performed with phenol saturated with TE buffer, phenol was removed with ether, and plasmid was isolated by ethanol precipitation.
Collect DNA.

Example (1) Preparation of plasmid pLS01 (4.4kb) Yeast Satucharomyces cerevisiae (S.
cerevisiae) IFO 1136 chromosomal DNA 500μg
500μ buffer [100mM Tris-HCl (PH
7.5), 50mM NaCl, 10mM magnesium chloride, and 1mM dithiothreitol] was treated with 15 units of EcoR at 37°C for one night, concentrated, and subjected to sucrose density gradient centrifugation to obtain around 2kb. Collected DNA fragments. This was ligated with 1 μg of a fragment obtained by cutting plasmid pUC13 using EcoR using 2 units of T4 ligase.
Escherichia coli strain JM83 was transformed with the obtained plasmid, and ampicillin ( ^Apr ) resistant strains were selected.

Colony hybridization was performed using the synthetic oligonucleotide 5′GGCCAACCAATGTACT3′ as a probe, positive colonies were selected, plasmid DNA was isolated, and analysis with restriction enzymes and nucleotide sequence determination were carried out in Cell, Vol. 30, p. 937 ( Plasmid pLS01 containing yeast α-factor DNA with a base sequence consistent with that described in 1982)
(4.4kb) was selected.

(2) Preparation of plasmid pREl032 (5.8 kb) Plasmid YRp7 (5.7 kb) was partially cut with EcoR, sticky ends were filled in, and then ligated with T4 ligase to create pREl032 (5.8 kb) in which one EcoR site of YRp7 was removed. kb) was prepared.

(3) Preparation of plasmid pUS8-βE (2.9kb) Plasmid pYT3-24 was cut with Hae,
160bp Hae containing β E DNA (93bp) ~
Hae fragments were collected.

Meanwhile, the RF DNA of Fuage M13mp7
Fragments cut with Hinc and dissolved in ethanol by adding ethanol
After removing 5′GACCTGCAGGTC3′ (Hinc~Hinc), the above βE
T4 the 160bp Hae-Hae fragment containing DNA
It was ligated with ligase. This was introduced into Escherichia coli HB101 strain. DNA was prepared from the obtained transformed strain and cut with BamH.
BamH to BamH fragment, plasmid pUC8
pUC8−βE by inserting it into the BamH site of
(2.9kb) was obtained.

(4) Preparation of plasmid pREl046 (7.4 kb) (a) pLS01 (4.4 kb) was cut with EcoR and Hind to obtain an EcoR-Hind fragment (1.4 kb) containing a promoter sequence and a leader sequence.

(b) pUC8−βE (2.9kb) by Hind and EcoR
Hind~EcoR fragment (0.2kb)
I got it.

(c) pREl032 (5.8 kb) was cut with EcoR and ligated with the DNA fragments obtained in (a) and (b) above using T4 ligase to obtain pREl046 (7.4 kb).

(5) Preparation of plasmid pREl052 (5.2 kb) (a) pREl032 (5.8 kb) was cut with EcoR and Pst to obtain an EcoR-Pst fragment (0.8 kb) containing TRP1.

(b) Cut the 2μm plasmid with EcoR, fill in the sticky ends to make blunt ends, and then insert Pst
Pst~EcoR containing the replication origin by cutting with
A fragment (2kb) was obtained.

(c) The DNA fragments obtained in (a) and (b) above,
Pvu obtained by cutting pBR322 with EcoR and Pvu
~T4 DNA with EcoR fragment (2.3kb)
After ligating with ligase to create pREl051 (5.1 kb) and partially cutting with EcoR, the sticky ends were filled in and blunt ends were ligated with T4 ligase to delete one EcoR fragment site.
pREl052 (5.2kb) was obtained.

(6) Preparation of plasmid pREl059 (6.8kb) (a) Cut pREl046 (7.4kb) with EcoR and Sma, resulting in EcoR containing the promoter sequence, leader sequence, and β E DNA sequence.
~Sma fragment (1.6kb) was collected.

(b) Hinc containing the terminator sequence by cutting pLS01 (4.4kb) with Hinc and EcoR
EcoR fragment (0.3kb) was collected.

(c) Cut pREl052 (5.2kb) with EcoR,
After adding BAP to remove the terminal phosphate group,
The DNA fragments obtained in (a) and (b) above were ligated using T4 ligase to create plasmid pREl059.
(6.8kb) was obtained.

(7) Preparation of plasmid pREl066 (6.6kb) (a) pREl059 (6.8kb) was prepared by Hinc, Ban and
Cut each with EcoR and cut the Ban to Hinc fragment (1.5kb, of which the part upstream from the Hinc site)
66 bases is the signal peptide sequence shown in Figure 1), Hinc~EcoR fragment (0.5kb)
A fragment and a Ban~EcoR (4.6kb) fragment were prepared, and these were ligated with T4 ligase.
Plasmid PREl066 (6.6 kb) in which the Hinc to Hinc site of pREl059 was deleted was obtained.

(8) Confirmation of the base sequence of plasmid pREl066 pREl066 DNA was cut with BamHI, the 5-terminal phosphate was removed by BAP treatment, and γ-P-
The 5-end was labeled with 32P using ATP and polynucleotide kinase. This was cleaved with Ava, and a fragment of approximately 320 bp obtained was eluted from a polyacrylamide gel, and the base sequence was determined by the Maxam-Gilbert method, confirming that the splicing method was as expected.

(9) Transformation of the yeast Satucharomyces cerevisiae strain YNN27 using plasmid pREl066 The yeast Satucharomyces cerevisiae (S.
cerevisiae) YNN27 strain was cultured overnight in YPD medium (consisting of 1% yeast extract, 2% peptone, and 2% glucose). 0.5 ml of culture solution.
were planted in 20 ml of YPD medium and cultured with shaking at 30°C until reaching 60 units. The bacterial cells obtained by centrifuging this 10 ml were washed with 10 ml of TE buffer,
Suspend in 1 ml of TE buffer. 0.5 to this 0.5ml
Add ml of 0.2M lithium acetate, 10mM Tris-HCl (PH7.5) and 1mM EDTA and incubate at 30°C.
After holding for an hour, cool in ice water.

Add 10μ of the DNA solution to 100μ of the obtained bacterial cell suspension.
was added and kept at 0℃ for 30 minutes, then 200μ
70% polyethylene glycol 4000 solution was added, and the bacteria were kept at 30°C for 1 hour and then at 42°C for 5 minutes. The bacteria were collected, washed with 0.5ml of water, suspended in 0.3ml of water, and 0.1% per plate was added. mlplant.

(10) Secretion amount of β-endorphin After culturing the yeast containing the plasmid pREl066 obtained in (9) above for 2 days, it was centrifuged, and the supernatant was collected using a β-endorphin [RIA] kit.
England Nuclear, catalog number NEK−
As a result of performing radioimmunoassay using 003) according to the method described in the catalog, the amount of β-endorphin in the supernatant was 73.7 ng/ml.

In addition, the above plasmids pREl046 and pREl052
Similarly, Saccharomyces cerevisiae
After transforming the YNN27 strain and culturing, the supernatants were centrifuged and the amounts of β-endorphin measured by the same method as above were 18.3 ng/ml and 0.2 ng/ml, respectively.

[Brief explanation of the drawing]

FIG. 1 shows the amino acid sequence of the polynucleotide of the present invention and the base sequence of the DNA encoding the amino acid, and FIGS. 2 to 5 are schematic diagrams showing the construction route of the plasmid in the present invention.
E stands for EcoR, H stands for Hind, S stands for Sal,
P stands for Pst, B stands for BamH, Pv stands for Pvu,
Sm stands for Sma, Hc stands for Hinc, Bn stands for Ban,
x indicates Xba, respectively.

Claims (1)

[Scope of Claims] 1. The promoter sequence of the S. cerevisiae α-factor gene is linked to the upstream of the structural gene and a polynucleotide encoding a peptide consisting of the following amino acid sequence, and the S. cerevisiae α-factor gene is linked downstream of the structural gene. and the terminator sequence of the plasmid.
A plasmid comprising a region containing the ampicillin resistance gene and replication origin of pBR322, a region containing the replication origin of a 2 μm plasmid, and a region containing the tryptophan synthase gene of Satucharomyces cerevisiae. Met Arg Phe Pro Ser lle Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser Ala Leu Ala
Ala Pro Val