JPH0336516B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to complex plasmids. More specifically, it relates to a complex plasmid that utilizes the yeast invertase gene.

(Structure of the Invention) Yeast has cell structures and functions that are characteristic of higher organisms, and can also be used as a raw material for foods, medicines, feeds, etc.
They are useful microorganisms that are closely related to human daily life, and are expected to be developed as hosts in genetic engineering.

However, because the yeast cell surface layer has a cell membrane and a strong outer cell wall, it is often difficult to purify useful substances (heterologous proteins) produced by genetic recombination technology. There is a desire to develop a system that secretes the product outside the bacterial cell.

The present inventors have achieved the present invention as a result of searching for plasmid vectors that can efficiently secrete useful substances produced within bacterial cells in genetic recombination technology using yeast as hosts.
The gist is that EcoR is located approximately 900 bp upstream of the invertase translation start codon in the Succalomyces cerevisiae invertase gene (SUC2).
From the N-terminal Ser of mature invertase
DNA up to the Ava site at the GTC position that encodes Val, the 12th amino acid downstream
The DNA from the Sau3A site approximately 70 bp upstream of the invertase stop codon to the Alu site approximately 500 bp downstream of the invertase gene (SUC2) of Satucharomyces cerevisiae is combined with the Ava site of Sau3A.
DNA directly linked to the site via a linker
A complex plasmid characterized by containing.

exists in

To explain the present invention in detail below, invertase is known as a typical secreted enzyme of the yeast Saccharomyces cerevisiae, but this enzyme is localized between the cell membrane and the cell wall, and a small portion of it is It just leaks out. This is thought to be due to the large molecular weight of this enzyme, which prevents it from passing through the cell wall.

The present invention provides a complex plasmid that can secrete useful substances produced within bacterial cells by utilizing a specific region of the invertase gene.

The SUC2 gene is known as the invertase gene of Satucharomyces cerevisiae, and its nucleotide sequence and corresponding amino acid sequence have been elucidated [Nucleic Acids research, Vol. 11,
No. 6, pages 1943-1953, especially its pages 1948-1949].

In the composite plasmid of the present invention, the invertase gene utilization region includes the invertase gene promoter sequence and signal sequence (consisting of 19 amino acid residues) from EcoR to Ava
EcoR region, approximately 900 bp upstream of the translation initiation codon of invertase in the SUC2 gene mentioned above.
From the N-terminal Ser of mature invertase
The base sequence from (TCA) to the Ava site located at the 12th Val (GTC) position downstream is used. In addition, the above region containing a terminator sequence is
In the SUC gene, the Sau3A to Alu region from the Sau3A site approximately 70 bp upstream of the invertase stop codon to the Alu site approximately 500 bp downstream is used.

The complex plasmid of the present invention is
Ava site in the EcoR~Ava region,
It contains a base sequence directly linked to the Sau3A site in the Sau3A-Alu region via a linker.

Note that plasmid 2 μm DNA and origins of replication such as pBR322 are used, and selection markers such as TRP1 and Ap ^r are used.

Below, the method for preparing the complex plasmid of the present invention,
The utility of inserting the mouse amylase gene into this plasmid and transforming yeast will be explained.

1 [Preparation of plasmids pSMF2, 3, 4 (3.8 kb)] Plasmid pRB58 containing the invertase gene SUC2 [Cell, Vol. 28, pp. 145-154 (1982)]
was cut with Xho and BamH to contain the SUC2 promoter sequence and signal peptide sequence.
Isolate the Xho~BamH fragment (approximately 2.9 kb),
Further cleavage with Ava yields a DNA fragment of approximately 1.3 kb. After filling both ends, BamH linkers (8mer, 10mer, 12mer) are connected.

The three types of DNA fragments obtained were incubated with EcoR and
After cutting with BamH, approximately 1kb containing the promoter sequence and signal peptide sequence of SUC2 was obtained.
Obtain the EcoR to BamH fragment.

This was then transformed into plasmid pUC38 [Bethesda
BRL Catalog (August 1, 1983) published by Research Laboratories, Inc., listed in Cat/No. 5359 SA] is inserted into the EcoR to BamH site.
Plasmids pSMF2, 3, and 4 are obtained, respectively. (1st
(See figure) 2 [Preparation of plasmid pSMF6 (3.3kb)] The above plasmid pRB58 was incubated with Xba and EcoR.
The Xba to EcoR fragment containing the terminator sequence of the invertase gene was isolated by cutting with
Next, cut this with Sau3A and Dde.
After obtaining the Sau3A~Dde fragment and filling both ends of this,
Connect BamH linker (10mer) and BamH
and Alu, and the resulting DNA fragment (approximately 0.55 kb) was added to BamH of plasmid pUC8.
~Hinc site to obtain plasmid pSMF6. (See Figure 1) 3 [Preparation of pSMF12, 15, 18 (3.9kb)] (1) Cut plasmid pBR322 with Pvu,
Connect the Hind linker to create the Pvu site.
After changing to Hind site, EcoR and Hind
(2) Cut the plasmids pSMF2, 3, and 4 into EcoR to create an EcoR-Hind fragment containing the ARS and ampicillin resistance genes of E.
and EcoR~BamH by cutting with BamH
(3) Transfect the plasmid pSMF6 with BamH and
Cut with Hind to create a BamH-Hind fragment, and the three DNA fragments of (1), (2) and (3) above,
pSMF12, ligated using T4 DNA ligase
15 and 18 are made. (See Figure 1) 4 [Preparation of pSMF32T, 35T, 38T (6.6kb)] (1) Cut yeast plasmid 2μm DNA with EcoR and Pst to create EcoR~Pst containing ARS.
Get the fragment.

(2) On the other hand, plasmid YRp7 [Nature, vol. 282,
39-43 (1979)], including TRP1 and ARS1.
Cut the EcoR~EcoR fragment with Pst,
Prepare an EcoR~Pst fragment containing only TRP1.

The EcoR-EcoR fragment containing the 2 μm DNA replication origin and TRP1 of YRp7 obtained by ligating the fragments of (1) and (2) was added to pSMF12,
pSMF32T by inserting into the EcoR site of 15 and 18,
Get 35T, 38T. (See Figure 1) The pSMF32T, 35T, and 38T obtained above are
Contains the promoter sequence, signal sequence and terminator sequence of Satucharomyces cerevisiae invertase gene, and contains 2 μm DNA and
It is a shuttle vector that has an origin of replication derived from pBR322, and has selection markers such as TRR1 and Ap ^r .

For this reason, for example, on this BamH site,
By inserting the aforementioned mouse amylase gene and transforming yeast, amylase can be secreted into the medium.

Note that similar results can be obtained by using plasmids 5 and 6 below instead of pSMF32T, 35T, and 38T described above.

5 [Preparation of pSMF32, 35, 38 (5.4 kb)] Cut YRp7 in 4 above with EcoR,
EcoR to EcoR containing TRP1 gene and ARS1
The fragment is isolated and inserted into the EcoR site of pSMF12, 15, 18 to obtain pSMF32, 35, 38.

6 [Preparation of pSMF32S, 35S, 38S (5.8kb)] (1) Cut plasmid 2μm DNA with Ava,
Ava ~ After collecting and filling Ava fragments
Cut with EcoR and include REP3 gene
Obtain an 850bp blunt DNA fragment.

(2) On the other hand, YRp7 is cleaved with EcoR and Rsa to prepare an EcoR-Rsa fragment containing TRP1 and ARS1.

By combining the fragments of (1) and (2)
EcoR~ including TRP1, ARS1 and REP3
Prepare an EcoR fragment and add it to the pSMF12,
pSMF32S by inserting into the EcoR site of 15 and 18,
Get 35S, 38S.

7 [Construction of secretion vector] (1) Introduction of mouse amylase gene (a) Plasmid pMSa104 [Cell, 179
vol., p. 21 (1980)] with Pst, and the resulting Pst-Pst fragment was transformed into a plasmid.
pUC13 [Pharmacia catalog, P-
LBiochemicals, p. 27, 27-4973]
Plasmid pRE1075 (4.3kb) containing the mouse amylase gene inserted into the Pst site.
Create.

pRE1075 was cut with Apa and the obtained
The Apa-Apa fragment is treated with S1 nuclease to make it blunt-ended and then cut with Dra to obtain an Apa-Dra fragment encoding an amylase protein lacking the first 15 amino acids of the amylase precursor. (This gene lacks the amylase secretion signal). A BamH linker (eg, 12mer) is attached to this and cut with BamH to obtain a DNA fragment with BamH sites at both ends.

(b) On the other hand, pSMF32T, 35T, 38T
(pSMF32, 35, 38 or pSMF32S, 35S,
38S) is cleaved with BamH, and then treated with alkaline phosphatase derived from calf small intestine (ClAP) to remove the phosphate group.

Plasmid pRE1100 containing the mouse amylase gene by combining the DNA fragments obtained in (a) and (b)
(8.1 kb). (See Figure 2) (Effect of the invention) Composite plasmid pREl100 constructed as described above
is introduced into the yeast Saccharomyces cerevisiae by a transformation method using, for example, lithium acetate,
When the transformed strain is cultured, amylase can be secreted into the medium 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 Examples below were performed in accordance with the following methods, unless otherwise specified.

[Cutting and recovery of DNA using restriction enzymes] Use the following three types of buffers for cutting with restriction enzymes.
Bacterial Genetics (1981) (Cold spring
(Hirbor, New York). The cutting conditions were as follows: using a restriction enzyme of 2 units/μg DNA;
Process at 37°C or 65°C 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.

[Plasmid DNA from E. coli
Preparation] (1) Mini preparation method (mini prep method) [Nucleic
Acids Res. Vol. 7, pp. 1513-1523 (1979)] E. coli containing plasmid DNA was
L-broth (10g peptone, 5g yeast extract, 1g glucose, 5g NaCl/
PH7.2) consisting of 1) overnight.
The bacterial cells collected by centrifugation were added to 100μ of solution A [50mM glycose, 10mM
EDTA, 25mM Tris-HCl (PH8.0) and 2mg/ml of lysozyme] and left in ice water for 30 minutes.

Then add 200μ of solution B [1%
0.2N with SDS (sodium dodecyl sulfate)
NaOH] and shake to denature the DNA at the same time.

Add 150μ of 3M sodium acetate solution (PH4.8), cool on ice, and centrifuge. Add cold ethanol to the supernatant, cool to -70°C, centrifuge, and collect the precipitate. The precipitate was dissolved in 400μ of solution C [consisting of 50mM Tris-HCl (PH8.0) and 0.1M sodium acetate], and after removing insoluble materials, cold ethanol was added to wash the precipitated DNA, and the solution was dissolved under reduced pressure. Dry and store at -20℃.

(2) Large-scale preparation method E. coli containing plasmid DNA is cultured using 100 ml of L-broth (containing drugs in the case of drug-resistant plasmids), and after collecting and washing,
3ml of solution A (50mM glucose, 10mM
EDTA, 25mM Tris-HCl (PH8.0) and 2mg/ml of lysozyme),
Leave in ice water for 30 minutes.

Then add 4 ml of solution B [1% SDS
(sodium dodecyl sulfate) containing 0.2N
NaOH] and shake to simultaneously denature the DNA.

Add 3 ml of 3M sodium acetate solution (PH 4.8), cool on ice, centrifuge, add 0.6 volume of isopropanol to the supernatant, and leave at -20°C for 20 minutes.

The precipitate is collected by centrifugation, washed with ethanol, and then suspended in 2 ml of TE (PH7.5) buffer.
Next, 10μ of RNase A (10mg/ml) is added, and after standing at 37°C for 30 minutes, phenol extraction is performed. 0.2 volume of 5M NaCl and 1/3 volume of 30% in the aqueous layer
Add polyethylene glycol 6000 and heat to -20℃
for 40 minutes, then at 4°C for 40 minutes. Collect the precipitate by centrifugation, dissolve in 400 ml of water,
Precipitate the DNA with ethanol and wash with 100μ
Dissolve in TE buffer.

[Legation using T4 DNA ligase] The two DNA fragments to be ligated are 1μg/10μ
Add the ligation buffer [66mM Tris-HCl (PH7.5), 6.6mM magnesium chloride,
consisting of 10mM dithiothreitol] and treated at 65℃ for 10 minutes, followed by 66μM dithiothreitol at 4℃.
Add ATP (adenosine triphosphate) and T4 ligase at 0.1 unit/μg DNA for sticky ends or 1 unit/μg DNA for blunt ends, and heat at 4°C for 18 hours.
After reacting for an hour, treat at 65°C for 10 minutes.

[Transformation of E. coli] [Advanced Bacterial
Genetics (1981) (Cold Spring Harbor, New
York)] Inoculate E. coli into 5 ml of Reebros and culture overnight. 0.2 ml of this was inoculated into 20 ml of L-broth, and cultured with shaking at 37°C until the number of cretunate units reached 60. Collect bacterial cells and cool on ice with 50mM calcium chloride and 10mM Tris-HCl (PH8.0)
Suspend in 10 ml of buffer consisting of and cool on ice for 30 minutes. The centrifuged cells were suspended in 1 ml of calcium chloride solution, 0.1 ml of this was mixed with 10μ of DNA solution, and incubated at 0°C for 30 minutes and 42°C for 2 minutes, then 1.5ml of L-broth was added. ℃
Incubate for 30 minutes and inoculate 0.1 ml of this onto an agar medium.

[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
of S1 nuclease at 20 units/μg DNA.
The mixture was incubated at 22°C for 40 minutes, extracted with phenol saturated with TE buffer, the phenol was removed with ether, and cold ethanol was added.
Cool to ℃ and precipitate by centrifugation.
Collect DNA.

[Filling of sticky ends] 1μg of DNA was added to 30μ of 50mM Tris-HCl (PH7.2), 10mM magnesium sulfate, 0.1mM
dithiothreitol, 50 μg/ml bovine serum albumin (BSA) and 0.2 mM dNTP, add 1.25 units of Klenow fragment (a large fragment of E. coli DNA polymerase), and react for 30 minutes at room temperature. Then 1μ 0.5M EDTA
(PH8.0) to stop the reaction, extract sequentially with phenol and ether to remove phenol, and collect DNA by ethanol precipitation.

[Phosphorylation of Bam H linker] Bam H linker (manufactured by BRL) 8mer: 5′CGGATCCG 3′, 10mer:
5′CCGGATCCGG 3′ 12mer: 5′CCCGGATCCGGG 3′ (only one strand of the duplex is shown) does not have a phosphate group at the end, so T4
Phosphorylation was performed with kinase.

3 n mol of BamH linker, 41 μ 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 was added and reacted 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.

[Treatment of DNA fragments with calf small intestine alkaline phosphatase (ClAP)] In order to prevent self-ligation of plasmid DNA by the ligase reaction, the restriction enzyme-cleaved fragments of plasmid DNA are treated with ClAP prior to the ligase reaction. .

Plasmid DNA (10p) cut with restriction enzymes
mol5' end) was dissolved in 50μ of ClAP buffer [consisting of 50mM Tris-HCl (PH9.0), 1mM magnesium chloride, 0.1mM zinc chloride and 1mM spermidine], and per 1pmol end,
After adding 0.01 units of ClAP and reacting at 37℃ for 30 minutes,
10μ 0.1M Tris-HCl (PH8.0), 1M Nacl,
Add 10mM EDTA, 5μ of 10% SDS, 40μ of water and hold at 65°C for 15 minutes. After cooling, extract with phenol saturated with TE buffer, remove phenol with ether, and collect plasmid DNA by ethanol precipitation.

[Transformation of yeast (KU method) Transform 10 ml of the yeast Satucharomyces cerevisiae into
Culture the cells overnight at 30°C in YEPD medium, collect the bacteria, wash once with TE buffer, and suspend in the same buffer until the number of cells is 2 x 10 ⁸ cells/ml.

Add the same amount of 0.2M lithium acetate (PH7.5) to 500μ of this suspension and hold at 30°C for 1 hour.
Dispense the DNA into 100μ test tubes at 0°C.
and mix for 30 minutes at 0°C. 100μ
Add TE buffer containing 70% polyethylene glycol 4000, mix thoroughly, and then hold at 30°C for 1 hour, then at 42°C for 5 minutes, centrifuge to collect bacteria, and wash with sterile water.

Suspend this in 500μ of sterilized water, inoculate 100μ onto a selective medium, and culture at 30°C for 3 to 4 days to obtain a transformed strain.

Example 1 [Preparation of plasmid pSMF4 (3.8 kb)] Plasmid pRB58 containing the invertase gene SUC2 was cut with Xho and BamH.
Xho to BamH fragment (approximately 2.9kb) containing the promoter sequence and signal peptide sequence of SUC2
was isolated and further fragmented with Ava to create approximately 1.3kb
After obtaining the DNA fragment and filling both ends of this BamH
A linker (12mer) was connected.

The obtained DNA fragment was digested with EcoR and BamH.
About 1kb of EcoR containing the promoter sequence and signal peptide sequence of SUC2 is cut with
BamH fragment was obtained.

This was then converted into plasmid pUC8 EcoR
~Insert into the BamH site and make the plasmid
pSMF4 was obtained.

2 [Preparation of plasmid pSMF6 (3.6kb)] The above plasmid pRB58 was incubated with Xba and EcoR.
The Xba to EcoR fragment containing the terminator sequence of the invertase gene was isolated by cutting with
Next, cut this with Sau3A and Dde.
After obtaining the Sau3A~Dde fragment and filling both ends of this,
Connect BamH linker (10mer) and BamH
and Alu, and the resulting DNA fragment (approximately 0.55 kb) was added to BamH of plasmid pUC8.
~Hinc site was inserted to obtain plasmid pSMF6.

3 [Preparation of pSMF18 (3.9kb)] (1) Cut plasmid pBR322 with Pvu,
Connect the Hind linker to create the Pvu site.
After changing to Hind site, EcoR and Hind
(2) Cut the plasmid pSMF4 into EcoR and Hind fragments containing E. coli ARS and ampicillin resistance genes.
Cut with BamH to create an EcoR-BamH fragment, (3) cleave the plasmid pSMF6 with BamH and
Cut with Hind to create a BamH-Hind fragment, and the three DNA fragments of (1), (2) and (3) above,
pSMF18 was created by ligation using T4 DNA ligase.

4 [Preparation of pSMF38T (6.6kb)] (1) Cut yeast plasmid 2μm DNA with EcoR and Pst to create EcoR~Pst containing ARS.
Got a piece.

(2) Contains TRP1 and ARS1 of plasmid YRp7
Cut the EcoR~EcoR fragment with Pst.
An EcoR~Pst fragment containing only TRP1 was prepared.

Origin of replication of 2 μm DNA and TRP1 of YRp7 obtained by combining fragments (1) and (2)
The EcoR~EcoR fragment containing pSMF18
It was inserted into the EcoR site to obtain pSMF38T.

The pSMF38T obtained above has the promoter sequence, signal peptide sequence, and terminator sequence of the S. cerevisiae invertase gene, and contains 2 μm DNA and
It is a shuttle vector that has an origin of replication derived from pBR322, and has selection markers such as TRR1 and Ap ^r .

7 [Construction of secretion vector] (1) Introduction of mouse amylase gene (a) Cleave plasmid pMSa104 containing amylase gene derived from mouse pancreas with Pst, and insert the obtained Pst-Pst fragment into the Pst site of plasmid pUC13. do,
Plasmid containing mouse amylase gene
pRE1075 (4.3kb) was created.

pRE1075 was cut with Apa and the obtained
The Apa-Apa fragment was treated with S1 nuclease to make blunt ends and then cut with Dra to produce an amylase protein lacking the first 15 amino acids of the amylase precursor (this gene lacks the amylase secretion signal). )did. Code Apa~Dra
Collect fragments. A BamH linker (12mer) was attached to this and cut with BamH to obtain a DNA fragment with BamH sites at both ends.

(b) On the other hand, after cutting pSMF38T with BamH,
Phosphate groups were removed by treatment with calf small intestine alkaline phosphatase (ClAP).

Plasmid pREl100 containing the mouse amylase gene by combining the DNA fragments obtained in (a) and (b)
(8.1 kb) was obtained.

8 Confirmation of amylase activity (secretion) Plasmid pREl100 constructed by the above method
Yeast Saccharomyces cerevisiae using
YNN27 strain was transformed by the KU method.

The resulting transformed strain was inoculated onto a YEP agar medium containing 1% starch as a carbon source and cultured at 30°C for 3 days. As a result, remarkable halo formation was observed due to secretory production of amylase.

In addition, in the same manner as above using the plasmid pSMF38T that does not contain the amylase gene.
The YNN27 strain was transformed, and the transformed strain was inoculated onto a YEP agar medium containing 1% starch as a carbon source.
No halo formation was observed when cultured at 30°C for 3 days.

[Brief explanation of the drawing]

FIG. 1 and FIG. 2 are schematic diagrams showing the construction route of the complex plasmid of the present invention, and the symbols A, Ap, Au, B, D, Dr, E, H, Hc, P,
Pv, S, X, Xh, etc. each represent the following restriction enzymes. A: Ava, Ap: Apa, Au: Alu, B:
BamH, D: Dde, Dr: Dra, E: EcoR
, H: Hind, Hc: Hinc, P: Pst,
Pv: Pvu, S: Sau3A, X: Xba, Xh:
Xho.

Claims (1)

[Claims] 1 Approximately 900 bp upstream of the translation start codon of invertase in the invertase gene (SUC2) of Satucharomyces cerevisiae
DNA from the EcoR site to the Ava site at the GTC position encoding Val, which is the 12th amino acid downstream from the N-terminal Ser of mature invertase, and the stop codon of invertase in the S. cerevisiae invertase gene (SUC2). DNA from the Sau3A site approximately 70 bp upstream to the Alu site approximately 500 bp downstream from the Sau3A site with the Ava site.
DNA directly linked to the site via a linker
A complex plasmid characterized by containing.