JPS626685A - Plasmid - Google Patents

Abstract

PURPOSE:A plasmid, containing a promoter and reader sequences of an alpha factor gene of a specific yeast and capable of secreting useful substances produced in microbial cells. CONSTITUTION:A plasmid pRE1086 (6.8kb) obtained by cleaving chromosome gene of Saccharomyces italicus with EcoRI, inserting the resultant cleaved fragment into a plasmid, carrying out operation, e.g. transformation, cultivation, separation of DNA, etc., to give a plasmid pLS11 (4.4kb) (A), containing alphafactor of the Saccharomyces italicus and shown in the figure, treating a DNA fragment derived from Saccharomyces cerevisiae (B) to give a plasmid pRE1059 (6.8kb) and linking the three DNA fragments of EcoRI-HindIII fragment obtained from the component (A), EcoRi-ArtII fragment and HindIII-AatII fragment obtained from the component (B).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention resides in a plasmid containing the promoter and leader sequence of the yeast α-factor gene.

(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, since 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. It is desired to develop a system that secretes products outside the bacterial cells. In addition, continuous culture is possible for secretory production, which is expected to significantly increase the production amount, and furthermore, it has great advantages such as preventing the decomposition of the product by intracellular proteases.

The present inventors have achieved the present invention as a result of searching for promoters and leader sequences that can efficiently secrete useful substances produced within bacterial cells in genetic recombination technology using yeast as a host. Its gist consists in a plasmid containing the promoter and leader sequences of the yeast Saccharomyces italicus α-factor gene.

To explain the present invention in detail, the α-factor gene in the present invention is derived from the yeast Saccharomyces italicus strain (Sacc
haromyces 1talicus, IFO02
53) and can be isolated from the chromosomal genes of Saccharomyces italicus.

The α-factor gene of Saccharomyces italicus has been identified as a result of research by the present inventors, for example, Ce1l, Vol. 30, 93.
The well-known α-factor gene of Satucharomyces cerevisiae, described on p. It was found that there were differences in the genes (consisting of four repeats) and in the base sequences, such as a two base pair difference.

Furthermore, when a foreign gene encoding a desired protein is inserted into a suitable plasmid vector after the promoter sequence and leader sequence, which occupy the main part of the Saccharomyces italicus α-factor gene, and the gene is expressed in the host, It has been found that the target protein can be secreted efficiently.

Below, the isolation of the α-factor gene of Saccharomyces italicus and the human β-endorphin gene (hereinafter β
The preparation method and usefulness of the composite plasmid of the present invention, which uses E DNA) as a marker and contains the promoter sequence and leader sequence of this α-factor gene, will be explained in detail.

[Single* gene containing alpha factor] Preparation details of plasmid pLslI (4,4 kb) are described in the Examples below, but the chromosomal gene of Saccharomyces italicus was cut with EcoRI, and the DNA fragment of around 2 kb was inserted into the plasmid. ρLICI3 (Ph-arm
acia catalog, P-L Biochewica
ls, p. 27, 27-4973)
transform E. coli, collect white colonies containing the inserted DNA, and add the following 16-base nucleotide 5 GGCCAAACCAATGTACT 3 (C-terminal of α-factor) homologous to the α-factor genes MFα1 and MFα2 of S. cerevisiae. -Gly-Gin-Pro-Net-Tyr on the side
(equivalent to ) as a probe, perform colony hybridization and select positive colonies. Next, the plasmid DNA was isolated and the α of Saccharomyces italicus was isolated.
Plasmid ρLS11 (4,4kb) containing the factor gene
Select and collect.

By cutting this plasmid with EcoRI, α
A gene (1.7 kb) containing the factor is obtained (see Figure 1).

[Construction of secretion vector] (1) Preparation of plasmid pLsOI (4,4 kb) containing the α-factor gene of Satucharomyces cerevisiae The chromosomal gene of the yeast Satucharomyces cerevisiae was
Cut with RI and transform the DNA fragment of around 2 kb into plasmid pUc13 (Pharmacia catalog, P-L
Biochemicals, 2? pp. 27-4973
) into the EcoRI site of the large 1i? lr was transformed, white colonies containing the inserted DNA were collected, and the following 16-base nucleotide 5 GGCCAAACCAATGTACT 3 (-Gly-Gln on the C-terminal side of the α-factor) homologous to the yeast α-factor gene MFal and liver α2 was transformed. -Corresponding to Pro-Met4yr) as a probe, colony hybridization is performed and positive colonies are selected.

Plasmid DNA was then isolated and added containing the S. cerevisiae α-factor gene.

Mid pLsO1 (4.4 kb) is selectively collected.

(2) Preparation of plasmid pRE1032 (5,8 kb) Plasmid YRρ7 [5,7 kb, yeast TRP
A plasmid in which a DNA fragment containing I and pBR322 were ligated, Na-ture, Vol. 282, pp. 39-43 (1
pREI032 (5.8 kb) in which one EcoRI site of VRp7 has been removed is prepared by partially cleaving the pREI032 (described in 979) with EcoRI and filling in sticky ends, and then ligating.

(3) Plasmid ρUC8-βE (2,9kb)
Preparation of plasmid pVT3-24 [JP-A-58-9
No. 2696, page 2, containing βE DNA]
Cut with f a e m and make B E DNA (93b
A 160 bp Haem~Hae■ fragment containing p) is collected.

On the other hand, Phage old 3mp? (Nucleic Ac1
RF I DNA (double-stranded DNA) of
Cut with ncII and dissolve in ethanol old nclI
~)Iinall fragment 5'GACCTGCAGGTC3
The 160-lap DNA fragment containing the βE DNA described above is ligated to the old ncII site excluding ', and E. coli is transformed. Prepare RF I DNA from the transformed strain,
This was cut with Bawl (I) to obtain 172 bp
The Bam)II to BamHI fragment of plasmid p
Uc8[Bethesda Re5earcl+ La
BRL Catalog (August 1゜1983) published by BO-Ratories, Inc., Cat/
No. described in 5359SA] into the BamHI site to obtain pUc8-βE (2.9 kb).

(4) Plasmid pREI046 (7.4kb)
Preparation of pLsO1 (4,4 kb) containing the promoter and leader sequences from EcoRI to old ndm (+,4
kb) and ptJc8-βE (2,9kb) old nd
~EcoRI fragment (0.2 kb, βE DNA
) was ligated to the EcoRI cleavage site of pRE1032 (!5.s kb) to create pREI046 (7,4 kb).
kb). (See Figure 2) (5) Preparation of plasmid pRE1052 (5,2 kb) E containing TRPI of pRE1032 (5,8 kb)
coRI-Pst 1 slice (0.8kb) and 2μ
PstI~EcoR containing the replication origin of the m plasmid
I fragment (2kb) and Pvu II of bBR322
~ EcoRI fragment (2,3 kb) was ligated to p
After producing RE+051 (5,1 kb), EcoR1
pRE 1 was partially cut, filled with sticky ends and ligated.
ρREI0 lacking one EcoRI cleavage site of 051
52 (5,2 kb). (See Figure 3) (6)
Preparation of plasmid pRE1059 (6,8 kb) EcoRI~S containing the promoter sequence, leader sequence and βE DNA sequence of pRE 046 obtained in (4) above.
ma I fragment (1,6 kb) and pLsOl (4,4
kb), old ncII to Ec including the terminator sequence
The oRI fragment (0.3 kb) is collected.

On the other hand, pRE1052 (5,2 kb) was cut with EcoRI, and then bacterial alkaline phosph, atase (Bacterial alkaline pho
After removing the terminal phosphate group by adding sphatase (BAP), this is ligated with the above two DNA fragments to obtain plasmid ρREI059 (6.8 kb). (
(See Figure 4) (7) Preparation of plasmid pRE1086 (6,8kb) EcoRI~Aat■ fragment obtained by cutting pREI059 with EcoRI and AatII, pREI0
Old n obtained by cutting 59 with old ndm and Aatll
dlII to AatI [fragment and 1) LSOllt
E obtained by cutting with EcoRI and 1lindl[
The three DNA fragments of coRI-)1indm fragment are ligated to obtain plasmid ρREI086 (6,8 kb'). (See Figure 5) (Effects of the Invention) Plasmid pRE1086 combines the βE DNA sequence with the promoter sequence and leader sequence (1,1
kb). This plasmid can be used, for example, in the yeast Saccharomyces cerevisiae YNN2.
When introduced into the T strain and culturing the transformed strain, β-endorphin can be cultivated with high efficiency, as shown in the Examples below! ! It can be secreted into fluid.

(Example) The present invention will be explained in more detail with reference to Examples below.

In addition, the operations in the following examples were performed according to the following methods 1 to 2, unless otherwise specified.

■ [Cleavage and recovery of DNA using restriction enzymes] Use the following four types of buffers for cutting with restriction enzymes (1) to (3).
How to use Advanced Bacte-ri
al Genetics (+981) (Cold s
Pring Harbor, New York). The cutting conditions include using a restriction enzyme of 2 units/μg DNA at 37°C or 65°C for 30 minutes.

Then, TE buffer (10mM Tris-HCl pH 8)
, extracted once with phenol saturated with 0 and 1 mM E [consisting of lTA], removed the phenol with ether,
After adding 2 times the volume of ethanol and leaving the mixture at -20°C for 30 minutes, centrifugation is performed to recover the DNA.

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

(2) Medium concentration buffer 50mM NaCl, 10mM Tris-HCl (pi(
7,4) Consisting of lomM magnesium sulfate and 1mM dithiothreitol.

(3) High salt buffer consisting of 100 ra NaCl, 50 mM Tris-HCl (pH 7,4) and 10 mM magnesium sulfate.

(4) Restriction enzyme Sma I buffer: 20 mM KCI, 10 mA Tris-HCl (all
8.0), +01H magnesium sulfate and IIIM
dithiothreitol.

■ [Preparation of plasmid DNA from large mMc E, coli) IIBIOI] (1) Mini preparation method (m1ni prep method) Nucl
eic Ac1dsRes, vol. 7, 1513-1523
(1979)] using E. coli 118 I 01 as a host,
0.51 Noshiichi Broth (10g peptone, 5g yeast extract, Ig glucose, 5g NaCl/
Pi 7.2) consisting of 100 μm solution A (50
mM glucose, 10 RIH EDTA, 25 mM Tris-HCl (pH 8,0) and lysozyme 2B/a+l
(consisting of) and left at room temperature for 30 minutes.

Then 200 μm of solution B[:IX 5O5(
0.28 NaOH containing (sodium dodecyl sulfate)]
is added and shaken, and DNA is denatured at the same time.

Add a 150 μm 3M sodium acetate solution, cool with water, centrifuge, add cold ethanol to the upper groove, cool to −20° C., centrifuge, and collect the precipitate.

The precipitate was dissolved in solution C (consisting of 50 mM Tris-HCl and 0.1 part sodium acetate), and after removing insoluble matter, cold ethanol was added to precipitate. The DNA was washed, dried under reduced pressure, and heated at -20°C. Save with .

(2) Large-scale preparation method 2001 Noshiichi broth (contains drug in case of drug-resistant plasmid) is inoculated with E. coli HBIOI, cultured overnight, and after harvesting, inoculate with 15 ml of 5TES buffer [TES buffer ( 25×
saccharose added], suspended in EDTA,
Lysozyme and ribonuclease A (manufactured by Sigma) at 30 a+M, 60071 g/a+l and 50, respectively.
Add μg/ml and further add 500 μg/ml of pronase E in ice water.
Add μg/ml. Next, add SDS to tX, shake at 37°C, return to ice water, and add NaCl.
was added to a final concentration of IM, centrifuged, and twice the volume of cold ethanol was added to the supernatant, kept at -20°C, centrifuged to collect DNA as a precipitate, and dried under reduced pressure. , store at -20°C.

m[Ligation using T4 DNA ligase] Two DNA fragments to be ligated were mixed with ligation buffer [66mM Tris-HCl (+) so that the size of the two DNA fragments was 710μg.
87.5), 6.6 n+M magnesium chloride, 1
0 mM dithiothreitol] dissolved in 65
After treatment for 10 min at 4°C, 66 μH of ATP (
Adenosine triphosphate) and T4 ligase at 0.1 unit/μg DNA for sticky ends or 1 unit/μg DNA for blunt ends, and at 4°C! After reacting for 8 hours, it is treated at 65°C for 10 minutes.

■Transformation of E. coli (Advanced Bacter
ial Ge-netics (1981) (Col.
d Spring Harbor, New York)
] 51 L-Pros was inoculated with Escherichia coli HBIOI, -
Cultivate overnight. This 0.21 was planted in 2On+1 L-broth and cultured with shaking at 37°C until the Klett unit reached 60. The bacterial cells are collected, suspended in ice-cold buffer solution 10 n+1 consisting of 50 mM calcium chloride and 10 mM Tris-HCl (pH 8,0), and cooled on ice for 30 minutes.

The centrifuged facepiece was suspended in a calcium chloride solution of 11, and this 0.11 was mixed with a 10 μm DNA solution and incubated at 0°C.
After incubation for 30 min at 42°C and 2 min at 42°C,
．． Add Noshiichi Broth No. 51 and incubate at 37°C for 30 minutes, then inoculate 0.1 ml of this onto an agar medium.

Removal of DNA sticky ends by vcst nuclease]
DNA with sticky ends was prepared in 200 μm high salt buffer [30 units of sodium acetate (p)I 4.25), 0.0 μm.
3H NaCl and 4mM zinc sulfate], 20 units/μg of St nuclease was added, treated at 22°C for 40 minutes, extracted with phenol saturated with TE buffer, and extracted with phenol in ether. , add cold ethanol, cool to -20°C, and collect the precipitated DNA by centrifugation.

Vl [Ram Hl linker nophosphorylation] BamHI
Linker (manufactured by 8, R, L) 5' CCGGATCCG
Since G 3' GGCCTAGGCC does not have a phosphate group at its end, it was phosphorylated with T4 kinase.

3 n mol of BamHI linker was added to 41μ+8
Dissolve in 0mM 1-Lis-HCl (pH 7.5) and 12mM magnesium chloride, incubate at 60°C for 10 minutes, add 10mM 2-mercaptoethanol and 20mM ATP at 37°C, and add 10 units. After adding T4 kinase and treating at 37°C for 30 minutes,
Store at -20°C.

■ [Linking BamHI linker to blunt end] Blunt-end DNA fragment < 1.2 p mol end) and BamHI linker phosphorylated as described above (100p n+ol
ends) in ligation buffer [66mM Tris-HCl (pH 7)
, 5) , 6.6 n+M magnesium chloride, 10
1 unit of T4 ligase was added, and after reacting at 4°C for 18 hours, fla
Excess BamHI linker was removed by treatment with mHI, and T
E) i! DNA is extracted with phenol saturated with ffi solution, phenol is removed with ether, and DNA is recovered by ethanol precipitation.

■ [Bacterial alkaline phosphatase (BAP) treatment of plasmid DNA] In order to prevent self-ligation of plasmid DNA, plasmid DNA is treated with RAP before ligation of the restriction enzyme site of plasmid DNA and DNA fragment.

Plasmid DNA cut with restriction enzymes (10p an
a15 end) in 200 μM BAP buffer [10 μM Tris-HCl (pH 8,0) and 0.1 a+M E[
100 units of BAP were added, and after reacting at 65°C for 1 hour, extraction was carried out with phenol saturated with TEII stock solution, phenol was removed with ether, and the plasmid was isolated by ethanol precipitation. Collect the DNA.

Example (1) Preparation of plasmid pLsO1 (4,4 kb) 500 μg of the chromosomal gene of Satucharomyces cerevisiae IFo 1136 was mixed in 500 μm buffer [100 mM Tris-HCl (pH 7,5), 50 mM NaCl 1,10
o+M magnesium chloride and In+M dithiothreitol] at 37°C with 15 units of EcoRI.
The fragments were treated overnight, cut, concentrated, and subjected to sucrose density gradient centrifugation to collect [lNA fragments of approximately 2 kb]. This was ligated with 1 μg of a fragment obtained by cutting plasmid ρUCl3 using EcoRI using 2 units of T4 ligase, and the resulting plasmid was used to create large I! Bacterium JM83 strain was transformed and ampicillin-resistant strains were selected.

Colony hybridization was performed using synthetic oligonucleotide 5 GGCCAAACCAATGTACT 3' as a probe, positive colonies were selected, plasmid DNA was isolated, and analysis with restriction enzymes and nucleotide sequence determination revealed Ce1l,
A plasmid ρLSOI (4.4 kb) containing the α-factor gene of Satucharomyces cerevisiae having a base sequence consistent with that described in Vol. 30, p. 937 (1982) was selected and collected.

f2) Preparation of plasmid pRE1032 (5,8 kb) Plasmid YRp7 (5,7 kb) was incubated with EcoRI
pRE1032 (5.8 kb), in which one EcoRI site of YRp7 was removed, was prepared by ligating with T4 ligase and filling in sticky ends.

(3) Plasmid pUc8-βE (2,9kb)
Preparation of plasmid pYT3-24 was cut with HaeII [+60
The Hae[[I~Haem fragment of bl) was collected.

On the other hand, Phage old 3mp? RFI DNA of HincI
Fragment 5' GACCTGCAGGTC ((formerly nc■ ~
After removing Hinc■), 160 bp of Haem to Haem containing the above βE DNA was inserted into the Hinc11 site.
The fragments were ligated with T4 ligase, and this was transformed into E. coli 88101.
introduced into the stock. DNA was prepared from the obtained transformed strain, which was cut with Bam+HI, and the resulting BamHI
-Baa+ If E fragment was added to the plasmid pucs
puca-βE (2,
9kb) was obtained.

(4) Preparation of plasmid pRE1046 (7,4kb) (a) IILsOI (4,4kb')
The EcoRI-Hindul fragment (1.4 kb) containing the promoter sequence and leader sequence was obtained by cutting with coRI and old nijm.

(b) puca-βE (2.9kb) old ndlI
The old ndm to EcoRI fragment (0,2 kb') was obtained by cutting with I and 1coRI.

(c) Digest pREI032 (5.8 kb) with EcoRI, and ligate it with the DNA fragments obtained in (a) and (b) above for T4 recursor L/tepRE1046.
(7,4kb') was obtained.

(5) Preparation of plasmid pRE1052 (5,2kb) (a) pREI032 (5,8kb)
EcoRI containing TRPI by cutting with RI and Pstl
~Pst I fragment (o, 8 kb) was obtained.

(b) Cut the 2 μm plasmid with EcoRI, fill in the sticky ends to make blunt ends, and then cut with PstI to create a blunt end. ! A PstI=EcoRI fragment (2 kb) containing the intervening start point was obtained.

c) The DNA fragments obtained in (a) and (b) above are
P obtained by cutting BR322 with EcoRI and Pvu II
T with vul~EcoRI fragment (2,3 kb)
pRE1051 (5,1k
pREI052 (h) was prepared and partially cut with EcoRI, the sticky ends were filled in and blunt ends were ligated with T4 ligase, and one EcoRI cleavage site was deleted, pREI052 (
5.2kb) was obtained.

(6) PlasmiF pREI 059 (6,8kb
(manufactured by OR) pRE1046 (7,4kb)
was digested with EcoRI and SmaI, and the resulting EcoRI-Smal fragment (+, 6 kb) containing the promoter sequence, leader sequence, and βE DNA sequence was collected.

(b) Cut pt, sot (4,4kb) with HincIi and EcoRI to obtain Hin containing the terminator sequence.
A cII-EcoR1 fragment (0.3 kb) was collected.

(c) EcaR pRε+052 (5.2kb)
After cutting with I and adding BAP to remove the terminal phosphate group, the [)NA fragment obtained in (a) and (b) above was ligated with T4 ligase to create plasmid pREI059 (6
, 8kh) was obtained.

(7) Preparation of plasmid pLsll (-4,4kb) The chromosomal gene soug of Satucharomyces italicus IFO0253 was dissolved in 500 μ+ buffer [100 m
M Tris-HCl (pH 7,5), 50 mM NaCl
, 10 mM magnesium chloride, and 1 mM dithiothreitol] was treated with 15 units of EcoRI at 37°C overnight, concentrated, and subjected to sucrose density gradient centrifugation to obtain approximately 2 kb DNA fragments. collected. This was ligated with 1 μg of a fragment of plasmid pUc13 cut with EcoRI using 2 units of T4 ligase.
Transform Escherichia coli JM83 strain with the obtained plasmid,
Ampicillin-resistant strains were selected.

Colony hybridization was performed using synthetic oligonucleotide 5 GGCCAAACCAATGTACT 3# as a probe, positive colonies were selected, and plasmid DNA was isolated.

This plasmid DNA was analyzed by restriction enzymes and the base sequence was determined.
Unlike the α-factor gene of Sacca 50 Myces cerevisiae described in 2'), the spacer consisting of 63 base pairs and the unit of mature α-factor are repeated five times, and it is different from the α-factor gene of Saccharomyces cerevisiae. teeth,
Plasmid pLs11 (4,4k
b) was selected and collected.

(8) Preparation of plasmid pREI088 (6,8 kb) pREI059 was digested with EcoRI and Aatll to obtain an EcoRI to AatII fragment (5, Okb). In addition, pRE+059)1indlI[and A
Cut with atII to obtain a HindIII-AatII fragment (
0.6kb) was obtained. Additionally, pLsll was labeled with EcoRI
and cut with 11indlll and EcoRI ~ old ndl
[I fragment (1,2 kb') was obtained. These three types of DN
A fragment was ligated with T4 ligase to create plasmid pRE10.
86 (6,8 kb) was obtained.

(9) Transformation of yeast Satucharomyces cerevisiae YNN27 strain with plasmid pRE108B Satucharomyces cerevisiae YNN27 strain was transformed into YP oral medium (IK
of yeast extract, 2x peptone and 2x glucose) - 0.5 ml ft of overnight culture
20 ml) in YPD medium and culture at 1 tilt to 30'C chiclet unit 60. This 10
The bacterial cells obtained by centrifuging 10 ml of TE)
ii buffer and suspend in 11 TE buffer. To this 0.5 ml, add 0.51 of 0.2M lithium acetate, 10
mM Tris-HCl (pH 7,5) and 1 mM (
7) Add EDTA, hold at 30'C for 1 hour, and then cool in ice water.

10 μg of pRE + was added to 100 μm of the obtained bacterial cell suspension.
After adding 10 μ+ of TE buffer containing 086 and holding at θ°C for 3 minutes, 70 μm of 200 μm was added. Add polyethylene glycol 4000 solution and heat at 30°C for 1 hour, then at 42°C
After holding for 5 minutes, collect bacteria, wash with 0.5 ml of water, suspend in 0.3 ml of water, and add 0.1 microorganisms per plate.
Plant 1.

(10) Amount of secretion of β-endorphin After culturing S. cerevisiae YNN27 strain containing plasmid pRE1086 obtained in (9) above for 2 days, it was centrifuged, and the supernatant was collected using β-endorphin [RIA] kit New England Nucl.
As a result of performing radioimmunoassay according to the method described in the catalog using Ear Inc., Catalog No. NEK-003'), the amount of β-endorphin in the superior groove was 24.
It was 1.0 ng/ml.

In addition, Myotscharomyces cerevisiae VNN27 strain was transformed into the same machine using the above plasmid pRE1059.
However, the amount of β-endorphin measured using the same method as above for the supernatant obtained by centrifugation after culturing was 233.7.
It was ng/ml.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing the sequence of the Saccharomyces ubarum α factor gene, and FIGS. 2 to 5 are schematic diagrams showing the construction route of the complex plasmid in the present invention.
E stands for EcoRI, H stands for old ndm, S! ,tSall
, P is Pstl, 8 is BamHI, Pv is Pv
u U, Sm is Sma I, Hc is HincI
I, × indicates Xba I, and A indicates Aatll, respectively. Applicant: Director of the Agency of Industrial Science and Technology, etc. Rapid Fire Figure 2 I-I

Claims (1)

[Claims] (1) A plasmid containing the promoter sequence and leader sequence of the yeast Saccharomyces italicus α-factor gene.