JP2507874B2 - 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) The present inventors have searched for a signal peptide capable of efficiently secreting a useful substance produced in the bacterial cell in a gene recombination technique using yeast as a host. The invention has been achieved.

To explain the present invention in detail, the DNA sequence encoding the secretory signal peptide of the present invention consists of 60 base pairs (bp) encoding the following 20 amino acids.

Kluyveromyces lacti
The yeast having the linear plasmid pGKL1 from s) kills certain yeasts that do not have pGKL1 by the killer toxin protein that it produces and secretes.

This is because the killer resistance gene exists together with the killer gene on pGKL1 and shows resistance to the killer toxin produced by itself, and the killer toxin is produced by the N-terminal signal peptide of the biosynthetic precursor of the killer toxin. Is thought to be released into the medium (Japanese Patent Laid-Open Publication No. Sho.
60-12983).

The DNA sequence encoding the signal peptide of the present invention is
Among the killer toxin proteins in pGKL1 described above, it can be isolated from the regions encoding the signal peptides of 97 kilodalton (kd) and 3kd subunits. 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 secretory vector] (1) Construction of plasmid pNW28 (3.0 kb) The regions encoding the promoters and signal peptides of the 97kd and 31kd genes of killer toxin were inserted into plasmid pUC13 (P
harmacia company catalog PL Biochemicals, 27 pages, 27-49
73) to prepare the plasmid pNW028 (3.0 kb).

That is, the plasmid pGKL1 (8.9 kb) was cleaved with Taq I to give a Taq I-Taq I fragment (81 containing the signal sequence of the present invention.
3 bp) was collected, inserted into the Cla I site of pBR322, cut with Cla I, and further cut with S1 nuclease to give a blunt end. BamHI linker (pCCGGATCCGG)
Bound and then cut with BamH I and Hind III to Bamh I
-Hind III fragment is obtained, and this fragment is cleaved with Alu I to obtain an Alu I-BamH I fragment from which the promoter sequence has been removed.

This fragment was inserted into the Hinc II-BamHI site of plasmid pUC13 to obtain plasmid pNW containing the signal sequence of pGKL1.
028 (3.0 kb) is obtained (Fig. 1).

(2) Construction of plasmid pNW032 (2.9 kb) A plasmid pNW032 (2.9 kb) containing regions coding for 97 kd and 31 kd signal peptides of killer toxin is constructed.

That is, pNW028 is cleaved with Hind III, and DNA is nuclease BAL31 from the cleaved site (hereinafter referred to as DNA for convenience).
Bacterial alkaline phosphatase (BAP) treatment, and then the Bgl II linker (pCAGATC
TG) and pNW032 containing the signal sequence of pGKL1
(2.9 kb) is obtained (Fig. 1).

(3) Construction of plasmid pNW033 (6.7 kb) A plasmid pNW033 (6.7 kb) containing the signal sequence of pGKL1 and the promoter sequence of the yeast phosphoglycerate kinase gene (PGK) is constructed.

Since the promoter of pGKL1 is said not to work in a circular vector, the plasmid pMA91 (9.5 kb) containing the promoter sequence of the phosphoglycerate kinase gene (PGK) of yeast (S. cerevisiae) [Gene, 24, 1 , Page 14 (1983)], the region containing the PGK promoter sequence is ligated to the upstream (5 'side) of the signal sequence of the above plasmid pNW032, and a plasmid containing this sequence is linked.
Create pNW033 (6.7kb).

That is, pMA91 (9.5 kb) was digested with EcoR I and Bgl II to obtain the EcoR I-Bgl II (1.4
kb) fragment and the fragment containing the signal sequence of pGKL1 obtained by cutting pNW032 with EcoRI and BglII were inserted into the EcoRI site of separately prepared plasmid pRE1052 (5.2 kb) to obtain pNW033 (6.7). kb) is created. (Fig. 2) The plasmid pRE1052 is shown in Fig. 3 and Japanese Patent Application No. 60-1.
1228), plasmid YRp7 [a plasmid in which a DNA fragment containing yeast TRP1 and ARS1 is bound to pBR322, Nature, 282, 39-43 (1979)]
A yeast and E. coli shuttle vector constructed from plasmid pRE1032 containing RP1 and ARS1, 2 μm plasmid and plasmid pBR322, and containing TRP1, Ap ^r and the origin of replication of both pBR322 and 2 μm plasmid.

The pNW033 (6.7 kb) prepared in this manner has the signal sequence of the present invention and has PG upstream (5 ′ side) thereof.
It has a K promoter sequence. In addition, downstream of the signal sequence (3 'side), there are cleavage sites such as BamHI, SmaI and SstI, and the desired protein in charge is expressed by inserting an arbitrary structural gene into these sites. Can be secreted.

(4) Construction of plasmid pNW037 (8.1 kb) A plasmid pW037 having a mouse amylase gene downstream of the signal sequence in pNW033 is constructed.

That is, pNW033 is cut with BamHI, and the BamHI site is blunt-ended with the Klenow fragment of DNA polymerase.

On the other hand, the separately prepared plasmid pRE1075 (4.3 kb) containing the trout amylase gene is cleaved with ApaI. Apa
After treating the I cleavage site with S1 nuclease to make it blunt-ended
Apa I-D that cuts with Dra I and encodes an amylase protein lacking the first 15 amino acids of the amylase precursor
Take the ra I fragment (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.

This fragment and the BamHI fragment of pNW033 described above are blunt-ended and then ligated to construct a plasmid pNW037 (8.1 kb) containing the mouse amylase gene (second).
Figure).

The plasmid pRE1075 (4.3 kb) is a plasmid pMSa104 [C containing the amylase gene derived from mouse salivary membrane.
[ell, 179, 21 (1980)], and the resulting Pst I to Pst I fragment containing the amylase gene
It is created by inserting it into the Pst I site of pUC13.

(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 m
Extraction is performed once with phenol saturated with M 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 / 1 (PH7.2) was used for overnight culture, and the collected cells were centrifuged to collect 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, in ice water, 4 ml of solution B [0.2% NaOH containing 1% SDS (sodium dodecyl sulfate)] was added and shaken.
At the same time, the DNA is denatured.

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.
Leave at 0 ° C 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 of RNase
Add A (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 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 and add 4 ° C
After reacting for 18 hours at 65 ° C., treat 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 S1 nuclease] 200 μl of high sticky buffer solution [30 mM
Of sodium acetate (pH4.25), 0.3M NaCl and 4mM zinc sulfate], and S1 nuclease 20 units / μgD
NA was added, treated at 22 ° C for 40 minutes, and extracted with phenol saturated with TE buffer, then phenol was removed with ether, cold ethanol was added, the mixture was cooled to -20 ° C, and the precipitated DNA was centrifuged. to recover.

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, 5
A klenow fragment of 1.25 units (large fragment obtained by treating E. coli DNA polymerase I with trypsin) dissolved in 0 μg / ml bovine serum albumin (BSA) and 0.2 mM dNTP.
Is added and reacted at room temperature for 30 minutes. Then 1 μl of 0.5M
The reaction is stopped by adding EDTA (pH 8.0), phenol and ether are sequentially extracted to remove phenol, and DNA is recovered by ethanol precipitation.

VIII [Phosphorylation of BamHI and BglII linkers] BamHI linker (5'CCGGATCCGG3 ') and BglII
Since the linker (5'GAGATCTG 3 ') (only one of the double chains is shown) has no phosphate group at the end, it was phosphorylated 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 [ligation of BamH I and Bgl II linkers to blunt end] A ligation buffer [66 mM Tris-HCl (pH 7) at which blunt-ended DNA fragment (1.2 pmol end) and linker DNA phosphorylated by VIII above (100 pmol end) were ligated .5), consisting of 6.6 mM of manesium chloride and 10 mM of dithiothreitol].
After adding T4 ligase as a unit and reacting at 4 ° C for 18 hours, remove excess linker by treating with BamHI or BglII, extract DNA with fail saturated with TE buffer, remove phenol with ether, and precipitate with ethanol. To recover the DNA.

X [Treatment of DNA fragment with bacterial alkaline phosphatase (BAP)] In order to prevent self-ligation of plasmid DNA by ligase reaction, prior to ligase reaction, brasmid DNA
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 after reacting at 60 ° C for 1 hour, 10 μl of
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 the 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) Construction of plasmid pNW028 (3.0 kb) Plasmid pGKL1 (8.9 kb) was cleaved with Taq I to give a Taq I-Taq I fragment (813b) containing the secretory signal sequence of the present invention.
p) was collected and inserted into the Cla I site of pBR322,
It was cut with Cla I and further cut with S1 nuclease to give a blunt end. BamH I linker (pCCGGATCCGG) was ligated to this, and then cut with BamH I and Hind III to obtain BamHI-Hi.
A nd III fragment was obtained and this fragment was cleaved with Alu I to give a 01 kb Alu I-BamH I fragment containing the secretory signal region.

This fragment was inserted into the Hinc II-BamHI site of plasmid pUC13 to obtain plasmid pNW028 (3.0 kb) containing the secretory signal sequence of pGKL1.

(2) Construction of plasmid pNW032 (2.9 kb) pNW028 was cleaved with Hind III, DNA was treated with nuclease BAL31 from the cleavage site, and then Bgl II linker was ligated to pNW032 (2.9 containing the secretory signal sequence of pGKL1.
kb) was obtained.

(3) Construction of plasmid pNW033 (6.7 kb) PG obtained by cutting pMA91 (9.5 kb) with EcoR I and Bgl II
An EcoR I-Bgl II (1.4 kb) fragment containing the promoter sequence of K, and pNW0.32 obtained by cutting with EcoR I and Bgl II
The fragment containing the signal sequence of GKL1 was inserted into the EcoRI site of the plasmid pRE1052 (5.2 kb) prepared by the method described below to prepare pNW033 (6.7 kb).

(4) Construction of plasmid pRE1052 (b) Plasmid YRp7 (5.7 kb) was partially cleaved with EcoRI and filled in with sticky ends, then ligated with T4 ligase to VRp7
PRE1032 (5.8 kb) in which one EcoR I site was removed was prepared, and then pRE1032 was cut with EcoR I and Pst I.
An EcoR I to Pst I fragment (0.8 kb) containing TRP1 was obtained.

(B) On the other hand, the 2 μm plasmid was cleaved with EcoR I, the sticky end was filled in to make a blunt end, and the fragment was cleaved with Pst I to obtain a Pst I to EcoR I fragment (2 kb) containing the replication origin. .

(C) The DNA fragment obtained in (a) and (b) above was transformed into pBR322
Was digested with EcoR I and Pvu II to obtain a Pvu II to EcoR I fragment (2.
Ligated with T4 DNA ligase together with pRE1051 (5.
1 kb), partially cut with EcoRI, filled in with sticky ends and ligated with T4 ligase as a blunt end.
pRE1052 (5.2 kb) lacking the oR I cleavage site was obtained.

(5) Construction of plasmid pNW037 (8.1 kb) pNW033 was cleaved with BamHI and the BamHI site was blunt-ended with a Klenow fragment of DNA polymerase.

On the other hand, the separately prepared plasmid pRE1075 (4.3 kb) containing the mouse amylase gene was cleaved with ApaI. Apa
The I-cleavage site was treated with S1 nuclease to make it blunt-ended and then cut with Dra I to encode Apa, which encodes an amylase protein lacking the first 15 amino acids of the amylase precursor.
The I to Dra I fragment was collected. A BamH I linker was ligated to this and cut with BamH I to obtain a DNA fragment having BamH I sites at both ends.

Insert this fragment into the BamHI site of pNW033 above,
Plasmid pNW037 (8.
1 kb) was constructed.

Construction of plasmid pRE1075 (4.3 kb) The plasmid pMSa104 containing the amylase gene derived from mouse salivary gland was digested with Pst I to obtain Pst I to Pst I.
The fragment was inserted into the Pst I site of the above plasmid pUC13 to obtain the plasmid pREl075 (4.3k containing the mouse amylase gene).
b) was created.

(6) Confirmation of amylase activity (secretion) Saccharomyces cerevisiae 20B12 strain was transformed by the KU method using the plasmid pNW037 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 pNW032, pNW037 and pNW037, respectively.
FIG. 3 is a schematic diagram showing a constitution route of pRE1052.

Claims (1)

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