JPS63133986A - Dna sequence to code secretory signal peptide - Google Patents

Abstract

PURPOSE:To obtain a DNA sequence to code secretory signal peptide containing a specific base sequence and capable of efficiently secreting a useful substance produced in a cell. CONSTITUTION:A range to code 97kd of killer toxin, promoter of gene 31kd and signal peptide is inserted into plasmid pUC13 to give plasmid pNWO28, the plasmid is scissored with HindIII, treated with an enzyme and bonded to linker BglII to give pNW033. EcoRI-BgIII fragment containing signal sequence of pGKL1 of the plasmid and EcoRI-BglII fragment containing promoter sequence of pGK of pMA91 are inserted into EcoRI site of pRE1052 to prepare pNW033. pNW037 containing mouse amylase is constructed at the downstream of secretory signal sequence shown by formula I of the plasmid and the plasmid is introduced into yeast to produce amylase.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to DNA sequences encoding secretory signal peptides.

(Conventional technology and problems) Yeast is a useful microorganism whose cell structure and function have the characteristics of higher organisms, and which is closely related to human daily life as a raw material for foods, medicines, feed, etc. It is expected to be developed as a host in genetic 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. It is desired to develop a system that secretes the microorganisms outside the bacterial body.

In addition, since continuous culture is possible for secretory production, a significant increase in production is expected, and furthermore, it has great advantages, such as preventing the decomposition of the product by intracellular proteases.

(Means for Solving the Problems) The present inventors conducted a search for a signal peptide that can efficiently secrete useful substances produced within the bacterial cells in genetic recombination technology using yeast as a host. This is an invention that has been achieved.

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.

MET ASN ILE PHE TYR
ILEATG AAT ATA TTT
TACAT, to PHE LEU PHE L
EU LEU 5ERTTT T T G
TTT TTG CTG TCAPHE
VAL GLN GLY LEU
GLNTTCGTT CAA GGT TT
G GAGHIS THR CAT ACT Kluyheromyces lactis (Kluyveron+
pGKL, a linear plasmid derived from P. yceslactis
The killer toxin protein that it produces and secretes kills certain yeasts that do not have pGKLI.

This is because pG has a killer resistance gene as well as a killer gene on Ll, and is resistant to the killer toxin produced by itself, as well as a signal peptide at the N-terminus of the biosynthetic precursor of the killer toxin. It is thought that this is due to the release of killer toxin into the culture medium (see Japanese Patent Application Laid-Open No. 12983/1983).

The l8JA sequence encoding the signal peptide of the present invention is described above! Among the killer toxin proteins in IGKLI,
It can be isolated from the region that coats the signal peptide of the 97 kilodalton (kd) and 31 kd subunits. In addition, it can also be prepared by chemical synthesis.

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

[Construction of secretion vector (1) Creation of plasmid Nen028 (3,0 kb) The promoter and signal peptide-coating regions of the 97 kd and 31 kd genes of the killer toxin were transferred to plasmid pUc13 (Pharmacia Catalog P-L). B
iochen+1-cals, 27 pages, 27-4973
) and insert it into the plasmid LeNen028 (3, Okb
).

That is, plasmid pGKLl (8,9 kb) was transformed into Taq
Taq I containing the signal sequence of the present invention by cutting with Taq I
-Taq 1 fragment (813bp) was collected and p
After inserting into the C1a I site of BR322, Cla
It was cut with I and further cut with S1 nuclease to make blunt ends. This was added with a BamHI linker (pCCGGATCC
GG), then Ram)II and HindI
This fragment is then cut with Alu I to obtain an Alu I-Bawl I fragment from which the promoter sequence has been removed.

This fragment was added to the old nclI − of plasmid pLIc13.
By inserting into the Ban+H■ site, plasmid plasmid N028 (3, Okb) containing the signal sequence of pGKLl is obtained (Fig. 1).

(2) Creation of plasmid pNWO32 (2,9 kb), which contains regions encoding the 97 kd and 31 kd signal peptides of the killer toxin.
9kb).

That is, pNWO28 was cut with Hindm, and DNA (hereinafter referred to as D for convenience) was extracted from the cut site with nuclease BAL31.
After treatment with Bg
l ■ Linker (pCAGATCTG) is attached to pc
pN032 (2,9 kb) containing the signal sequence of Ll in
) is obtained (Figure 1).

(3) Creation of plasmid virus Nen033 (6,7kb) p
A plasmid pNWO33 (6,7 kb) containing the signal sequence of GKLl and the promoter sequence of the yeast phosphoglycerate kinase gene (±) is constructed.

Since the promoter of pGKLI is said to not work in circular vectors, yeast (S, cerevisiae)
) Plasmid pMA91 (9.5kb) containing the promoter sequence of the phosphoglycerate kinase gene (mountain)
[Gene, Vol. 24, pp. 1-+4 (1983)! li
] to insert the region containing the promoter sequence of PGK above the signal sequence of plasmid pNWO32 (ff1 (
s' side) and a plasmid ρ containing this sequence.
Create NWO33 (6,7kb).

That is, pMA91 (9.5 kb) was incubated with EcoRI and Bg
Ec containing the scale promoter sequence obtained by cutting with lII
oRI-BgI n (+, 4 kb) fragment and the p
p obtained by cutting NWO32 with EcoRI and Bgln
A fragment containing the signal sequence of GK1 was added to EcoR of the separately prepared plasmid pRE+052 (5.2 kb).
1 site to create pNWO33 (6,7 kb). (Fig. 2) As described in Fig. 3 and Japanese Patent Application No. 6O-11228), plasmid pRE+052 is obtained by ligating plasmid YRp7 [yeast yeast and HA fragment containing AI'lSI with pBR322]. plasmid, NatLI
res Volume 282, pages 39-43 (+979) ■■ and plasmid pRE1032 containing AR5I, and 2
It is a yeast and E. coli shuttle vector constructed from the μm plasmid and the plasmid pBR322, and contains the replication origins of both the μm, Apr, and pBR322 and 2Bta plasmids.

pNI+1033 (6,7k
b) has the signal sequence of the present invention and its upstream (
It has a small promoter sequence on the 5' side). Further, downstream (3' side) of the signal sequence, BamHI, SmaI
and Sst■, etc., and by inserting arbitrary structural genes into these sites, the corresponding desired proteins can be expressed and secreted.

(4) Creation of plasmid pNWO37 (8,1 kb) p
A plasmid pH037 having a mouse amylase gene downstream of the signal sequence in Nv033 is created.

That is, pNWO33 was cut with Bamfl I, and Bam
111 site [INA polymerase kle
now) fragment and make blunt ends.

On the other hand, a separately prepared plasmid pRE+075 (4.3 kb) containing the mouse amylase gene was incubated with Apa I
Cut. The Apa I cleavage site is treated with 51 nuclease to make the ends blunt, and then cut with Dra I to collect an Apa I to Dra I fragment encoding an amylase protein lacking the first 15 amino acids of the amylase precursor. (This gene lacks the amylase secretion signal). Add to this a BamHI linker (e.g. 12m
er), cut with Bamfl I, and attached Ba to both ends.
A DNA fragment having the IIIH■ site is obtained.

This fragment and the Bamfl I cut fragment of pNWO33 described above were respectively made blunt-ended and ligated to form a plasmid plasmid N037 (8,1k) containing the mouse amylase gene.
b) Construct (Figure 2).

Naofura 7. mit' pRE1075 (4,3kb)
is a plasmid 'pMsa+04 [ceI, vol. 179, containing the amylase gene derived from mouse salivary gland].
21 (1980)] with Pst 1 and inserting the resulting PstT to PstI fragment containing the amylase gene into the Pst 1 site of the pUc13.

(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 I-XI, unless otherwise specified.

■ [Cleavage and recovery of DNA using restriction enzymes] Use the following three types of buffers for cutting with restriction enzymes (1) to (3).
) is used as Advanced Bacteria.
lGenetics (1981
g Harbor, 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 p)l 8
．． Extract once with phenol saturated with 0 and 1 mM EDTA), remove the phenol with ether, add 2 volumes of ethanol, leave at -20°C for 30 minutes, and collect the DNA by centrifugation. .

(1) Low salt concentration buffer IQ mM (1) ) Liss-HCl (pH 7,4), 10
Consists of mM magnesium sulfate and 1 mM dithiothreitol.

(2) Raw salt concentration buffer 50 mM NaCl, 10 mM Tris-HCl (pH
7,4), consisting of 1°l magnesium sulfate and 1mM dithiothreitol.

n [Preparation of plasmid DNA from E. coli (1) Mini preparation method (m1ni prep method) N
ucleic Ac1dsRes, vol. 7, 1513~+
p. 523 (+979)] E. coli containing plasmid DNA was added to 5 ooμm of L-broth (10 g of peptone, 5 g of
yeast extract, 1g glucose, 5g NaCl/l
pfl 7.2) consisting of PFL 7.2) was cultured overnight, and the bacterial cells collected by centrifugation were collected in 100 μ+ m M A [5
0mM glucose, 10mM EDTA, 25mM
of Tris-HCl (pHs, o) and lysozyme 2 mg/m
1] and left in ice water for 30 minutes.

Next step: Ice water test 200μ1 (7) i8? ff B[I
X (IDsDs (sodium dodecyl sulfate) containing 0.
2N NaOH] and shaking to simultaneously denature the DNA.

Add 150 μm of 3M sodium acetate solution (pH 4,8), cool with water, and centrifuge. Add 2 volumes of cold ethanol to the supernatant, cool to -70°C for 10 minutes, and then centrifuge to collect the precipitate. The precipitate was dissolved in 400 μm of dissolved iαC [consisting of 50n+H Tris salt m (pH 8,0) and 0.1 M sodium acetate], and after removing insoluble matter, cold ethanol was added to wash the precipitated DNA, Dry under reduced pressure at -20°
C and save.

(2) Large-scale preparation method Escherichia coli containing plasmid DNA was cultured using 1001 L-broth (containing drugs in the case of drug-resistant plasmids), and after collection and washing, 31 solution A (50 mM glucose, 10mM EDTA, 25mM Tris-HCl (
pH 8.0) and lysozyme 2 mg/ml)
and incubate in ice water for 30 minutes.

Then add 4 ml of solution B [l % 5DS (
0.2N NaOH containing sodium dodecyl sulfate]
is added and shaken to denature the DNA at the same time.

Add 3 ml of 3M sodium acetate solution (p 14.8), cool with water, centrifuge, add 0.6 volume of isopropanol to the supernatant, and leave at -20°C for 20 minutes.

The precipitate was collected by centrifugation, washed with ethanol, and then washed at 21 T.
E'(p)l 7.5) Suspend in buffer. Next, 1
Add 0 μ m RNase A (10 mg/ml),
After standing at 37°C for 30 minutes, phenol extraction is performed. Add 0.2 volumes of 5M NaCl and 173 volumes of 30x polyethylene glycol 6000 to the aqueous layer and let stand at -20°C for 40 minutes, then at 4°C for 40 minutes. The precipitate is collected by centrifugation and dissolved in 400 ml of water, and the DNA is precipitated with ethanol, washed and then dissolved in 100 μm TE buffer.

III [Legation using T4 DNA ligase] The two DNA fragments to be ligated were prepared using a ligation buffer [66 mM Tris-HCl (pH 7.5) to give a concentration of 1 Mg/10 μm.
, 6.6 mM magnesium chloride, 10 n+H dithiothreitol] and treated at 65°C for 10 minutes, then 66μ of ATP (adenosine triphosphate) was added at 4°C, and T4 ligase was added. Add 0.1 unit/μg DNA for sticky ends, or 1 unit/μg DNA for blunt ends, and add 1 unit/μg DNA at 4°C.
After reacting for 8 hours, it is treated at 65°C for 10 minutes.

■[Escherichia coli transformation co[advanced Bact]
Erial Genetics (+981) (Col
d Spring Harbor, New York)]
51 L-broth is inoculated with E. coli and cultured overnight.

This 0.21 strain was planted in 20 ml of Shiichi broth and cultured with shaking at 37°C until the Klett unit reached 60. Collect the facepieces and add ice-cold 50 mM calcium chloride and lom.
10 m of a buffer solution consisting of M Tris-HCl (pH 8,0)
1 and cooled in water for 30 minutes. Centrifuged bacterial cells
ml of calcium chloride solution, and this 0.1 ml
was mixed with a 10 μm DNA solution and incubated at 0°C for 30 minutes at 42
After incubating at 37°C for 2 minutes, add 1.5 ml of Shiichi Pross and incubate at 37°C for 30 minutes.
ml onto agar medium.

Add 20 units/μg of DNA, treat at 22°C for 40 minutes, extract with phenol saturated with TE buffer, remove phenol with ether, add cold ethanol, cool to -20°C, D precipitated by centrifugation
Collect NA.

VI [BAL31 processing] In order to digest both strands from both ends of the DNA, BAL31
Perform processing. That is, 15 μg of ethanol-precipitated DN
A was dissolved in 20 μM of sx BAL31 loose solution [3M sodium chloride, 60 mM calcium chloride, 60 mM magnesium chloride, 5 mH EDTA], and 79 μM of water and 1 μM (2 units) of BAL31 enzyme were added.
Incubate at 20°C for 30 minutes. After the reaction, 3 times with phenol,
After extraction with ether three times, ethanol precipitation is performed.

■ [Filling of sticky ends] Iug DNA was added to 30μm of 50mM) lithium-hydrochloric acid (p
)l 7.2), IQ mM magnesium sulfate, 0.
Dissolve in 1 mM dithiothreitol, 50 μg/ml bovine serum albumin (BSA), and 0.2 mM dNTPs, and collect 1.25 units of klenow fragment (E. coli DNA polymerase) by trypsin ethanol precipitation. .

■[BamHT and 8glll linker phosphorylated co-Ba
mtll linker (5'CCGGATCCGG 3')
and Bgl II linker (5'CAGATCTG 3
') (only one strand of the double strand is shown) does not have a phosphate group at the end, so it was phosphorylated with T4 kinase.

3 nmol of linker DNA was added to 41μ+8011
Dissolved in IM Tris-HCl (pH 7.5) and 12 mM magnesium chloride, incubated at 60°C for 10 minutes, then added 10 mM 2-mercaptoethanol and 20 mM ATP at 37°C, and further added 10 units of T4. After addition of kinase and treatment at 37°C for 30 min, -20
Store at °C.

IX[8am)II and 8gllll linker ligated to the blunt end, a blunt end DNA fragment (1.2 pmol end) and the above 1
The linker DNA (100 pmol end) phosphorylated with
), 6.6 mM magnesium chloride, and 10 mM dithiothreate. After removal, DNA is recovered by ethanol precipitation.

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

Plasmid DNA cut with restriction enzymes (IQ pmol
5' end) in 10 μ+ IOX BAP buffer [10
0mM Tris-HCl (p)l LO), 1mM El
) dissolved in TAI, 88.5 µm water and 1.5 µm + (
After adding 0.75 units of BAP and reacting at 60°C for 1 hour,
Add 0 μm of 0.25 M EDTA. After that, phenol-chloroform extraction treatment is performed twice and phenol extraction treatment is performed twice, then phenol is removed with ether, and plasmid DNA is recovered by ethanol precipitation.

XI [Transformation of yeast (KU method) IQ ml YEPD of Satucharomyces cerevisiae
1 g was cultured in the ground at 30°C overnight, harvested, washed once with TE buffer, suspended in 1 drop of the same grade, and the number of cells was 2?
Adjust to IQ7 cells/ml.

ν 12! ! 1lii solution 500μ liquid port 008m, 2M lithium acetate 4.9p147.5) was added and kept at 30°C for 1 hour, then dispensed into 100μm test tubes and added D to this at 0°C.
Add NA and mix for 30 minutes at 0°C. 100μm T
o! Add TE buffer containing polyethylene glycol 4000 and mix well.
Hold at ℃ for 5 minutes, centrifuge to collect bacteria, and wash with sterile water.

Turn this into 500μm sterile water! 3, inoculate 100 μi onto a selective medium, and culture at 30° C. for 3 to 4 days to obtain a transformed strain.

Example (1) Creation of plasmid pNWO28 (3.0 kb) Plasmid pGKLl (8.9 kb) was cut with Taq I to obtain Taq T- containing the secretion signal sequence of the present invention.
Collect the Taq I fragment (813bp) and transform it into pBR.
After inserting into the Cla 1 site of 322, it was cut with C1a I and further cut with S1 nuclease to make blunt ends.

This was added with a BamHT linker (pCCGGATCCGG
) and then cut with Bamtl T and old ndm to obtain the Bam)I I-11indm fragment, which was cut with Alul to generate the 0.0.
A 1 kb Alu I-BamHI fragment was obtained.

This fragment was added to 1IincI of plasmid pLIc+3 [
-BamH■ site to obtain plasmid plasmid N028 (3.0 kb) containing the secretory signal sequence of pGKLl.

(2) Creation of plasmid pNν032 (2,9kb) p
Nw028 was cleaved with Hindlll, DNA from the cleavage site was treated with nuclease BAL3+, and a BglII linker was ligated thereto to obtain pNen032 (2.9 kb) containing the secretion and signal sequence of pGKLl.

(3) Creation of plasmid virus Nen033 (6,7kb) p
EcoRI containing the P. aeruginosa promoter sequence obtained by cutting MA91 (9.5 kb) with EcoRr and BglII
-Bgl II (1,4 kb) fragment and the pNWO3
pGKL obtained by cutting 2 with EcoRI and BglI [
A fragment containing the signal sequence of plasmid pRE+052 (5.2 kb), which was created by the method described below, was
It was inserted into the coR1 site to create pNWO33 (6,7 kb).

(4) Creation of plasmid pRE+052 (a) Plasmid YRp7 (5,7 kb) was partially cut with EcoRI,
After filling in the sticky ends, ligation with T4 ligase creates YRp.
pRE103, in which one EcoRI site of 7 was removed.
2 (5,8 kb), and then cut pRE1032 with EcoRI and PstI to create E containing TRPI.
A coRI to Pst I fragment (0.8 kb) was obtained.

(b) On the other hand, cut the 2 μm plasmid with EcoRI,
After filling in the sticky end to make a blunt end, it was cut with PstI and the Pstl~EcoRI cut containing the replication origin (
c) Convert the DNA fragments obtained in (a) and (b) above into pBR
Pvu 322 was cleaved with EcoRI and Pvu II
T4 with II ~ EcoRI fragment (2,3 kb)
It was ligated with DNA ligase to create pREIO5+(5,1
pRE+052 (kb) was prepared, partially cut with EcoRI, filled with sticky ends, and ligated with T4 ligase to make blunt ends, with one EcoRI cleavage site deleted.
5.2kb) was obtained.

(5) Boo 5 Smid pNWO37 (8,1 kb) 0) Created pNWO33 was cut with BamHI, Bal1lt (
The I site is replaced by DNA polymerase Klenow.
) and cut it into blunt ends.

On the other hand, a separately prepared plasmid pRE+075 (4.3 kb) containing the mouse amylase gene was incubated with Apa I
I cut it with. The Apa I cleavage site was treated with St nuclease to make blunt ends, and then cut with Dra I to collect the Apa I-Dra I fragment encoding an amylase protein lacking the first 15 amino acids of the amylase precursor. did. To this, bind BamH[linker,
DNA cut with m1ll and having BamH1 sites on both ends
Got a piece.

Plasmid pMSa 104 containing the mouse salivary gland-derived amylase gene was digested with PstI, and the resulting Pstl to Pstl fragment was used as the plasmid ptJc.
13 into the Pst1 site to create plasmid pRE+075 (4.3 kb) containing the mouse amylase gene.

(6) Confirmation of amylase activity (secretion) Using the plasmid virus Nen037 prepared by the above method, Ktl.
Transformation was performed by the method.

The obtained transformed strain was cultured at 30°C for 20 hours using a medium containing 2zcasamino acids, lx yeast extract, and 2x glucose, and the centrifuged supernatant was subjected to Methodsin.
Enzymology, volume 1, page 499 (+955)
As a result of measurement according to the method described in , the amount of amylase in the supernatant was 1000 μg/l.

(Effects of the Invention) As shown in the above example, Satucharomyces cerevisiae into which a plasmid containing a DNA sequence encoding the secretory signal peptide of the present invention has been introduced can efficiently separate and filter out heterologous proteins into the culture medium. pNν037 and p
It is a schematic diagram showing the configuration route of RE+052.

Applicant: Director of the Agency of Industrial Science and Technology Yuki Iizuka Mitsuhaya 1 Figure , G.K. T soil Illustration

Claims (1)

[Claims] (1) A DNA sequence that encodes an amino acid sequence containing a secretion signal peptide shown by the following formula [gene sequence is available].