JPS6152289A - Plasmid - Google Patents

Abstract

PURPOSE:A plasmid for expressing fused protein having tryptophan promoter/ operator sequence and leader sequence and part of tryptophan E structure gene and further multi-purpose cloning sites linked to the part of the gene. CONSTITUTION:A plasmid containing tryptophan promoter/operator sequence and leader sequence and part of tryptophan E structure gene, and having multi- purpose plural cloning sites linked to the tryptophan E, e.g. pBR322-trpEm, etc. The plasmid can be used as a vector for expressing fused protein utilizing the cloning sites. Thus, physiologically active peptides, e.g. beta-endorphin or cardionatrin, can be produced with a high expression efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION (Eleventh Field of Industry) The present invention relates to plasmids. More specifically, the present invention provides colonic FJ trp (), which is suitable for expressing fusion proteins.
Lyptophan) operon-containing plasmids.

(Prior Art) Conventionally, various studies have been conducted on plasmids for protein expression, and the present inventors have developed an improved plasmid containing the trpE#I gene. As a result of various studies, we have arrived at the present invention.

That is, the gist of the present invention is that the trp F! Structural gene 1
This plasmid has a plurality of versatile cloning sites connected to each other, and a DNA fragment containing a gene encoding cardionatrin is inserted into these cloning sites.

(Invention @Kin) Below, I will explain the book @ Ming in detail.

An example of a method for producing a plasmid according to the present invention will now be described.

An example of a raw material plasmid is a plasmid derived from Bacillus Bacteria and having a large copy number, that is, a so-called Escherichia coli multi-copy plasmid. Such a plasmid has E as the insertion site. Those having an OR1 site are preferred.

For example, plasmid pBR322, pBR3, 23%
pBR327, etc., but pBR322 is most preferred.

The insertion site (hereinafter referred to as Ec) of this raw material plasmid.

The R work part will be explained. ), trp promoter/
A fragment containing the operator and leader sequences and 7 parts of the trp h:structural gene is inserted. This fragment was obtained by digesting the gene containing the Escherichia coli tryptophan operon with the restriction enzyme ``C'' and extracting the tryptophan promoter/operator sequence and leader sequence.
A DNA fragment containing a partial region is obtained, and Kc and □R engineering sites are formed at the ends of this fragment.

Big II! Examples of the gene containing the bacterium tryptophan suberon include the Escherichia coli chromocyme gene. Examples are phage λtrp E A46-@ and plasmid R8F, 7" lasmid (/ri77) from 2/2 Hiro,
/II/ -/,! r2) is preferably used in view of the advantageous location of the Btl 11 site upstream of the promoter region.

This plasmid R8F, 2/24t-trp was digested with restriction enzyme syty, and 2.3. Kb Bgt fJ
Get fragment. This fragment contains promoter/operator and leader sequences and a partial region of the trpKm gene.

Then, a Be OR engineering site is formed at the end of this fragment. Although the KcoRI site can be formed by conventional methods, the following method is also suitably employed.

That is, restriction enzyme B of phage M/jmp7 (RF)
The above fragment was introduced into the amH engineering site, E. coli was transformed into E. coli, a replicative form (R1'e) was obtained from the transformed strain, and the entire restriction enzyme EcOFI was extracted.
All of the above objectives can be achieved by digesting with I and accessing the Kc-OR fragment.

Next, using pBR322 as a raw material plasmid, the above FIcORI fragment having an Ec QR engineering site at the end is introduced into the Be□RI site of pBR322, and the resulting plasmid is used to transform E. coli.

The resulting plasmid (Figure 1) contains the promoter (PT)
Since the entire EcmR engineering site is located in the upper bV and downstream of 1111, EcoI (I) is used to eliminate the ytl position.

This plasmid f was partially digested with EcORI and TμI).
treated with NA polymerase and then T 4' D N
After combining with A IJ case, E. coli was transformed into J case to obtain the desired A, & KbO norasmid (p
DR3,1! r - trp E ) obtain f (Figure 1)
.

The cleavage map of this plasmid is shown in Figure 2 (λPTI is the λ phage promoter, Ap is the ampinin promoter, Tc
indicates tetracycline resistance. ).

This plasmid pBRJ, 2.2-trp
Partially digested with 1'r-HpaE and further digested with gcOR to obtain Hpa' I-Eic containing trp F25.
oR efragment "-m=" is obtained.

On the other hand, DNA with a versatile initial cloning site
To obtain fragments, e.g. plasmid pUOI
Digested with WQoR engineering and Sat l and 8ma T and B
A KQOR Ame L fragment (, 20 bp) with an am H restriction site is obtained.

At least seven of the versatile multiple cloning sites are in phase with the E structural gene.

Furthermore, plasmid pBR32,2 was digested with EcORI, made blunt-ended with H,T≠DNA polymerase, and then digested with Sa
Digested with zI, large (1!1cORI)-8a
l l fragmentra get ( (Fico) ? engineering) 0,
Represents the smooth end above. ).

Then, the above three 7-ragment, namely Hpa
I EcoRr 7 Ragment, I! ! The cOR Nicho-17 and (EcoR)-8at17 ragments were ligated with iT 4' DNA ligase and injected into the intestine.
After converting the clothes to p, we made Grasmid pBR322-trpEm.
(@Figure 3).

This plasmid pBR32,7-trp Em is tr
p promoter/operator sequence and leader sequence and t
Contains -1911 of the rp E Surizuke gene and tr
Is it multipurpose by connecting to pE? N gJ cloning site, i.e., R, K1, Bam1 (I and L, t
1 part ke■1"ru.

Here, "connect" means to connect the entire sequence (bow 2, not including the stop codon), and the sequence encodes up to 70 amino acid residues.

This plasmid can be used as a vector for expressing a fusion protein by making use of its multi-purpose loning site, but the polypeptide to be expressed is not particularly limited. Bioactive peptides can be mentioned, and fused fresh white can be produced with high expression efficiency.

The case of cardioonatrin as the polypeptide will be explained below.

Caldeionatrin 7 (0ard, 1onatrin
) is a peptide consisting of 1.2r amino acids (Ser-
Based on Leu-Arg precursor, Na diuretic effect,
It is known to have a blood vessel relaxing effect.

An example of a method for producing a DNA fragment containing a gene encoding cardionathrin, which is used in the present invention, is shown below.

Human heart fragments were homogenized with guanidinyl thiocyanate, and then subjected to oligo(clT) cellulose column chromatography using a Cf1ICt equilibrium density gradient -7 monomer.
MRNA A ke, in, *do (mRNAJl fee).

/70. cDNA library can be obtained by /RIZA2). A vector primer and an oligo(dG) tail linker were obtained using pBR32,2, Svl, and tO hybrid plasmids.

cDNA is synthesized by reacting reverse transcriptase in the presence of this vector primer and the above mRNA, and the restriction enzyme H
1st Ill, and then the above linker-f#+1
and cyclize. After that, t in the mRNA part '1 DNA.
The cDNA fragment-containing plasmid was incubated at 1A for 44 hours.
p.ru.

Next, this was transformed into Escherichia coli (Kscheri-
chia coli), etc., to obtain Ambinrino-resistant strains.

-day One, Mst-Asp-Arg of cardioonatrine
-1'1e-G], a nucleotide complementary to the nucleotide encoding y was synthesized as a probe.

The above-mentioned cDNA library is screened using the probe at T OT T OT , and the base sequence is determined by complementary cloning.

The resulting DNA fragment pHANFAt is a DNA fragment containing the base sequence encoding the precursor of cardioonatrin, which sequence includes the base sequence encoding the entire amino acid sequence of cardioonatrin (
Figure μ).

Then, Haθ1m of this fragment pHANFJ4
All 4q fragments.

That is, 1st Pst is internalized at 1, and then Haθ■
A Haofll fragment (/70 bp) containing the clear gene encoding cardioonatrin is obtained.

This 9 was introduced into the Sma 1 site of pBR32,2-trpFtn, and it was large! I! I: Perform bacterial transformation and transform the target plasmid of the present invention T) BR3 coco-trp
EmANF (Figure 5) is obtained.

By growing a transformed strain containing this plasmid, cardionathrine fusion protein can be produced.

(Effects of the Invention) The plasmid according to the present invention is suitable for expressing a fusion protein, and can stably and efficiently produce a carteionatrin fusion protein in bamboo.

The target protein (cardionathrin%) can be isolated from the resulting fusion protein by a conventional method.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

The enzyme used in the examples was NθW (Japan).
The gauze liquid etc. were obtained from the manufacturer according to the instructions.

Example 1 <Plasmid pBR32,2-trp Bm
(Ia) Preparation of plasmid pBR322-trpE (@/shi1) (li77), /hiro/-/jλ) was incubated at 37°C for 7 hours with restriction enzyme Shi+1, and then incubated with 7% agarose. A 2.3 Kb Byt I fragment was obtained by electrophoresis. on the other hand,
Phage M/3mp? (7J31 Bethesda
Research 2 Bondries BP ) (Bethe
sda Research: c+aboratori
esInc, (US 1-Ii+) acquired)
This was digested with Bft '1 at 37°C for 1 hour, and the above Byz ■ fragment was ligated using 74 (DNA ligase) at 75°C for 1 hour. '/4 JM/03 (manufactured by Bethesda Research) f transformed, and X gal (ra
A transformed strain was selected using the color of No. 13 (change from blue to white) as an indicator, and RF was obtained from this, which was then digested with the restriction enzyme EcORI at 37°C/hour I'1-11, and F3
The cmR fragment was obtained.

That is, this 7-ragment contains a promoter/operator sequence, a leader sequence, and a portion of the trp B structural gene, with a BcoRI site at the end.

Next, plasmid pBR31,2 was treated with restriction enzyme EcOR.
Digested with IccoI for 1 hour at 37°C and digested with the above IccoI (effragment iT4'DIJA lycase/s°C).
, /≠ When concave and curved, they were connected. Next, this was transformed into Escherichia coli HB/'0/'/(l), and the transformed strain was transformed with K E:t2qR1 upstream and downstream of the promoter (]'T).
A plasmid (Fig. 7) containing the site was obtained. This plasmid was then partially digested with RI at 37°C and T4'DN
Treated with A polymerase for 30 minutes at 37°C, then treated with T4 polymerase.
7 with tDNA ligase! The mixture was incubated for 4 hours at 0.degree. C. for binding. Next, E. coli HB101 was transformed, and the target plasmid pBR3,2,2-trp was extracted from the transformed strain.
B was obtained (Fig. 1). .

This plasmid consists of 4. &Kb, and has a KcoR1 site downstream of FT. The restriction map is shown in Figure 2.

(Jb) Hpa l -F2cm containing trp R
Construction of R fragment (Fig. 3) (Plasmid pBR322-trp obtained in [a)
Fj f, partial digestion with restriction enzyme Hpal (37°C, 3
0 minutes) and then digested with EcOR (37''°C, 2 hours) and Hpa I Ffc containing trpE.

The RII fragment was obtained.

(It) Creation of KcoRI-Jiku■ fragment (-20 bp) of pUoza; Digested with Zu2sumid p110♂ Zenku R1, ShikuI (37°C
, 2 hours) (-1 Target multipurpose cloning site f: Obtain a fragment with multiple fragments ≠ 10 (III) (Fi
conI)-5atl fragment; plasmid pBR322 was digested with EcoRI (37°C,
2 hours), treated with T4' DNA polymerase for 1 hour at 37°C to make the JficoRI site blunt, then digested with box 1 (37°C, 2 hours), and injected with large (EcoRI
) -SaA I fragment (36 Jbp) was obtained.

(■), Creation of pBR322-trp Km; Next, the three fragments obtained in (Ib), (If), and (■1) above were ligated with 74' DNA ligase.
°C, /44 hours) and cyclized.

Next, this was transformed into Escherichia coli HB10/, and plasmid DNA was prepared from the resulting transformant according to Helensky's method 7 (7).

The nucleotide sequence of this DNA was analyzed using the method of Maxam-Gilno (-), and it was confirmed that it was the desired DNA (plasmid pBRJ+2+2-trp Km
).

Example λ <Plasmid pBR32,2-trp]!
Preparation of imANF> (Ia) Preparation of a DNA fragment containing the gene encoding carthionatrin; (1) Human heart fragments were crushed in liquid nitrogen and then homogenized by adding an aqueous solution of guanidinium thiocyanate. The obtained homogenate was subjected to the method of OhirgWin et al. (Biochemistry/I, JJ Rihiro-
Total RNA was isolated by cesium chloride-equilibrium density gradient ultracentrifugation according to 3,2, 7, and 7). Next, this was mixed with oligo(aT) + Reulose Scarano by the usual method.
The poly(A)-containing RNA was purified by chromatography and used as an mRNA raw material.

(2) On the other hand, Okayama and Berg's method (Mo1ecul
A vector primer and an oligo(dG) tail linker were obtained by combining pBR3,22 and SV4'o high frit plasmids with Ar and.

hybrid plasmid μOθμ? was digested with the restriction enzyme Kpnl in a buffer containing bovine serum albumin at 37°C for an hour.

Next, DNA was recovered by jetanol precipitation using a conventional method, dissolved in a buffer containing clTTP, terminal deoxynucleotidyl transferase was added, and the reaction was carried out at 37°C for 30 minutes to obtain the restriction enzyme Kpn.
After adding about 60 dT details to the digestion site of
D'NA was recovered by ethanol precipitation. This DNA was then digested with the restriction enzyme Hpal in an IP buffer containing bovine serum albumin (37°C, 5 hours).

Larger DNA fragment) k, agarose gel electromethod 4/! -A/ri, lli7li). Thereafter, this DNA was applied to an oligo(dA) cellulose column at θ°C, eluted with water, and recovered with ethanol.
A vector primer with an oligo(dT) tail was obtained.

On the other hand, p B R3,2-! 100 µ2 of a hybrid plasmid of SV4'O and SV4'O (0.3-2 MAP units) was digested with the restriction enzyme Pstr in a buffer containing bovine serum albumin (37°C, 1 hour i.
ff and a half). Then, the DNA was collected, dissolved in a super buffer solution containing all of dGTP, terminal deoxynucleotedyl transferase was added, and the mixture was reacted at 37°C for 20 minutes to add about 10~/j of dG detail.

The collected &DNA'i was digested with the restriction enzyme H1ndl[l in a buffer containing bovine serum albumin (37°C
, 7 hours) and then subjected to agarose gel (/, %) electrophoresis to collect the small oligo(dG) tail linker-DNA.
'z-extracted and collected.

(3) Okayama and Berg's method (Molecular
and Oelular BiFuji-BiO], Ogy, 2, /Otsu/-/70. /rif
, 2), a cDNA library was obtained.

That is, Tris-HCI (pH 1,3), MgC
l2, KOl, dithiothreitol, dATP%dTT
P.

(To the aqueous solution containing iGTP and (P]dOTP)
) and 10 μv of the vector primer obtained in (2) above were added and reacted at 37°C for 20 minutes in the presence of reverse transcriptase to transform the plasmid-
cDNA:mRNA was synthesized, precipitated with ethanol, and passed through pellets (7) to cattle. CoCl
2, dithiothreitol, poly(A), (P)dOT
Dissolved in a buffer containing P and terminal deoxynucleotidyl transferase, reacted at 37°C for 70 minutes, and per terminal (i.

Residues 70 to /j of MP were added.

Then, the transformed oligo(dC) tail plasmid-
cD'tJA: Pellet containing mRNA) k Dissolved in a buffer containing bovine leprosy albumin, digested with restriction enzyme H1ndIll at 37°C for 7 hours, and precipitated with ethanol to obtain the fH,ndm digested oligo. (dC)
The tail cDNA:mRNA plasmid was recovered.

This is the oligo (dG) obtained with NiT Nishi (2).
Dissolve in buffer containing tail linker DNA and incubate at ts°C.
The mixture was incubated at 1.2°C for 30 minutes, and then cooled to 0°C.

Then, Escherichia coli (E. coli) DNA ligase was added to β-NAD in the presence of cotin adenine dinucleotide, and the mixture was incubated overnight.

Then, dATP, rlTTP% dGTP, dCTP,
β-NAD% Ei, coal, DNA ligase, B, coli DNA polymerase and B, co], 1RNaeθH were added and the mixture was incubated at 72°C for 7 h and then at room temperature for 7 h. The reaction was stopped by cooling to obtain a plasmid containing the desired cDNA fragment.

Next, using this plasmid, E. coli (
L coli ) HB10/ was transformed.

(4) On the other hand, Met-Asp-A of cardionatrine
rg-10-a], nucleotides f complementary to the nucleotides encoding yk were synthesized as two groups.

T OT T OT Next, use this oligo I as a cDNA probe.
The library was screened and 72 complementary clones were selected from po and ooo transformants. When cDNA fragments were extracted from these 12 clones and a restriction enzyme map was created, it was found that a common DNA fragment existed. /910), the Yotte base sequence was determined. FIG. V shows the nucleotide sequence and the assumed amino acid sequence of this DNA fragment, and in the figure, the opening shows the sequence portion of Cardiona IJ (also shows the restriction enzyme cleavage site).

(Ib) pHANFAJ gloss ■ fragment (/9
0bp) creation; pHANFAt! 0 μt was digested with Pst I (37° C. for 2 hours) and separated by polyacrylamide gel electrophoresis. Seven fragments of approximately 700 bp were extracted from the gel. Then, it was digested on a shelf (at 37°C for 2 hours), separated by polyacrylamide gel electrophoresis, and then diluted with Obp.
The fragment was extracted (Figure 5).

After digestion with enzyme 1111ma I (37°C, 3 hours), digestion with alkaline phosphatase (BAP) at 37°C, 30
Processed for minutes. This was repeated twice (Figure 5).

(ill) pBF 32. ．． 2-trp Bm
A N F department (Fig. 5) 7 fragments of λ obtained from m1 (lb) and (U) were heated at 75°C in the presence of Tl7 DNA ligase, l≠Tokiishi 1.
It was reacted and cyclized. This circular DNA was injected for 3 hours.
p4HB10/, and the resulting transformant was transformed into siblasmid DNA by a conventional method.
+/, the base sequence was confirmed by the method of Maxam and Gilbert.

Reference example/゛Plasmid pBR, 3,22-trp Bm A N
E. coli transformed with F was transferred to M9OA medium (Molθ
The cells were cultured at Cu1arl gλ, 4'4'/negative), and the culture solution was run on a 70% 5DS-polyacrylamide gel to analyze whether the fusion protein was expressed. A band of a supposed peptide consisting of 371 amino acid residues (approximately 110 Daltons) was recognized by Coomassie.

1. Brief description of the drawings 1, 1 and 5. FIG. Shows the restriction map.

Furthermore, FIG. 5 shows the base sequence of a DNA fragment containing the gene encoding carteionatrin used in the present invention and the amine sequence assumed therefrom.

Sender: Sanshoku Kasei Kogyo Co., Ltd. Representative Patent Attorney Hase - Others/Names

Claims (2)

[Claims] (1) Contains a trp promoter/operator sequence, a leader sequence, and a part of the trpE structural gene, and the trp
A plasmid with multiple versatile cloning sites connected to a portion of the E structural gene. (2) Contains a trp promoter/operator sequence, a leader sequence, and a part of the trpE structural gene, and the trp
In a plasmid that is connected to the pE structural gene and has multiple versatile cloning sites, this cloning site contains a D containing the gene encoding cardioonatrin.
A plasmid with an inserted NA fragment.