JPS62104584A - Novel plasmid - Google Patents

Abstract

PURPOSE:A plasmid, having a base sequence, corresponding to an amino acid at a specific position from the N terminal of human growth hormone (hGH) and consisting of TCT and useful for producing hGH having the activity equal to that of natural hGH. CONSTITUTION:0.62kb fragments obtained by transforming a plasmid pGH-L9 (A) for expressing natural hGH into Escherichia coli are linked to 7.24kb fragments obtained from Escherichia coli phage vector to give a template single stranded DNA (B). A 5'-phosphorylated 15-mer oligonucleotide having a base sequence expressed by the formula and the component (B) are annealed to afford a recombinant single stranded DNA, which is then treated with polymerase and oriented into a recombinant double stranded DNA. The resultant DNA is then cleaved to give 0.17kb fragments (C). Fragments (E) obtained by treating the component (A) and the fragments (C) are linked to give a plasmid having the ability to express a novel polypeptide in which cysteine that is an amino acid at the 165th position from the N terminal of hGH is converted into serine and further a base sequence of TCT corresponding to the above-mentioned amino acid at the 165th position.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel plasmid for expressing a novel polypeptide in which the amino acid at position 165 from the N-terminus of human growth hormone is mutated.

[Conventional technology and its problems]

Human growth hormone (hereinafter referred to as hGH) is present in a relatively large amount in the pituitary gland, and is a polypeptide of molecule-1121500 consisting of 191 f[ill] amino acids.

Its structure has S-8 bonds between positions 53 and 165 and between positions 182 and 189 from the N-terminus, and the amino acid at position 165 is cysteine. Are known.

It is also known that the base sequence corresponding to the 165th position of the gene expressing hGH is TGT.

However, by partially changing the structure of hGH, h
No attempt has yet been made to obtain a new polypeptide with GH-like activity.

[Means for solving problems]

Therefore, as a result of intensive research to obtain a gene for producing a highly active hG-like polypeptide, the present inventors found that
Base sequence CT corresponding to positions 163 to 167 of the gene base sequence corresponding to around position 165 of GH
G TACTGT 15 chain length of TTCCGT (merl
They discovered that this nucleotide sequence exists only at this position, and that there is no other location that has this nucleotide sequence. Furthermore, they mutated the base at position 165 of this 15-strand long nucleotide sequence and created the natural h
The present invention was completed based on the discovery that a novel plasmid for producing a novel polypeptide in which only position 165 of the amino acid sequence of hGH is converted from cysteine to serine can be obtained by integrating the present invention into a GH plasmid.

That is, the present invention provides a gene for expressing a novel polypeptide in which the 165th amino acid cysteine from the N-terminus of hGH is converted to serine, and the 16th amino acid cysteine is converted to serine.
The present invention provides a plasmid in which the base sequence corresponding to the amino acid at position 5 is TCT.

The novel plasmid of the present invention is produced, for example, as follows.

(1) pGI(-), a plasmid for expressing natural hGH
L9 is transformed into E. coli HBIOI, which is cut using a specific restriction enzyme to obtain a 0.62 Kb fragment.

(2) E. coli M13mpH phage vector is cleaved using a specific restriction enzyme to obtain a 7.24 Kb fragment.

(3) Next, the 0.62 Kb fragment of (1) above and (
The 7.24 Kb fragment of 2) was ligated to create template single-stranded D
Get NA.

(41) 5 having the base sequence CTG TACTCT TTCCGT obtained by the solid-phase synthesis method of the ester method
The 1-phosphorylated 5-strand oligonucleotide and the template single-stranded DNA are annealed to obtain recombinant single-stranded DNA.

(5) extending the bases of the recombinant single-stranded DNA by a method using a normal polymerase, and then arranging it into a recombinant double-stranded DNA;
Cut with a specific restriction enzyme to obtain a 0.17 Kb fragment.

(6) Cut the pGH-L9'Jt recombinant double-stranded DNA with the same restriction enzyme used to cut it to obtain a large fragment.

(7) Combine the 0.17 Kb fragment and the large fragment to obtain the desired new plasmid.

The above method will be explained in detail below.

To obtain the 0.62 Kb fragment, a plasmid for expression of natural hGl (pGH-L9) is transformed into Escherichia coli HB 101 cells, the cells are cultured in a medium containing ampicillin, and the cells are treated using conventional methods such as alkaline. Law [Rapid]
Isolation Op Plasmid Or Bacteriophage λDNA In 1 Molecular Cloning, Laboratory Manual”, Cold Spring Bar Laboratory, Cold Spring
Harbor.

New York (Rapid solution of
plasmid orbacteriophageλ
DNA in″Molar Cloning,
A Laboratory mnual”, Co1d
Spring Harbor Laborator
y, Co1d Spring Harbor, Ne
York), p. 355, 1982], and this was digested with restriction enzyme 1 (pal,
This is carried out by cutting with saline and collecting with low melting point gel electrophoresis. In addition, natural hG) (expression plasmid pGl(-L9),
National Academy of Sciences of the United States of America (Proc, Nat4. Acad,
Sci.

USAI, Vol. 81, p. 5956, 1984.

The 7,24 Kb fragment is obtained by cleaving the M13mpH phage vector with the restriction enzymes Sma i and Sad [ and recovering it by gel electrophoresis.

To obtain template single-stranded DNA, a 0.62 Kb fragment and 7
．． The 24 Kb fragment is ligated using DNA ligase, such as T41J gauze obtained from T4 phage E. coli, and then infected and cultured in E. coli JMIOI. This cultured phage DNA was transferred to M13mp11 using hGHD.
X-ga/(
5-Bromo-4-chloro-3-indolyl-β-galactosidase) containing YT medium (agar containing Bacto tryptone and crude yeast extract), 3
The cells were cultured overnight at 4°C to 37°C, preferably at 37°C. As a result, colorless plaques appeared as evidence that hGHDNA had been integrated. To further confirm that this plaque contained 0.62 Kb double-stranded DNA, the following operation was performed.

Take one part of the colorless plaque and add it together with JM101m cells.
Culture in double concentration YT medium (hereinafter referred to as 2xYT). After culturing, the culture solution was taken and centrifuged to separate the supernatant and precipitate. This precipitate was treated with an alkaline method to isolate recombinant double-stranded 7age DNA, and then the restriction enzyme Ec
Cut with oR (and San I, perform gel electrophoresis and remove 0
．． A band was confirmed at the 62 Kb position.

After confirmation, polyethylene glycol is added to the supernatant and left to stand for several minutes in a cold place, preferably around 4° C., followed by centrifugation to precipitate the template single-stranded DNA.

This precipitate is dissolved in a buffer containing EDTA, for example, Tris-HCl buffer (pH 8), treated with phenol, and then centrifuged to separate 21-. The aqueous layer is separated, and sodium acetate and ethyl alcohol are added to precipitate purified template single-stranded phage DNA.

The 15-chain oligonucleotide used a compound in which 5'-(4°4'-dimethoxytrityl)thymidine was bonded to polystyrene via succinic acid as a solid support.
It is obtained by sequentially condensing each nucleotide dimer onto the support using a solid phase nucleic acid synthesizer and then releasing it from the support.

Phosphorylation of the 5' end of this 15-chain oligonucleotide is carried out by a conventional method, for example, by using T4 polynucleotide kinase.

Annealing of the template single-stranded phage DNA and the 57-phosphorylated 15-strand oligonucleotide was carried out at 60-70°C.
This is preferably carried out by reacting at 65° C. for several hours and then slowly returning the temperature to room temperature, thereby obtaining complementary sequenced DNA. This is divided into four types of nucleotides: dATP, dGTP, dCTP, and dT.
The chain length is sequentially extended using TP and ligated with DNA IJ gauze to obtain recombinant double-stranded phage DNA. This double-stranded DNA is also treated with CaC, infected with JMlol, and left overnight. The cells cultured overnight are plated on YT medium to purify plaques and fixed on a filter. Thereafter, a pretreatment for hybridization is performed, and then a hybridization treatment is performed. Then, a 15-chain oligonucleotide labeled with 4p at the 5' end was added to the phage on the filter, and after being left at room temperature for several tens of hours, the temperature was gradually increased. Search for DNA arranged in

Based on this result, plaques with complementary sequences are found and each is taken out from the YT medium. Each plaque is JM
IOl was cultured separately in 2XYT together with cells grown overnight, and the culture solution after culture was separated into a supernatant portion and a cell precipitate portion by centrifugation. Polyethylene glycol 6000 is added to each supernatant, and after discharge in a cold place for several tens of hours, centrifugation is performed to obtain a precipitate of recombinant single-stranded phage DNA.

Each of the precipitates is dissolved in a buffer solution containing El:DTA, treated with phenol, and further centrifuged to separate into two layers. The separated aqueous layer portions are separated, and purified recombinant single-stranded phage DNA is isolated using sodium acetate and ethyl alcohol. The base sequence of the isolated single-stranded phage DNA is confirmed by the dideoxy method.

contains a portion with a mutated base sequence.

On the other hand, PGH-L9 Grasmid was mixed with Bal I and S&
0.17 Kb corresponding to the C-terminus of hGH cut with tl
The fragment lacking the is recovered by gel electrophoresis. This fragment contains ld, the avian f (trp) f promoter region for expression of vector portion 1 derived from pBR322, and the N-terminal region of hGH.

This fragment contains the mutated portion of the above base sequence.
After incorporating the 7 icb fragment using DNAIJ gauze, it is treated by conventional methods and transformed into HBIOI.

After culturing these cells in a medium containing ampicillin, they were treated with an alkaline method to produce a plasmid (pG) I-L9/c in which the base sequence corresponding to the amino acid at position 165 of hGH was mutated.
a3-1) is isolated.

(used for producing Grasmid of the present invention)
(Bl 01, JMIOI, limited #cum, M 13 rn
p117age vector, DNA ligase, X-gtJ
, YT medium, dATP, dGTP, dCTP,
dTTP and polymerase are all commercially available and can be easily obtained.

[Action and effect of invention]

The novel plasmid of the present invention can be introduced into Escherichia coli etc. by a normal transformation method and cultured to produce hGH 16
A novel peptide in which only Cys at position 5 is converted to Ser can be obtained. This new peptide has S at position 165.
Since it is an er, it does not have an S-8 bond between positions 53 and 165, and is structurally very different from hG)l, and its activity is also higher than that of natural hGH.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 Isolation of hGH structural gene HB 101 cells transformed with pGH-L9, a plasmid for hGH expression, were cultured overnight in LB medium containing 1.51 nl of ampicillin, and the plasmid was isolated by the alkaline method. 0.62K obtained from the isolated plasmid by treatment with restriction enzymes Hpa I and 5aI3I
The fragment b was isolated and recovered by low melting point agarose gel electrophoresis.

1 pP 0M13 mpH (Bethesda Re5
double-stranded DN of searchLab, Inc, l)
A is the restriction enzyme Sma[.

After treatment with Sag I, low melting point agarose gel electrophoresis was performed and a 7.24 Kb fragment was isolated and recovered.

This vector (0.1 μm) and the amount of V3 of the previously isolated hGH structural gene 0.62 Kb fragment were mixed with 50 mM Tris-HCl, pH 8.0, and 10 mM Mg (J, , 1).
mM EDTA.

6 mM β-mercaptoethanol (β-EtSI (l
.

0, smM ATP, 0.11n97-gelatin, 200 units T4 DNA ligase (manufactured by Zenshuzo), 2
Binding was carried out by reacting in 5μ2 for 16 hours. 8 μk was sampled from this reaction mixture and infected into CaC4-treated colon IMJMIO1, JMIQII late culture cells Q
, 'l ml, 100 mM ingropyrthio β-
Galactoside (IPTG l 10 μ2.2 5-bromo-4-chloro-3-indolyl-β-D-galacto/
8111i1 was taken from the colorless plaques produced by culturing it at 37°C overnight and cultured in JMIO11 overnight. 25 μk of cells were added and cultured in 1.5 d of double concentration YT medium.
After centrifugation at rpm for 5 minutes, the W8 double-stranded DNA set was isolated from the precipitate using an alkaline method. EcoRl, Sad
All eight phages treated with r and counted by agarose gel electrophoresis were treated with 0,62 phage of the target hGH.
It was confirmed that the Kb fragment was incorporated.

Preparation of Template Single-Stranded DNA One of the isolated phages was selected, and the supernatant of the culture solution obtained from it was used to isolate recombinant single-stranded DNA. 20% polyethylene glycol 60 on top fft1 rnl
00 (Hani Chemicals ■ Released) 2. , 5MNa (420
After adding 0μ2 and leaving it for 30 minutes at 49C, 1s 000
The 7age was precipitated by centrifugation at rpm for 10 minutes. The precipitate was diluted with 10mM Tri, Z, H (J pH 8,0, 1mM
Dissolved in 100μ2 of EDTA, 50μp of pheno-#
(10mM TrisH(J pH8,0,1mM
After treatment with EDTA (saturated), the two layers were separated by centrifugation and the aqueous layer was collected. Add to this 76 amounts of 3MNa0A
c, DNA was precipitated by adding 2.5 times the volume of EtOH. DNA was prepared in 50 μi of 10 mM TrisH (
J pH 8.0, 1rn Dissolved in MEDTA and heated to -20°C.
Saved with.

It was synthesized by solid-phase synthesis using the triester method.

As a solid support (resin), a compound (Wako Pure Chemical Industries Ltd.) in which 5'-(4,4'-dimethoxytrityl) thymidine/ was bonded to polystyrene via a succinic acid spacer was used. As each nucleotide (dimer) (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd. (Kikki)), those listed in Table 1 below were used.

Table 1 DMTr: dimethoxytrityl, bz: benzoyl group.

1buH isobutyryl group A; adenosine, C; cytidine, G; guanosine, T;
Thymidine (1) condensation reaction Using a nucleic acid solid phase synthesizer, resin 3μmoJ3 and condensation agent (
Using 2,4,6-drimethylbenzenesulfonyl-3-nitrotriazole (MSNTII), the operations in Table 2 were repeated for each addition of dimer to be coupled.

(11) Separation of 15mar oligonucleotide from resin ■ 0.5 M 2-pyridine aldoxime, 1°1
．． 3.3-Tetramethylguanidine-1-dioxane:
Pyridine:water=5:4:1 20 mg of the dimer condensate obtained from (1) was added to 1d and stirred at room temperature for 24 hours.

■ After distilling off the solution in ■, the residue contains concentrated ammonia water (arn
After heating in separate portions at 55°C for 5 hours, the solution of (1) and (2) was washed with ethyl acetate (6dX31), and the aqueous layer was concentrated.

The residue was applied to a reverse phase (C+sl silica gel column (Waters Co., Ltd. 55-105 μ) (φ0.7 x 5 cT!11, 0.1 M) ethyl ammonium acetate pH
7 in 10 to 30 degrees of acetonitrile (total 100 m
Elute with the concentration gradient of 1). 20% of acetonitrile
A fraction of the dimethoxytrityl compound eluted around the concentration is collected and concentrated.

(2) Add 80% aqueous acetic acid (5 rnl 1) to the residue and let stand at room temperature for 15 minutes. Wash the reaction solution with ether (5 rnl x 3) and concentrate. Completely deprotected oligomer of 1200D26Q is obtained.

■ Part of the above oligomer is reverse phased” I8 + 5μ
) It was purified by silica gel high performance liquid chromatography. Elution is carried out with a concentration gradient of 10.6% to 14% acetonitrile in 0.1 M triethylammonium acetate (1)f (7) at a flow rate of 1 rnl in 7 minutes over 30 minutes. The central part of the peak was collected, and after $41, it was azeotropically distilled with water-ethanol to desalt it.

Properties: Water-soluble white powder UVλH2O260ft, 10,000/base
1ax high performance liquid chromatography R, T, 22 minute mold 1
Cys TGT was recombined with the original pDNA at one site.
165 f< Ser TCT 15mer oligonucleotide CT designed to mutate to 185
G TACTCT TTCCGT 100 pmo,,
g'e 50mM TrisH (Jp) (8,0,
10mM MgC4, 1mM EDTA, 6mM βEtS
H,0,1q/-gelatin.

500 pmoI3ATP, lny”) T4hol
J5Z Riv Otide Kinase (released by Takara Shuzo), 25μ
5' phosphorylation was carried out by reacting for 1.5 hours at 37'C: in p.p.

For hybridization, the 5' position of 15 mar of 20 pmo4 is 30 /j Ci (D (r
-''pl ATP (3 in 3000 CiAnmo) was highly labeled under the same conditions as above without 500 pmo4 ATP as the sole ATP source.

Template single-stranded recombinant DNA 5' phosphorylated 1 to 2μp
Add 5 mer 6 pmoA, l Q mM T
risH(J pH7, 5, 10mM Mg(J2.5
After heating at 65° C. for 1 hour in 0 mM Na (J 10 pl), annealing was performed at room temperature for 30 minutes.

The Kono solution was diluted with a final concentration of 10 mM TrisH (J p
H7,5゜10mM MgC4, 20mM NaC[
, 6mM βEtSH.

0.6mM ATP, 0.25mM dATP
, 0.25 mM dGTP.

0.25mM dCTP, 0.25mM dTT
P (all sold by Pharmacia), prepared to a total volume of 25 μp, and 2 units of Escherichia coli DNA polymerase Klenow fragment (sold by Pehringer Mannheim).

Add 200 units of T4 DNA ligase to 14
Sequential ligation was performed to direct primers for 16 hours at °C. 6μ
Sample 2 and infect Ca(J, treated JMIol, JMIOII late culture cells 0.2 d.

The mixture was spread on YT medium together with 3 ml of 0.8% soft agar and cultured at 37°C overnight.

After the infected medium was kept at 4° C. for about 2 hours, a nitrocellulose filter (Millipore HAo, 45 μm) was brought into contact with the surface of the plaque on the medium, and the phages were adsorbed onto the filter. After fixing the phages on the filter by heating at 80°C for 2 hours, 20ml of 5x concentration ss
c (5Xsscl [: 1xssc = 0.15
M NaC (3,0,015M sodium citrate,
1 mM EDTA], 5x Denhardt's solution (
5x Denhardt's 5solution l
[l Q Q times = 2% bovine serum albumin, 2% polyvinylpyrrolidone, 2% ficony] Preno-hybridization was performed in 0.1% sodium pyrophosphate at 65° C. for 1 hour.

20 rLl of 5X ssc, 5X De/Hart solution, 0.1
1 obtained by highly labeling the 5' position of sodium tibirophosphate
10μ2 of a 5mer probe was added and hybridization was performed at room temperature for 15 hours. While checking the filter with a Geiger counter, check the 20m 5 x 9
After washing at 80° C. at 30° C., 45° C., and 55° C., an autoradiogram was taken, and positive plaques containing the desired mutation were selected.

Sixteen 7-age samples corresponding to positive plaque hybridization sites were collected and cultured for 12 hours in 5 ml of double-concentration YT medium with 25 μp of JMIOI overnight cultured cells. Cells were pelleted by centrifugation at 6,000 rpm for 5 minutes, and recombinant single-stranded DNA was isolated from the supernatant. Sequencing was performed using the dideoxy method, and it was confirmed that 15 of the 16 samples collected were the desired recombinants. The precipitate obtained from the phage containing the desired recombinant was treated with an alkaline method to obtain recombinant double-stranded DNA. Restriction enzyme B
g, e II, Sa[[h containing the recombination site after cutting with
A 0.17K bilfr piece corresponding to the C-terminus of GH was separated by gel electrophoresis and recovered.

Cloning hG into a plasmid for expressing mutant hGH
] (The expression plasmid pGH-
L9 was treated with Bg-g If, Sad [te, hG
H) A large fragment lacking 0.17 Kb corresponding to the c-terminus was separated and recovered by agarose gel electrophoresis.

This large fragment containing the trp promoter region for expression of the vector part 9 derived from pBR322 and the N-terminal site of hGH and the 0,17 Kb fragment corresponding to the C-terminus of hG) containing the previously isolated recombination site was transformed into T4. After ligation with DNA ligase, it was transformed into HBIOI which had also been treated with CaC. A plasmid was isolated from the resulting transformant by an alkaline method, and treated with 8g6 [, Sad I to confirm that the insertion had the correct length.

Additionally, the recombinant Ser TCT 165 plasmid and the original Cys TGT 165 pGH-
The L9 plasmid was spotted on a nitrocellulose filter, heated at 80°C for 2 hours, prehybridized at 65°C for 1 hour, and then subjected to the same conditions as the previous plaque hybridization using 15rner corresponding to 5erTCT165 as a probe. Hybridization was performed. After washing the filter with 5Xssc at 37°C, autoradiography revealed that the recombinant S
The plasmid of er TCT 165 gave a very strong positive spot, whereas the plasmid of CysTGT 165 gave a very strong positive spot.
GH-L9 gave only a very weak spot.

This confirmed that the recombinant Ser TCT 165 fragment was reflown into pGH-L9.

This plasmid was named pGH-L9/C83-1, whereas the original pGH-L9 was named pGH-L9/C83-1.
It will also be referred to as L9/CCl-1.

Example 2 Expression of hGH recombinant plasmid in E. coli pG)l-L9
I(B101) transformed with /C83-1 or pGH-L9 /CC1-1 was expressed in M9 casamino acid medium containing Ambicily/. 50μ6e of cells cultured overnight
Add 5 ml of fresh ampicillin-M9-casamino cough medium and incubate at 37°C for 3 hours. Add IAA (indolyl-3-acrylic acid) to a final concentration of 40 μg and apply induction for another 12 hours. Cultivation continued. At this time, as a control, cells that were not induced with IAA were also cultured. 0.2 m each for cells with and without IAA induction
Centrifuge and collect 62.5 mM TrisHC [pH
6,8,2Z SDS, 5mM EDTA, 1
0% glycerol, 5 μEtSH, 0.1 μBFB
The cells were suspended at 20μ2 and treated at 1000C for 15 minutes to disrupt the cells. Centrifuge the whole cell extract at 1000 rpm for 10 minutes to remove insoluble matter and dilute it with 15% SDS.
Complete polyacrylamide gel electrophoresis, molecular agreement 21
,000, and a protein corresponding to hGH was found to be highly expressed.

that's all Procedural correction band (voluntary) November 25, 1985

Claims (1)

[Claims] 1. A gene for expressing a novel polypeptide in which cysteine, an amino acid at position 165 from the N-terminus of human growth hormone, is converted to serine, and the base sequence corresponding to the amino acid at position 165 is TCT. A plasmid.