JPS6248383A - C-ras synthetic gene segment - Google Patents

Abstract

NEW MATERIAL:A c-ras synthetic gene segment, consisting of a base sequence expressed by formula I (A, C and G are respective residues of deoxyadenylic acid, deoxycytidylic acid, deoxyguanylic acid and thymidylic acid; X is G or T; Y is C when X is G, and A when X is T; abbreviations of amino acids are amino acid sequence coded by the base sequence), etc. having a restriction enzyme BssH II part in a DNA sequence part coding serine, alanine and leucine at the 17--19-positions of the amino and coding c-ras protein. USE:Production of c-ras proteins and diagnosis of diseases related to cancers. PREPARATION:A group of combination having mutual complementation, e.g. an oligonucleotide expressed by formula II is selected as shown in formula III, and the respective complementary chains are subjected to hydrogen bonding. Several double-stranded DNA fragments are prepared using a ligase, and then linked with a ligase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a human C-las synthetic gene segment, and more particularly to a novel human C-las synthetic gene segment and a recombinant plasmid containing the same. The present invention relates to a microorganism transformed with this recombinant plasmid and a method for producing C-ras protein using this microorganism.

[Prior art 1 Activated cell C-ras genes (C-11a-ra
S.

C-Ki-ras and N-ras) are also found in human cancers as well as in HΦ tumors in experimental animals. It has been found that it is involved in the transformation of (Reference 1) c-ras genes are molecules ff121
,000 Dalton protein (ρ21). Activation of C-las III genes occurs with a single base substitution, resulting in a single amino acid substitution at the 12th or 61st codon. Activation of the human C-ras gene in bladder cancer occurs with a single base change from G to ■, resulting in the substitution of glycine at position 12 by valine. (References 2-6) The specific characteristics and physicochemical properties of mutated p21 proteins have been previously discussed, and (References 7-9) some biochemical and biological studies of 1121 mutated at the 12th codon. The scientific properties have been reported.

In order to elucidate the molecular biological mechanism of activated p21 protein in cell transformation, it is necessary to isolate human p21 protein. In addition, such normal or mutant p21
If the protein is obtained in large quantities, these can be
Through the identification of the C-ras gene by the protectonic method, it can be expected to be used as an antigen for the diagnosis of cancer-related diseases and for the preparation of monoclonal antibodies against the protein produced by the C-ras gene, so it is also used in the field of disease diagnosis. It is considered to be extremely useful.

Contains cDNA derived from natural mRNA [
.

Recombinant techniques using the E. coli host vector system were previously employed for the synthesis of p21 encoded by the normal or activated c-11a-ras gene.

[Problems to be solved by this invention] However, depending on the recombinant technology using cDN8, p21
It has not always been possible to prepare proteins manually.

[Means for Solving the Problems] The present invention aims to prepare p21 protein in humans, and for this purpose, it provides useful human C-las synthetic gene segments, and constructs containing this segment that can self-replicate in microorganisms. A recombinant plasmid, a microorganism that can be transformed with this recombinant plasmid and express the target p21 protein, and a p21 protein using this microorganism.
The present invention provides a method for producing proteins.

[Structure and operation of the invention 1] The first invention encodes a C-ras protein, and injects the restriction enzyme YussH [1 of DNA encoding fulginine, aspartic acid, and glutamine at positions 68 to 10
The restriction enzyme Bcl I site is placed in the sequence part, and the first
Restriction enzyme Ec was added to the DNA sequence encoding glycine, isoleucine, and proline at positions 38 to 140.
This provides a C-ras synthetic gene segment having an oRI site and a restriction enzyme HindIII site in the DNA sequence encoding lysine and leucine at positions 110 and 111.

Such C-las synthetic gene segments include the formula % formula % Lys Arg Val Lys Asp
Scr Asp Asp Vat Pr.

AAA CGI GTT AAA GACTC
T GAT GACCTT CCG Ding HGCA
CAA 1-cho T CTG AGA C-cho^
CTG CAA GGCMet Vat Le
u Val Gly Asn Lys Cy
s Asp LeuATG GTT CTG
GTT GGT AACAAA TGCGAC
TTG■To 8C CAA GACCAA CCA
rchoG r1cho 8CG CTG AACAla
Ala 8rg Thr Val Glu
Ser Arg Gln AlaGCA
GCG CGT ACT GTT GAA
TCT CGT CAG GCTCGT CG
CGCA TGA CAA CTT ＾GA
GCA GTCCGAGln Asp Leu
Ala Arg Ser Tyr Gly
Ile Pr.

CAG GAT CTG GCT CGT
TCT TACGG'A 8TT CCGEco
Rr Dir Ile Glu Thr Ser
Ala Lys Thr Arg GinTA
C8 [To CG^ 8CCTCr GCT 8AA
ACT CGT CA8 1G 1^G CT
T TGG AGA CGA 1 Ding■ Ding GA
GCA GTTGly Vat Glu A
sp 8 la Phc Tyr Thr L
eu VatArg Glu Ilc Arg G
ln 1lis Lys Leu Arg L
ysGCA CrT TAG GCA GTC
GTG TTCGACGCA TTC Old nd Leu Asn Pro Pro Asp Glu S
er Gly Pro Gly-1” II Cys Hat Ser Cys Lys
A C-las synthetic gene segment consisting of the base sequence represented by Cys Val Lcu Scr [hereinafter referred to as formula (1), where A is deoxyadenylic acid, C is deoxycytidylic acid, G is deoxyguanylic acid, D represents each base of thymidylic acid, X represents G or T, Y
When × is G, C represents 1 togihachi (the same applies below).

An example of the amino acid abbreviation on a base sequence is 1, which indicates the amino acid sequence encoded by this base sequence.

This human C-las synthetic gene segment preferably has a restriction enzyme CoI site at the upstream end, and a stop codon and subsequent restriction 11-3alI site at the downstream end. Such sequences added to the upstream side J3 and the downstream side are "'CGA ding 3° [A . . . (2) and 5° TG^TAGG", respectively.

ACICTGCAGCT ・ ・ ,
An example of this is the array represented by (3). In formula (2) above, the upstream end is "I", and in formula (3), the downstream end is each cleavage end of Giroux (respectively, they are hollowed out).

The C-ras synthetic gene segment of the present invention is identical in its encoded amino acid sequence to that of the cDNA described above, but its codons are significantly different.

C-ras synthetic gene segments of the present invention (X is r,
If Y is 8), use the following formula, Ul to U2o and Ll to '19, CT to 5' UI CGA DING ATGACCGAATAC 8A''.

5゜02 GGTTGT8GTTG to CGCTG
TT ”5゜113 GGTGTAGGGCA8^ΔGCG
To CG-TGACCATTCAGTTG8■C3゜5゜14 To CAG8ACACTDing CGT^GA
T-GAGTACGACCCGACT 8cho■3゜υ5
G to AGAC CTTACCGTAAGC3
゜5゜116 AGGTTGTT/1rcGAcG
G-cho 3'5゜07 GAGACCTGTTTGCTGGACAT
C3'5゜t18 C Ding TGATACCGCAGGCC
AAG 3'5゜5゜09 AAGAArACTCTGCchohachiTG
C-GTG 8T to A TAT^TGCG ACC3゜U
lo ”GGCGAAGGCTTCCTGTGC-G
TTTTCGC ding ATCAACAACAC3゜Old 1 5
°CAAADTTTTTGAAGACAT-cc^TC
A＾TACCGTGAACAGATC3゜1112
``'AAACGDingGrrAAAGAcDingcTGAT
"013" GACGTTCCGATGGTTCT
GGTT 3゜u14 5゜GGTAACAAAATGC
GACT Ding G-GCAGCGCGTACTGTTGA^
Ding C3゜U155°TCGTCAGGCTCAGGAT
CT-GGCTCG TCTTACGGA＾TC3゜u16 5°CGTACATCGAAAACCTCTG-
CTAAAACTCGTCAAAGGCGDingT3゜ull
5°GAAGACGCDTACACC-TT
GGTTCGTGAAATCCG-CAGCAC3゜u
185°AAGCTGCGTAAGCTTAAC-CC
GCCGGACGAATCTGGCCC"t119
AGGCrGCATGTCCTGCA8ATG 3°
Oyohi 5゛ u205 to CGTTCTGTCTTGATAGG 3゜ and 5゜11 TCGACCTArCAAGACAGA-^
CGCATTTGCAG3゜5゜L2 GACATGCAGCCTGGGCCA-G
ATTCGTCCGGCGGGTDing^^GCT3゜5゛L3 TACGCAGCTTGTGCTGACG
3゜1.4 GATTTCACGAACC^^GG
TO”5゛5゜L5 TAGAAAGCGTCTTCAACG-C
CTTGACGAGTTTTAGCAGAGGTT”5
' 16 TCGA-GTACGGAATTCC
G-TAAGAACGAGCCAGADCTG3゜5
゜L7 AGCC dingGACGAG＾TTCAA
C-AGT^CGCGCTGCC^^GTCGC”18
5'ATTTGTTACCAACCAG^^-CCA
TCGGAACGTCATCAGAGTC3゜5' L9 TTTAACACGTTTGATCTG
-TTCACGGT8TTGATG^TGTCTTC
3'[105°8AAAGATTTGGTGTTGTT
-GATAGCGAAAACGCACAG3'[115
°GAAGCCTTCGCCGGTACG-CAT^T
ACTG Haccho C8 CGCA to AG.

[125°CAGAGTATTCTTCTTGGCCT
G 3° L13 5° CGGT 8 TCAA GATGT
CCA-GCAAACAGGTCTCACCGTCGA
T” [145°A8 CAACCTGCTT^CGGTA
-^GAGTCTTCAAT8GTCGG3'[Go to 15 5°GTCGrACTC^DCTACGAAG 3
゜[165°TGGTTCTGG8TC88CTG
”[175°8ArGGTCAGCGCGCTTT
3゜118 “TGCCT＾CACCAACAGCG
CC3' and Li2 "AACTAC8^CCA
Select a group of mutually complementary combinations of oligonucleotides represented by TTTGTΔ-TTCGGTCAT8 and 3, hydrogen bond each complementary strand, and combine them in the presence of ligase to form several two-stranded oligonucleotides. It can be prepared by preparing stranded DNA fragments and further ligating them in the presence of ligase. Groups of such oligonucleotides include Group I 111f 111'
MU-U U5~U8 U9~U12151
9 1″″L L ~shi・shi12~'14, 911, IV group V group U13~U16 U17~U20L -
Examples include L, and the groups L1 to L4. An example of preparing the C-ras synthetic gene segment of the present invention using this group is shown in FIG.

Oligonucleotide or double-stranded DNA using ligase
When binding the A fragment, the 5° ends of the components whose 5° ends participate in the reaction are phosphorylated in advance.

In FIG. 1, these components are indicated by horizontal lines. A black dot at one end indicates that the 5' end is phosphorylated.

All phosphorylation and ligase conjugation can be performed using conventional techniques and conditions. The DNA sequences of the two iDN^ fragments obtained in the first ligation shown in Figure 1 are as follows.

-ri''■ Box 11 1 1 dingCTACTC^TGCTG ``GAAGACTCTTACCGTAAGCAGGGGG
CTGAT＾ACTTCDingGAG８＾TGGC８TTC
GTCC"' Ding Ding GTT 8TCGACGG Ding G
AGACCTGTTTGCTGG88 CAATAGCi
GCCACTCrGGAc8^^CGACCTGT8G
GAACT＾DingGOCGICCGGTTCTDingCTTA
TGAGAC5' ”AAGAATACTCTGCTHacchoGCGTGAchoC
AGTA Ding AG 8 TAC GCACT 8 GTCAT 8 B
cl I TGCGTACCGGCGAAGGCTDingCCTGTG
CGdingTTACGCATGGCCGCTTCCGAAG
GACACGC88^8GCGATAGdingTGTTGT
GGTTT^GAAAACTTCAC^TCCATCA
AT8CCGIGAACAGHacchoCAAACTGTAG
GTAGTT^TGGCACT rGTcTAGdingTT
GGTGTTAAAGACTCTGAT 3゜CACA
ATTOTT [co RI GACGHATCHOTOTTGAGCHAGTTCCGCAACT
TCTGCGAAAGAr 5' GTGGAACCAAGCACTT 11indllll CCGCCGGACGAACTGGCCCAGGCCATGGGCGGCCTGCTTAGACCGGG
TCCGACGTAC ding CCTGC 8^8 TGCG ding T
C-cho G-cho CTTG 8 TAGG 3 “AGGACGTTTA
It is preferable that each of the eight double-stranded DNA fragments produced be purified and isolated by a conventional gel electrophoresis method and used for the next ligation.

By attaching these five types of double-stranded oligonucleotide fragments with a ligator pin, it is possible to obtain a C-las gene segment of the present invention having a cleaved end of Ko on the upstream side and Sal on the downstream side. can.

In the C-ras synthetic gene segment of the present invention, X is G,
Those where Y is C are replaced with U2 and [18, respectively. 5゜ 3゜ [2 GGTTGTAGTTGGCGCTGG
and 118' ”゜TGCCT8C8CCACC
A similar preparation can be made using an oligonucleotide consisting of AGCGCC 3'. In addition, the above double-stranded DN
From the end of Cla I upstream of I of A fragment as
It is also possible to prepare a partial fragment of ① containing the s still site, digest it with ass HI, and replace it with the corresponding portion of the above fragment I.

The obtained C-las synthetic drug segment is inserted into an appropriate plasmid (for example, inserted into a plasmid containing a gene encoding drug resistance) and transformed into E. coli, etc., and used as an indicator of drug resistance. can be cloned and amplified using techniques such as , colony hybridization, etc.

Each oligonucleotide used as a starting material in the method of the present invention can be prepared by the method described below.

First, each single-stranded portion of these oligonucleotides is synthesized. This synthesis can be carried out, for example, by a solid phase method. A commercially available DNA synthesizer may be used.

When using the solid phase method, the carrier used is a divinylbenzene crosslinked polystyrene resin having p-(aminomethyl)phenyl groups [Hiyoshi et al.
), Nuclef Assilas Research (Njc le
i cAcids Res, ), Volume 8, 5507 (1
980)] or silica gel [(H, D, Hatte
ucci ct al,) dit! Null of the American Chemical Society (J, 8m, Chem
, Soc, ), p. 103.3.3185 (1981)] is preferred.

Four types of deoxynucleosides constituting the 3° end of each fragment, namely deoxycytidine, deoxyadenosine, deoxyguanosine, and thymidine, are each converted into 5°-dimethoxytritylated 3°-succinate esters by a conventional method [Proki et al. (Broka C,et a
l, ), Nucletsk Assilas Research (Nucle
ic Ac1ds Res, ), vol. 8, 5461-5
471 (1980)].

This is bonded to the 7-mino group of the carrier (nucleoside carrier) and used at the 3° end of the oligonucleotide. Next, for oligonucleotides consisting of an even number of deoxyribonucleotides, these 3° terminal nucleosides are treated with the mononucleotide corresponding to the second from the 3° terminal [^ and N-benzoylated for C, and N-benzoylated for G. Protected by N-isobutyrylation. For oligonucleotides consisting of an odd number of deoxyribonucleotides, the 3° end is The second and third corresponding dinucleotides (with each protection as before) are coupled using a condensing agent. As a condensing agent, mesitylenesulfonyl-3-
Nitrotriazolide (H3NT), 2,4゜6-dolyisobrobylbenzenesulfonyl-3-nitrotriazolide (TPSNT), methicytidinesulfonyl-tetrazolide (H3Te), 2,4,6-dolyisobrobyl Benzenesulfonyl tetrazolide (TPSTe)
) etc. are appropriate.

Furthermore, for the former oligonucleotide, similarly protected dinucleotides corresponding to the third and fourth oligonucleotides,
Dinucleotides corresponding to the fifth and sixth nucleotides...
..., and for the latter oligonucleotide, the fourth and fifth dinucleotides, the sixth and seventh dinucleotides, and so on, are sequentially linked in the 5' direction in the same manner. However, if necessary, mononucleotides, trinucleotides, tetranucleotides, etc. may be used in the middle.

This preparation method is shown in FIG. In the figure, P is a copolymer of polystyrene with 1x divinylbenzene, bz and i
b is penzoyl and isobutyryl groups, 8 is bzA
, ibG, bzC or T, Bo is A, G,
C or ■, DHTr is 4,4-dimethoxytrityl group, Ar is orthochlorophenyl group, BSA is methylene dichloride-methanol a of benzenesulfonic acid
Combined W (capacitance 7:3) solution (2x) was added to DHAP Ha4
- A 0.14) 1 pyridine solution of dimethylaminopyridine was transferred to THG-P.〇 is a dioxane-water mixed solvent (capacity 9 :1) Shows solution (0.5M).

Sixteen types of the above-mentioned dinucleotides are required depending on the permutation of the four types of nucleotides, and they can be synthesized, for example, by the method shown in FIG. 3 below.

However, DHTr is 4', 4-dimethoxytrityl group, 8 and 8' are 6-tobenzoyladenine-9-yl group, 4-N-benzoylcytosin-1-yl group, 2-toisobutyrylguanine- They are selected from a 9-yl group and a thymin-1-yl group, and may be the same or different. H3NT represents mesitylenesulfonyl-3-nitrotriazolide.

The introduction of protecting groups into these dinucleotides is carried out using known methods [
For example, Protein Nuclear M Enzyme, 26(4), 259 (1981
)].

"Plasmid and its preparation" The second invention provides a recombinant plasmid containing the C-ras synthetic gene type 1 segment of the present invention. That is, human C-ras synthetic gene 1 of the base sequence represented by formula (1)
The object of the present invention is to provide a recombinant plasmid containing a similar segment and capable of self-replication in microorganisms. and preferably C-
A promoter-operator system that controls the expression of this gene upstream of the C-ras synthetic gene, and a portion and a drug that enable the plasmid to self-replicate between the upstream of the promoter-operator system and the downstream of the C-ras synthetic gene. It contains a part that codes for resistance.

As a promoter-operator system, the bacterial tryptophan promoter, which lacks a portion of the attenuator, is used.
An example is an operator system.

The recombinant plasmid of the present invention can be produced in WA by integrating the C-ras synthetic gene type 1 segment of the present invention into an appropriate vector plasmid by a conventional method. In particular, upstream and downstream of Cla I and Sa
The C-las synthetic gene class 1 segment of the present invention with a tI restriction M cleavage end can be easily integrated between the corresponding cleavage sites of such a plasmid.

A plasmid with such a sequence is, for example, pGll-L.
The plasmid of the present invention is a pGII-L9 plasmid of the C+at and Sal
The larger fragment of the two DNA fragments produced is digested with r and the C- of the present invention has c+a I and Sat T υ restriction enzyme cleavage ends on the upstream and downstream sides, respectively.
It can be prepared by adhesive ring closure of a class 1 synthetic gene segment. This preparation is illustrated in FIG. pGI
The I-191 lasmid is disclosed in reference 15.

The plasmid thus prepared can be transformed into a suitable host such as E. coli, and can be easily cloned and amplified using appropriate traits such as drug resistance (susceptibility) as an indicator. Plasmids can be extracted and purified from host cells by conventional methods.

The recombinant plasmid of the present invention can be transformed into a microorganism such as E. coli to produce the product of the C-ras synthetic gene of the present invention directly or in the form of a fusion protein.

For example, the C-las synthetic gene type 1 segment of the present invention has an end represented by the formula (2) on the upstream side, and a terminal represented by the formula (3) on the downstream side.
) has the restriction enzyme c+a I after the S0 sequence of the promoter-operator system.
This C-ras synthetic gene segment can be directly expressed by transforming E. coli into a vector plasmid having a restriction PIII and Sal I site downstream thereof.

In such a system using a bacterial tryptophan promoter operator system, the DN^ sequence from the metsenger RN^ transcription start site of the promoter-operator system to the C-ras synthesis gene is 5゛^AGTTCACGrA.
AAAAGGGT^A Ding CG^■3゜TTCAAGTG
Preferably, it is expressed as CATTTDINGTCCCATT8GCT^.

As such a vector/plasmid, a patent application published in 1983
PGI containing the above-mentioned PGII-L9 disclosed in No. 2084 and Japanese Patent Application No. 60-45773
I-1, series plasmids can be exemplified. These plasmids encode human growth hormone as a heterologous protein to be expressed between the restriction enzyme cleavage sites HA and SAT 1, and this portion is modified according to the present invention with the upstream and downstream ends described above. C-ras p by replacing with the C-ras synthetic gene segment of
Plasmids for direct production of the 21 protein can be constructed. FIG. 5 shows such a preparation method. Plasmid pR-12V can be prepared in this way.

In addition, the C-las synthetic gene type 1 segment of the present invention encodes another protein and is inserted into a gene that is expressibly integrated into a plasmid so that the open reading frame coincides with the C-las synthetic gene segment 1.
Plasmids can be constructed that are capable of producing the rasp21 protein in the form of a fusion protein.

The C-las synthetic gene class 1 segment of the present invention prepared from the aforementioned double-stranded ON8 fragments, ■ to V, in which X is ■ and Y is eight in formula (1), can be used with a plasmid containing this. Restriction enzymes - Cla T and ass H! Digest with to remove the short Cla I-ass tll fragment and replace it with 5°CGATATGACCGAATACA^^CdingGG
TrGTAGTTATACTGGCTT＾TGTTTG
ACCAACATCA” It■ TGGCGCTGGTGGrGTAGGCAAAAG3
゜8CCGCGACCACCACATCCGI T-T
By integrating the double-stranded DNA fragment represented by CGCGC box 111I by a conventional method, a plasmid containing the normal C-ras synthetic gene can be easily obtained. Such a preparation is shown in FIG. Moreover, the reverse can be easily performed in the same manner.

In this manner, plasmid pR-12G containing the normal C-ras synthetic gene can be prepared.

In addition, coli strain carrying plasmid pGII-L9,
L. coli pHG-L9 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as Microtechnology Research Institute No. 7606.

"Transformant" The recombinant plasmid of the present invention can be prepared by conventional methods [eg, Cohen et al., Proceedings of the National Academy of Sciences (Pro.

Na mouth, Acad, Sci,) U, S, A,,
69, p. 2110 (1972) 1, and can be easily transformed into microorganisms such as E. coli.

Thus, with plasmid pR-12G, E. coli/11
Strain obtained by transforming B101 strain, E. coli/pR
-12G has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and has been given the deposit number, Microbiological Research Institute No. 8425.

The mycological properties of these strains are no different from the host strain other than that they have acquired ampicillin resistance and that they express and produce C-ras p21 protein, and they can be easily cultured under similar conditions. . Also, a medium supplemented with ampicillin can be used.

By culturing the transformed L coli strain of the present invention in a nutrient medium, the C-ras synthetic gene can be efficiently amplified. Furthermore, the C-ras p21 protein has a promoter-operator system upstream of the C-ras synthetic gene of the present invention that controls and enables its expression, especially a tryptophan promoter-operator system lacking a part of its attenuator. directly expresses
A significant amount of ffl accumulates within the bacterial body. In this case (tryptophan promoter operator system) C-rasp
21 protein production ←mRNA synthesis) is strongly induced by the addition of indole acrylic acid.

The C-ras p21 protein accumulated in the bacterial cells can be separated and recovered by a conventional physiologically active protein recovery method after the bacterial cells are lysed. For example, after culturing, the Kokutai cells are collected by centrifugation, and treated with ultrasound, lysozyme, and French press in a phosphate buffer.

The desired protein fraction is obtained by ammonium sulfate fractionation or gel filtration of the supernatant obtained by treatment with Dynomil or the like. If even higher purity is required, it can be purified using an antibody column method. The C-ras p21 protein can be completely extracted by treatment with guanidine from cells in which it is absorbed into the cell wall, and can be reactivated and recovered in high yield.

[Example J Hereinafter, the present invention will be explained in more detail with reference to Examples. The reagents indicated by abbreviations in the examples are as follows.

Standard buffer: 50+gHTris-FA acid pH 7,
535 mHH (lc12 35 sH2-mercaptoethanolTr
is-NaCI: 10sHTris-Salt M buffer, 0.
14HNaCl, pH 8°TE-Sucrose; 25% sucrose (Ken/V), 50114 Tris-HCl buffer, 1sHEDTA E18 8° Lysodeme:
10Ily/m! Lysozyme, 0.25HTris-HCl buffer, pH 8° 0.58 EDTA: pl+ 8
゜PNase: 1 in 0.04M acetic acid buffer solution h5)
04/d and heat-treated at 100°C for 5 minutes.

tyttc Mixture: 0.1% nonionic surfactant (Triton

[Solution B/Ethidium bromide to water: 4.67
Rg/#! i! Prepare by dissolving it so that

TE: 10sHTris-j! 2 acid buffer, 1+nH
[DT8, pH 7, 4°TE-sarkosyl: 10sHTirs-HCl buffer, IIaHEDTA, 0.38χ Sodium
IN-Lauroyl 5arkosinate
.

pH 7, 4°TEN: 10+eHTirs-Salt M buffer, 1sHE
D.T.A.

0, IHNaCl, pH 7.4゜Example 1 [Synthesis of oligonucleotide] (1) AA to dCG^TADINGGACCG8ATAC
CT(Lll) Synthesis 5°-(4,4-dimethoxytrityl)thymidine carrier (100 μn+ol/g) The polystyrene resin 501(7) was left in pyridine overnight at room temperature, and a condensation reaction was carried out by the following procedure.

Procedure 1) Dichloromethane-methanol (7:3, V/V
) Washed 3 times with 21!1J2.

2) After treating with 2% benzenesulfonic acid solution (solvent dichloromethane-methyl alcohol, 7:3) for 2 minutes with 2mj2, the process of washing three times with the same mixed solvent was repeated to confirm that no color developed.

3) Wash 3 times with pyridine 2a+It.

4) Pyridine solution of 5°-(4,4°-dimethoxytrityl)-N-penzoyldeoxyadenyl p(o-chlorophenyl)-N-benzoyldeoxycytosine 3°(0-di[corophenyl)phosphate] (0 ,2ml
1, containing 20+ ng of the same wave cleotide) was added, and the solvent was distilled off under reduced pressure.

5) Condensing agent, mesitylenesulfonyl-3-nitrotriazolide (2011 g) in pyridine solution (0,2nj!
) and left open at 40°C for 20 minutes.

3) Wash twice with pyridine 2 aft.

7) Add 0.1M dimethylaminopyridine in pyridine solution (C8lll) and acetic anhydride (0.2mA).
Leave it for a minute.

B) Wash 3 times with 2 lll of pyridine. Next, the above 2
) Subsequent operations were changed to 5'-(4,4°-dimethoxytrityl)-tobenzoyldeoxyadenylyl-p-(o-chlorophenyl)deoxyadenosine-3'-(o-chlorophenyl)phosphate to give 5'-( 4,4-dimethoxytrityl)-N-penzoyldeoxyadenylyl p-(0-chlorophenyl)deoxyadenosine-3
dAC, dAT, dG using °-(0-chlorophenyl)phosphate and further having similar protecting groups
A.

The DMA chain was sequentially extended in the 5° direction using dCC, dGA, dAT, dAT, and dCG.
5M trimethylguanidium-pyridine-2-aldoximate i:c, B, Reese et al.
, Tetrahedron Lett,, 272
7 (1978)] in a dioxane-water (9:1) solution (IIIi) for 16 hours. The resin was washed with 50% pyridine water, the furnace solution and washing solution were combined, and concentrated under reduced pressure.
Add concentrated ammonia water (15 m) to the residue and seal the bottle.
It was heated at ℃ for 5 hours. Ammonia was distilled off, and the oligonucleotide having a dimethoxytrityl group was purified by C18 silica gel chromatography. Elution was carried out using a gradient of acetonitrile in 501H tritactylamine bicarbonate MvijJ solution.

Dry one fraction containing the target product under reduced pressure to a residue of 8.
After adding 0% vinegar M1d and keeping at 25°C for 30 minutes, the FB medium was distilled off, and the residue was dissolved in water and ethyl acetate.

After concentrating the aqueous layer, HPLC (C18 silica gel carrier)
It was purified to 12,6^260 units (630μQ
) was obtained. HPLC (C,8 silica gel carrier) and -
Purity was confirmed by single dimensional homochromatography.

(2) (IGGTGTAGGCAAAAGCGCGC
Synthesis of TGACCATTCAGTTGATC (U3) O
It was prepared by the phosphoramidite method using an N8 hemogen, model 380^ (manufactured by Applied Biosystems). Namely, dedimethoxytritylation with trichloroacetic acid, condensation using a 7-osphoramidite reagent of about 15 molecules per cycle, oxidation with iodine, and acetylation of unreacted 5'-hydroxyl groups with acetic anhydride and dimethylaminopyridine. After automatically deprotecting and cutting out from the carrier, purification was carried out in the same manner as in ``Synthesis of r(1)LJl'' to obtain 8.8A26o units (440 μg) of the target product.

(3) Synthesis of oligonucleotides shorter than 30 bases
U2, U2°, U5~U8 in the same way as in case 1
, LJl2. L113. L119. U3O,L
3, 14.

L12.115-L18. Oligonucleotides constituting L18° and Ll9 were combined with a 5′-(4,4°-dimethoxytrityl)deoxyguanosine carrier and a 5′-(4
, 4°-dimethoxytrityl)deoxyadenosine carrier.

Using a 5°-(4,4-dimethoxytrityl)deoxycytidine carrier and a 5°(-4,4-dimethoxytrityl)thymidine carrier, the sequence sites listed in Tables 1-1 and 7-2 are indicated by underlining. In each unit, dinucleotides each having a protective base are sequentially condensed from the 3' end toward the 5° side in the same manner as in the case of Ul, and the protection 'i! ! ! U4 was produced by base and purification separation.

(4) Synthesis of oligonucleotides of 30 bases and longer In the same manner as in the case of U3, Ll3. U4, LI9~U
11. U14-U18. Ll, L2, L5~L1
1°Ll3 and Ll4 were prepared using a DNA synthesizer, model 380 manufactured by Applied Biosystems.

The oligonucleotides thus obtained are shown in Tables 1-1 and 1-2.

Table 1-1 5-11 CGATATGACCGAATACA
8 ACT 3゜12 GGT to GTAGTTGG
CGCTGTT 3'5゛02 G (dchoGchoAGTTGGcGCTG
GT ”55゜5゜03 GGTGrAGGCA8AAAGCG
To CG-TGACCATTCAGTTG＾TC3゜5゛Ll4 CAGAACCACTTCGTAG
AT-GAGTACGACCCGACTATT3゜5゜Ll9 AAGAATACTCdingGCTAT
GC-GrGATCAGTA Chohachi TGCGTACC Old 0 5° GGCG^^GGCTTCCTG Chohachi GC-GT
TCGCTATCACAACAAC”.

Old 1 “CAAATCT Ding T Ding GAAGAC Haccho-
To CCA CAATAACCGTGAACAGATC” old 4
”GGTAAC^^ATGCGACTTG-GC
AGCGC(dACTGTTGA^TC3'old 5 5°
DingCGTCAGGCTCAGGGHacchoCT-GGCrC(
iTTCrrACGGAATrC3゜116 ”C
GTACATCGAAAACCTCTG-CTAAAAC
TCG Ding CAA GGCGT Ding 3゜ January 7 “GAA
GACGCT ding ding CT^CACC-TTGGTTCGT
GAAA CCGTCAGAC3゜1118 5°
EightheGCTGCGTAAGCdingTAAC-CCGCCG
GACGA＾TCTGGCCC3'5゜Ul9 AGGCTGCATGTCCTGCAAA
TG 3°, 15. JltF020 CG
TTCTGTCTTGATAGG 3゜5゜Table 1-2 5' 11 TCGACCTATCAAAGACAG
A-ACGCA TTGCAG” 5゜L2 GACATGCAGCCTGGGCCA-GA
TTCGTCCGGCGGGTT8AGCT”13
To TACGCGCTTGTGCTG/li, G 3゜5゛5゜1.4 GATTTCACGAACCAAG
GTG 3゜5゜shi5 TAGAAAGCGTCTTC＾8C
G-CCTTGACGAGTTT^GCAGAGGT
T3゜5゜L6 TCGATGT＾CGGAATTCCG-TA
AGAACGAGCCAGATCCTG3゜5゛[78GCCrGACGAGATTCAAC-AGT8CCGCTGCCAAGTCGC”[85°A-T
rG Ding TACCAACCAGAA-CCATCGGAA
CGTCArCAGAGTC”5゜L9 rTTAAcAcGdingrTG8TCT
G-Ding TACGGTATTG^TGG^Ding GTCTT
C3'L105°AAAAG^TTTGGTGTTGT
T-G＾TAGCGAAAACGCACAG3゜Lll
5°GAAGCCTTCGCCGGTACG-CA
DingATACrGATCACGCADingAG”.

L12 “CAGAGTATTCTTCTTGGCCT
G 3゜Ll3 ``CGGTATCAAGG8TG
TCC^-GC to AACAGGTCTCACCGrCG
AT3゜Ll4 ”AAC^^cc GCTTAC
GGTA-AGAGTCDingTCA＾■＾GTCGG3゜L15 5°GTCGTACTC8TCTACGAAG
3'116 ``TGGT Goose υsu G3゜shi1
1 5°AALΩ(dcAGcGcGβTIT 3'
118 ”LGCCTACACCAACAGCGCC
3゜L18°5°TGCCTACACCACCAGCG
CC''a J: Figures 6 and 7 show chromatograms of 38-mar obtained by the phosphoramidite method during purification by anion exchange chromatography.

Figure 8 shows the analysis results of this isolated 38-1iOr by anion exchange high performance liquid chromatography (
In both cases, the elution buffer is ammonium formate).

Synthesis of active C-ras gene segments 11 groups
+1 group III group U1~U4 tJ5~
U8 U9~(J12L15~L19, L12~L
14, Shi9-L11, rv group
V group IJ13~IJ 16 U 17~U2
Double-stranded DNA fragments were prepared for each group of 0-5 to L8 and L1-L4 using the oligonucleotides that should constitute each group as follows.

150 pmol of [γ-32P] ATP, 0.5 p of each oligonucleotide to constitute each of the aforementioned groups.
mol (excluding Ul and Ll) and 0.5 units of ■4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.)
10 μA of caination buffer [50 mH Tris-
HCl pH8,0/10a N +4 (IcI2/1
Mix 1 nM spermine and incubate 1 at 31°C.
time phosphorylated. 10 mH of ATP was added to the reaction mixture, and the reaction was further continued for 1 hour. It was further treated at 90°C for 3 minutes.

Phosphorylated oligonucleotide mixture (however,
Group and v81! For, after adding o and spmol of both UIJ3 and Ll oligonucleotides,
) in ligation buffer (66IIIH Tris-II
CI pl+ 7.8/ 6.6mHHoCl2)
Annealed by heating to 75° C. for 2 minutes in 150 μm,
It was then cooled to room temperature in a water bath for 1 hour. The following is the Proceedings of the National Academy of Sciences';1. (Pro, Natl, Acad,
Sci, USA), vol. 81, pp. 5956-5960 (
Ligation was performed according to the method described in (1984) (Reference 15), and the following fragment was first obtained.

C1a T BssllllI Ding CTACTCA Ding GCTG 5°GAAGACTCTTACCGTAAGCAGGG
GCTGATAACTTCTGAGAATGGC^TT
CGTCCrGAGAc5' cl T CACAATTT "GACGTTCCGATGGTTCTGGTTGCT
GAGACTACTGCAAGGCTACCAAGAC
CAACEco R1 GAAAGAT S' Hindll! at 1 These fragments were further subjected to ligation according to the method described in Reference 15 to obtain a C-ras gene segment represented by the following formula (1)-(a).

”CGAT A Glu 1 Net Trer Glu Tyr Lys
Leu Val Val Vat GlyAl
a Val Gly Vat Gly Ly
s Ser 8 la Leu Thr1ie
Gln Leu Ile Gln Asn His P
he Val^5pATT CAG TTG A
TCCAG AACCACTTCGT^ GATT8^ To TCAAACTAG GTCrTG GTG
^^G CA Ding CT^Glu Tyr Asp
Pro Thr Ile Glu Asp Ser
CCG ACT to TyrGAG T^CG8C
ATT GAA GAC CT TACCTCAT
G CTG GGC ding GA TAA CTT
CTG AGA ^rGArg Lys Gi
n Vat Val Ile Asp Gly G
lu ThrCGT 八＾G CAG GTT
GTT ATCGAC GGT GAG AC
CGC^ TTCGTCCAA CAA TAG
CTG CCA CTC Ding GGCys Leu
Leu Asp Ile Leu Asp
Tt+r Ala GlyTGT TTG CTG
GACATCCTT GAT ACCGCA
GGC^CA AACGACCTG TAG
GAA CTA TGG CGT CCGGin
Glu Glu Tyr Ser Ala Ne
t Arg Asp GlnC^^ GAA GAA
TACTCT GCT ATCCGT GAT
CAGGT ding crr crr ATG A
GA CGA TACGCA CTA GTC
clI Tyr Met Arg Thr Gly Glu
Gly Phe Leu CysVal P
he Ala Ile Asn Asn Thr
Lys Ser PheGTT TTCGCT
ATC8^CAACACC^hehe TCT TTT
C^^ AAG CGA TAG rTG T
TG TGG TTT ACA 8AAAGlu
Asp Ile His Gln Tyr Arg G
lu Gln IleLys Arg Val L
ys Asp Ser Asp ASD Vat P
r.

2O Net Val Leu Val Gly A
sn Lys Cys Asp LeuATG
GTT CTG GTT GGT AACAA
A TGCGACTTGTACCAA GACCA
A CCA TTG TTT ACG CTC
AACAla Ala Arg Thr Val
Glu Ser Arg Gln Ala4G Gln Asp Leu Ala Arg S
er Tyr Gly lie Pr.

CAG GAT CTG GCT CGT
TCT TCGGA ATT CCGGTCC
TA GACCGA GCA ACA ATCCG
T Ding AA GGCEco R1 Tyr Ile Glu Thr Ser Ala
Lys Thr Arg GlnTACATCGA
A ACCTCT GCT AA^ ACT
CGT CAAATG TAG C7 Ding GG
ACA CGA TTT TGA GCA
GT Ding Gly Val Glu Asp
Ala Phe Tyr Thr Leu
VatArg Glu Ile Arg Gl
n Hi, s Lys Leu Arg L
ysLau Asn Pro Pro 8sp
Glu Ser Gly Pro Gly
[11 Cys Met Ser Cys Lys Cy
s Val Leu Ser Term
m Ding AG G 3' ArCCAGCT Sat I Example 2 [Preparation of plasmid with active C-las synthetic gene segment! ! I] Escherichia coli carrying plasmid pGll-19 p
GH-19 (Feikoken Bibori No. 7606) was added to an agar medium (B
acto-Tryptonelg, Yeast ex
tract O, 5g, NaCl 10,5g.

j; IItlCO3e 0. After culturing on IQ with 1.5 g of agar and 31 LJ of ampicillin), collect the bacteria and add 5TET buffer (8% 5ucrose,
50 mHTris-+l (J(p) 18.0).

50mHEDTA, 5% rriton x 10
0).

Risoboom (10110/ 1al 0.25HTri
s-HCj (pH8,0)) Add 20μA? A
! After heating for 60 seconds in Ill water, 13.00Orp
Centrifuged for 10 minutes at m.

Add 0.5al of 1sopropanol to the supernatant and leave at -20°C for 30 minutes, 13. After centrifugation at OOOrpm for 10 minutes, the precipitate was dissolved in 50μ7 of TEI solution,
Sopropanol 8 (IμJ) was added, the mixture was left at -20°C for 30 minutes, centrifuged at 13,000 rpm for 10 minutes, and the precipitate was dried.

The precipitate was dissolved in 23.5 μJ of water, 6 μm of 8 uHcr and 0.5 ul of 10 u/μn were added and reacted at 37° C. for 60 minutes, followed by 58 NaCJ 1.
Add 2uO and 1.5μm of 5alI (8u/μI)
The reaction was continued for an additional 90 minutes at 37°C.

The obtained liquid was treated with phenol, and after ethanol precipitation, 5%
After electrophoresis using PAGE, about 4kbp of SatI
and a large double 1ID with a cleaved end of -C1a I
N^ was made of W4.

This large double-stranded DNA O,04p sol was converted into the synthetic C-las gene segment 0,6pi prepared in Example 1.
o l and 50 mW Tris-HCI (pH 7,4
)-10s HATP-5 units were mixed in 100 µL of *W solution containing T4 DNA ligase and incubated at 4°C for 12 hours.

Calcium-treated E. coli IIB 101 cells (5 x 108 cells) were added to 50μ of this ligation reaction mixture.
1, spread on an agar plate containing 50 μq of ampicillin, and incubated at 37° C. for 18 hours to obtain several dozen ampicillin-resistant cells. Of these, 12 independent clones were treated in the same manner as for E. coli/DGII-L9 to isolate plasmids, and the plasmids were
E mill R[, C1at ~ Hind III and "Ro r-
When each second-generation cleavage fragment of Sdan I was prepared and analyzed by polyacrylamide electrophoresis, the presence of the cleavage fragment expected from the C-ras synthetic gene of the present invention was confirmed in all of them. The present inventors converted this plasmid into pR
-12V and E. coli II B 101 waiting for this again
Stock [, Cori/HBIO1/pl? -12V
It was named.

In order to roughly confirm the DNA sequence of the C-ras synthetic gene of plasmid pR-12V,! , II limited enzyme” sealed! −[
co Itl and Eco Rr-8al [te] were each double digested, and each of the seven fragments was added to phage vector H1.
3I! 1D18/19 and cloned.
The ON^ sequence was determined using the standard procedure of the deoxy chain termination method (Reference 1γ), and this was determined by the above formula (1).
- It was confirmed that it has the sequence shown in (a).

Example 3 [Preparation of plasmid having normal type C-las synthetic gene] Ul, U2-1L17 and L18゛0.2 each
0f) 0.30D (A) were mixed and lyophilized. This oligonucleotide mixture was prepared in Example 1 [
In the same manner as in the case of [Synthesis of active C-ras gene segment], the 5' end of each oligonucleotide was phosphorylated by kination reaction, and further ligation was performed to obtain a dimeric fragment as shown below. A reaction solution was obtained.

ssHII TACACCACCAGCGCCAAACTACAACC
AGTTTATGTGGTGGTCGCGGTTGAT
GTTGGTCAAAGTATTCGGTCATATC
'GATATGACCGAATAACCA
G CCA G T A T A G C;! A T
A CT G G CT T A T G Kodan I AAAACTGGTTGTAGTTGGCGCTGGTG
GTGTATTTGACCAACATCAACCCGCG
ACCACCACATGGCAAAAGCGCGCTG
ACCATTCAGTTGATC5゜CCGTTTTC
GCGCGACTGGTAABss) III This dimeric fragment was digested with restriction enzyme fragment 1 to obtain the following fragment (fragment 1').

5°CGATATGACCGAATACAAAACTGG
TTGTAGTTATACTGGCTTATGTTTG
ACCAACATCAko1 TGGCGCTGGTGGTGTAGGCAAAAAGC
GCGCT1': ACCGCGACCACCACA
TCCGTTTTCGCGCGGAOikawa GACCATTCAGTTGATC3゜CTGCTAA On the other hand, obtained in Practical Example 1, E. coli / ) IB 101 /
pR-12V was transformed into L containing ampicillin 0 and 1 tags.
- cultured overnight at 37°C in 5 ml of broth;
It was added to 300 ml of culture medium and cultured with shaking at 37°C for 4 hours. 45 mg of chloramphenicol was added to this and cultured with shaking overnight. The bacteria were collected by centrifugation at 8,000 rpm for 10 minutes, and the cells were diluted with 50 mM Tris-11.
cI (pHs, o ), 1 mM EDTA, and 25% sicisugar in a buffer containing 50 μ m of ribonuclease A (10 mg/ml) and lysozyme (10
mg/ml) 1 ml was added, digested at 0°C for 5 minutes, and then 0.5 M EDTA (pif 8.0) 2
ml was added and kept at 0°C for 10 minutes.

50 sM Tris-HCI (pH (8)
,0), 82.5 iM EDTA and 0.1
%Tr! Add 16ml of buffer containing ton
Centrifuge for 40 minutes, and add 2.9 g of polyethylene glycol 8000 and 2.9 g of 5M NaCl to the supernatant (approximately 29 ml).
Add 9 ml and leave at 4°C overnight to give 1G, 0 (IOr
The precipitate was collected by centrifugation at pm for 30 minutes.

The collected precipitate was diluted with 1 mM Trls-tlcl (pH
7,5) and 1 aM EDTA, add 4.00 g of cesium chloride and 400 ul of ethidium bromide solution (4,87a + g/ml), and centrifuge at 40.00 rpm for 72 hours to isolate the ecDNA band. was collected, washed four times with isopropanol, and then dialyzed against water 21 for 4 hours. After performing ethanol precipitation twice from the dialyzed fluid, the precipitate was dissolved in water to obtain a plasmid solution. Yield: 4.500 (225μg/
200 μl).

Add 20μl of this solution to 6μl of ligation buffer, 1μl of water.
l and C1a, mixed with I solution (9 LJ/μl), 3
Digestion was performed at 7°C for 2 hours.

Add 8 μl of the above Hn solution (4 U/μm) to this.

1 μl of 1 M-HCl and 11 μl of bovine serum albumin solution (250 μg/ml) were added and the mixture was digested at 50° C. for 2 hours.

After phenol treatment, ethanol precipitation was repeated twice, and the precipitate was purified by 10% polyacrylamide gel electrophoresis to prepare a large double-stranded DNA with cleaved ends of C1a I and Bss IIB.

This large double-stranded DNA and the previously prepared fragment 1° were ligated in the same manner as in Example 1, and purified by 5% polyacrylamide gel electrophoresis.

This was digested with Bss III, purified by 5% polyacrylamide gel electrophoresis, and closed by ligation. The reaction solution was treated with phenol, precipitated twice with ethanol, dried, and dissolved in 40 Ll of water. Using this solution, E. coli HB 101 was transformed in the same manner as in Example 1, and screening was performed. Among the clones obtained, the rapid boiling method (r
apid boiling method) 5
Clones of interest were selected by detecting restriction enzyme fragments on a % polyacrylamide gel. One of these clones is E,
coli/IIB 101/pR-12G. E, coli/HB lot/I) R-12V (7
) A plasmid was isolated from this strain and its Cla I/Sat I fragment was prepared. This fragment was subjected to sequencing using the deoxy chain termination method described above, and it was confirmed that this fragment consisted of the following DNA sequence.

“CGAT TA Glu 1 Met Thr Glu Tyr Lys Leu V
al Val Vat GlyAla Gly Gly
Val Gly Lys Ser Ala Leu
Thr1ie Gin Leu lie Gin As
n Hls Phe Val AspGlu Tyr
Asp Pro Thr ie Glu
Asp Ser Tyr Arg Lys
Gin Val Val lle Asp
Gly Glu ThrCys Leu Leu
Asp Eye Leu Asp Thr Ala Gly
Gin Glu Glu Tyr Ser
Ala Met Arg Asp Gincl
I Tyr Met Arg Thr Gly
Glu Gly Phe Leu CysVa
l Phe Ala lie Asn As
n Thr Lys Ser PheGlu
Asp IIe) Its Gin Tyr Arg G
lu Gin lieLys Arg Vat
Lys Asp Ser Asp Asp
Val Pr.

AAA CGT GTT AAA CACTC
T CAT GACCTT C00TTT G
CA CAA TTT CTG AGA C
TA CTG CAA GGCMet Vat
Leu Vat Gly Asn Lys
Cys Asp LeuAla Ala
Arg Thr Val Glu Ser
Arg Gin AlaGin, Asp Le
u Ala Arg Ser Tyr Gl
y lie Pr.

Tyr lie Glu Thr Ser
Ala Lys Thr Arg GinTA
CATCGAA ACCTCT GCT AAA
ACT CGT CAAATG TAG
CTT TGG ACA CGA TTT
TGA GCA GTTGly Val Gl
u Asp Ala Phe Tyr Th
r Leu Va1Arg'GIu lie Ar
g Gln l1ls Lys Leu Arg Ly
sCOT GAA ATCCGT CAG C
ACAAG CTG CGT AAGGCA
CTT TAG GCA GTCGTG TT
CGACGCA TTC11nd Leu Asn Pro Pro Asp
Glu Ser Gly Pro GlyCT
T AACCCG CCG GACGAA T
CT GGCCCA GGCGAA TTG
GGCGGCCTG CTT AGA CCG
GGT CCGI1 Cys Met Ser Cys Lys
Cys Val Leu Ser Tertx
TGCATG TCCTGCAAA TGCGTT
CTCTCT TGAACG TACAGG
ACG TTT ACG CAA GACA
GA ACTTar TAG G 3' ATCCAGCT al I Example 4 [Expression of active C-ras synthesis gene and p21 ffl
Production of white matter] E. coli/HB 101/pR-12V in literature 1
with 0.2% casamino acids according to the method described in Section 5.
Grown in M9 medium containing 50 Mg/ml ampicillin. 40 μz/ml of 3-indoleacrylic acid was added to this culture solution at the beginning of the logarithmic growth phase. After this induction, bacterial cells were collected over time (each 0.06A
unit), 82.5 IIM Tris-11CI
(pit B, 8), 2% SDS, 5mM E
Bacteria were lysed in a saline solution containing DTA, 10% glycerin, 30IIM mercaptoethanol and o,ooi% bromophenol blue.

This sample was heated at 100° C. for 5 minutes and then analyzed according to the method of Remilli (Lacmm 11, Reference 1g).

The resulting migration pattern is shown in FIG. The symbol on the left side of the figure indicates the electrophoresis position of p21 protein, and the number on the right side indicates the Dalton value of the electrophoresis position. Further, the symbols and numbers in the upper row indicate the presence or absence of induction by 3-indoleacrylic acid for each lane and the elapsed time from addition to sampling. A dark protein band appears only when 3-indole acrylic acid is added between the markers Dalton at 31 and Dalton at 22, and its intensity reaches its maximum 20 hours after the start of induction. The size of this protein matches the value described in Reference 10. As a result of scanning with a densitometer, it was found that the amount was more than 10% of the total amount of bacterial protein.

Example 5 [Large-scale expression of active C-ras synthetic gene and purification of p21 protein] Using E. coli/JIB 101/pR-L2V, production of pal protein was performed essentially according to the method described in Reference 13. It was done in the same way. That is, this transformant was cultured in medium 21. Bacterial cells were collected 11 hours after the start of induction and treated with 50 mM Tris-11CI (pif
7.5) and buffer 100 containing 25% sucrose
Washed with ml.

The bacterial cells were treated with 5011M Tris-11cI (pH 7,
5), the cells were resuspended in a buffer containing 25% sucrose and 0.01% NP-40, and 100 μg/ml lysozyme was added and lysed at 0 °C for 15 minutes. The lysate was mixed with 0.5% NP-40, 5atM MgCt2 and I
It was transferred to a solution containing QOμz/ml deoxynuclease and kept on ice for 15 minutes. This liquid was heated to 15.00O
Clearate lysate was prepared by centrifugation at rpm for 10 minutes and 50 mM II IEP E S -Na0
11 (pH 7,5), 1 mM EDTA, I
DEAE-3cph diluted twice in buffer containing IIIM DTT and 0.01% n-octylgelcond.
accl column chromatography (2X 3B c
m) of 500 ml of the above dilution buffer and 0 to 0.6
Fractionation was performed using a linear gradient of NaCl up to M. Both fractions were analyzed by polyacrylamide electrophoresis, and the p21-enriched fraction was analyzed using 5ephadex.
G-75 column chromatography (2,6X
100 c+n). Approximately 15mg
(21A units) Glue 21 was obtained with a purity of 95% or more.

The amino acid sequence near the N-terminus of the p21 protein thus obtained was analyzed by gas phase sequencing. That is,
The p21 protein was analyzed using an automatic gas phase sequencer (model 470A).
, Applied Biosystems Co., Ltd.) using the reference 1
9 Subjected to Edman degradation according to the described procedure, the resulting amino acid residues were subjected to high performance liquid chromatography apparatus SP
8700 and an integrator 5P4270 (Spectro Physics). In this way, the N-terminal up to five amino acid residues are Met-Thr-Glu-Tyr.
-Lys was clearly determined. This matches the N-terminal sequence of p21 protein described in References 2 and 3.

Example 6 [Expression of normal C-ras synthesis gene] Example 4 (1) E, coIJ /IIB 101/
pR-12V l: Instead of E, coli/JIB lot
Example 4 was repeated using /pR-12G (Feikoken Bibori No. 8425) and substantially the same results were obtained.

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(1983) Science 2
22.771-778゜(2) Tabin et al.
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[Brief explanation of the drawing]

FIG. 1 is a diagram illustrating the preparation of the C-las synthetic genes of the present invention, and FIGS. 2 and 3 are diagrams illustrating the preparation of oligonucleotides used for the preparation of the C-las synthetic genes of the present invention. FIGS. 4 and 5 are diagrams explaining the preparation of plasmids containing the C-las synthetic genes of the present invention, and FIGS. 6 to 8 are diagrams illustrating the preparation of plasmids containing the C-las synthetic genes of the present invention. FIG. 9 is a diagram illustrating the purification and analysis of oligonucleotides used for preparation, and FIG. 9 is a diagram showing the electrophoretic pattern of p21 protein produced by the method of the present invention. .

Claims (31)

[Claims] (1) Restriction enzyme \Bs is applied to the part of the DNA sequence that encodes the c-ras protein and encodes serine, alanine, and leucine at positions 17 to 19 of the encoded amino acids.
s\HII position is arginine at position 68 to 70,
The restriction enzyme \Bcl\ I site was added to the DNA sequence encoding aspartic acid and glutamine at position 138.
Restriction enzyme￥Eco￥ in the DNA sequence encoding glycine, isoleucine and proline at positions 1 to 140
The R I site is inserted into the DNA sequence encoding lysine and leucine at positions 170 and 171 using a restriction enzyme.
c-ras synthetic gene segment with Hind\III site. (2) The DNA sequence represented by the formula, [There is a gene sequence] (in the formula, X represents G or T, and Y
represents a complementary base of X). (3) Claim 2 in which X is G and Y is C
The c-las synthetic gene segment described in Section 1. (4) Claim 2 in which X is T and Y is A
The c-las synthetic gene segment described in Section 1. (5) Place the restriction enzyme\Cla\I site on the upstream side, and the stop codon and subsequent restriction enzyme\Sal site on the downstream side.
The c-las synthetic gene segment according to any one of claims 1 to 4, which has a portion of \I. (6) On the upstream side, there is a DNA sequence expressed as [There is a DNA sequence], and on the downstream side, there is a DNA sequence expressed as [There is a DNA sequence] c- Las synthetic gene segment. (7) Restriction enzyme \Bs is applied to the DNA sequence that encodes the c-ras protein and encodes serine, alanine, and leucine at positions 17 to 19 of the encoded amino acids.
s\HII position is arginine at position 68 to 70,
The restriction enzyme \Bcl\I site was added to the DHA sequence encoding aspartic acid and glutamine at position 138.
Restriction enzyme￥Eco￥ in the DNA sequence encoding glycine, isoleucine and proline at positions 1 to 140
The R I site is inserted into the DNA sequence encoding lysine and leucine at positions 170 and 171 using a restriction enzyme.
A recombinant plasmid that contains a c-ras synthetic gene segment with a Hind\III site as a structural gene and is capable of self-replication in microorganisms. (8) The DNA sequence represented by the formula, [There is a gene sequence] (in the formula, X represents G or T, and Y
represents a complementary base of X) as a structural gene.
Recombinant plasmids as described in Section. (9) The recombinant plasmid according to claim 8, wherein X is G and Y is C in the c-ras synthetic gene segment structural gene portion, and is capable of producing normal c-ras protein in microorganisms. . (10) The recombinant plasmid according to claim 8, wherein X is T and Y is A in the c-ras synthetic gene segment structural gene portion, and is capable of producing a mutant c-ras protein in a microorganism. . (11) c-ras synthetic gene segment structure The gene part is a DNA sequence represented by other DNA sequence parts, and on the upstream side, there is a DNA sequence, and on the downstream side, there is a DNA sequence. Claim 7 continues with the DNA sequence represented.
Recombinant plasmid according to items 1 to 10. (12) The recombinant plasmid according to any one of claims 7 to 11, which has a bacterial tryptophan promoter/operator upstream of the c-ras synthetic gene segment structural gene. (13) A DNA sequence capable of expressing ampicillin resistance and a recombinant plasmid were placed between the upstream end of the bacterial tryptophan promoter operator and the downstream end of the c-ras synthetic gene segment structural gene, and the recombinant plasmid was placed in Escherichia coli. 13. The recombinant plasmid according to claim 12, which comprises a DNA sequence that enables self-propagation. (14) Claim 12, wherein the base sequence from the messenger RNA transcription start point of the bacterial tryptophan promoter operator to the c-las synthetic gene segment structural gene is a DNA sequence represented by [There is a DNA sequence] Recombinant plasmid according to Section 1 or Section 13. (15) As a structural gene, a restriction enzyme\Bss\HII site is inserted into the DNA sequence that encodes c-ras protein and encodes serine, alanine, and leucine at positions 17 to 19 of the encoded amino acids. Restriction enzyme￥Bcl￥ in the DNA sequence encoding arginine, aspartic acid and glutamine at positions 68 to 70
The I site is inserted into the DNA sequence encoding glycine, isoleucine, and proline at positions 138 to 140, and the restriction enzyme\Eco\R I site is inserted into the DNA sequence encoding lysine and leucine at positions 170 and 171. c- which has restriction enzyme \Hind\III site in the part
ras synthetic gene segment as a structural gene,
A microorganism that has a recombinant plasmid in its cells that can self-replicate. (16) The c-las synthetic gene segment of the structural gene has the formula, [There is a DNA sequence] (in the formula, X represents G or T, and Y
represents a complementary base of X). (17) Claim 16, which has a recombinant plasmid in which X is G and Y is C in the c-ras synthetic gene segment structural gene portion, and is capable of producing normal c-ras protein. Microorganisms mentioned. (18) Claim 16, which has a recombinant plasmid in which X is T and Y is A in the c-ras synthetic gene segment structural gene portion, and is capable of producing a mutant c-ras protein. Microorganisms mentioned. (19) c-ras synthetic gene segment structure The gene part is a DNA sequence represented by other DNA sequence parts, and on the upstream side, there is a DNA sequence, and on the downstream side, there is a DNA sequence. 19. A microorganism according to any one of claims 16 to 18, which is E. coli carrying a recombinant plasmid followed by the DNA sequence represented. (20) A recombinant plasmid having a tryptophan promoter and operator derived from bacteria upstream of the c-ras synthetic gene segment structural gene, according to any one of claims 16 to 19. microorganisms. (21) A DNA sequence capable of expressing ampicillin resistance and self-propagation in E. coli is present between the upstream end of the bacterial tryptophan promoter operator and the downstream end of the c-las synthetic gene segment structural gene. The microorganism according to any one of claims 16 to 20, which has a recombinant plasmid containing a DNA sequence. (22) Contains a recombinant plasmid whose nucleotide sequence from the bacterial tryptophan promoter operator messenger RNA transcription start point to the c-las synthetic gene segment structural gene portion is a DNA sequence expressed as [DNA sequence exists] The microorganism according to claim 20 or 21, which is a microorganism. (23) As a structural gene, a restriction enzyme\Bss\HII site is inserted into the DNA sequence that encodes c-ras protein and encodes serine, alanine, and leucine at positions 17 to 19 of the encoded amino acids. Restriction enzyme￥Bcl￥ in the DNA sequence encoding arginine, aspartic acid and glutamine at positions 68 to 70
Place the I site on the restriction enzyme \EcoR\I site on the part of the DNA sequence that encodes glycine, isoleucine, and proline at positions 138 to 140. Place the I site on the part of the DNA sequence that encodes lysine and leucine at positions 170 and 171. c- which has restriction enzyme ￥ Hind ￥ III site in
A transformed microorganism harboring in its cells a recombinant plasmid containing a c-ras synthetic gene segment and capable of self-replication and expression of the c-ras synthetic gene segment structural gene in the microorganism. 1. A method for producing c-ras protein, which comprises culturing in a medium to accumulate c-ras protein in bacterial cells, and collecting it. (24) The c-las synthetic gene segment of the structural gene has the formula, [There is a DNA sequence] (in the formula, X represents G or T, and Y
24. The production method according to claim 23, which uses a microorganism that contains in its cells a recombinant plasmid in which ``x'' represents a complementary base of X. (25) Claim 23 or 24 in which a normal c-ras protein is produced using a microorganism having a recombinant plasmid in which X is G and Y is C in the c-ras synthetic gene segment structural gene portion. Manufacturing method described in section. (26) A mutant c-las protein is produced using a microorganism having a recombinant plasmid in which X is T and Y is A in the c-ras synthetic gene segment structural gene portion Claim 23 or 24 Manufacturing method described in section. (27) c-ras synthetic gene segment structure The gene part is a DNA sequence expressed by other DNA sequence parts, and on the upstream side, there is a DNA sequence, and on the downstream side, there is a DNA sequence. 27. The production method according to any one of claims 23 to 26, which uses Escherichia coli transformed with a recombinant plasmid following the indicated DNA sequence. (28) Claims 23 to 2 use Escherichia coli transformed with a recombinant plasmid having a bacterial tryptophan promoter/operator upstream of the c-ras synthetic gene segment structural gene.
The manufacturing method described in any of Section 7. (29) A DNA sequence capable of expressing ampicillin resistance and self-propagation in E. coli is present between the upstream end of the bacterial tryptophan promoter operator and the downstream end of the c-ras synthetic gene segment structural gene. 29. The production method according to claim 28, which uses Escherichia coli transformed with a recombinant plasmid containing the DNA sequence. (30) Transformation with a recombinant plasmid in which the base sequence from the bacterial tryptophan promoter operator messenger RNA transcription start point to the c-ras synthetic gene segment structural gene portion is a DNA sequence represented by [DNA sequence is available] Claim 28, which uses transformed E. coli.
29. The manufacturing method according to item 29. (31) The production method according to claims 23 to 30, wherein indole acrylic acid is added during the culture.