CH670654A5 - - Google Patents

Description

DESCRIPTION The invention relates to a new ß-urogastron gene, corresponding recombinant plasmids, corresponding trans-65 formants and their production and the production of ß-urogastron.

ß-Urogastron is a polypeptide hormone that is synthesized in the salivary glands of humans, etc. (cf.

5

670 654

for example, Heitz et al., Gut, 19, 408-413 (1978)). It has a primary structure comprising 53 amino acids in the following sequence (cf. Gregory et al., Int. J. Peptide Protein Res., 9, 1997-118 (1977)).

Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gin Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg

In the description below, amino acids are represented by the following symbols:

Ser: Serine

Glu: glutamic acid

Pro: proline

His: histidine

Tyr: Tyrosine Met: Methionine

Ala: Alanine

Gin: glutamine

Trp: tryptophan

Asn: asparagine

Asp: aspartic acid

Cys: cysteine

Leu: Leucine

Gly: glycine

Val: valine

Ile: isoleucine

Lys: lysine

Arg: arginine

Phe: phenylalanine

ß-Urogastron has physiological activities such as suppressing gastric acid secretion and promoting cell growth (see Eider et al., Gut, 16, 887-893 (1975)), and is therefore useful in the treatment of ulcers and wounds useful.

Since ß-urogastron is excreted in small amounts in human urine, this compound is currently produced by extraction from urine, separation and purification. However, this method has a problem that large amounts of the compound cannot be easily obtained because the compound is a minor component of human urine.

On the other hand, published European patent application No. 0 046 039 describes an attempt to manufacture β-urogastron by means of a genetic engineering process using a synthetic β-urogastron gene. While the above publication describes a gene with a specific nucleotide sequence, it does not teach whether there are other genes capable of expressing ß-urogastron by a similar method, nor does it mention such a gene with one special nucleotide sequence.

A large number of nucleotide sequences can code for the amino acid sequence of β-urogastron. Nonetheless, it is difficult to predict which of these genes will be able to express β-urogastron by a genetic engineering process, or which gene is most suitable for use in genetic engineering processes. 15 Therefore, many experiments and inventive efforts are required to determine the most suitable nucleotide sequence.

The object of the invention is to create a new ß-urogastron gene which is completely different in nucleotide sequence from the gene described in 20 of the aforementioned publication and is capable of expressing ß-urogastron by a genetic engineering method, furthermore to create a gene , which is suitable for expressing ß-urogastron by genetic engineering methods, as well as to create new recombinant plasmids and transformants corresponding to the new ß-25 urogastron gene, and finally to create a process which enables the production of ß-urogastron in considerable quantities and made possible with a high degree of purity using the new gene according to a genetic engineering method.

These and other objects of the invention will appear from the following description.

The inventors have carried out many experiments and found that a gene I with the subsequent nucleotide sequence achieves the object of the invention.

Gen I:

A

A

T

A

G

C.

G

A

T

T

C.

T

G

A

G

T

G

C.

C.

C.

A

C.

T

G

T

T

A

T

C.

G

C.

T

A

A

G

A

C.

T

C.

A

C.

G

G

G

T

G

A

C.

T

C.

T

C.

A

C.

G

A

T

G

G

C.

T

A

T

T

G

T

C.

T

G

C.

A

C.

A

G

A

G

T

G

C.

T

A

C.

C.

G

A

T

A

A

C.

A

G

A

C.

G

T

G

G

A

C.

G

G

T

G

T

T

T

G

C.

A

T

G

T

A

C.

A

T

C.

G

A

A

C.

T

G

C.

C.

A

C.

A

A

A

C.

G

T

A

C.

A

T

G

T

A

G

C.

T

T

G

C.

T

T

T

G

G

A

T

A

A

A

T

A

C.

G

C.

G

T

G

T

A

A-

C.

C.

G

A

A

A

C.

C.

T

A

T

T

T

A

T

G

C.

G

C.

A

C.

A

T

T

G

T

G

T

G

T

A

. G

T

G

G

G

T

T

A

T

A

T

C.

G

G

T

G

A

A

A

C.

A

C.

A

T

C.

A

C.

C.

C.

A

A

T

A

T

A

G

C.

C.

A

C.

T

T

C.

G

C.

T

G

T

C.

A

A

T

A

C.

C.

G

T

G

A

T

C.

T

G

A

A

A

G

C.

G

A

C.

A

G

T

T

A

T

G

G

C.

A

C.

T

A

G

A

C.

T

T

T

T

G

G

T

G

G

G

A

A

T

T

G

C.

G

T

—R 3

O"

A

C.

C.

A

C.

C.

C.

T

T

T

T

C.

G

C.

A

5?

The letters stand for the purine and pyrimidine bases that form the nucleotide sequence. The symbols used here for the bases mean the following: A is adenine, G is guanine, C is cytosine and T is thymine.

65 Gen I is completely new and not in itself obvious. It is obtained by determining the specified nucleotide sequence from a very large number of possible nucleotide sequences.

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Gen I has the following characteristics:

(1) ß-Urogastron can be expressed in a very advantageous manner by genetic engineering methods.

(2) The trinucleotide codons that make up gene I are all acceptable to host cells, particularly Escherichia Coli, which is easily and safely available, ensuring a high level of expression.

(3) Specific restriction enzyme recognition sites can be provided within the gene and at both ends thereof, and the recognition sites can be manipulated as desired to facilitate coupling with other genes and insertion into the plasmid vector.

(4) To produce the gene I, its forming oligonucleotides can be coupled into blocks and the blocks into subunits easily, as desired and essentially free of undesired couplings.

(5) When ß-urogastron is expressed as a fused protein, means are available with which an unnecessary part can be easily removed to give the desired ß-urogastron.

6

If β-urogastron is actually to be expressed using gene I, restriction enzyme recognition sites can be provided at the front and / or rear end of the gene to couple with the promoter required for expression, Shine-Dalgarno sequence (hereinafter, occasionally referred to as “SD sequence”), vector, etc. In addition, if necessary, a start codon and / or a stop codon or up and down from the gene can be provided. The choice of recognition sites, start codon and stop codon is not particularly restricted, the desired types can be used will.

An example of a gene having an expanded sequence (hereinafter referred to as "Gen II") is given below. This gene includes a restriction enzyme recognition site and a start codon located upstream from gene I, as well as a stop codon and a restriction enzyme recognition site located downward from gene I, the recognition sites and the Co-20 dons in are arranged in the order given and the gene II also comprises other restriction enzyme recognition sites.

Gen II:

■ 15

! A A T

Ti C S ~ G C

A A [G_ TTC

Ta

A T C

DAY

T G A C

Bg (S)

■ 1,

C G

Start codon

A T G] A A T A G C T A | C T T A T C G

Mb

10th

Gj A T C T A

T C T AGA

H-f

GAG C T C

20 T G C A C G

C C A G G T

CTG G A C

30 T C T AGA

C A C G T G

40

GAT C T A

G G C C C G

TAT A- T A

T G T A C A

CTG GAG

50 C A C G T G

G A C CTG

G G T C C A

60 70 - 80

GTT T G C A T G TAC A T [_C G AI A G C T T T G

C A A A C G TAC A T G T AG El T T C G A A A C

Ta

Hd

90

GAT A A A T A] C G C G C T A T T T A T G CGC

100

T G T A A C T G T G T A ACA T T G ACA CAT

Ml (Th)

7

670 654

G T 8 C A C

110 G G T C C A

TAT ATA

A T C DAY

12 G G T C C A

G A A GTT

CGC G C G

T G T ACA

130 C A A GTT

TAC A T G

140

CGTjGAT CTG A A A G C A C T A GTA C T T T

150 T G G ACC

T G G ACC

160

S

Stop codon

170

B | A A T T G C G T T A A DAY T G A AIG A T C T

C T T A A C G C A ATT A T C ACT T C T AGA

1

Bg (S)

G

C C T AG

Ba

The symbols which represent the restriction enzymes in the above sequence have the following meaning:

E: EcoRI Bg: Bglll Mb MboII Ba: BamHI Ml: Mlul

Ta: Taql S: Sau3AI Hf: Hinfl Hd: Hindlll Th: Thal

The present invention relates to the ß-urogastron gene according to claim 1 and, according to the dependent claims, genes and gene subunits derived from the ß-urogastron gene according to the invention, recombinant plasmids containing a gene according to the invention, a transformant having a recombinant plasmid, methods for the production of a recombinant plasmid and a transformant, and a method for the production of ß-urogastron.

In the synthetic production of the gene I or II, it is advantageous to construct the gene I or II as a gene divided into an anterior and a posterior half. For example, it is possible to make one subunit that has the front half of the nucleotide sequence of gene I or II, and make another subunit that has the back half of the nucleotide sequence thereof because it is roughly divided in the middle, and these two To couple subunits of the gene I or II to each other to form gene I or II. The subunit which has the front half of the nucleotide sequence of gene II can also have a restriction enzyme recognition site at its rear end, and another subunit which has the rear half of the nucleotide sequence of gene II can also have a restriction enzyme. Have recognition site at their front end, and these two sub-40 units are coupled together to form Gen II.

Specifically, for purposes of illustration, the former subunit may be a subunit A, which comprises the front half of the gene II and has a restriction enzyme (BamHI) recognition site at its rear end, and the latter subunit a subunit B. , which comprises the rear half of the gene II and is provided with a restriction enzyme (HindIII) recognition site at its front end. These subunits are given below:

50

Subunit A:

A

A

T

T

C.

G

A

A

G

A

T

C.

T

G

C.

A

T

G

A

A

T

A

G

C.

G

C.

T

T

C.

T

A

G

A

C.

G

T

A

C.

T

T

A

T

C.

G

G

A

T

T

C.

T

G

A

G

T

G

C.

C.

C.

A

C.

T

G

t

C.

....

r

A

C.

C.

T

A

A

G

A

C.

T

C.

A

C.

G

G

G

T

G

A

C.

A

G

A

G

T

G

G

A

T

G

G

C.

T

A

T

T

G

T

C.

T

G

C.

A

C.

G

A

C.

G

G

T

C.

T

A

C.

C.

G

A

T

A

A

C.

A

G

A

C.

G

T

G

C.

T

G

C.

C.

A

G

T

T

T

G

C.

A

T

G

T

A

C.

A

T

C.

G

A

A

G

C.

T

T

C.

G

C.

A

A

A

C.

G

T

A

C.

A

T

G

T

A

G

C.

T

T

C.

G

A

A

G

C.

C T A

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Subunit B:

8th

5 *

A

G

C.

T

T

T

G

, ^ i '

A

A

C.

A

A

A

T

A

C.

G

C.

G

T

G

T

A

A

C.

T

G

T

G

T

A

T

T

T

A

T

G

C.

G

C.

A

C.

A

T

T

G

A

C.

A

C.

A

T

G

G

T

T

A

T

A

T

C.

G

G

T

G

A

A

C.

G

C.

T

G

T

C.

C.

A

A

T

A

T

A

G

C.

C.

A

C.

T

T

G

C.

G

A

C.

A

T

A

C.

C.

G

T

G

A

T

C.

T

G

A

A

A

T

G

G

T

G

G

A

T

G

G

C.

A

C.

T

A

G

A

C.

T

T

T

A

C.

C.

A

C.

C.

T

T

B

C.

G

T

T

A

A

T

A

G

T

G

A

A

G

A

T

C.

T

A

A

C.

G

C.

A

A

T

T

A

T

C.

A

C.

T

T

C.

T

A

G

A

A G

G

A

T

C.

T

A

G

T

G

C.

A

C.

C.

A

A

G

T

T

G

A

A

C.

T

T

G

C.

C.

T

The subunits A and B are synthesized as follows, for example. Oligonucleotides with 11, 13 or 15 bases are synthesized (A-1 to A-16 and B-1 to B-16, i.e. 32 oligonucleotides). Then 4 to 6 of these oligonucleotides

Block 1:

kleotide assembled and coupled into blocks (block 25 1 to block 7, i.e. 7 blocks). These oligonucleotides and blocks are given below.

(A-l) (A-2) (A-3)

5 'AATTCGAAGAT CTGCATGAATAGC GATTCTGAGTG 3 "

3 'GCTTCTAGACGTA CTTATCGCTAA GACTCACGGGTGA 5'

(A-16) (A-l5) (A-14)

Block 2;

(A-4) (A-5) (A-6)

5 'CCCACTGTCTCAC GATGGCTATTG TCTGCACGACGGT 3'

3 'CAGAGTGCTAC CGATAACAGACGT GCTGCCACAAA 5'

(A-13) (A-12) (A-ll)

Block 3:

(A-7) (A-8)

51 GTTTGCATGTA CATCGAAGCTTCG 31

3 * CGTACATGTAGCT TCGAAGCCTAG 5 '

(A-10) (A-9)

Block 4:

(B-l) (B-2)

5 'AGCTTTGGATA AATACGCGTGTAACT 3'

3 'AACCTATTTATGC GCACATTGACACA 5'

(B-16) (B-15)

Block 5:

(B-3) (B-4)

5 'GTGTAGTGGGT TATATCGGTGAACGC. 3 '3' TCACCCAATATAG CCACTTGCGACAG 5 '

(B-14) (B-13)

9

670 654

Block 6:

(B-5) (B-6)

5 'TGTCAATACCG TGATCTGAAATGGTG 3'

3 'TTATGGCACTAGA CTTTACCACCCTT 5'

(B-12) (B-ll)

Block 7:

(B-7) (B-8)

5 'GGAATTGCGTT AATAGTGAAGATCTG 3

3 'AACGCAATTATCA CTTCTAGACCTAG 5

(B-10) (B-9)

Blocks 1 to 3 are then coupled together to form subunit A and blocks 4 to 7 are coupled together to form subunit B.

The present invention will now be described in detail with reference to the accompanying drawings and photographs.

Fig. 1 shows schematically the synthesis of an oligonucleotide by the solid phase method;

Figure 2 shows a method of coupling oligonucleotides A-1 through A-16 to subunit A and inserting the subunit into a plasmid pBR322 derived from Escherichia Coli to give a recombinant plasmid pUGl;

Figure 3 shows a similar method for producing a recombinant plasmid pUG2 by inserting a subunit B into a plasmid pBP322;

4 shows a method for the production of a recombinant plasmid pUG3 from pUG1 and pUG2;

5 shows the result obtained by analyzing the nucleotide sequence of oligonucleotide A-3 with the aid of two-dimensional fractionation by electrophoresis and homochromatography;

Fig. 6 shows a process for the production of a recombinant plasmid pGH37;

Fig. 7 shows a process for the production of a recombinant plasmid pGH35;

Fig. 8 shows a process for the production of a recombinant plasmid pEK28;

FIG. 9 shows methods for the production of the recombinant plasmids pUG102 to pUG122 and the recombinant plasmids pUG103-E to pUGl 17-E;

Fig. 10 shows methods for producing the recombinant plasmids pBRH02 and pBRH03;

Figure 11 shows a MboII restriction map of pUG3 comprising an H fragment (179 base pairs) containing the present β-urogastron gene;

Fig. 12 shows a process for the production of the recombinant plasmids pUG2301 and pUG2303;

Fig. 13 shows a process for the production of the recombinant plasmids pUG2101 and pUG2105;

Fig. 14 shows a process for the production of the recombinant plasmids pUG2701 and pUG2703;

15 shows a method for the production of the recombinant plasmids pUGl 102 and pUGl 105;

Fig. 16 shows a process for the production of the recombinant plasmid pUG1004;

Fig. 17 shows a process for the production of the recombinant plasmid pUG1201;

Fig. 18 shows a process for the production of the recombinant plasmid pUG1301;

Photographs 1 to 5 each show analysis results for nucleotide sequences of

xam-Gilbert method obtained recombinant plasmids.

The procedures for forming gene II according to the invention are known per se. The oligonucleotides for the formation of the gene II can be produced by known methods, for example by the solid phase method described briefly below (see, for example, H. Ito et al., Nucleic Acids Research, 10.1755-1769 (1982)).

When the solid phase method is used, the oligonucleotide is synthesized as shown in Fig. 1 by sequentially coupling mononucleotides or dinucleotides with a nucleoside attached to a polystyrene resin to give the predetermined nucleotide sequence.

For example, the resin on which the nucleoside is attached can be made using a partially cross-linked polystyrene resin by reacting N- (chloromethyl) phthalimide with the resin and the product with hydrazine to give aminomethylated polystyrene resin. and thereafter a nucleoside is coupled with its amino group, its 5'-hydroxyl group is free and its amino group is protected, using succinic acid as an interval former.

On the other hand, various methods are known for producing mononucleotides or dinucleotides (see, for example, C. Broka et al., Nucleic Acids Research, 8, 5461-5471 (1980)). For example, a mononucleotide can be produced by reacting o-chlorophenylphosphorodichloridate, triazole and a nucleoside whose 5'-hydroxyl group is protected with a dimethoxytrityl group (DMTr) in the presence of triethylamine, then the monotriazolide obtained with β-cyanoethanol in the presence of 1-methylimidazole is reacted as a catalyst and the reaction product is eluted in a silica gel column chromatography with chloroform-methanol. This procedure provides a fully protected mononucleotide.

A dinucleotide can be prepared by treating the fully protected mononucleotide obtained above with benzenesulfonic acid or a similar acid to give the mononucleotide with a free 5'-hydroxyl group, reacting it with the above-mentioned monotriazolide and reacting the reaction product in a silica gel column chromatography is eluted with chloroform-methanol. This procedure provides a fully protected dinucleotide.

The solid phase synthesis of oligonucleotides is advantageously carried out by using an agent for DNA synthesis, for example the agent for DNA synthesis available from Bachem Inc. in the USA. The resin on which the nucleoside is attached is placed in a reaction vessel and mixed with dichloromethane

25th

30th

35

40

45

50

55

60

670 654

Washed isopropanol, and a solution of zinc bromide in dichloromethane-isopropanol is added to the resin to cleave the dimethoxytrityl group at the 5 'position. This procedure is repeated several times until the color of the solution disappears. The resin is washed with dichloromethane-isopropanol and then with a solution of triethylammonium acetate in dimethylformamide to remove remaining Zn2 +, then washed with tetrahydrofuran and exposed to a stream of nitrogen gas to dry for a few minutes. Regardless, the fully protected mononucleotide or dinucleotide is dissolved in pyridine, whereupon triethylamine is added, and the resulting solution is shaken, then left to stand at room temperature for a few hours and then evaporated under reduced pressure. The resulting triethylammonium salt is dissolved in pyridine and azeotroped to dryness several times using pyridine.

The salt of the nucleotide is dissolved in a solution of mesitylenesulfonyl-5-nitrotriazole (MSNT, a condensation reagent) in pyridine. The resulting solution is added to the dried resin and left to stand at room temperature. The liquid portion of the reaction mixture is removed and the resin portion is washed with pyridine and then reacted with acetic anhydride to mask the unreacted hydroxyl groups in tetrahydrofuran pyridine using dimethyl aminopyridine as a catalyst. Finally, the resin is washed with pyridine, which completes a cycle of solid-phase synthesis. One cycle extends the nucleotide sequence by 1 or 2 chain lengths. The above procedure is repeated to successively couple mononucleotides or dinucleotides with the resin to the desired length, whereby a fully protected oligonucleotide attached to the resin can be obtained.

A solution of tetramethylguanidine-2-pyridinaldoximate in pyridine-water is added to the resulting resin, and the mixture is allowed to stand with heating. _ The resin is then filtered off and washed alternately with pyridine and ethanol. The washing liquid and the filtrate are combined and concentrated under reduced pressure. The concentrate is dissolved in an aqueous solution of triethylammonium bicarbonate (TEAB) and then washed with ether. The aqueous solution is subjected to column chromatography with Sephadex G-50 using triethylammonium bicarbonate (TEAB) as the eluent. The fractions are aggregated. melt and the optical density of each fraction is measured at 260 nm. A fraction is concentrated which contains the first elution peak. The concentrate is purified, for example by high speed liquid chromatography, until a single tip is obtained. In the oligonucleotide thus obtained, the 5 'end is still protected by a dimethoxytrityl group, so that the product is treated with an aqueous solution of acetic acid to remove the protecting group, and then again purified by high-speed liquid chromatography or the like until a single tip is obtained.

The desired oligonucleotides are produced by the method described above, then individually examined for the presence of the nucleotide sequence by a two-dimensional fractionation method using electrophoresis and homochromatography and / or by the Maxam-Gilbert method, and then used to produce blocks and subunits .

The two-dimensional fractionation method for examining the nucleotide sequence can be carried out according to the procedure of Wu et al. (E. Jay, R. A. Bambara, R. Pad-manabhan and R. Wu, Nucleic Acids Res., 1,331 (1974)).

In order to use this method, the lyophilized oligonucleotide is dissolved in distilled water to a concentration of approximately 0.1% / year 1. Part of this solution is treated with y-32P-ATP and Xt polynucleotide kinase to label the 5 'end with 32P and then partially digested with snake venom phosphodiesterase. The product is spotted on a sheet of cellulose acetate and subjected to electrophoresis for the first dimensional development in order to separate the product according to the different bases. The developed products are transferred to a plate of diethylaminoethyl cellulose (DEAE cellulose) and subjected to the second dimensional development using a solution of partially hydrolyzed RNA called "homomixing" (this procedure is referred to as "homochromatography"). In this way, the oligonucleotide is separated according to the chain length. The nucleotide sequence of the oligonucleotide is then read autoradiographically starting from the 5 'end.

If the sequence is difficult to examine by this method, the Maxam-Gilbert method is used as needed. (A.M. Maxam and W. Gilbert,

Proc. Nati. Acad. Sei., USA. 74, 560 (1977); A.M. Maxam and W. Gilbert, Methods in Enzymol., Vol. 65, p. 499, Academic Press 1980).

This process, also called chemical decomposition process, uses a reaction specific for a particular base to cleave the oligonucleotide at the site of the base, and the bands developed by electrophoresis are used to sequence from 5 ' - or to read 3 'end. The base specific reactions are as follows. Guanine is specifically methylated by dimethyl sulfate. Guanine and adenine undergo a depurination reaction in the presence of an acid. Thymine and cytosine both react with hydrazine at a low salt concentration, but at a high salt concentration only cytosine reacts with hydrazine. After the completion of the reactions for the four bases, each reaction mixture is reacted with piperidine in order to split off the bases opened in the ring and to catalyze the β-elimination of both phosphates from the sugar, and finally the DNA chain is cleaved at the site of this base . The resulting reaction mixtures are each subjected to polyacrylamide gel electrophoresis to confirm the nucleotide sequence, depending on which of the reactions each band had generated.

The oligonucleotides are then coupled using a T4 DNA ligase, as shown in FIG. 2. To achieve correct coupling, the 16 oligonucleotides A-1 to A-16 corresponding to subunit A are divided into three groups and coupled to one another, i. H. Block 1 comprising Al, A-2, A-3, A-14, A-15 and A-16, Block 2 comprising A-4, A-5, A-6, Al 1, A-12 and A-13 , and block 3 comprising A-7, A-8, A-9 and A-10 as shown in FIG. 2. Blocks 1 to 3, which have the correct sequences, are obtained by electrophoresis and are then coupled together to form subunit A.

In more detail, some of the 5 'ends of the 16 oligonucleotides A-1 through A-16 are labeled with 32P using y-32P-ATP and T4 polynucleotide kinase and the hydroxyl groups of the remaining 5' ends are phosphorylated with ATP. To form each of the three

10th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

11

670 654

Blocks are joined and coupled to the oligonucleotides using T4 DNA ligase and the product is subjected to polyacrylamide gel electrophoresis to isolate the desired block. The three blocks thus obtained are coupled using T4 DNA ligase to give subunit A. Although a dimer structure can be produced in the coupling reaction, it is easily cleaved with the restriction enzymes EcoRI and BamHI to give subunit A. 2, a known plasmid vector, pBR322, which is readily available from Escherichia Coli, is cleaved with EcoRI and BamHI, and subunit A is inserted into the vector to give a recombinant plasmid pUGl.

The same procedure as above is also followed with respect to subunit B. As in the case of subunit A, the 16 oligonucleotides B-1 to B-16 are divided into four groups and coupled together, as shown in Fig. 3, and the blocks are coupled together to give subunit B. The dimer which may be produced is cleaved with the restriction enzymes HindIII and BamHI to give subunit B. A plasmid vector, pBR322, is digested with HindIII and BamHI and subunit B is inserted into the vector to give a recombinant plasmid pUG2, as shown in FIG. 3.

4, pUGl is cleaved with restriction enzymes Hindlll and Sali and a fragment removed from pUG2 using the same restriction enzymes is inserted into pUGl to produce a recombinant plasmid pUG3 which has a structural ß-urogastron gene (gene II) .

pUGl, pUG2 and pUG3 are recombinant plasmids, each of which comprises pBR322 and subunit A, which is the front half of the structural ß-urogastron gene, and subunit B, which is the rear half of the gene, that is, the entire structural gene . These recombinant plasmids can be multiplied in large quantities, namely by introduction into a host such as the HB 101 strain from Escherichia Coli, which is known and is readily available as a transformation method according to the calcium method (E. Lederberg and S. Cohen, J Bacteriol., 119, 1072 (1975)).

Whether pUGl, pUG2 and pUG3 are present in the host, for example in the HB 101 strain of Escherichia Coli,

can be examined using the following methods. After the plasmids have been collected by an alkaline extraction method, pUG1 and pUG2 are examined for the presence of the BglH recognition site which is not present on the vector pBR322. In the same way, pUG2 and pUG3 are examined to determine whether they can be cleaved with Mlul, which is not present on pBR322.

After the alkaline extraction procedure, Escherichia coli harboring the plasmid are incubated, the cells are then collected and exposed to the action of lysozyme to dissolve the cell wall. A mixture of sodium hydroxide and sodium lauryl sulfate is used to disassemble the cell and then denature the DNA, after which it is neutralized with sodium acetate buffer. At this point, the chromosomal DNA remains denatured, but the plasmid, which consists of extra-chromosomal DNA, again forms the initial double-chain form. Plasmids are collected using these properties. Thereafter, to purify the plasmids, they are subjected to density gradient ultracentrifugation with cesium chloride and ethidium bromide and then passed through a 50 m column of Biogel A to

To remove RNA. Thus plasmids can be obtained in large quantities with high purity. The β-urogastron gene (gene II) according to the invention can be obtained in this way.

The method for introducing the β-urogastron gene into the host cells will now be described.

The host cells to be used in the present invention are not particularly limited, and any of the known host cells can be used, for example, those of Escherichia Coli, Bacillus, Pseudomonas, yeasts, etc., among which the cells Escherichia Coli are preferred.

Expression of the ß-urogastron gene using Escherichia Coli includes a system for direct expression of the ß-urogastron gene and a system in which it is expressed as protein fused with ß-lactamase or another protein other than it.

To express the ß-urogastron gene directly, it is necessary to introduce a promoter and a Shine-Dalgarno sequence into the recombinant plasmid upstream of the ß-urogastron gene. While the promoter is not particularly limited, the advantageous promoters are those that ensure a high level of expression, such as WPL, which is the left-handed promoter from A. phag, lac UV5, which is upward from the β-galactosi-dase gene from Escherichia Coli, etc. If WPI is used as the promoter, the Shine-Dalgarno sequence is not particularly restricted, but it is advantageous to use the four-base sequence from AGGA. On the other hand, if lac UV5 is used as the promoter, it is advantageous to use the Shine-Dalgarno sequence lying downward from the lac UV5 promoter or the chemically synthesized sequence.

The system for direct expression of the β-urogastron gene will now be described with reference to the case in which a ^ PL-Shine-Dalgarno sequence β-urogastron gene is used.

Although XPL is a strong promoter (J. Hedgpeth et al., Molecular and General Genetics, 163, 197-203 (1978)), the fully activated X.PL promoter has a lethal effect on the Escherichia coli host cell, so there is a need to allowing the cell to multiply under conditions free from lethal action and then trigger the function of AJ? l. On the other hand, cI857, a gene within the A. phag, is one of the mutated genes of the cI repressor that acts on the operator for X.Pl. At low temperatures> (up to about 30 ° C) cI857 couples with the operator to completely inhibit the activity of XPL as a promoter, which consequently allows the multiplication of Escherichia Coli. Thus, the host cells can multiply in this state and they are then brought to a high temperature (not below 37 ° C), which triggers the function of X, Pl. In addition, the plasmid vectors with an energetic replication mechanism, such as pSCIOl, which are known and readily available, and the plasmid vectors with a loose replication mechanism, such as pBR322, are not incompatible with one another, but rather can coexist within the same Escherichia Coli cell.

Accordingly, it is suitable to construct a recombinant plasmid pGH37 in which a gene cI857 is introduced into a plasmid vector pSCIOl (which has a lac UV5 promoter upstream of it for effective expression of cI857), as can be seen in FIG. 6, and the recombinant plasmid Introduce plasmid into Escherichia Coli (strain HB101) to obtain a transformant (strain ECI-2) which can be used as a host for the vector for expressing β-urogastron under the control of the promoter X.Pl.

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According to the invention, the À.PL-Shine-Dalgarno sequence-ß-urogastron gene is introduced into pBR322, for example, to give a ß-urogastron-expressing vector which is used to transform the strain ECI-2, resulting in a so-called two-plasmid System is created in which two useful plasmids coexist in an Escherichia Coli cell.

In this system, the pIH7-encoded cI857 repressor couples with the operator for the XPL promoter on the second plasmid when the cell is cultured at 30 ° C, for example, which allows the cell to multiply. After the cell has fully grown in this state, the temperature is raised to 40 ° C., for example, whereupon the operator disassociates the cI857 repressor, which enables the activity of the XPL promoter to express β-urogastron.

Although a similar concept has been applied to the expression of fibroblast interferon, SV-40 small-t antigen, etc., an A, lysogen in which the DNA of an X carrying the cI857 gene is used as the host in these cases Phags are introduced into the host's chromosome (R. Derynck et al., Nature, 287, 193-197 (1980); C. Derom et al., Gene, 17.45-54 (1982); K. Küpper et al. , Nature, 289: 555-559 (1981)).

With the system according to the invention, however, the gene cI857 is introduced into another plasmid which is resistant to tetracycline. Accordingly, this system has the advantages that the X, phag introduced into the host chromosome is unlikely to be induced to multiply, and the strain is easy to control. Of course, the two-plasmid system is used for the first time for systems for expressing β-urogastron.

In another system, part of some other protein gene, such as a β-lactamase gene, is coupled to the β-urogastron gene to express the β-urogastron gene as a fused protein. This method has the advantage that the fused protein is less sensitive to protease decomposition within Escherichia Coli, providing protection for ß-urogastron. Another advantage is that the fused protein migrates in the cell from Escherichia Coli to the periplasm and accumulates there (SJ Chan et al., Proc. Nati. Acad. Sei., USA, 78, 5401-5405 (1981)), there locally and is therefore easy to separate and clean.

In more detail, a gene encoding two basic amino acids that can cleave a cleavage site for removing β-urogastron from the fused protein by cleavage with an enzyme is inserted into the ß-lactamase gene at an appropriate cleavage site for a restriction enzyme , and a ß-urogastron gene is coupled with the ß-lactamase gene.

Preferably the sequence of the two basic amino acids is -Lys-Arg- or -Arg-Lys-. Examples of enzymes for recognizing the sequence of amino acids for cleaving β-urogastron from the fused protein are kallikrein, trypsin, etc. Examples of restriction enzymes for cleaving the β-lactamase gene are XmnI, HincII, Seal, Pvil, Pstl, Bgll, Banl etc.

The ß-lactamase-ß-urogastron recom binant plasmid thus produced can express a fused protein within Escherichia Coli for mass production. The resulting fused protein is treated with kallikrein or the like, whereby β-urogastron can be obtained.

The expression system can be determined by direct analysis of the nucleotide sequence of the gene using the Maxam-Gilbert method, by confirmation of the insertion of the gene and its direction according to the mini-production method or imaging method (HC Birnboim et al., Nucleic Acids Research, 7.1513-1523 (1979)), or still be examined by radioimmunoassay for ß-urogastron.

The transformant according to the invention thus obtained is cultivated by the conventional method, so that ß-urogastron can be collected in large quantities of high purity.

The invention is described in more detail below with reference to the following example, the invention being in no way restricted to this example.

example

1) Preparation of the resin attaching the nucleoside

Various nucleoside attaching resins were made by the following procedure.

A quantity of 1% by weight of crosslinked polystyrene resin "S-Xl" (a product of Biorad Laboratories, USA, with a particle size corresponding to a sieve with a mesh size of 0.074 to 0.037 mm) was mixed with 2.41 g of N- (chloromethyl) - phthalimide, 0.22 ml trifhioromethanesulfonic acid and 50 ml dichloromethane mixed with stirring for 2 hours at room temperature. After the reaction was completed, the resin was filtered out, washed successively with dichloromethane, ethanol and methanol, dried under reduced pressure and then heated under reflux with 50 ml of a 5% by weight solution of hydrazine in ethanol overnight. The resin was filtered out and washed successively with ethanol, dichloromethane and methanol, and then dried under reduced pressure. The mixture of aminomethylated polystyrene resin (2.5 g) obtained by the above method, 0.75 mM monosuccinic acid ester of 5'-o-dimethoxytrityl nucleoside, 1.23 mM dicyclohexylcarbodiimide and 1 mM dimethylaminopyridine was added with the addition of 30 ml dichloromethane left at room temperature overnight. The resin was filtered out, washed successively with dichloromethane, methanol and pyridine, then immersed in pyridine-acetic anhydride (90:10 by volume) and left at room temperature for 30 minutes. The obtained nucleoside-fixing resin was filtered out, washed with pyridine and dichloromethane, and dried under reduced pressure for use in the solid-phase synthesis reaction.

2) Synthesis of the dinucleotide

The synthesis of a fully protected dinucleotide with the base sequence TA is described as an example. Adenosine (13.14 g), the 5'-hydroxyl group of which was protected with a dimethoxytrityl group (DMTr) and the amino group of which was protected with a benzoyl group, and 6.34 g of triazole were dissolved in anhydrous dioxane. While cooling with ice, 8.35 ml of triethylamine was added, then 6.86 g of o-chlorophenylphosphorodichloridate was added dropwise to the mixture over 10 minutes, and the resulting mixture was stirred at room temperature for 2.5 hours.

The triethylamine hydrochloride formed was filtered out, the filtrate was concentrated to about 2/3 of its volume and 3.6 g of β-cyanoethanol and 4.8 g of 1-methyl-imidazole were added to the concentrate with stirring at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed three times with 0.1 M aqueous sodium diphosphate solution and twice with water and then concentrated under reduced pressure,

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which gave 19.96 g of crude product. The product was purified by chloroform-methanol (98: 2 by volume) as the eluent by silica gel column chromatography. The purification procedure was repeated to give 15.12 g of fully protected adenosine mononucleotide.

The adenosine mononucleotide (7.81 g) thus obtained was added to a 2% by weight solution of benzenesulfonic acid in chloroform-methanol (70:30 in volume ratio), and the mixture was stirred under ice-cooling for 20 minutes and then with an aqueous Solution neutralized by sodium hydrogen carbonate. The deposited chloroform layer was washed with water and concentrated under reduced pressure to give 7.11 g of a crude product. The product was subjected to silica gel column chromatography and eluted with chloroform-methanol (97: 3 by volume) to give 4.31 g of adenosine mononucleotide with a free 5'-hydroxyl group.

Thymidine (1.64 g), the 5'-hydroxyl group of which was protected with a dimethoxytrityl group (DMTr), and 0.95 g of triazole were dissolved in 21 ml of anhydrous dioxane, 1.25 ml of triethylamine were added to the solution, then 69 ml of o-chlorophenylphosphorodichloridate are added dropwise to the mixture over 5 minutes, with stirring and ice-cooling. The resulting mixture was stirred at room temperature for 1 hour. The triethylamine hydrochloride formed in the reaction was filtered out and the filtrate was stirred with 1.1 ml of aqueous pyridine solution (IM) for 10 minutes. To the solution was added a solution of 1.17 g of the adenosine mononucleotide having a free 5'-hydroxyl group in dioxane (10 ml) and 0.72 ml of 1-methylimidazole obtained as described above, and the mixture was stirred at room temperature stirred for 3 hours. The obtained reaction mixture was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 0.1 M aqueous sodium diphosphate solution and then with water, and then concentrated under reduced pressure to give 2.39 g of a crude product. The product was subjected to silica gel column chromatography and eluted with chloroform-methanol (98: 2 by volume) to give 2.39 g of fully protected dinucleotide TA.

Different nucleotides were produced by the same method as above.

3) Synthesis of oligonucleotides

The solid phase synthesis of oligonucleotide A-1, i. H. the undecanucleotide AATTCGAAGAT is described.

A resin (40 mg) prepared by method 1) described above with nucleoside T bound thereon was introduced into a reaction vessel, washed three times with dichloromethane-isopropanol (85:15 by volume) and then with a solution (IM) of zinc bromide treated in dichloromethane-isopropanol to split off the dimethoxytrityl group (DMTr) in the 5 'position. This procedure was repeated several times until the color of the solution had disappeared. The resin was washed with dichloromethane and then to remove the remaining Zn2 + with a solution of triethylammonium acetate (0.5 M) in dimethylformamide, then with tetrahydrofuran and then dried by passing nitrogen gas through the reaction vessel for a few minutes.

The fully protected dinucleotide GA (50 mg), prepared according to method 2) described above, was dissolved in 1 ml of pyridine, protected with 1 ml of triethylamine 670 654

and then left to stand at room temperature for a few hours. The solution was then evaporated under reduced pressure. The residue was azeotropically evaporated with pyridine several times to convert the nucleotide to a triethylammonium salt. The salt was dissolved in 0.3 ml of a solution of mesitylenesulfonyl-5-nitrotriazole (0.3 M) in pyridine. The solution was added to the dried resin and the reaction was carried out at room temperature for 60 minutes. The liquid portion was filtered out from the reaction mixture and the solid portion was washed with pyridine and then for 5 minutes in a mixture of 0.2 ml of acetic anhydride and 0.8 ml of a solution of dimethylaminopyridine (0.1 M) in tetrahydrofuran pyridine left to mask the unreacted hydroxyl group. Finally, the resin was washed with pyridine, completing a solid phase synthesis cycle. One cycle extends the nucleotide chain by 2 base lengths. The same procedure as above was repeated to sequentially couple the dinucleotides AA, CG, TT and AA to the resulting nucleotide by condensation to produce the fully protected undecanucleotide AATTCGAAGAT attached to the resin.

The resin obtained (20 mg) was left to stand at 40 ° C. for one hour with 0.6 ml of a solution of tetramethylguanidine-2-pyridine doximate (0.5 M) in pyridine-water (90:10 by volume). The resin was then passed through a Pasteur pipette stuffed with cotton and thereby filtered out. The resin was washed alternately with pyridine and ethanol. The washing solution and the filtrate were combined and concentrated at 40 ° C under reduced pressure. The residue was dissolved in 2 ml of an aqueous solution of triethylammonium bicarbonate (TEAB, 10 mM). The solution was washed three times with ether. The aqueous phase was loaded onto a column (2 × 100 cm) of Sephadex G-50 and eluted with 10 mM of a solution of triethylammonium bicarbonate (TEAB). The fractions were examined for absorption at 260 nm. The fraction containing the first elution tip was concentrated. The residue was subjected to high speed liquid chromatography (Type 6000A pump; Type 440 detector; Waters Associates, USA products) to give a purified single peak fraction. The column used for high-speed liquid chromatography was of the type | i-Bondapak C] 8 (a product of Waters Associates, USA), and a solution of triethylammonium acetate (0.05%) was used as eluent for gradient elution (5-40% by volume). 1 M) used in acetonitrile water. The undecanucleotide so purified still had a 5 'end protected with a dimethoxytrityl group, so the compound was treated with an 80 vol% aqueous solution of acetic acid for 15 minutes to cleave the dimethoxytrityl group, and then again by high speed Liquid chromatography was cleaned until a single tip was obtained. For this purpose the same column was used as in the previous one and a solution of triethylammonium acetate (0.1 M) in acetonitrile-water was used as eluent for gradient elution (5 → 25% by volume).

In the same manner as in the foregoing, oligonucleotides A-2 to A-16 and B-1 to B-16 were synthesized.

Table 1 shows the yield of each oligonucleotide. The determination was made using 20 mg

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of the resin resulting from the solid-phase synthesis, Ab- The yield was determined from the absorption

cleavage of the oligonucleotide from the resin and subsequent tion of the purified end product at 260 nm and the sum

Removal of the protective group and cleaning. Absorption values for the nucleotide bases were calculated.

Table 1

A-l

80 ed

A-5

140 (ig

A-9

100 (ig

A-13

50 [ig

B-l

60 (ig

B-5

100 (ig

B-9

110 ed

B-13

130 ed

A-2

120 ed

A-6

70 ed

A-10

90 ed

A-14

40 ed

B-2

100 Hg

B-6

90 ed

B-10

100 Hg

B-l 4

60 ed

A-3

90 ed

A-7

80 ed

Alles

110 ed

A-15

60 ed

B-3

50 Hg

B-7

130 ed

B-ll

110 ed

B-l 5

70 ed

A-4

50 Hg

A-8

90 ed

A-12

40 ed

A-16

150 Hg

B-4

90 ed

B-8

100 Hg

B-l 2

110 ed

B-16

50 Hg

4) Examination of the sequence of the oligonucleotide bases The sequence was carried out according to the above-mentioned method of two-dimensional fractionation with the aid of electrophoresis and homochromatography by Wu et al. examined.

It was found that each of the oligonucleotides A-1 to A-16 and B-1 to B-16 had the expected nucleotide sequence. FIG. 5 shows the result obtained with the analysis of oligonucleotide A-3, it being found that A-3, read from the 5 'end, had the sequence GATTCTGAGTG.

The nucleotide sequence of each of the oligonucleotides was also examined according to the Maxam-Gilbert method mentioned above.

It was confirmed that each of the oligonucleotides A-1 to A-16 and B-1 to B-16 had the expected nucleotide sequence.

5) Formation of the oligonucleotide blocks and the subunits The blocks and subunits were produced by the method shown in FIG. 2 and described in the following.

First, about 5 jig of each of the oligonucleotides Al, A-2, A-3, A-14, A-15 and A-16 were dissolved in distilled water (50 jj.1) to make a solution with a concentration of about 0 To give 1 ng / nl. The six different aqueous solutions were each individually introduced in an amount of 10 μl (1 ng calculated DNA) in one of six Ep-pendorf glasses. A mixed solution (6 (il) containing 250mM TRIS-HC1 (pH 7.6), 50mM magnesium chloride, 10mM spermine and 50mM DTT was added to each glass and then 0.5 (il of an aqueous solution of y- 32P-ATP (a product of Amersham International Ltd., UK), 0.5 | il of Tj polynucleotide kinase (a product of Takara Shuzo Co., Ltd., JP) and 13 (il distilled water added to make 30 (il The mixture was reacted for 30 minutes at 37 ° C. and then after the addition of 1 ml of an aqueous 30 mM solution of ATP for a further 30 minutes. The reaction was carried out by heating to 100 ° C. for 2 The reaction mixture was rapidly cooled with ice and the oligonucleotides Al, A-2, A-3, A-14, A-15 and A-16 thus phosphorylated at 5 'end were individually added in amounts of 10 1 each in a 1.5 ml Eppendorf glass, into which 40 (1 l of an aqueous 250 mM solution of TRIS — HCl (pH 7.6), 40 (T of a 50 mM -L Solution of magnesium chloride and 35 ml of distilled water are added to make a total of 175 ml. The mixture was heated to 90 ° C over 2 minutes, then gradually cooled to room temperature. After adding 10 μl of a 200 mM solution of DTT, 10 μl of an aqueous 20 mM solution of

ATP and 5 p.1 (100 units) of T4 DNA ligase (a product of Nippon Gene Co., Ltd., JP) the mixture was reacted overnight at 4 ° C. to form block 1 of coupled oligonucleotides Al, A-2, A-3, A-14, A-15 and A-20 16.

Similarly, blocks 2 and 3 were coupled by coupling A-4, A-5, A-6, Al 1, A-12 and A-13 and by coupling A-7, A-8, A- 9 and A-10.

The reaction mixture with the blocks 25 thus formed was added twice with its volume of ethanol, and then the mixture was allowed to stand at -80 ° C for 30 minutes to precipitate DNA. Electrophoresis was then carried out on a 12.5% by weight polyacrylamide gel and autoradiography. This resulted in tapes at the 72 and 36 base pair positions for block 1, a tape at the 36 base pair position for block 2, and tapes at the 48 and 24 base pair positions for block 3.

This band was then cut out and a mixture of 10 mM TRIS-HCl (pH 7.6) and an aqueous solution of 10 mM EDTA (TRIS-EDTA) was added. The mixture was then left overnight for extraction at room temperature. The resulting mixture was centrifuged, the supernatant was separated, shaken with phenol which had been saturated with TRIS-EDTA, and finally centrifuged to separate the lower layer. The same procedure was repeated twice with phenol which had been saturated with TRIS-EDTA. Finally, the upper layer was passed through a 1 cm diameter and 20 cm long column coated with Sephadex G-50 to remove phenol and acrylamide. The eluate was then concentrated to 200 ml and then left at -80 ° C for 30 minutes in the presence of twice the volume of the concentrate in ethanol to precipitate DNA.

The three blocks obtained were coupled together. 50mM TRIS-HC1 (pH 7.6), 10mM magnesium chloride, 20mM DTT, 1mM ATP and 5mL (100 units) of TrDNA ligase were added to the mixture C. The mixture was added twice with its volume of ethanol and then allowed to stand at -80 ° C. for 30 minutes to precipitate DNA, followed by electrophoresis on an 8% by weight polyacrylamide gel and autoradiography resulted in bands at positions of 96 and 192 base pairs, each band was cut out and TRIS-EDTA was added, and the mixture was then left to extract at room temperature overnight 65. The mixture was centrifuged to remove the supernatant , the supernatant was shaken with phenol saturated with TRIS-EDTA and the bottom layer was separated.

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the procedure was repeated twice with phenol which had been saturated with TRIS-EDTA. The top layer was passed through a column coated with Sephadex G-50, the eluate was concentrated, and then twice the volume of ethanol was added to the concentrate and allowed to stand at -80 ° C for 30 minutes to precipitate DNA. The resulting product was digested with EcoRI and BamHI to give subunit A.

The same procedure as above was repeated, as shown in Fig. 3, to move oligonucleotides B1, B-2, B-15 and B-16 to block 4, oligonucleotides B-3, B-4, B-13 and Coupling B-14 to block 5, oligonucleotides B-5, B-6, B1 and B-12 to block 6 and oligonucleotides B-7, B-8, B-9 and B-10 to block 7. The blocks corresponding to 26 and 52 base pairs were collected and coupled in the same manner to give 104 and 208 base pair products which were cleaved by HindIII and BamHI. Subunit B was thus obtained.

6) Cloning the subunits and analysis of the recombinant plasmids

Referring to Figure 2, pBR322 was cleaved by EcoRI and BamHI and the phosphate groups were cleaved from the 5 'ends by alkaline phosphatase (a product of Takara Shuzo Co., Ltd., JP) to return to the original state to prevent. The pBR322 thus split and dephosphorylated and the subunit A were then at 4 ° C. overnight in a mixture of 50 mM TRIS-HC1 (pH 7.6), 10 mM magnesium chloride, 20 mM DTT and 1 mM ATP with the addition of 5 | Il Xj DNA ligase was left standing, whereby they were coupled together. The reaction mixture was added twice its volume of ethanol, and then the mixture was allowed to stand at -80 ° C for 30 minutes. The mixture was then centrifuged, the precipitate was dried and dissolved in 100 ml of distilled water, whereby a plasmid pUGl was obtained in which the subunit A had been inserted into pBR322.

The HB 101 strain from Escherichia Coli was transformed with the plasmid pUGl according to the calcium method.

The strain HB101 serving as the host was cultivated at 37 ° C. in 50 ml of LB culture medium (1% by weight of bactotrypton, 0.5% by weight of yeast extract and 0.5% by weight of sodium chloride). When the absorption at 610 nm reached 0.25, a portion of 40 ml of the culture broth was transferred to a centrifuge tube and centrifuged at 6000 rpm for 10 minutes at 4 ° C. The supernatant was removed, the precipitate suspended in 20 ml of an ice-cooled solution of 0.1 M magnesium chloride, the suspension centrifuged again under the same conditions and the supernatant removed. The precipitate was suspended in 20 ml of an ice-cooled solution of 0.1 M calcium chloride and 0.05 M magnesium chloride and ice-cooled for 1 hour. The suspension was centrifuged, the supernatant removed, the precipitate suspended in 2 ml of an ice-cooled solution of 0.1 M calcium chloride and 0.05 M magnesium chloride. A portion of 200 μl of the suspension was added 10 μl of an aqueous solution of pUGl and the mixture was ice-cooled for 1 hour and then heated in a water bath to 43.5 ° C. for 30 seconds. Then 2.8 ml of the LB culture medium was added to the mixture, followed by incubation at 37 ° C for 1 hour. The culture was then spread on an LB plate containing 50 μg / ml ampicillin in an amount of 200 μl / glass and overnight at 37 ° C.

subjected to incubation. The growing colonies were examined by further transplantation on an LB plate containing 50 μg / ml ampicillin and also on an LB plate containing 20 μg / ml tetracycline and subjected to incubation overnight at 37 ° C. The colonies only resistant to ampicillin were separated to give a transformed cell. Plasmids were obtained from the cell on a small scale using the alkaline extraction method and examined for the presence of a cleavage site for BglII. One of the cells harboring the plasmid containing the BglII cleavage site was cultured on a large scale to give a purified plasmid obtained in the same manner by the alkaline extraction method.

The nucleotide sequence of subunit A inserted into the resulting pUGl was analyzed on both strands by the Maxam-Gilbert method mentioned above.

Photographs 1 and 2 show the results of the analysis. Lanes 1 to 4 are the result of electrophoresis for the EcoRI-Sall fragment and lanes 5 to 8 that for the BamHI-PstI fragment. Lanes 1 and 5 show the reaction products for guanine, lanes 2 and 6 the reaction products for guanine + adenine, lanes 3 and 7 the reaction products for thymine + cytosine, and lanes 4 and 8 the reaction products for cytosine. Photograph 2 shows the results obtained with the same samples as in the above, with an area on the higher molecular weight side (corresponding to the upper part of Photograph 1) enlarged. In this way the nucleotide sequence of subunit A was confirmed.

With reference to Figure 3, plasmid pBR322 was cleaved by HindIII and BamHI and the larger fragment isolated by electrophoresis and coupled to subunit B. A plasmid pUG2 in which subunit B had been inserted into pBR322 was thus obtained in the same way as in the case of pUGl. Using the resulting plasmid pUG2, strain HB 101 was transformed and the colonies only resistant to ampicillin were selected. Plasmids were collected from the colonies and then examined for the presence of a cleavage site for BglII and a cleavage site for Mlul. The cells harboring the plasmid with the two cleavage sites were selected. One of the selected cells was cultured on a large scale to give a purified plasmid pUG2. The nucleotide sequence of subunit B in pUG2 was analyzed on both strands by the Maxam-Gilbert method.

Photograph 3 shows the results of the analysis. Lanes 1 to 4 show the result achieved with the Hindll-Sall fragment. Lanes 5 to 8 show the result achieved with the same sample, with an area on the higher molecular weight side (corresponding to the upper part of lanes 1 to 4) being enlarged. Each lane shows the same reaction product as in the corresponding lane in Photograph 1. In this way, the nucleotide sequence of subunit B was confirmed.

With reference to Fig. 4, pUGl was cleaved by Hindlll and Sali and a larger fragment was isolated with a 1.5m column from Biogel. pUG2 was digested by HindIII and Sali and then subjected to electrophoresis to obtain a smaller fragment. The two fragments were combined and treated for coupling with T4 DNA ligase, whereby a plasmid pUG3 was obtained in which the subunits A plus B, i.e. H. the ß-urogastron gene that had been inserted into pBR322. Using the resulting plasmid pUG3 was 5

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en the HB 101 strain from Escherichia Coli was transformed. The transformant was awarded to the Fermentation Research Institute, Agency of Industriai Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-Chome, Yatabe- under the Budapest Treaty on the international recognition of the deposit with the deposit number FERM BR-543. machi Tsubuka-gun, Ibaraki-ken 305-JP, deposited on June 22, 1984.

In the preceding case too, the cells harboring the plasmid pUG3 and only resistant to ampicillin and having a cleavage site for Mlul were selected. One of the selected cells was cultured on a large scale to give a purified plasmid pUG3. The nucleotide sequence of the ß-urogastron gene in pUG3 was analyzed by the Maxam-Gilbert method on both strands.

Photograph 4 shows the results of the analysis. Lanes 1 to 4 show the result achieved with the BamHI-Pstl fragment. Lanes 5 to 8 show the result achieved with the same sample, with an area on the higher molecular weight side (corresponding to the upper part of lanes 1 to 4) being enlarged. Each lane shows the same reaction product as in the corresponding lane in Photograph 1. The analysis confirmed the nucleotide sequence of the β-urogastron gene.

7) The promoter IP1 advertising the expression vector

To express β-urogastron, the promoter A.Pl, the left-hand promoter from A, -Pague, was used as described in the following.

First, the production of a strain ECI-2 derived from the HB 101 strain from Escherichia Coli is described. ECI-2 served as a host for plasmids expressing A.PL. Then the cloning of a DNA fragment will be described which contains the promoter WPI, which was obtained from the DNA of ICI857S7, a mutant of the X-phag. The expression of the β-urogastron gene by the promoter 1PL in the host strain ECI-2 will then be described.

7-1) Formation of the strain ECI-2

The ECI-2 strain is Escherichia Coli HB 101 and harbors a plasmid pGH37 for expressing a CI857 gene.

pGH37 was produced by the method shown in FIG. 6. First, DNA from XCI857S7 was cleaved by BglII. Then the cohesive ends at the cleavage site were digested using Sl nuclease. 1 Hg of the BglII-digested DNA from A.CI857S7 was in 30 ml in 100 ml of a 200 mM sodium chloride, 30 mM sodium acetate and 5 mM sodium chloride aqueous solution (pH 4.5) with 200 units of S1 nuclease at 20 ° C implemented. The blunt-ended DNA fragments thus obtained were subjected to electrophoresis with 1.0 wt% agarose gel to isolate a 2385 base pair fragment having the entire structural gene CI857. The fragment was inserted into the PvuII site of plasmids pGLlOl to produce a plasmid pGH36 that expresses the gene CI857 under the control of a promoter, lac UV5. Subsequently, pGH36 was digested by two restriction enzymes, EcoRI and Pstl, to give a fragment with 1193 base pairs, which was inserted into a plasmid pSCIOl between the cleavage sites for EcoRI and Pstl to form a plasmid pGH37.

Then the HB101 strain from Escherichia io Coli was transformed with pGH37 according to the calcium method mentioned above. One of the resulting strains was named ECI-2. The strain ECI-2 was registered under the Budapest Treaty on the international recognition of the deposit with the deposit number FERM BR-i5 542 at the Fermentation Research Institute, Agency of Industriai Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1- Chome, Yatabe-machi Tsubuka-gun, Ibaraki-ken 305-JP, deposited on June 22, 1984. This strain is resistant to tetracycline, expresses the 2o gene CI857 and allows the simultaneous presence, by transformation, of other plasmids derived, for example, from pBR322. For these reasons, the strain ECI-2 was subsequently used as a host for plasmids expressing A.Pl.

25th

7-2) Cloning of the promoter A-Pl and production of expressing plasmids

As shown in Fig. 7, pGH35 was first formed. DNA from X, CI857S7 was digested by EcoRI and Sali 30 to give a 5925 base pair fragment comprising both promoter A, PL and the gene CI857 and promoter ÄPr. The fragment was inserted into a plasmid pBR322 between the EcoRI and Sali cleavage sites to form a plasmid pGH25. 3s Then pGH25 was cleaved by BamHI and coupled with pBR322 cleaved by BamHI in the same way to form pGH34.

Thereafter, pGH34 was cleaved by Aval and BglII and then processed by S1 nuclease to give a fragment of about 4500 base pairs with blunted ends. The fragment was closed by T4 DNA ligase to form a plasmid pGH35.

PEK28 was then formed, as shown in FIG. 8. Synthetic oligonucleotides C-1-1 and C-1-2, comprising a Shine-Dalgarno sequence and having the nucleotide sequence shown below, were coupled to the fragment obtained by cleaving pGH35 by Hpal. The composition was further coupled with the BamHI digested plasmid pMC1403 to form plasmid pEG2. pEG2 comprises two genes of ampicillin resistance and, under the control of the promoter XPl, expresses a β-galactosidase gene derived from pMC1403, where it uses a start codon and the Shine-Dalgarno sequence contained in the adapter.

Fragments C-1-1 and C-1-2 have the following nucleotide sequence.

60

Shi ne-Dalgarno sequences

Start codon

C-l-i C-l-2

U -T 7

A G G A A C AG A T C T A T G

TCCTTGTCTAGATACCTAG

Bgl II

17th

670 654

To confirm the expression of β-galactosidase in the host ECI-2 harboring the plasmid pEG2, the Miller method was used (J. Miller, "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, New York (1972) , Pp. 352 - 355). This process is based on the reaction of ß-galactosidase with a synthetic substrate (o-nitrophenylgalactoside), which releases a yellow compound o-nitrophenol. The Miller method is described in more detail. An amount of 0.1 ml of the culture of a bacterial sample, the absorption of which was measured at 610 nm, is mixed with 1.9 ml of an assay buffer (0.1 M sodium phosphate, pH 7.0; 1 mM magnesium sulfate; 0.1 M ß-mercaptoethanol) mixed and shaken for 15 seconds with 0.1 ml of toluene to increase the permeability of the bacterial sample. The toluene is then evaporated using an aspirator. After adding 0.2 ml of the solution of o-nitrophenylgalactoside (solution of 400 mg of o-nitrophenylgalactoside in 100 ml of the assay-5 buffer), the mixture is subjected to incubation at 30 ° C. until a yellow color develops, whereupon 0 , 5 ml of 1 M sodium carbonate are added to stop the enzyme reaction. The absorption of the reaction mixture is determined at 420 nm and 550 nm, io. The activity of β-galactosidase is defined according to the following equation in units of 1 ml of the liquid culture, the absorption at 610 nm being set to 1.0.

Activity from 0D42O ~ 1 s 75 * DD55D

ß-Gal actosi dase = x 1000

(Units) t> •: v x where t = incubation period (minutes)

v = amount of sample added to the reaction system OD6io = absorption of the sample at 610 nm

This procedure gave the following results. When the ECI-2 harboring the pEG2 was incubated at 30 ° C, the activity of β-galactosidase was 98 units. However, if the culture was further incubated at 42 ° C for 1 hour, the 1PL promoter was activated, with the result that the activity of β-galactosidase was now 9637 units. This confirms that the sequence from the promoter AJP1 to the β-galactosidase is in the expected order.

Although pEG2 has two cleavage sites for Bglll, only the cleavage site for Bglll that is immediately after the Shine-Dalgarno sequence of the β-galactosidase is required, while the other site is undesirable. Accordingly, the plasmid was cleaved by BamHI and coupled again to remove a fragment with about 770 base pairs. The formation of pEK28, an expressing plasmid using the AJPl promoter, was then completed.

7-3) Expression of the fused gene of the front half of β-urogastron and expression of the β-galactosidase. FIG. 9 shows schematically the series of procedures which are described below.

A plasmid pUGlOl with a fused front half gene of β-urogastron and with β-galactosidase was formed in the following manner. In particular, pUGl, which has a fused front half gene of β-urogastron, and pMC1403, which has a β-galactosidase gene, were cleaved by BamHI and then coupled to form pUGlOl. In this plasmid, the front half of the ß-urogastron gene and the ß-galactosi-dase gene are coupled in the same structure. Accordingly, the plasmid expresses the amino acid sequences of the two as a fused protein.

To express with this plasmid under the control of promoter A, PI, pUGlOl and pEK28 were both cleaved by BglII.

The cleavage by Bglll results in a DNA fragment with an outstanding 5 'end in the form a \

... TCTAG

However, if the DNA fragment is reacted with a large fragment of DNA polymerase I from Escherichia Coli (Klenow fragment) in the presence of the four types of de-25 oxyribonucleotide triphosphates dGTP, dATP, dTTP and dCTP, the corresponding ones are filled in Nucleotides get a blunt end by putting an end in the shape

30th

AGATC TCTAG

is formed. If only dGTP is added as nucleotide component 35, an end in the form will result because of the termination of the reaction

/ ... AG

[... TCTAG

educated. Now when Sl nuclease is used to digest the remaining single strand, a blunt end in the shape

45

AG TC

so educated. In the same way, if dGTP and dATP are added in the Klenow reaction and then digested with Sl nuclease, an end will be in the form

55

AGA TCT

educated. If the Klenow reaction is carried out with the addition of dGTP, dATP and dTTP and then digested with 60 μl nuclease, an end in the form

/. . . AGAT I ... TCTA

65

educated. When digesting with Sl nuclease only, without performing the Klenow reaction, there is an end to the form

670 654

18th

a formed. Thus, five kinds of blunt ends with lengths different from each other by a base pair are formed when a DNA fragment with one end in the shape

. . ag \

.. tctag)

subjected to the Klenow reaction using various nucleotides and / or the reaction with Sl nuclease.

The Klenow reaction and the reaction with S1 nuclease were carried out under the following conditions.

*** Klenow reaction:

In 50 μl of a reaction medium containing 40 mM potassium phosphate buffer (pH 7.4), 1 mM β-mercaptoethanol and 10 mM magnesium chloride, 1 Hg of DNA was added as substrate with 1 mM of each of the deoxyribonucleotide triphosphates at 12 ° C. for 30 minutes in the presence of 1 unit of the enzyme (Klenow fragment) and 1 mM of ATP.

*** Sl nuclease reaction:

In 100 h1 of a reaction medium containing 200 mM sodium chloride, 30 mM sodium acetate and 5 mM zinc sulfate (pH 4.5), 10% DNA as the substrate was reacted with 200 units of the enzyme at 20 ° C. for 30 minutes.

pEK28 and pUGlOl were both cleaved by BglII and then subjected to various combinations of the Klenow reaction and the S1 nuclease reaction, resulting in fragments with 5 types of blunt ends. In combination with this, the two types of these fragments 21 result in different combinations of one another in the number and in the sequence of the nucleotides arranged between the Shine-Dalgarno sequence and the start codon of the fused protein, as can be seen in Table 2.

Indeed, the DNA fragments thus obtained were digested by Sali to isolate fragments containing the gene encoding the protein fused from the front half of ß-urogastron and ß-galactosidase from pUGlOl, and to isolate fragments , which contained the promoter Ä, PL from pEK28. These fragments were coupled by T4 DNA ligase in the combinations shown in Table 2.

As a result, the recombinant plasmids listed in Table 3 were obtained. The β-galactosidase activity of the 25 expressed fused proteins was determined according to the Miller method. Table 3 shows that all plasmids expressed a fairly high level of β-galactosidase activity. In particular, remarkable results were achieved with pUG103, pUG104 and pUG117.

30th

Table 2

Nucleotide sequence up from the start codon (ATG) ATCTGC- GATCTGC-

Nucleotide sequence down from the SD sequence (AGGA)

-ACA

-ACAG

-ACAGA

-ACAGAT

-ACAGATC

-ACAATCTGC- (pUG105)

-ACAGAATCTGC- (pUGl 13) -ACAGATATCTGC- (pUG117) -ACAGATCATCTGC- (pUG121)

-ACAGATCTGC- (pUG106) -ACAGGATCTGC- (pUGllO) -ACAGAGATCTGC- (pUG114) -ACAGATGATCTGC- (pUG118) -ACAGATCGATCTGC- (pUG122)

Table 2 (continued)

Nucleotide sequence up from the start codon (ATG) TGC- CTGC-

TCTGC-

-ACATGC-

(pUG102)

-ACAGTGC-

(pUG107)

-ACAGATGC-

(pUGlll)

-ACAGATTGC-

(pUG115)

-ACACTGC-

(pUG103)

-ACAGCTGC-

(pUG108)

-ACAGACTGC-

(pUG112)

-ACAGATCCTGC- (pUG119)

-ACATCTGC- (pUG104) -ACAGTCTGC- (pUG109)

-ACAGATTCTGC- (pUG116)

-ACAGATCTCTGC- (pUG120)

Nucleotide sequence down-ACA from the SD sequence

-ACAG -ACAGA -ACAGAT -ACAGATC

Next, a vector for expressing β-urogastron was made from pUG103 and pUG117. The plasmid (pUG103 or pUGl 17) was cleaved by Hindlll and Pvu-II to give a fragment of 1.2 Kb (Kb = kilo base pairs), which extends from the promoter A.Pl to the front half of the ß-Urogastron -Gens extending area included. In addition, pUG2 was replaced by EcoRI

cleaved, filled in with the Klenow fragment, and cleaved by HindIII to give a 4.1 Kb fragment. The two fragments were coupled by Tt DNA ligase and the Escherichia Coli strain ECI-2 was transformed by the calcium method mentioned above to give a recombinant which was used to express the combination of the front half and the

19th

670 654

Table 3

Number of nucleotide recombinant β-galactosidase between the SD sequence plasmid (units)

and the start codon

6

pUG102

1674

7

pUG103

2533

7

pUG107

2260

8th

pUG104

2802

8th

pUG108

1835

9

pUG105

1018

9

pUG109

1942

10th

pUG106

973

11

pUGllO

1764

11

pUG113

1950

11

pUG119

1862

12

pUG114

946

12

pUG117

2332

12

pUG120

1374

13

pUG118

2041

13

pUG121

1678

14

pUG122

1814

rear half of the ß-urogastron gene, d. H. of the entire ß-urogastron gene under the control of the promoter Ä.Pl, which contained plasmid pUG103-E or pUGl 17-E.

8) Vector for expressing the fused protein As described below, a β-lactamase gene present on the plasmid pBR322 and a β-urogastron gene were coupled to each other to express β-urogastron as a fused protein.

8-1) Donor of the β-lactamase gene By cleavage of pBR322 by Aval and PvuII and subsequent Klenow reaction and coupling by T4 DNA ligase, pBRH02 is obtained. This plasmid has genes for resistance to ampicillin (ApR) and to tetracycline (TcR) as markers. By cleavage of pBR325 by Aval and Hindlll and subsequent Klenow reaction and coupling, pBRH02 is obtained, which has genes for resistance to ampicillin (ApR) and chloramphenicol (CmR) as markers. Figure 10 shows these plasmids.

8-2) Donor of the β-Urogastron Gene The pUG3 formed as already described was cleaved by MboII to give 13 types of DNA fragments which, as shown in FIG. 11, in the order of their size «A» to «M »Were named. Among these fragments, it was found that the H fragment, beginning with a nucleotide coding for asparagine at the N-terminus of ß-urogastron and ending with 16 bases down from the stop codon, was composed of 179 base pairs and the entire structural ß-urogastron -Gen showed. To isolate the H fragment, the fragments were subjected to electrophoresis with a 6% by weight polyacrylamide gel, and then the fragment was purified.

8-3) adapter

To form adapters, the oligonucleotides listed in Table 4 were prepared by the same method as described above. These

Adapters have been designed to code for the basic amino acid pair of Lys-Arg or Arg-Lys to enzymatically cleave the β-urogastron gene from the expressed fused protein.

Table 4

Adapter 5 'end 3' end

D-1-3

CCGTAAG

D-1-4

TTACGG

D-2-1

CGTAAG

D-2-2

TTACG

D-3-2

TTACGGAT

D-4-2

TTACGTGCA

E-l

CGCTAAACGG

E-2

CGTTTAGCG

E-3

GACAAACGG

E-4

CGTTTGTC

E-5

CGTTTAGCGAT

E-6

CGTTTGTCTGCA

E-7

CGGCTAAACGG

E-8

CGTTTAGCCG

E-9

CAAACGG

E-10

CGTTTG

8-4) System for expressing the fused protein of ß-lactamase and ß-urogastron coupled by Lys-Arg

A vector for expressing the protein fused from ß-lactamase and ß-urogastron was formed so as to generate a restriction enzyme recognition sequence in the region containing an adapter.

8-4-a) Formation of p2301 to pUG2303 The procedure given in Fig. 12 was used. The plasmid pBRH02 was completely cleaved by the action of XmnI at 37 CC for 3 hours.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

670 654

20th

Thereupon, in a single step of 15 hours at 12 CC, 3 ng of the vector, about 0.1 Hg of the β-urogastron fragment of 179 base pairs and in each case about 1 Hg of E-1 and E-2 (serving as adapters) with non-phosphorylated 5 'ends) to give plasmids as expression vectors. The HB101 strain was transformed using the plasmids according to the calcium method.

Of the 499 TcR colonies obtained, 168 (33.7%) were Aps colonies. These colonies were examined for the size of the plasmid DNA using the mini-production method. 13 plasmids were about 200 base pairs larger than the vector and it was considered that a ß-urogastron gene was inserted therein. All of them, which had a cleavage site for Mlul, were cleaved by Hinfl and examined for the orientation of the insertion of the β-urogastron gene by electrophoresis with a 1.5% by weight agarose gel. Three of the examined revealed fragments of approximately 1050 base pairs and approximately 800 base pairs. This indicated that the ß-urogastron gene was inserted with the same orientation as the ß-lactamase. These three plasmids were named pUG2301 to pUG2303.

8-4-b) Formation of pUG2101 to pUG2105

The procedure given in Fig. 13 was used. The plasmid pBR322 was used as a plasmid vector with a single cleavage site for Pvul in the β-lactamase gene. An expression vector was formed according to the procedure described in 8-4-a) using E-1 and E-5 serving as adapters.

When the TcR colonies obtained in 1626 were examined for Ap sensitivity, they were found to be 31 (1.9%) Aps colonies. The mini-preparation procedure was applied to 22 colonies of TcR and Aps and the plasmids were examined for the insertion of the β-urogastron gene by cleavage by Mlul. It was found that 20 plasmids had a cleavage site for Mlul. The orientation was examined by cleavage by Hinfl or BamHI. It was found that the plasmids pUG2101 to pUG2105 had a β-urogastron gene inserted therein with the same orientation as the β-lactama-io se gene.

8-4-c) Formation of pUG2701 to pUG2703

Expression plasmids were formed according to the same procedure as in 8-4-a) using pBR322 as a vector and E-7 and E-8 as an adapter, as shown in Fig. 14.

Of the 217 TcR colonies obtained, 106 (48.8%) were Aps colonies. The mini-production process was applied to 25 of these colonies. 8 of these plasmids were about 200 base pairs larger than the vector and had a β-urogastron gene inserted therein, so that these 8 plasmids were cleaved by BamHI and examined for the orientation of the gene. As a result, it was found that in three plasmids the ß-urogastron gene with the same orientation as the ß-lactamase was inserted, these three plasmids were named pUG2701 to pUG2703.

The plasmid pUG2301 obtained according to procedure 8-4-a) above produces a fused protein of 30 parts of β-lactamase and β-urogastron. The expected sequence of amino acids and the corresponding nucleotide sequence are given below.

Met Ser

ATG AGT

Ala Leu

GCC CTT

Phe Cys

TTT TG C

Per glu

C CA GAA

Asp Ala

GAT GCT

Arg Val

CGA GTG

Leu Asn

CT C AAC

Ile GI il

ATT CAA

I le Pro

ATT CCC

L eu P po

CTT CCT

Thr Leu

A CG CTG

Glu Asp

GAA GAT

Gly Tyr

GGT TAC

Ser Gly

AGC GGT

His Phe

CAT TTC

Phe Phe

TTT TTT

Val Phe

GTT TTT

Val Lys

GTG AAA

Gin Leu

CAG TTG

Ile Glu

ATC GAA

Lys I le

AAG ATC

A rg Val

CGT GTC

Ala Ala

G C G G C A

Ala His

GCT CAC

Val Lys

GTA AAA

Gly Ala

GGT GCA

Leu Asp

CTG GAT

Leu Glu

CTT GAG

21 670 654

Ser Phe Arg Pro Glu Glu Arg Ala

AGT TTT CGC CCC GAA GAA CGC GCT

Lvs Arg Asn Ser Asp Ser Glu Cys

AAA CGG AAT AGC GAT TCT GAG TGC

Pro Leu Ser His Asp Gly Tyr Cys

CCA CTG TCT CAC GAT GGC TAT TGT

Leu His Asp Gly Val Cys Met Tyr CTG CAC GAC GGT GTT TGC ATG TAC

Ile Glu Ala Leu Asp Lys Tyr Ala ATC GAA GCT TTG GAT AAA TAC GCG

Cys Asn Cys Vai Val Gly Tyr lie TGT AA C TGT GTA GTG GGT TAT ATC

Gyl Glu Arg Cys Gin Tyr Arg Asp GGT GAA CGC TGT CAA TAC CGT GAT

Leu Lys Trp Trp Glu Leu Arg (stop) CTG AAA TGG TGG GAA TTG CGT T AA

TAGTGAAGATCTGGATCCGTTTAGCGTTTTCCA ATGATGAGCACTTTTAAAGTTCTGCTATGTGGC

GCGGTATTATCCCGTGTTGACGCCGGGCAAGAG

CAACTCGGTCGCCGCATAC

In the same way, the plasmid pUG2101 obtained according to procedure 8-4-b) above produces a fused protein with the following primary structure.

Met Ser Ile Gin His Phe Arg Val ATG AGT ATT CAA CAT TTC CGT GT C

Ala L eu

GCC CTT

Phe Cys

TTT TGC

Ile Pro

ATT CCC

L eu P ro

CTT C CT

Phe Phe

TTT TTT

Val Phe

GTT TTT

Ala Ala

GCG GCA

Ala His

GCT CAC

670 654 22

Pro Glu Thr Leu Val Lys Val Lys C CA GAA A CG CTG GTG AAA GTA AAA

Asp Ala Glu Asp

GAT GCT GAA GAT

Arg Val Gly Tyr

CGA GTG GGT TAC

Leu Asn Ser Gly

CTC AAC AGC GGT

Ser Phe Arg Pro

AGT TTT CGC CCC

Per Met Met Ser

CCA ATG ATG AGC

Leu Leu Cys Gly

CTG CT A TGT GGC

Arg Val Asp Ala

CGT GTT GAC GCC

Leu Gly Arg Arg

CTC GGT CGC CGC

Gin Asn Asp I le

CAG AAT GAC TTG

Pro Val Thr Glu

CCA GTC ACA GAA

Asp Gly Met Thr

GAT GGC ATG ACA

Cys Ser Ala Ala

TGC AGT GCT GCC

Asp Asn Thr Ala

GAT AAC ACT GCG

Gin Leu Gly Ala

CAG TTG GGT GCA

Ile Glu Leu Asp

ATC GAA CTG GAT

Lys Ile Leu Glu

AAG ATC CTT GAG

Glu Glu Arg Phe

GAA GAA CGT TTT

Thr Phe Lys Val

ACT TTT AAA GTT

Ala Val Leu Ser

GCG GTA TT A TCC

Gly Gin Glu Gin

GGG CAA GAG CAA

Ile His Tyr Ser

ATA CAC TAT TCT

Val Glu Tyr Ser

GTT GAG TAC T CA

Lys His Leu Thr

AAG CAT CTT ACG

Val Arg Glu Leu

GTA AGA GAA TTA

I le Thr Met Ser

ATA ACC ATG AGT

Ala Asn Leu Leu

GCC AAC TTA CTT

Leu CTG

Ser AGC

H is CAC

Gly GGT

Leu TTG

Val GTA

Cys TGT

T rp TGG

Thr ACA

Asp GAT

Asp GAT

Val GTT

Asp GAT

Val GTG

Gin CAA

Glu GAA

Thr ACG

Ser TCT

Gly GGC

Cys TGC

Lys AA A

Gly GGT

Tyr TAC

Leu TTG

I le ATC

Glu GAG

Tyr TAT

With ATG

Tyr TAC

Tyr TAT

Arg CGT

Arg CGT

Ala GCT

Cys TGC

Cys TGT

T yr TAC

Ala GCG

I le ATC

Asp GAT

Lys A A A

Per CCA

Leu CTG

I le ATC

Cys TGT

Gly GGT

Leu CTG

Arg CGG

Leu CTG

His CAC

GI and GAA

Asn AAC

GI and GAA

Lys A A A

Asn AAT

Ser TCT

Asp GAC

Ala GCT

Cys TGT

Arg CGC

T rp TGG

(stop)

T AA TAGTGAAGATC

TGGATCCGTTTAGCGATCGGAGGACCGAAGG AGCTAACCGCTTT TTT G C A C A

In the same way, the plasmid pUG2701 obtained according to procedure 8-4-c) above produces a sionated protein with the following primary structure.

Met Ser Ile Gin His Phe Arg Val ATG AGT ATT CAA CAT TTC CGT GTC

Ala L eu

GCC CTT

Phe Cys

TTT TGC

Per glu

CCA GAA

Ile Pro

ATT CCC

Leu Pro

CTT C CT

Thr Leu

ACG CTG

Phe Phe

TTT TTT

Val-Phe

GTT TTT

Val Lys

GTG AAA

Ala Ala

GCG G C A

Ala His

GCT CAC

Val Lys

GTA AAA

670 654 24

Asp Ala Glu Asp Gin Leu Gly Ala GAT GCT GAA GAT GAG TTG GGT GCA

Arg Val Gly Tyr Ile Glu Leu Asp

CGA GTG GGT TAC ATC GAA CTG GAT

Leu Asn Ser Gly Lys I le Leu Glu

CTC AAC AGC GGT AAG ATC CTT GAG

Ser phe Arg Pro Glu Glu Arg phe

AGT TTT CGC CCC GAA GAA CGT TTT

Per Met Met Ser Thr phe Lys Val

CCA ATG ATG AGC ACT TTT AAA GTT

Leu Leu Cys Gly Ala Val Leu Ser

CTG CTA TGT GGC GCG GTA TTA TCC

Arg Val Asp Ala Gly Gin Glu Gin

CGT GTT GAC GCC GGG CAA GAG CAA

Leu Gly Arg Arg Ile His Tyr Ser

CTC GGT CGC CGC ATA CAC TAT TCT

Gin Asn Asp Ile Val Glu Ser Ala

CAG AAT GAC TTG GTT GAG TCG GCT

Lys Arg Asn Ser Asp Ser Glu Cys

AAA CGG AAT AGC GAT TCT GAG TGC

Pro Leu Ser His Asp Gly Tyr Cys

CCA CTG TCT CAC GAT GGC TAT TGT

Leu His Asp Gly Val Cys Met Tyr

CTG CAC GAC GGT GTT TGC ATG TAC

Ile Glu Ala Leu Asp Lys Tyr Ala

ATC GAA GCT TTG GAT AAA TAC GCG

Cys Asn Cys Val Val Gly Tyr Ile

TGT AAC TGT GTA GTG GGT TAT ATC

25 670 654

Gly Glu Arg Cys Gin Tyr Arg Asp GGT GAA CGC TGT CAA TAC CGT GAT

Leu Lys Trp Trp Glu Leu Arg (stop) CTG AAA TGG TGG GAA TTG CGT TAA

TAGTGAAGATCTGGATCCGTTTAGCCGACTCAC

CAGTCACAGAAAAGCATCTTACGGAT

The nucleotide sequence coding for the fused proteins in the plasmids pUG2101, pUG2301 and pUG2701 was analyzed by the Maxam-Gilbert method.

Photograph 5 shows the results of the analysis. Lanes 1 to 4 show the result achieved with the Mlul-Pstl fragment (224 base pairs) from pUG2101, lanes 5 to 8 that with the MluI-EcoRI fragment (721 base pairs) from pUG2101, lanes 9 to 12 with the Mlul -BamHI fragment (452 base pairs) from pUG2301, lanes 13 to 16 the result achieved with the Mlul-PstI fragment (335 base pairs) from pUG2701 and lanes 17 to 20 the result achieved with the MluI-EcoRI fragment (610 base pairs) from pUG2701. Lanes 1, 5, 9, 13 and 17 show the reaction products for guanine, lanes 2, 6, 10, 14 and 18 show the reaction products for guanine + adenine, lanes 3, 7, 11, 15 and 19 show the reaction products for thymine + Cytosine, and lanes 4, 8, 12, 16 and 20 the reaction products for cytosine. The part marked with "}" is an adapter.

8-5) System for expressing the fused protein of ß-lactamase and ß-urogastron coupled by Lys-Arg

8-5-a) Formation of pUGl 102 and pUGl 105

A ß-urogastron gene was inserted into the ß-lactamase gene of pBR322 at its only restriction enzyme recognition site for Pvul to give vectors for expressing the fused protein of ß-lactamase and ß-urogastron, as in 15.

The plasmid pBR322 was cleaved by the action of Pvul at 37 ° C for 3 hours. Some of the plasmids were examined by electrophoresis with a 1% by weight agarose gel to confirm that they had been completely digested. The adapters Dl-3 and D-3-2 were coupled to the fragment at 12 ° C for 15 hours, and the coupled product was then subjected to electrophoresis with a 1 wt% agarose gel to obtain a DNA fragment isolate. The β-urogastron fragment and the vector were then mixed with one another in a molar ratio of approximately 5: 1 and coupled at 12 ° C. for 15 hours. After coupling, strain HB 101 was transformed with the resulting plasmid and the colonies selected for TcR.

71 of the colonies transformed with TcR were obtained and examined for their Aps sensitivity. Plasmid DNA was prepared from 20 Aps colonies (28.2%) and then examined for the presence of a restriction enzyme recognition site for Mlul to confirm the insertion of the β-urogastron gene. It was found that

5 of the 20 plasmids had a cleavage site of the β-urogastron gene for 20 ml. The DNA was digested by Hinfl and then subjected to electrophoresis with a 1.5% by weight agarose gel to examine the orientation of the insertion of the β-urogastron gene. Two of the plasmids, namely pUGl 102 and pUGl 105, had genes in the correct orientation.

8-5-b) Formation of pUG1004, pUG1201 and pUG1301 The procedure of 8-5-a) was repeated using Pstl, HincII and XmnI instead of Pvul to give pUG1004, pUG1201 and pUG1301 as in 16, 17 and 18 is shown.

9) Confirmation of ß-urogastron expression The expression plasmids thus formed were used 35 to transform Escherichia Coli HB 101 or ECI-2, and the cells were cultured according to the following procedure, then extracted and subjected to radioimmunoassay to say that To confirm expressions.

40 9-1) Cultivation of recombinant microorganisms provided with a β-urogastron gene and extraction of the protein

9-1-a) Expression system using the 45 promoter XPL

The strain ECI-2 harboring the expression plasmid pUG103-E and the same strain harboring the expression plasmid pUGl 17-E were each cultured in two flasks, each containing 1 liter of LB culture medium, at 25 ° C. When the culture in one of the flasks had an absorption of 0.3 at 660 nm, the culture was subjected to heat induction at 42 ° C for 1 hour. The culture in the other flask was continuously incubated at 25 ° C until the absorbance was 0.4. The cells of each flask were collected, washed with a PBS buffer (137mM sodium chloride, 2.7mM potassium chloride, 8.1mM sodium diphosphate and 1.5mM sodium monophosphate, pH 7.0), then in an amount of the PBS buffer , which corresponded to 3% by volume of the original amount of culture 60, resuspended and finally destroyed under ice cooling by sonication using a type 5202 sound device (a product of Ohtake Works Co., Ltd., JP) (three times for 30 seconds at 100 W ). The supernatant separated from the cell debris by ultracentrifugation (at 40,000 g for 16 hours) was dialyzed against a 0.01N aqueous solution of acetic acid and the dialysate was lyophilized and then subjected to a radioimmunoassay (RIA).

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26

9-1-b) System for expressing the fused protein

A strain Escherichia Coli HB101 harboring the plasmids pUG1004, pUG1301, pUG2101, pUG2303 or pUG2703 was incubated in a culture medium containing 50 | xg / ml tetracycline at 37 ° C., then diluted and cultured in a volume ratio of 1: 100 with the same medium, until it had an absorption of 0.4 at 660 nm. The cells were collected, washed with PBS buffer, then resuspended in an amount of the PBS buffer which corresponded to 3% by volume of the original amount of the culture and finally destroyed under ice cooling by sonication with the same sonic device (three times for 30 seconds 100 W). The supernatant separated from the cell debris by ultracentrifugation (at 40,000 g for 1 hour) was dialyzed against a 0.01N aqueous solution of acetic acid, lyophilized and then subjected to a radioimmunoassay.

To the accumulation of merged! To confirm protein in the peri-plasm, a periplasmic fraction was prepared by the method of S.J. Chan et al (Chan, S.J. et al., Proc. Nati. Acad. Sei., USA, 78, 5401-5405 (1981)). A part of the culture was mixed in a volume ratio of 1: 100 with a fresh e-culture medium (1 liter aqueous solution of 10 g potassium diphosphate, 3.5 g sodium ammonium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 2 g citric acid, 2 g glucose, 0.23 g L-proline, 39.5 g L-leucine, 16.85 mg thiamine and 20 mg tetracycline hydrochloride) diluted and then cultured at 37 ° C until an absorption of 0.4 at 660 nm was reached. The cells were collected (6000 revolutions per minute, 10 minutes) and washed twice with a mixture of 10 mM TRIS-HC1 (pH 8.0) and 30 mM sodium chloride. The cells (1 g) were resuspended in 80 ml of 20% by weight sucrose with 30 mM TRIS-HC1 (pH 8.0), whereupon EDTA was added to the suspension to a concentration of 1 mM. The mixture was shaken in a rotary shaker at 180 rpm for 10 minutes (24 ° C) and then centrifuged (13,000 g, 10 minutes) to collect the cells, which were then resuspended in 80 ml of distilled water. The suspension was left in ice for 10 minutes with occasional stirring and then centrifuged (13,000 g, 10 minutes). The supernatant was collected as a periplasmic fraction (O-Sup). The lozenge was suspended in a mixture of 10 mM TRIS-HC1 (pH 8.0) and 30 mM sodium chloride and treated with the same sonicator as in the previous to give a cytoplasmic fraction (O-Ppt). These samples were subjected to the radioimmunoassay.

9-2) Radioimmunoassay

9-2-a) Perform the radioimmunoassay

Rabbits were immunized with purified human ß-urogastron as the antigen to give antiserum. The ß-urogastron (300 [ig) was dissolved in 0.2 ml of distilled water, 1.5 ml of a 50% solution of polyvinylpyrrolidone was added to the solution and the mixture was stirred at room temperature for 2 hours. Complete Freund's additive (2.0 ml) was added to the mixture to give an emulsion which was subcutaneously injected into three rabbits in their breasts. After repeating the immunization four times a week, every other week, 50% of the antigen was injected intravenously, the whole blood was collected 3 days later and the serum was separated.

Then the following conditions for the radioimmunoassay were determined in view of the titration curve to determine the degree of dilution of the antiserum for the assay, the incubation period to optimize the assay conditions, the method for separating the bound radiolabeled antigen from the free radiolabeled antigen, etc. .

The dilution solution used was a phosphate buffer (10 mM, pH 7.4) containing 0.5% by weight bovine serum albumin, 140 mM sodium chloride and 25 mM disodium EDTA. The dilution solution (400 | il) and io 100 (xl of the sample or standard solution of human ß-Urogastron and 100 | il of the anti-human ß-Urogastron serum were mixed. After incubating the mixture at 4 ° C for 24 hours were added to the mixture 100 μl 1 of human ß-Urogastron-15 solution labeled with 125 I (approx. 5000 pulses per minute = cpm) After a further incubation of the mixture at 4 ° C. for 48 hours, the resulting mixture was added 100 μl .1 of a second antibody (anti-rabbit-y-globulin goat serum in 20-fold dilution with PBS buffer), 20 100 (il normal rabbit serum (in 200-fold dilution with PBS buffer) and 900 nl of 5 wt. % 10% PBS buffer containing polyethylene glycol was added and then the incubation was continued for 3 hours at 4 ° C. The culture was centrifuged at 3000 25 revolutions per minute for 30 minutes, the supernatant was separated off and the precipitate was counted Immunoreactive sample ver substance as human ß-urogastron was determined from the standard characteristic using standard human ß-urogastron.

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9-2-b) Confirmation of the β-urogastron productivity of the recombinant microorganism

Table 5 shows the result of the radioimmunoassay carried out on the expression system using 35 from the promoter XPl.

Table 5

Expression plasmid

40

Heat induction

Amount of ß-Urogastrons produced (ng / 1 culture)

pUG103-E

Yes

450.2

pUG103-E

No

3.2

pUGU7-E

Yes

388.4

"PUG117-E

No

3.2

comparison

(pBR322)

Yes not ascertainable

50

Table 6 shows the result of the radioimmunoassay performed on the expression system of the fused protein.

55 Table 6

Expression plasmid

Amount of ß-Urogastrons produced (Hg / 1 culture)

60 pUG1004 pUG1301 pUG2101 pUG2301 pUG2701

729.6

650.7 31.3

125.2 119.2

65

Table 7 shows the location of the expressed fused protein.

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Table 7

Expression plasmid Amount of ß-urogastrons produced (Hg / 1 culture)

Periplasmic cytoplasmic

Fraction (O-Sup) Fraction (O-Ppt)

pUG1004 326.0 4.0

pUG1301 347.4 2.8

pUG2101 79.9 10.2

pUG2301 118.8 4.1

pUG2701 65.7 3.6

Tables 5 and 6 show that the system for expressing β-urogastron directly with the promoter A.Pl and the system for expressing the compound as a fused protein both expressed β-urogastron immunoreactivity in Escherichia Coli. Table 7 shows that in the case le of the fused protein, the expressed β-urogastron immunoreactivity is almost completely localized in the periplasm.

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10 sheets of drawings

Claims (24)

670 654 2nd PATENT CLAIMS 1. ß-Urogastron gene with the following nucleotide sequence: A \ T A G C. G A T T C. T G A G T G r C. C. K r T r T T A T C. G C. T A A G A C. T C. A C. G G G T G A C. T C. T C. A C. G A T G G C. T A T T G T C. T G C. A C. A- G A G T G C. T A C. C. G A T A A C. A G A C. G T G G A C. G G T G T T T G C. A T G T A C. A T C. G A A C. T G C. C. A C. A A A C. G T A C. A T G T A G C. T T G C. T T T G G A T A A A T A C. G C. G T G T A A C. C. G A A A C. C. T A T T T A T G C. G C. A C. A T T G T G T G T A G T G G G T T A T A T C. G G T G A A A C. A C. A T C. A C. C. C. A A T A T A G C. C. A C. T T C. G C. T G T C. A A T A C. C. G T G A T C. T G A A A G C. G A C. A G T T A T G G C. A C. T A G A C. T T T T G G T G G G A A T T G C. G T 3 ' f A C. C. A C. C. C. T T T T C. G C. A 5 ' 1 2. Gene according to claim 1, characterized by a restriction enzyme recognition site arranged at the front and / or rear end of the nucleotide sequence according to claim 1. 3. Gene according to claim 1, characterized by a restriction enzyme recognition site and start codon and / or start codon arranged upwards from the nucleotide sequence according to claim 1. 5 ' 3 ' or the stop codon and restriction enzyme recognition site located downward from the nucleotide sequence, the codons and the recognition sites being arranged in the aforementioned order. 4. Gene according to claim 3 with the following nucleotide sequence: 5 -1.1 A A T T C G A A G A T C T G C A T G A A T A G C GC TTC TAG A C G TAC TT A T C G 20 T G C A C G C C A G G T CTG G A C 30 T C T A G A C A C G T G T G T A C A CTG G A C 50 C A C G T G G A C CTG G G T C C A 60 GTT C A A T G C A C G A T G TAC TAC A T G 70 A T C DAY G A A C T T G C T C G A 80 T T G A A C 10th GAT T C T GAG C T A AGA C T C 40 GAT G G C TAT C T A C C G ATA 90 100 GAT A A A TAC G C G T G T A AC T G T G T A C T A T T T A T G CGC A CA T T G AC A CAT 110 120 G T G G G T TAT A T C G G T G A A CGC T G T CAC C C A ATA TAG CCA C T T G C G ACA 130 140 150 C A A TAC C G T GAT CTG A A A T G G T G G GTT A T G G C A C T A GAC T T T ACC ACC 3rd 670 654 160 170 GAA T T G C G T TA A DAY TG A C T T A AC G C A ATT A T C ACT A G A T C T T C T AGA G 3 ' C C T A G 5 ' 5. gene as a subunit for a ß-urogastron gene according to claim 1, characterized in that it has approximately the front half of the nucleotide sequence defined in claim 1. 6. gene according to claim 5, characterized in that A A T T C G A A G G C T T C. G A T T C T G A G C. T A A G A C. T C. G A T G G C T A T C. T A C. C G A T A G T T T G C A T G C. A A A C G T A C. 3 ' C. T A G 5 ' 8. gene as a subunit for a ß-urogastron gene according to claim 1, characterized in that it has approximately the rear half of the nucleotide sequence defined in claim 1. 9. gene according to claim 8, characterized in that 10th it has a restriction enzyme recognition site at its rear end. 7. The gene of claim 6 with the following nucleotide sequence: 15 T G C. A T G A A T A G C. A C. G T A C. T T A T C. G C. C. A C. T G T C. T C. A C. G G T G A C. A G A G T G C. T G C. A C. G A C. G G T G A C. G T G C. T G C. C. A A T C. G A A G C. T T C. G T A G C. T T C. G A A G C. it has a restriction enzyme recognition site at its front end. 10. The gene of claim 9 with the following nucleotide sequence: A T C DAY T G C A C G T G T A C A TAC A T G G A T C. T A G T G C. A C. C. A A G T T G A A C. T T G C. C. T A A A T T T G G T C C A TAC A T G T T G A A C A G TAC A T G TAT ATA C G T G C A C G T G C A 3 ' 5 ' G C G CGC A T C DAY GAT C T A T A A ATT T G T A C A G G T C C A CTG G A C DAY A T C 5 'A 3' A A C T T G G A A C T T A A A T T T T G A ACT G C T DEAD A C A CGC G C G T G G ACC A G A T C T T T G A A C G T \ C A T T G T A C A T G G ACC T C T AGA 11. Recombinant plasmid which has a gene according to claim 4. 12. A method for producing the recombinant plasmid according to claim 11, characterized by the insertion of the gene according to claim 7 and the gene according to claim 10 at an insertion site of a plasmid vector. 13. Recombinant plasmid with a ß-urogastron The gene of claim 4 and further comprising a promoter located upstream of that gene to control expression of the gene and a Shine-Dalgarno sequence linked to promoter 65. 14. Recombinant plasmid with the sequence of the fourth and subsequent base pairs of the β-urogastron gene according to claim 4, namely 670 654 4th 5 'A A T A G C 3' T T A T C G GAT C T A T C T AGA GAG C T C T G C A C G C C A G G T CTG G A C T C T A G A C A C G T G GAT C T A G G C C C G TAT A T A T G T A C A CTG G A C C A C G T G G A C CTG G G T C C A GTT C A A T G C A C G A T G TAC TAC A T G A T C DAY G A A C T T G C T C G A T T G A A C GAT C T A A A A T T T TAC A T G G C G CGC T G T A C A A A C T T G T G T A C A G T A C A T G T G C A C G G T C C A TAT ATA A T C DAY G G T C C A G A A C T T CGC G C G T G T A C A C A A GTT TAC A T G C G T G C A GAT C T A CTG G A C A A A T T T T G G ACC T G G ACC G A A C T T T T G A A C C G T G C A T A A ATT DAY A T C T G A ACT AGA T C T T C T AGA G C CT AG 3 '5 " and with a combination of a promoter, a Shine-Dalgarno sequence and another gene located upwards from this gene. 15. Recombinant plasmid according to claim 14, characterized in that said other gene is a β-lacta-mase gene. 16. Recombinant plasmid according to one of claims 13 or 14, characterized in that the promoter is 1PL or lac UV5. 17. Recombinant plasmid according to one of claims 13 or 14, characterized in that the plasmid vector is pBR322. 18. A transformant comprising a host cell with a recombinant plasmid according to one of claims 11, 13 and 14. 19. Transformant according to claim 18, characterized in that the recombinant plasmid is the recombinant plasmid according to claim 13. 20. Transformant according to claim 18, characterized in that the recombinant plasmid is the recombinant plasmid according to claim 14. 21. Transformant according to claim 18, characterized. that the host cell is Escherichia Coli. 45 22. Transformant according to claim 18, characterized in that the host cell is transformed with a recombinant plasmid having a TcR gene and a cI857 gene. 23. A method for producing a transformant according to claim 18 by transforming a host cell with a recombinant plasmid according to one of claims 11, 13 and 14. 24. A method for producing ß-urogastron, ge-55 characterized by cultivating the transformant according to claim 18 and collecting the expressed ß-urogastron. 60