NL192116C - Beta-urogastron genes, corresponding recombinant plasmids, corresponding transformants and their preparation, and of beta-urogastron. - Google Patents

Description

1 192116 β-urogastron genes, corresponding recombinant plasmids, corresponding transformants and their preparation and of β-urogastron

The present invention relates to novel β-urogastron genes, corresponding recombinant-5 plasmids, corresponding transformants and their preparation, and of β-urogastron.

β-urogastrone is a polypeptide hormone, which is synthesized in the salivary glands of humans, etc. (see, e.g., Heitz et al., Gut, 19,408-413 (1978)), and has a primary structure with 53 amino acids in the following order (see H. Gregory et al., Int. J. Peptide Protein Res., 9, 107-118 (1977)).

Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp 10 Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr lie Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr He Gly Glu Arg Cys Gin Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg.

In the description, amino acids are represented by the following symbols.

15 Asn: Asparagine Ser: Serine

Asp: Aspartic Acid Glu: Glutamic Acid

Leu: leucine His: histidine

Gly: glycine Tyr: tyrosine

Val: valine With: methionine 20 lie: isoleucine Ala: alanine

Lys: lysine Gin: glutamine

Arg: arginine Trp: tryptophan

Phe: phenylalanine Cys: cysteine

Pro: proline 25 β-urogastron has physiological activities such as suppressing gastric acid secretion and promoting cell growth (see Elder et al., Gut, 16, 887-893 (1975)) and is therefore useful for treating ulcers and wounds.

Since β-urogastron is excreted in human urine in small amounts, the compound was previously prepared from urine by extraction, separation and purification. However, this method has the drawback that large amounts of the compound cannot be easily obtained because the compound is a minor constituent of human urine.

European patent application No. 0.046.039 describes the preparation of β-urogastron using a synthetic β-urogastron gene with a specific nudeotide sequence, but does not disclose whether there are other genes capable of producing β-urogastron express. The nucleotide sequence described in this publication differs at 30 triplet codons from the nucleotide sequence of the novel synthetic β-urogastrone gene of the invention.

WO-A-830430 describes two nucleotide sequences of a synthetic urogastron gene, the first of which differs from 20 triplet codons and the second from 28 triplet codons from the nucleotide sequence of the present invention.

40 Proceedings of the National Academy of Sciences of USA, vol. 80, No. 24, December 1983, p.

7461-7465 describes a synthetic urogastrone gene with a nucleotide sequence, which differs from the nucleotide sequence of the invention at 25 triplet codons.

A large number of nucleotide sequences can encode the amino acid sequence of β-urogastron. Nevertheless, it is difficult to predict which such nucleotide sequences are capable of expressing β-urogastron 45 by gene manipulation, or which gene is most suitable for the application of gene manipulation techniques. As a result, many tests and inventive efforts are required to determine the most appropriate nucleotide sequence.

An object of the present invention is to provide a novel β-urogastron gene, which is completely different from the genes described in the above publications with respect to the 50 nucleotide sequence and is capable of expressing β-urogastron through gene manipulation.

Another object of the present invention is to provide new recombinant plasmids and transformants corresponding to the new β-urogastrone gene.

Yet another object of the present invention is to provide a method which allows for the production of β-urogastron in high purity, high-volume production using the new gene by gene manipulation.

These and other objects of the present invention will be explained in the following description.

192116 2

Many tests have been carried out by the applicant and it has been found that a gene I with the following nucleotide sequence makes it possible to achieve the objects of the invention:

Gen I:

5 'AAT AGC GAT TCT GAG TGC CCA CTG

5 3 'TT A TCG CTA AGA CTC ACG GGT GAC

TCT CAC GAT GGC TAT TGT CTG CAC

AGA GTG CTA CCG ATA ACA GAC GTG

10 GAC GGT GTT TGC ATG TAC ATC GAA

CTG CCA CAA ACG TAC ATG TAG CTT

GCT TTG GAT A A A TAC GCG TGT AAC

CGA AAC CTA TT T ATG CGC ACA TTG

15

TGT GTA GTG GGT TAT ATC GGT GAA

ACA CAT CAC CCA ATA TAG CCA CTT

CGC TGT CAA TAC CGT GAT CTG A A A

20 GCG ACA GTT ATG GCA CTA GAC TTT

TGG TGG GAA TTG CGT 3 'ACC ACC CTT AAC GCA 5'

The letters indicate the purine or pyrimidine bases, which form the nucleotide sequence. The symbols used herein for the bases are as follows: A for adenine, G for guanine, C for cytosine and T for thymine.

Gene I is completely new and as such is not obvious and is obtained by determining the specific nucleotide sequence from a very large number of possible nucleotide sequences. Other sequences may be present at the front and / or back of this sequence.

Gene I has the following properties: 1. β-urogastron can be very fully expressed by gene manipulation techniques.

2. The trinucleotide codons, which make up the gene I, are all acceptable to the host cells, in particular Escherichia coli (E.coli), which are readily available with certainty, ensuring a high degree of expression.

3. Specific recognition sites of the restriction enzyme are present in the gene and can be generated at either end thereof and manipulated as desired to facilitate coupling with another gene and incorporation into the plasmid vector.

4. For the preparation of the gene I, the constituent oligonucleotides can be linked into blocks and the blocks can be easily linked into subunits, which are substantially free of undesired coupling, as desired.

5. When expressing β-urogastrone as a fused protein, means are available, whereby an unnecessary portion can be easily removed to obtain the desired β-urogastron.

When β-urogastron is actually to be expressed using the gene I, 45 recognition sites of the restriction enzyme can be placed on the front and / or back of the gene for coupling to the promoter, Shine- Dalgamo sequence (hereinafter referred to as SD sequence), vector, etc. required for the expression. Furthermore, if necessary, a start codon and / or a stop codon can be placed on the front and back of the gene, respectively. The recognition sites, the start codon and the stop codon are not particularly limited, but can be selected as desired.

Below is an example of an extended sequence gene (hereinafter referred to as gene II) which has a restriction enzyme recognition site located at the front of gene I and a start codon and a gene coded at the back of gene I stop codon and restriction enzyme recognition site, wherein the sites and codons are arranged in the order listed, and the 55 gene II further contains other restriction enzyme recognition sites.

Gen II: 3 192116

Start codon -15 -1, 1_

5- Iaat t I c g a a 1g a t c tgc atgJaat agc 5 3- G cl T TC T A g | ACG T A [c TTA TCG

E Ta Bg (S) Mb 10 20 30

10 G I A T T C T GAG TGC CCA CTG TCT CAC

C T A A ~ | g A CTC ACG GGT GAC AGA GTG

Chapter 15 40 50

HOLE GGC TAT TGT CTG CAC GAC GGT

CTA CCG ATA ACA GAC GTG CTG CCA

60 70 80

20 GTT TGC ATG TAC AT | _C_G A Ia G C T T T G

CAA ACG TAC ATG TA G cl TT CGA AAC

Ta Hd 25 90 100

HOLE AAA TA l C G C G TGT AAC TGT GTA

CTA TTT ATG CGC ACA TTG ACA CAT

30 (Th) 110 120

GTG GGT TAT ATC GGT GAA CGC TGT

CAC CCA ATA TAG CCA CTT GCG ACA

35 130 140 150

CAA TAC CGT I G A T C T G AAA TGG TGG

GTT ATG GCA CTA G ~ j A C TTT ACC ACC

40 S

Stop codon 160 _ _ 170

gIa A T T G CGT TAA TAG TGA aIg A T C T

45 C T T A Ale GCA ATT ATC ACT TCT A G Ia E Bg (S) G | _ 3 '50 C C T A G 5'

Ba 55 192116 4

The symbols, which represent the restriction enzymes in the above order, have the following meanings: E: EcoRI, Ta: Taql

Bg: Bglll, S: Sau3AI

5 Mb-.Mboll, Hf: HinFI

Ba: BamHI, Hd: Hindlll

Ml: Mlul, Th: Thai

More specifically by way of illustration, the first subunit may have a subunit A with the front half of gene II and a restriction enzyme (BamHI) recognition site at the back thereof, and the latter subunit may have a subunit B with the back half of gene II. with a restriction enzyme (Hindlll) recognition site at the front thereof. These subunits are shown below.

Subunit A:

5 'AAT TCG AAG ATC TGC AT G AAT AGC

15 3 'GC TT C TAG ACG TAC TT A TCG

HOLE TCT GAG TGC CCA CTG TCT CAC

CTA AGA CTC ACG GGT GAC AGA GTG

20 HOLE GGC TAT TGT CTG CAC GAC GGT

CTA CCG ATA ACA GAC GTG CTG CCA

GTT TGC ATG TAC ATC GAA GCT TCG

CAA ACG TAC ATG TAG CTT CGA AGC

25 3 'CTA G 5'

Subunit B:

5 'AGCT TTG GAT A A A TAC GCG TGT

30 3 'AAC CTA TTT ATG CGC ACA

AAC TGT GTA GTG GGT TAT ATC GGT

TTG ACA CAT CAC CCA ATA TAG CCA

35 GAA CGC TGT CAA TAC CGT GAT CTG

CTT GCG ACA GTT ATG GCA CTA GAC

A A A TGG TGG GAA TTG CGT TAA TAG

TTT ACC ACC CTT AAC GCA ATT ATC

40 TGA AGA TCT G 3 'ACT TCT AGA CCT AG 5'

Subunits A and B are prepared, for example, in the following manner. 11, 13 or 15 base oligonucleotides are prepared (A-1 to A-16 and B-1 to B-16, i.e., 32 oligonucleotides). Then, 4 to 6 of these oligonucleotides are pooled and linked into blocks (block 1 to block 7, i.e. 7 blocks). These oligonucleotides and blocks are shown below.

Block 1: (A-1) (A-2) (A-3) 5 'AATTCGAAGAT CTGCATGAATAGC GATTCT G AGTG 3' 50 3 'GCTTCTAGACGTA CTTATCGCTAA GACTCACGGGTGA 5' (A-16) (A-15) (A-14)

Block 2: (A-4) (A-5) (A-6) 55 5 'CCCACTGTCTCAC GATGGCTATTG TCTGCACGACGGT 3' 3 'CAGAGTGCTAC CGATAACAGACGT GCTGCCACAAA 5' (A-13) (A-12) (A-11) 5 192116

Block 3: (A-7) (A-8) 5 'GTTTGCATGTA CATCGAAGCTTCG 3' 5 3 'CGTACATGTAGCT TCGAAGCCTAG 5' (A-10) (A-9)

Block 4: m (B-1) (B-2) 5 'AGCTTTGGATA AATACGCGTGTAACT 3' 3 'AACCTATTTATGC GCACATTGACACA 5' (B-16) (B-15)

Block5: (B-3) (B-4) 5 'GTGTAGTGGGT TATATCGGTGAACGC 3' 3 'TCACCAATATAG CCACTTGCGACAG 5' (B-14) (B-13)

Block 6: (B-5) (B-6) 5 'TGTCAATACCG TGATCTGAAATGGTG 3' 3 'TTATGGCACTAGA CTTTACCACCCTT 5' (B-12) (B-11)

Block ?: (B-7) (B-8) 5 'GGAATTGCGTT AATAGTGAAGATCTG 3' 3 'AACGCAATTAT CA CTTCTAGACCTAG 5' (B-10) (B-9) 30 Then blocks 1 to 3 are linked together to form the subunit A and the blocks 4 to 7 are coupled together to the subunit B.

The present invention will be described in more detail with reference to the drawings and photos.

Figure 1 schematically shows the synthesis of an oligonucleotide by the solid phase method; Figure 2 shows a method of coupling oligonucleotides A-1 to A-16 to a subunit A and incorporating the subunit into a plasmid pBR322 from E.coli to obtain a recombinant plasmid pUG1; Figure 3 shows a similar method of preparing a recombinant plasmid p1) G2 by incorporating a subunit B into a plasmid pBP322; Figure 4 shows a method for preparing a recombinant plasmid pUG3 from pUG1 and pUG2; Figure 5 shows the result obtained by analyzing the nucleotide sequence of oligonucleotide A-3 by two-dimensional fractionation by electrophoresis and homochromatography; Figure 6 shows a method of preparing a recombinant plasmid pGH37; Figure 7 shows a method of preparing a recombinant plasmid pGH35; Figure 3 shows a method of preparing a recombinant plasmid pEK28; Figure 9 shows methods of preparing recombinant plasmids pUG102 to pUG122 and recombinant plasmids pUG103-E and pUG117-E; Figure 10 shows methods for preparing recombinant plasmids pBRH02 and pBRH03; Figure 11 shows a Mboll restriction scheme of pUG3 with an H fragment (179 b.p.) containing the present β-urogastrone gene; Figure 12 shows a method of preparing recombinant plasmids pUG2301 through pUH2303; Figure 13 shows a method for preparing recombinant plasmids pUG2101 through pUG2105; Figure 14 shows a method of preparing recombinant plasmids pUG2701 through pUG2703; Figure 15 shows a method of preparing recombinant plasmids pUG1102 and pUG1105; Figure 16 shows a method of preparing a recombinant plasmid pUG1004; Figure 17 shows a method of preparing a recombinant plasmid pUG1201; Figure 18 shows a method of preparing a recombinant plasmid pUG1301; and 192116 6 photos 1 to 5 respectively show analytical results of nucleotide sequences of recombinant plasmids obtained, for example, by the Maxam-Gilbert method.

The procedures for building the gene II of the present invention are known per se. The 5 oligonucleotides for building the gene II can be prepared by known methods, for example, by the solid phase method, which will be briefly described below (see, e.g., H. Ito et al., Nucleic Acids Research, 10.1755 -1769 (1982)).

When using the solid phase method, the oligonucleotide as shown in Figure 1 is prepared by sequentially coupling mononucleotides or dinudeotides with a nucleoside supported on polystyrene resin to obtain a predetermined sequence of nucleotides.

For example, the resin for supporting the nucleoside can be prepared using a partially cross-linked polystyrene resin by reacting N- (chloromethyl) phthalimide with the resin, reacting hydrazine with the product to obtain aminomethyl-containing polystyrene resin, and amino group thereof to bind a nucleoside with the 5'-hydroxyl group in free form and the amino group 15 protected using succinic acid as spacer.

On the other hand, various methods of preparing mononucleotides or dinudeotides are known (see, e.g., C. Broka et al., Nucleic Adds Research, 8, 5461-5471 (1980)). For example, a mononucleotide can be prepared by reacting o-chlorophenylphosphorodichloridate, triazole and a nucleoside, the 5'-hydroxyl group of which is protected with a dimethoxytrityl group (DMTr) in the presence of triethylamine, and the resulting monotriazolide is reacted with β cyanoethanol in the presence of 1-methylimidazole as a catalyst and the reaction product elutes with chloroform-methanol by column chromatography over silica gel. This method produces 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 obtain the mononucleotide with a free 5'-hydroxyl group, which is reacted with the previously obtained monotriazolide, and eluting the reaction product with chloroform-methanol by column chromatography on silica gel. This method produces a fully protected dinucleotide.

The synthesis of solid phase oligonucleotides is advantageously performed using a DNA synthesizer, which is available, for example, as a DNA synthesizer from Bachem Inc., U.S.A. The nudeoside supporting resin obtained above is placed in a reaction vessel and washed with dichloromethane isopropanol, and a solution of zinc bromide in dichloromethane isopropanol is added to the resin to remove 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 dimethylfor-35 mamide to remove the remaining Zn2 *, then washed with tetrahydnofuran and exposed to a stream of nitrogen gas for several minutes to dry it. Separately, the fully protected dinucleotide or mononucleotide is dissolved in pyridine followed by addition of triethylamine and the resulting solution is shaken, left to stand at room temperature for several hours and then evaporated under reduced pressure. The resulting triethyl-40 ammonium salt is dissolved in pyridine and azeotroped several times with pyridine to dry it. The nucleotide salt is dissolved in a solution of mesitylene sulfonyl-5-nitrotriazole (MSNT, condensing agent) 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 using methyl aminopyridine as a catalyst in tetrahydrofuran pyridine to mask the unreacted hydroxyl group. Finally, the resin is washed with pyridine to complete one cycle of the solid phase synthesis. One cycle extends the nucleotide sequence by one or two chain lengths. The above procedure is repeated to successively couple mononucleotides or dinudeotides to the resin to the desired length, thereby obtaining a fully protected oligonucleotide supported on the resin.

A solution of tetramethylguanidine-2-pyridine aldoximate in pyridine water is added to the resulting resin and the mixture is allowed to stand under heating. The resin is then filtered off and washed alternately with pyridine and ethanol. The washings and the filtrate are combined and concentrated under reduced pressure. The concentrate is dissolved in an aqueous solution of 55 triethyl ammonium bicarbonate (TEAB) followed by washing with ether. The aqueous solution is subjected to Sephadex G-50 column chromatography using a TEAB solution as an eluent. The fractions are collected and the optical density of each fraction is determined at 260 nm 7 192116. A fraction with first elution peak is concentrated. The concentrate is purified, for example, by rapid liquid chromatography, until a single peak is obtained. The oligonucleotide thus obtained is still protected in the 5 'position by a dimethoxytrityl group, so that the product is treated with an aqueous solution of acetic acid to remove the protecting group, followed again by rapid liquid chromatography or the like for purification into a single peak is obtained.

The desired oligonucleotides are prepared according to the method described above and then individually examined for the nucleotide sequence by a two-dimensional fractionation method using electrophoresis and homochromatography and (or) the Maxarrt-10 Gilbert method and then used to prepare the blocks and subunits.

The two-dimensional fractionation method for determining nucleotide sequence can be performed according to the method of Wu et al. (E. Jay, R.A. Bambara, R. Padmanabhan and R. Wu, Nucleic Acids Res., 1, 331 (1974)).

For the performance of this method, the oligonucleotide is dissolved in freeze-dried state in distilled water to a concentration of about 0.1 pg / μΙ. A portion of this solution is treated with γ-32Ρ-ΑΤΡ and T4 polynucleotide kinase to label the 5 'end with 32P and then partially digested with snake venom phosphodiesterase. The product is dripped onto a cellulose acetate film and subjected to first dimensional development electrophoresis to separate the product from the difference in bases. The developed products are then transferred to a diethylaminoethyl cellulose (DEAE cellulose) plate and subjected to the second dimensional development using a solution of partially hydrolysed RNA called homomix. (This procedure is called homochromatography.) Thus, the oligonucleotide is separated according to the chain length. The nucleotide sequence of the oligonucleotide is then determined from the 5 'end by autoradiography.

25 When it is difficult to determine the sequence by this method, the Maxam-Gilbert method is used if necessary. (A.M. Maxam and W. Gilbert, Proc. Natl. Acad. Set., USA, 74, 560 (1977), A.M. Maxam and W. Gilbert, Methods in Enzymol., Vol 65, p. 499, Academy Press 1980.

This method, also referred to as a chemical decomposition method, uses a particular base-specific reaction to cleave the oligonucleotide at the site of the base, and the bands shown by electrophoresis serve to sequence from 5 'or 3 'end to be determined. 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 in a low salt concentration, but cytosine only reacts with hydrazine in a high salt concentration. After the completion of the reactions for the four bases, each reaction mixture is reacted with piperidine to replace the base where the ring is opened, and to catalyze the β-elimination of both phosphates from the sugar, and finally the DNA strand spliced at this base. The resulting reaction mixtures are respectively subjected to polyacrylamide gel electrophoresis to determine the nucleotide sequence depending on which of the reactions each band produced.

40 Next, the oligonucleotides are coupled using a T4 DNA ligase as shown in figure 2. For proper coupling, the 16 oligonucleotides A-1 to A-16 corresponding to the subunit A are divided into three sets, ie the block 1 with A-1, A-2, A-3, A-14, A-15 and A-16, block 2 with A-4, A-5, A-6, A-11, A-12 and A -13, and the block 3 coupled with A-7, A-8, A-9 and A-10 as shown in Figure 2. By electrophoresis, blocks 1 to 3 are obtained with the correct sequences and 45 are further coupled to the subunit A.

More specifically, some of the 5 'ends of the 16 oligonucleotides A-1 to A-16 are labeled with 32P using γ-32Ρ-ΑΤΡ and T4 polynucleotide kinase and the hydroxyl groups of the remaining 5' ends phosphorylated with ATP. To form each of the three blocks, the oligonucleotides are combined and coupled using T4 DNA ligase, and the product is subjected to polyacrylamide gel electrophoresis to isolate the desired block. The three btocks thus obtained are coupled using T4 DNA ligase to form the subunit A. Although a dimer structure can be formed in the coupling reaction, it is easily cleaved with the restriction enzymes EcoRI and BamHI to obtain the subunit A. Then As shown in Figure 2, a known plasmid vector, pBR322, which is from E. coli and is readily available 55, is cleaved with EcoRI and BamHI and Subunit A is included in the vector to obtain a recombinant plasmid pUG1.

The same procedure as hivivore is also applied for the subunit B. As in the case of 192116 8 of the subunit A, the 16 oligonucleotides B-1 to B-16 are divided into four pairs as shown in figure 3 coupled and the blocks are coupled together to form subunit B. The dimer, if formed, is cleaved with the restriction enzymes Hindlll and BamHI to obtain the subunit B. A plasmid vector, pBR322, is cleaved with Hindlll and BamHI and the subunit B5 is included in the vector to obtain a recombinant plasmid pUG2 as shown in Figure 3.

Furthermore, as shown in Figure 4, pUG1 is cleaved with restriction enzymes Hindlll and Sail and a fragment removed from pUG2 using the same restriction enzymes is incorporated into pUG1 to form a recombinant plasmid pUG3 with a β-urogastrone structure. gene (gene II).

PUG1, pUG2 and pUG3 are recombinant plasmids, each of which is pBR322 and the subunit A, which is the front half of the β-urogastron structural gene, the subunit B, which is the back half of the gene, or all of the structural gene. These recombinant plasmids can be multiplied in large quantities by incorporating them into a host such as the E. coli strain HB101, which is known and readily available by the calcium method as transformation method (E. Lederberg and S.

Cohen, J. Bacteriol., 119, 1072 (1974)).

Whether pUG1, pUG2 and pUG3 are present in the host, such as E.coli strain HB101, can be determined by the following methods. After the plasmids are collected by the alkaline extraction method, pUG1 and pUG2 are checked for the presence of the BglII recognition site, which is not present in the vector pBR322. Similarly, pUG2 and pUG3 are checked for whether they can be cleaved with Mlul, which is not present in pBR322.

According to the alkaline extraction method, E. coli, in which the plasmid is housed, is incubated, after which the cells are collected and lysozyme is allowed to act to dissolve the cell wall. A mixture of sodium hydroxide and sodium lauryl sulfate is used to break the cell and then denature the DNA, which is then neutralized with sodium acetate buffer. Hereby, the chromosome DNA remains denatured, but it acquires the plasmid, which is a non-chromosome DNA, in its original double strand form. Plasmids are collected using these properties. The plasmids are further subjected to ultracentrifugation with a density gradient with cesium chloride and ethidium bromide for purification and then passed through a biogel A 50m column to remove RNA. For example, high purity plasmids can be obtained in a large amount. In this way, the β-urogastrone gene of the invention (gene II) can be obtained.

Next, the method of incorporating the β-urogastrone gene into host cells will be described.

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

The ways of expressing the β-urogastron gene using E.coli includes a system for directly expressing β-urogastron, and a system in which it is expressed as a fused protein with β-lactamase or a other protein.

For the direct expression of β-urogastron gene, it is necessary to include a promoter and an SD sequence upstream of the β-urogastron gene 40 in the recombinant plasmid. Because the promoter is not particularly limited, desirable promoters are those that ensure a high level of expression, such as XPL, which is the left promoter of γ bacteriophage, lac UV5, which is present upstream of β-galactosidase gene from E.coli. , etc. When XPL is used as a promoter, the SD sequence is not particularly limited, but it is desirable to use the four base sequence of AGGA. Furthermore, when using LAC UV5 as a promoter, it is desirable to use the SD sequence occurring upstream of the LAC UV5 promoter, or the one that is chemically synthesized.

The system for direct expression of the β-urogastron gene will be described using the case where XPL-SD sequence γ-urogastron gene is used. Although λΡ ,. is a potent promoter (J. Hedgpeth et al., Molecular and General Genetics, 163,197-203 (1978)) causes the fully 50 activated λΡ ^ ρΓοπιοίοΓ effects on the E.coli host cell so that it is necessary to place the cell under a of any Iethal operation, multiply free circumstance and then run the XPL. On the other hand, CI857, which is a gene in λ bacteriophage, is one of the mutated genes of Cl repressor, which acts on the operator for λΡί. At low temperatures (up to about 30 ° C), the CI857 repressor binds to the operator, completely inhibiting the activity of λΡί as a promoter, allowing 55 to multiply E.coli. As a result, the host cells are allowed to multiply in this state and then brought to a high temperature (not lower than 37 ° C), allowing the XPL to operate. Furthermore, the plasmid vectors, such as pSC101, which are known and readily available, with a strict replication mechanism, and those such as pBR322 with a moderate replication mechanism, are not incompatible with one another, but may coexist in the same E. coli cell.

As a result, it is suitable to build a recombinant plasmid pGH37, in which a 5 CI857 gene has been incorporated into a tetracycline resistant plasmid vector pSC101 (with lac UV5 promoter arranged upstream of it for efficient expression of CI857) as shown in Figure 6 , and incorporating the recombinant plasmid into E.coli (strain HB101) to obtain a transformant (strain ECI-2) to host the vector for the expression of β-urogastrone under the λΡ | _ promoter .

For example, according to the present invention, the \ PL-SD sequence β-urogastron gene is incorporated into pBR322 to obtain a β-urogastron expressing vector, which is used for the transformation of the strain ECI-2, resulting in a so-called two plasmid system is obtained, in which two useful plasmids coexist in an E. coli cell.

With this system, the pGH37 encoded CI857 repressor binds to the operator for the XPL promoter on the second plasmid when, for example, the cell is grown at 30 ° C, allowing the cell to multiply. For example, after the cell has fully multiplied in this state, the temperature is raised to 40 ° C, whereupon the operator's CI857 repressor is separated, allowing the activity of the XPL promoter for the expression of β-urogastrone.

Although a similar design has been used for the expression of fibroblast interferon, SV-40 Small t antigen, etc., in these cases a λ lysogen is used as a host, incorporating the DNA of λ bacteriophage with a CI857 gene. in the host chromosome (R. Derynck et af,

Nature, 287, 193-197 (1980), C. Derom et al, Gene, 17, 45-54 (1982), K. Küpper et al, Nature, 289, 555-559 (1981)).

However, with the system of the present invention, the CI857 gene is incorporated into a different plasmid resistant to tetracycline. Consequently, the present system has the advantages that it is unlikely that the λ bacteriophage contained in the host chromosome will be propagated and the strain can be easily monitored. Naturally, the two-plasmid system is used for the first time for systems for the expression of β-urogastrone.

According to another system, a portion of another protein gene such as β-lactamase gene is linked 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 susceptible to decomposition by the protease into the E. coli and consequently provides protection for the β-urogastrone. Another advantage is that the fused protein migrates to and accumulates in the periplasm in the E. coli cell (SJ Chan et al., Proc. Natl. Acad. Sci., USA, 78, 5401-5405 (1981). ), is locally present, and is therefore easy to separate and purify.

More specifically, a gene coding for two basic amino acids, which can provide a cleavage site for the removal of β-urogastron from the fused protein by cleavage with an enzyme, is incorporated into the β-lactamase gene at an appropriate restriction. enzyme cleavage site, and a β-urogastron gene is linked to the β-lactamase gene.

Preferably, the order of the two basic amino acids is -Lys-Arg- or Arg-Lys-. Examples of enzymes for recognizing the amino acid sequence for cleaving β-urogastron from the fused protein are kallikrein, trypsin, etc. Examples of restriction enzymes for cleaving the 45 β-lactamase gene are Xmnl, Hindi, Seal, Pvul , Pstl, Bgll, Banl, etc.

The β-Ιθοίθπιββθ-β-υΗ ^ ββίΓοη recombinant plasmid thus prepared can express a fused protein in E.coli for production. The fused protein obtained is treated with kallikrein or the like, whereby β-urogastron can be obtained.

The expression system can be checked by direct analysis of the nucleotide sequence of the 50 gene by the Maxam-Gilbert method, by determining the gene uptake and its direction by the mini-preparation or design method (HC Bimboim et al., Nucleic Acids Research, 7, 1513-1523 (1979)), or by radioimmunoassay of β-urogastrone.

The transformant of the present invention thus obtained is cultivated by the conventional method, whereby high purity β-urogastron can be obtained in a large amount. The present invention will now be further described by way of the following example, to which the invention is in no way limited.

192116 10

Example 1. Preparation of the nucleoside bearing resin.

Various nucleoside-bearing resins were prepared according to the following method.

1% by weight of cross-linked polystyrene resin "S-XI" (product of BIO.RAD Laboratories, USA 5, 200 to 400 mesh) was mixed with 2.41 g of N- (chloromethyl) phthalimide, 0.22 ml of trifluorom -thanesulfonic acid and 50 ml of dichloromethane by stirring at room temperature for 2 hours. After the completion of the reaction, the resin was filtered off, washed successively with dichloromethane, ethanol and methanol, dried under reduced pressure and then refluxed overnight with 50 ml of a 5 wt% hydrazine solution in ethanol. The resin was filtered off, washed successively with ethanol, dichloromethane and methanol and then dried under reduced pressure. The mixture of polystyrene resin (2.5 g) containing aminomethyl groups thus obtained, 0.75 mmol of monobamic acid ester of 5'-o-dimethoxytrityl nucleoside, 1.23 mmol of dicyclohexylcarbodiimide and 1 mmol of dimethylaminopyridine was left overnight at room temperature with the addition of 30 ml of dichloromethane. The resin was filtered off, washed successively with dichloromethane, methanol and pyridine, then immersed in pyridine acetic anhydride (90:10 volume ratio) and left to stand at room temperature for 30 minutes. The resulting nucleoside-bearing resin was filtered off, washed with pyridine and dichloromethane and dried under reduced pressure for use in the solid phase synthesis reaction.

2. Synthesis of dinucleotide.

For example, the synthesis of a fully protected dinucleotide with the base order of TA will be described. Adenosine (13.14 g) with the 5'-hydroxyl group protected with a dimethoxytriethyl (DMTr) group and the amino group protected with a benzoyl group, and 6.34 g triazole were dissolved in anhydrous dioxane. Under ice-cooling, 8.35 ml of triethylamine was added to the solution, after which 6.86 g of o-chlorophenylphosford dichloridate 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 off, the filtrate was concentrated to about 2/3 by volume and 3.6 g of β-cyanoethanol and 4.8 g of 1-methylimidazole were mixed with the concentrate by 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 a 0.1 M aqueous solution of dibasic sodium phosphate and twice with water and then concentrated under reduced pressure to obtain 19.96 g of crude product. The product was purified by silica gel column chromatography using chloroform-methanol (98: 2 volume ratio) as eluent. Purification was repeated to obtain 15.12 g of fully protected adenosine mononucleotide.

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

Thymidine (1.64 g) with the 5'-hydroxyl group protected with a dimethoxytrityl group and 0.95 g of triazole were dissolved in 21 ml of anhydrous dioxane, 1.25 ml of triethylamine were added to the solution and the mixture was stirred for 5 minutes stirring and cooling with ice, 0.69 ml of 45 o-chlorophenylphosphorus dichloridate were added dropwise. The mixture was then stirred at room temperature for 1 hour. The triethylamine hydrochloride obtained in the reaction was filtered off and the filtrate was stirred with 1.1 ml of an aqueous pyridine solution (1 M) for 10 minutes. To the solution was added a dioxane solution (10 ml) of 1.17 g of the adenosine mononucleotide with the free 5'-hydroxyl group prepared as described above and 0.72 ml of 1-methylimidazole, and the mixture was stirred at room temperature for 3 hours. -50 stirred. The resulting reaction mixture was concentrated under reduced pressure, the residue was dissolved in ethyl acetate and the solution was washed with an aqueous solution of dibasic sodium phosphate (0.1 M) and then concentrated with water and under reduced pressure to give 2.39 g of crude product was obtained. The product was subjected to silica gel column chromatography and eluted with chloroform-methanol (98: 2 volume ratio) to obtain 2.39 g of fully protected 55 dinucleotide TA.

Different nucleotrides were prepared in the same manner.

3. Synthesis of oligonucleotide.

11 192116

The solid phase synthesis of the oligonucleotide A-1, i.e. undecanucleotide AATTCGAAGAT will be described.

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

The fully protected dinucleotide GA (50 mg) prepared as described above was dissolved in 1 ml pyridine, shaken with 1 ml triethylamine and left at room temperature for several hours. The solution was then evaporated under reduced pressure. The residue was azeotroped several times with pyridine to convert the nucleotide into a triethyl-15 ammonium salt. The salt was dissolved in 0.3 ml of a 0.3 M solution of mesitylene sulfonyl-5-nitrotriazole in pyridine. The solution was added to the dried resin and allowed to react at room temperature for 60 minutes. The liquid portion was filtered from the reaction mixture and the solid portion was washed with pyridine, then left to stand for 5 minutes in the mixture of 0.2 ml acetic anhydride and 0.8 ml 0.1M solution of dimethylaminopyridine in tetrahydrofuranpyridine. 20 dine 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 two bases. The same procedure was repeated to successively couple dinucleotides AA, CG, TT and AA by condensation with the resulting nucleotide, thereby preparing the fully protected undecanucleotide AATTCGAAGAT carried by the resin.

The resulting resin (20 mg) was allowed to stand at 40 ° C for 1 hour with 0.6 ml of a 0.5 M solution of tetramethylguanidine-2-pyridine aldoximate in pyridine water (volume ratio 90:10). The resin was then passed through a cotton-filled pasteur pipette and filtered through it. The resin was washed alternately with pyridine and ethanol. The washings and filtrate were combined and concentrated under reduced pressure at 40 ° C. The residue was dissolved in 2 ml of an aqueous solution of triethylammonium bicarbonate (TEAB, 10 mmol). The solution was washed three times with ether. The aqueous phase was transferred to a Sephadex G-50 column (2 x 100 cm) and eluted with 10 mM TEAB solution. The fractions were checked for the absorbance at 260 nm. The fraction with the first eluate peak was concentrated. The residue was subjected to rapid liquid chromatography (pump: model 6000A, detector: model 440, products of Waters Associates, U.S.A.) to obtain a purified fraction with a single ek. The column used for the rapid liquid chromatography was μ-Bondapak C18 (product of Waters Associates, USA), and a 0.1 M solution of triethylammonium acetate in aqueous acetonitrile was used as eluent for gradient elution (5 - 40 vol%). . The undecanucleotide thus purified was still protected in the 5 'position with the dimethoxytrityl group, so that the compound was treated with an 80 vol.% 40 aqueous acetic acid solution for 15 minutes to remove the dimethoxytrityl group and then purified again by rapid liquid chromatography until a single peak was obtained. Use was made for this purpose of the same column as in the foregoing, and a 0.1 M solution of triethylammonium acetate in aqueous acetonitrile was used for the gradient elution (5 -> 25% by volume).

Oligonucleotides A-2 to A-16 and B-1 to B-16 were synthesized in the same manner as before.

Table 1 shows the yield of each oligonucleotide determined using 20 mg of the solid phase synthesis resin, by separating the oligonucleotide from the resin, followed by deprotection and purification.

The yield was calculated from the determination of the absorbance of the final purified product at 50-260 nm and the sum of the absorbance values for the nucleotide bases.

TABLE 1 A-1 80 pg A-2 120 pg A-3 90 pg A-4 50 pg 55 A-5 140 pg A-6 70 pg A-7 80 pg A-8 90 pg A-9 100 pg A- 10 90 pg A-11 110 pg A-12 40 pg 192116 12 TABLE 1 (continued) A-13 50 pg A-14 40 pg A-15 60 pg A-16 150 pg B-1 60 pg B-2 100 pg B-3 50 pg B-4 90 pg 5 B-5 100 pg B-6 90 pg B-7 130 pg B-8 100 pg B-9 110 pg B-10 100 pg B-11 110 pg B-12 110 pg B-13 130 pg B-14 60 pg B-15 70 pg B-16 50 pg 10 4. Checking the order of the oligonucleotide bases.

The sequence was checked according to the aforementioned two-dimensional fractionation by electrophoresis and homochromatography from Wu et al.

Each of oligonucleotides A-1 to A-16 and B-1 to B-16 was found to have the desired nucleotide sequence. Figure 5 shows the result obtained by analysis of the oligonucleotide A-3, where it was found that A-3 had the sequence GATTCTGAGTG seen from the 5 'end.

The nucleotide sequence of each oligonucleotide was also checked by the aforementioned Maxam-Gilbert method.

Oligonucleotides A-1 to A-16 and B-1 to B-16 were each determined to have the desired nucleotide sequence.

20 5. Construction of oligonudeotide blocks and subunits.

The blocks and subunits were prepared in the manner illustrated in Figure 2 and described in more detail below.

First, about 5 µg of each of the oligonucleotides A-1, A-2, A-3, A-14, A-15 and A-16 were dissolved in distilled water (50 µl) to give a solution of a concentration of about 0.1 pg / pl.

The six kinds of aqueous solutions were each placed in six Eppendorf tubes in an amount of 10 µl (1 µg calculated as DNA). A mixed solution (6 µl) with 250 mM tris-HCl (pH 7.6), 50 mM magnesium chloride, 10 mM spermine and 50 mM DTT was placed in each tube, followed by addition of 0.5 µl aqueous γ-32p- ATP solution (product of Amersham International Ltd., England), 0.5 µl of T4 polynucleotide kinase (product of Takara Shuzo Co., Ltd., Japan) and 13 µl of distilled water, to give 30 µl of mixture. The mixture was allowed to react at 37 ° C for 30 minutes and then reacted for an additional 30 minutes adding 1 µl of aqueous 30 mM ATP solution. The reaction was stopped by heating at 100 ° C for 2 minutes. The reaction mixture was quickly cooled with ice. The oligonucleotides A-1, A-2, A-3, A-14, A-15 and A-16 with their thus phosphorilated 5 'sites were each in an amount of 10 µl in a single Eppendorf tube of 1, 5 ml. 40 µl of a 35 aqueous 250 mM tris-HCl solution (pH 7.6), 40 µl of 50 mM magnesium chloride and 35 µl of distilled water were introduced into the tube to give a total amount of 175 µl. The mixture was heated at 90 ° C for 2 minutes and then gradually cooled to room temperature. With the addition of 10 µl aqueous 200 mM DTT solution, 10 µl aqueous 20 mM ATP solution and 5 µl (100 units) T4 DNA ligase (product of Nippon GEne Co., Ltd., Japan) the mixture was left overnight at 4 ° C to prepare block 1 of 40 coupled oligonucleotides A-1, A-2, A-3, A-14, A-15 and A-16.

Blocks 2 and 3 were similarly formed by coupling A-4, A-5, A-6, A-11, A-12 and A-13 and by coupling A-7, A-8, A- 9 and A-10.

Ethanol was added in twice the volume by volume to the reaction mixture of the blocks thus prepared, and the mixture was allowed to stand at -80 ° C for 30 minutes to precipitate DNA, followed by electrophoresis at 12.5 wt% polyacrylamide gel and autoradiography. This led to 72 b.p. (base pair) and 36 b.p. for block 1, a band in place of 36 b.p. for block 2 and tires at the positions of 48 b.p. and 24 b.p. for block 3. Then, each band was cut and a mixture of 10 mM tris-HCl (pH 7.6) and 10 mM EDTA in aqueous solution (tris-EDTA) was added. The mixture was then left overnight at room temperature for extraction. The resulting mixture was centrifuged, the supernatant was separated and the supernatant was shaken thoroughly with tris-EDTA saturated phenol then centrifuged to remove the bottom layer. The same procedure was repeated twice with tris-EDTA saturated phenol. Finally, the top layer was passed through a Sephadex G-50 packed column with a diameter of 1 cm and a length of 20 cm to remove the phenol and acrylamide. The eluate 55 was then concentrated to 200 µl and left to stand at -80 ° C for 30 minutes with twice the volume of ethanol to precipitate DNA.

The three blocks obtained were combined. 50 mM tris-HCl (pH 7.6) 10 mM was added to the mixture

13 192116 magnesium chloride, 20 mM DTT, 1 mM ATP and 5 μΙ (100 units) T4 DNA ligase were added. The resulting mixture was left overnight at 4 ° C for coupling. Twice the volume of ethanol was added to the mixture and the mixture was allowed to stand at -80 ° C for 30 minutes to precipitate DNA, followed by electrophoresis on 8 wt% polyacrylamide gel and autoradiography, yielding bands at 5 96 b.p. and 192 b.p. were shown. Each band was cut and tris-EDTA was added, and the mixture was left overnight at room temperature for extraction. The mixture was centrifuged to separate the supernatant, the supernatant was shaken thoroughly with tris-EDTA saturated phenol, and the bottom layer discarded. This procedure was repeated twice with further addition of tris-EDTA saturated phenol. The top layer was passed through a 10 Sephadex G-50 column, the eluate was concentrated and twice the volume of ethanol was added to the concentrate, followed by standing at -80 ° C for 30 minutes to precipitate DNA. The obtained product was cleaved with EcoRI and BamHI to obtain the subunit A.

The same procedure as above was repeated as shown in Figure 3 to couple oligonudeotides B-1, B-2, B-15 and B-16 to block 4, oligonudeotides B-3, B-4, B-13 and B-15 to link to block 5, oligonudeotides B-5, B-6, B-11 and B-12 to link to block 6 and oligonudeotides B-7, B-8, B-9 and B-10 link to block 7. The blocks corresponding to 26 bp and 52 b.p. were collected and similarly coupled to form 104 b.p. products. and 208 b.p., which were split with Hind III and BamHI. Thus, the subunit B was obtained.

6. Cloning of subunits and analysis of recombinant plasmids.

Referring to Figure 2, pBR322 was cleaved with EcoRI and BamHI and phosphate groups with alkaline phosphatase (product of Takara Shuzo Co., Ltd., Japan) were removed from the 5 'ends so as not to restore the original state. Then the thus split and dephosphorylated pBR322 and the subunit A were left overnight at 4 ° C in a mixture of 50 mM tris-HCl (pH 7.6), 10 mM magnesium chloride, 20 mM DTT and 1 mM ATP with the addition of 5 μΙ T4 DNA ligase are standing, so they were linked. Twice volume of ethanol was added to the reaction mixture and the mixture was allowed to stand at -80 ° C for 30 minutes to precipitate. The mixture was then centrifuged, the precipitate dried and dissolved in 100 µl of distilled water to give a plasmid pUG1 in which the subunit A was contained in pBR322.

The E.coli strain HB101 was transformed with the plasmid pUG1 according to the calcium method.

The host strain HB101 was grown at 37 ° C in 50 ml LB culture medium (1 wt% bactotrypton, 0.5 wt% yeast extract and 0.5 wt% sodium chloride). When the absorbance at 610 nm reached 0.25, 40 ml of the broth was transferred to a centrifuge tube and centrifuged at 6000 rpm for 10 minutes at 4 ° C. The supernatant was discarded, the precipitate was suspended in 20 ml of ice-cooled 0.1 M magnesium chloride solution, the suspension was centrifuged again under the same conditions and the supernatant was discarded.

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 cooled for 1 hour with ice. The suspension was centrifuged, the supernatant was removed and the precipitate was suspended in 2 ml of an ice-cooled solution of 0.1 M calcium chloride and 0.05M magnesium chloride. To an amount of 200 μΙ of the 40 suspension was added 10 μΙ of an aqueous solution of pUG1 and the mixture was cooled with ice for 1 hour and then heated at 43.5 ° C in a water bath for 30 seconds. 2.8 ml of LB culture medium was then 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 at 200 µl / dish and incubated overnight at $ 37 ° C. The growing colonies were checked by further transplantation on an LB plate 45 with 50 µg / ml ampicillin and also on an LB plate with 20 µg / ml tetracycline and incubated overnight at 37 ° C. Only the ampicillin-resistant colonies were separated to obtain a transformed cell.

Plasmids were collected from the cell on a small scale by the alkaline extraction method and checked for the presence of a BGIII cleavage site. One of the cells containing the plasmid 50 with a BglII cleavage site was grown extensively to obtain similarly purified plasmid pUG1 using the alkaline extraction method.

The nucleotide sequence of the subunit A incorporated in the resulting pUG1 was analyzed on both strands using the aforementioned Maxam-Gilbert method.

Photos 1 and 2 show the results of the analysis. Lanes 1 through 4 are the result of the 55 electrophoresis for the EcoRI-Sall fragment and lanes 5 through 8 for the BamHI-Pstl fragment. Lanes 1 and 5 show the reaction products for guanine, lanes 2 and 6 show the reaction products for guanine + adenine, lanes 3 and 7 show the reaction products for thymine + cytosine, and lanes 4 and 8 show the reaction products for cytosine. Photo 2 shows the results obtained using the same samples, in which an area of the higher molecular weight portion (corresponding to the top portion of photo 1) is enlarged. Thus, the nucleotide sequence of the subunit A was determined.

Referring to Figure 3, the plasmid pBR322 was cleaved with HindIII and BamHI and the larger fragment was isolated by electrophoresis and coupled to the subunit B. Thus, a plasmid p (JG2 was obtained, in which subunit B was incorporated into pBR322 in a similar manner as in the case of pUG1. Using the obtained plasmid p (JG2, the strain HB101 was transformed and only the ampicillin resistant colonies were selected. Plasmids were collected from the colonies and then checked for the presence of a BglII cleavage site and a Mlul cleavage site Cells containing the plasmid with both sites were selected One of the selected cells was grown widely to obtain purified plasmid pUG2. The nucleotide sequence of the subunit B in pUG2 was analyzed on both strands using the Maxam-Gilbert method.

Photo 3 shows the results of the analysis. Lanes 1 through 4 show the result obtained by the Hindll-Sall fragment 15. Lanes 5 through 8 show the result obtained with the same sample, in which an area of the higher molecular weight portion (corresponding to the upper portion of lanes 1 through 4) is shown in an enlarged scale. Each lane shows the same corresponding reaction product as in photo 1. Thus, the nucleotide sequence of the subunit B was determined.

Then, referring to Figure 4, pUG1 was cleaved with Hindlll and Sail and a larger fragment was separated using a 1.5m biogol column. PUG2 was cleaved with Hindlll and Sail, followed by electrophoresis to obtain a smaller fragment. The two fragments were combined and treated with T4 DNA ligase before coupling to obtain the plasmid pUG3, in which the subunits A plus B, i.e., β-urogastron gene, were contained in pBR322. The E.ooli strain HB101 was transformed using the plasmid pUG3. The transformant has been deposited under the Budapest Treaty regarding international recognition of filings under the number FERM BP 543 with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

In the above case, cells containing only ampicillin resistant plasmid pUG3 were also used. with a Mlul housing splitting site, selected. One of the selected cells were grown widely to obtain purified plasmid pUG3. The nucleotide sequence of the β-urogastrone gene in pUG3 was analyzed on both strands using the Maxam-Gilbert method.

Photo 4 shows the results of the analysis. Lanes 1 to 4 show the result obtained with the BamHI-Pstl fragment. Lanes 5 to 8 show the result obtained with the same sample, in which a region of the higher molecular weight portion (corresponding to the upper portion of lanes 1 to 4) is shown in large scale. The reaction products of the lanes are the same as the corresponding products in photo 1. The analysis confirmed the nucleotide sequence of the β-urogastron gene.

7. Expression vector with XPL promoter.

The XPL promoter, the left promoter of λ bacteriophage, was used to express 40 β-urogastron, as will be further described below.

First, the preparation of a strain ECI-2 derived from E.coli strain HB101 will be described. ECI-2 hosted λ-PL expression plasmids. Next, donating a DNA fragment with XPL promoter from the DNA of λ-0Ι85737, which is a mutant of λ bacteriophage, and the construction of expression plasmids from the donated DNA will be described. Furthermore, the expression of 45 β-urogastron gene in the ECI-2 host strain will be described by the λΡί-ρπ> ιηοΙοΓ.

7-1. Construction of the strain ECI-2.

The strain ECI-2 is E.coli HB101, which houses a plasmid pGH37 for the expression of CI857 gene. pGH37 was prepared according to the method shown in Figure 6. First, DNA from λΟΙ85737 was cleaved with Bglll. Then, the cohesive ends were digested at the cleavage site using 50 S1 nudease. 1 µg of BglII cleaved DNA from XCI857S7 was reacted for 30 minutes at 20 ° C with 200 units of S1 nuclease in 100 µl of an aqueous solution (pH 4.5) containing 200 mM sodium chloride, 30 mM sodium acetate and 5 mM zinc sulfate. The blunt-ended DNA fragments thus obtained were subjected to an electrophoresis with 1.0 wt.% Agarose gel to obtain a fragment of 2385 b.p. with the entire CI857 structural gene. The fragment was recorded at the Pvull cleavage-55 site of plasmids pGL101 to form a plasmid pGH36, which expresses the CI857 gene under the direction of a promoter, lac UV5. Then pGH36 is cleaved with two restriction enzymes, EcoRI and Pstl, to obtain a fragment containing 1193 b.p., which was introduced into a plasmid pSC101 192116 between the EcoRI and Pstl cleavage sites to form a plasmid pGH37.

Then the E. coli strain HB101 was transformed with pGH37 according to the aforementioned calcium method. One of the strains obtained was named ECI-2. The ECI-2 strain has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, under number FERM BP 542 5, under the Budapest Convention on International Recognition of Depots. This strain is resistant to tetracycline, expresses the CI857 gene and allows for the simultaneous presence, by transformation, of other plasmids derived, for example, from pBR322. As a result, the ECI-2 strain was then used as a host for XPL expression plasmids.

10 7-2. Cloning of λΡ ^ ρΓοπκΛοΓ and preparation of expansion plasmids.

As illustrated in Figure 7, pGH35 was first formed. DNA of CI857S7 was cleaved with EcoRI and Sail to obtain a 5925 b.p. fragment containing the XPL promoter and the CI857 gene as well as PR promoter. The fragment was included in the plasmid pBR322 between the EcoRI and Sail cleavage sites to form a plasmid pGH25.

Subsequently, pGH25 was cleaved with BamHI and coupled to pBR322 similarly cleaved with BamHI to give pGH34.

Then pGH34 was cleaved with Aval and BGIII and then treated with S1 nuclease to form a blunt-ended fragment of approximately 4500 b.p. The fragment was ringed with T4 DNA ligase to form a plasmid pGH35.

Then, as shown in figure 8, pEK28 was built up. Synthetic oligonucleotides C-1-1 and C-1-2 as an adapter, containing an SD sequence and the nucleotide sequence listed below, were coupled to the fragment obtained by cleavage of pGH35 and Hpal. The combination was further coupled to the BamHI cleaved plasmid pMC1403 to obtain plasmid pEG2. The pEG2 has two ampicillin resistant genes and expresses pMC1403 derived β-galactosidase gene under the direction of XPL promoter using a start codon as well as the SD sequence in the adapter.

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

SD order Start codon.

30 C-1-1: 5 'A G G A A C A I G A T C T A T G 3' C-1-2: 3'TCCTTGTCTAGATACCTAG 5 '

Bglll 35 The Miller method was used to determine the expression of β-galactosidase in the host ECI-2 of the plasmid pEG2 (Miller, J. (1972) "Experiments in Molecular Genetics" New York, Cold Spring Harbor Laboratory pp352-355). This method is based on the reaction of β-galactosidase with a synthetic substrate ONPG (o-nitrophenylgalactoside) to release a yellow compound o-nitrophenol. Miller's method will be described in more detail. 0.1 ml of a culture of a 40 bacterial species, the absorbance of which is measured at 610 nm, is mixed with 1.9 ml of a test buffer (0.1 M sodium phosphate, pH 7.1 mM magnesium sulfate and 0.1 M β-mercaptoethanol) and shake vigorously with 0.1 ml toluene for 15 seconds to increase the permeability of the bacterial species. The toluene is then evaporated by means of an aspirator. With the addition of 0.2 ml ONPG solution (solution of 400 mg ONPG in 100 ml test buffer), the mixture is incubated at 30 ° C until a 45 yellow color develops, after which 0.5 ml 1 M sodium caibonate is added to react the enzyme reaction. stop. The absorbance of the reaction mixture is measured at 420 nm and 550 nm.

The activity of β-galactosidase is defined by the units in 1 ml of the broth according to the equation below, the absorbance at 610 nm being calculated as 1.0.

Activity of β-galactosidase 35 (units) = Ql ^ Pf20 ~~ x 9 ^ 55 ° x 1000 50 txvxOD610 t: incubation time (minutes) v: aliquots added to the reaction system (0.1 ml) OD610: absorbance at 610 nm of the sample.

When the above method was carried out, the following result was obtained. When the pEG2 55 harboring strain ECI-2 was incubated at 30 ° C, the β-galactosidase activity was 98 units. However, when the culture was further incubated for 1 hour at 42 ° C, the XPL promoter was activated resulting in a β-galactosidase activity of 9637 units. This confirms that the 19211 $ 16 sequence from the promoter to β-galactosidase is the desired sequence.

Although pEG2 has two BglII cleavage sites, only the BglII site present immediately after the SD sequence of β-galactosidase is necessary, while the other site is undesirable. As a result, the plasmid was cleaved with BamHI and coupled again to remove a fragment containing approximately 770 b.p. Thus, the construction of pEK28, which is an expression plasmid using XPL promoter, was completed.

7-3. Expression of fused gene from the anterior half of β-urogastrone and β-galactosidase.

Figure 9 schematically shows the sequence of the procedures described below.

A plasmid pUG101 was constructed in the following manner. This plasmid is a fused front half gene of β-urogastron and a β-galactosidase. More specifically, pUG1, which contains the front half of β-urogastron gene, and pMC1403 with a β-galactosidase gene was cleaved with BamHI and then coupled to form pUG101. The front half of the β-urogastron gene and the β-galactosidase gene are linked to this plasmid in the same frame. As a result, the plasmid expresses the amino acid sequence of the two as a fused protein.

For expression with this plasmid under the direction of the λΡι / ριοιηοΙοΓ, pUG101 and pEK28 were each split with BglII.

The BglII cleavage yields a DNA fragment with protruding 5 'ends in the form of (jcTAg). However, when the DNA fragment is reacted with a large fragment of E.coli DNA polymerase I (Klenow fragment) in the presence of the four types of deoxribonucleotide triphosphates dGTP, dATP, dTTP and dCTP, a blunt end is obtained by padding with the corresponding nucleotides, which is the end ("jctag) νοΓΤΤ1θη · When only dGTP is added as a nucleotide constituent, the end (" jctag) due to the termination of the reaction. Then, when 2 ^ S1 nuclease is used to digest the remaining single strand, a blunt end in the form of ("jq) is obtained. Similarly, when dGTP and dATP are added in the Klenow reaction, followed by digestion with S1 nuclease, ("jqj) obtained. When Klenow reaction is performed by addition of dGTP, dATP and dTTP, followed by digestion with (AGAT \ TCTA / veri <r®9en · Furthermore, when only digestion with S1 nuclease

is performed without performing the Klenow reaction, ("j"). Therefore, when the DNA

fragment with the end ("jctag) is subjected to the Klenow reaction using different nucleotides and / or to the S1 nuclease reaction, five kinds of blunt ends are obtained, differing from each other by the length of a base pair.

The Klenow reaction and S1 nuclease reaction were performed under the following conditions. xKlenow response:

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

XS1 nuclease reaction:

1 µg of DNA as substrate and 200 units of the enzyme were reacted for 30 minutes at 20 ° C in 100 µl of a reaction medium containing 200 mM sodium chloride, 30 mM sodium acetate and 5 mM 45 zinc sulfate (pH 4.5).

pEK28 and pUG101 were each cleaved with BglII and then subjected to different combinations of the Klenow reaction and the S1 nuclease reaction, yielding fragments with five kinds of blunt ends. Combining the two types of these fragments, 21 combinations were obtained, which differed in the number and sequence of the nucleotides between the 50 SD sequence and the starting codon of the fused protein, as listed in Table 2.

In practice, the DNA fragments thus obtained were cleaved with Sail to isolate fragments containing the gene encoding fused protein of the front β-urogastron half and β-galactosidase from pUG101, and fragments with λ-Ρ ^ promoter. from pEK28. These fragments were coupled by T4 DNA ligase in the combinations listed in Table 2.

As a result, the recombinant plasmids in Table 3 were obtained. The β-galactosidase activity of the expressed fused proteins was measured by Miller method. Table 3 shows that all expressed plasmids have a relatively high level of β-galactosidase activity. In particular pUG103, pUG104 and pUG117, 192116 achieved remarkable results.

TABLE 2 5 Nucleotide Sequence Upstream of the Start Codon (ATG)

ATCTGC- GATCTGC

Nucleotide -ACA -ACAATCTGC- -ACAGATCTGC order (pUG105) (pUG106) 10 downstream -ACAG - -ACAGGATCTGC- of SD order (pUG110) (AGGA) -ACAGA -ACAGAATCTGC- -ACAGAGATCTGC- (pUG114) - ACAGAT -ACAG ATATCTGC- -ACAGATGATCTGC- 15 (pUG117) (pUG118) -ACAGATC -ACAGATCATCTGC- -ACAGATCGATCTGC- (pUG122)

Nucleotide sequence upstream of the start oodon (ATG) 20 - TGC- CTGC- TCTGC-

Nucleotide -ACA -ACATGC- -ACACTGC- -ACATCTGC order (pUG102) (pUG103) (pUG104) downstream -ACAG -ACAGTGC- -ACAGCTGC- -ACAGTCTGC- 25 of the SD order <PUG107> <PUG109> (PUG109> (AGGA) -ACAGA -ACAGATGC- -ACAGACT GC- (pUG111) (pUG112) -ACAGAT -ACAGATTGC- - -ACAGATTCTGC- 30 (pUG115) (pUG116) -ACAGATC - -ACAGATCCTGC- -ACAGATCTCTGC- (pUG119) TABLE 3

Number of nucleotides between the SD sequence and the Recombinant plasmid β-Galactosidase start codon (units) 40 6 pUG102 1674 7 pUG103 2533 7 pUG107 2260 8 pUG104 2802 8 pUG108 1835 45 9 pUG105 1018 9 pUG109 1942 10 pUG106 973 11 pUG110 1764 11 11 pUG119 1862 12 pUG114 946 12 pUG117 2332 12 pUG120 1374 13 pUG118 2041 55 13 pUG121 1678 14 pUG122 1814 192116 18

Then a vector for the expression of β-urogastron was prepared from pUG103 or pUG117. The plasmid (pUG103 or pUG117) was cleaved with Hindlll and Pvull to obtain a 1.2 kb fragment containing the region of XPL promoter to the front half of β-urogastron gene. Furthermore, pUG2 was cleaved with EcoRI supplemented with a Klenow fragment and cleaved with HindIII to give a 4.1 kb fragment. The two fragments were coupled with T4 DNA ligase, and the E.coli strain ECI-2 was transformed according to the aforementioned calcium method to obtain a recombinant containing the plasmid pUG103-E or pUG117-E for the expression of the combination of the anterior and posterior halves of the β-urogastron gene, ie the entire β-urogastron gene, under the direction of APL promoter.

10 8. Vector for the expression of fused protein.

A β-lactamase gene on the plasmid pBR322 and a β-urogastron gene were linked to express β-urogastron as a fused protein, as will be described below.

8-1. Donor of β-lactamase gene.

pBRH02 is obtained by cleavage of pBR322 with Aval and Pvull followed by the Klenow reaction and coupling by T4 DNA ligase. This plasmid has ampicillin resistance (ApR) and tetracycline resistance (TcR) genes as markers. pBRH03 is obtained by cleaving pBR325 with Aval and Hindll1 followed by the Klenow reaction and coupling and contains ApR and chloramphenicol resistance (CmR) as markers. Figure 10 shows these plasmids.

8-2. Donor of β-urogastron gene.

PUG3 prepared in the manner already described was cleaved with Mboll to obtain 13 kinds of DNA fragments, named A to M in the order of size shown in Figure 11. These DNA fragments were found to H fragment was 179 bp starting with an asparagine-encoding nucleotide at the N-terminus of β-urogastron and ending with 16 bases downstream of the stop codon, the fragment containing the entire structural gene of β-urogastron. To isolate the H fragment, the fragments were electrophoresed with 6 wt% polyacrylamide gel and the fragment purified.

8-3. Adapter.

As adapters, the oligonucleotides listed in Table 4 were prepared in the manner previously described. These adapters were designed to encode the basic amino acid pair of Lys-Arg or Arg-Lys for the enzymatic cleavage of β-urogastron from the expressed fused protein.

TABLE 4 35 Adapter 5 'end - 3' end

D-1-3 CCG TAAG

D-1-4 TTACGG

D-2-1 CGTAAG

40 D-2-2 TTACG

D-3-2 TTACGGAT

D-4-2 TTACGTGCA

E-1 CGCTAAACGG

E-2 CGTTTAGCG

45 E-3 GACAAACGG

E-4 CGTTTGTC

E-5 CGTTTAGCGAT

E-6 CGTTTGTCTGCA

E-7 CGGCTAAACGG

50 E-8 CGTTTAGCCG

E-9 CAAACGG

E-10 CGTTTG

55 8-4. System for the expression of fused protein of β-lactamase and β-urogastron linked by Lys-Arg.

A vector for the expression of β-Ιβοίθητοβθ-β-υιχχϊθείΐΌη ^ θίυβθθκΙ protein was prepared such that 19 192116 would form a restriction enzyme recognition sequence in the region containing an adapter.

8-4-a. Construction of pUG2301 to pUG2303.

The procedure shown in Figure 12 was performed.

The plasmid pBRH02 was cleaved completely with Xmnl for 3 hours at 37 ° C. Then, 3 µg of the vector, approximately 0.1 µg of β-urogastrone fragment of 179 b.p. and coupled about 1 µg of both E-1 and E-2 (with nonphosphorylated 5 'end) as a single stage adapter for 15 hours at 12 ° C to obtain plasmids as expression vector. The strain HB101 was transformed using the plasmids according to the calcium method.

Of the 499 TcR colonies obtained, 168 colonies (33.7%) were Aps. These colonies were mini-checked for the size of the plasmid DNA. Thirteen plasmids were approximately 200 b.p. larger than the vector and were believed to contain a β-urogastrone gene contained therein.

All plasmids with a Mlul site were cleaved with Hinfl and checked for the orientation of the β-urogastron gene uptake by electrophoresis with 1.5 wt% agarose gel. Three of the controlled plasmids gave fragments of about 1050 b.p. and about 800 b.p. This showed that the β-urogastron gene was included with the same orientation as β-lactamase. These three plasmids were designated pUG2301 through pUG2303.

8-4-b. Construction of pUG2101 to pUG2105.

The procedure shown in Figure 13 was performed.

The plasmid pBR322 was used as a single Pvul plasmid vector site in the β-lactamase-20 gene. According to the method described in 8-4-a, an expression vector was generated using E-1 and E-5 as an adapter.

When the obtained 1626 TcR colonies were checked for Ap sensitivity, 31 colonies (1.9%) were Aps. A mini preparation was performed for the 22 TcR and Aps colonies and the plasmids were checked for the uptake of β-urogastron gene by cleavage of Mlul. Twenty plasmids were found to have an Mlul site. The orientation was checked by splitting with Hinfl or BamHI. The plasmids pUG2101 through pUG2105 were found to contain a β-urogastrone gene contained therein with the same orientation as the β-lactamase gene.

8-4-c. Construction of pUG2701 to pUG2703.

Expression plasmids were generated by the same method as 8-4-a using pBR322 as vector and E-7 and E-8 as adapter as shown in Figure 14.

Of the 217 TcR colonies obtained, 106 colonies (48.8%) were Aps. A mini preparation was carried out for 25 of these colonies. Eight of the plasmids were approximately 200 b.p. larger than the vector and were found to contain an β-urogastron gene contained therein, so that these eight plasmids were cleaved with BamHI and checked for gene orientation. Three plasmids were found to contain the β-urogastrone gene with the same orientation as β-lactamase and were designated pUG2701 through 2703.

The plasmid pUG2301 obtained by the above method 8-4-a yields a fused white of a portion of β-lactamase and β-urogastrone. The predicted amino acid sequence and the corresponding nucleotide sequence are listed below.

40 With Ser He Gin His Phe Arg Val

ATG AGT ATT CAA CAT TTC CGT GTC

Ala Leu lie Pro Phe Phe Ala Ala

GCC CTT ATT CCC TTT TTT GCG GCA

45

Phe Cys Leu Pro Val Phe Ala His

TTT TGC CTT CCT GTT TTT GCT CAC

Pro Glu Thr Leu Val Lys Val Lys

50 CCA GAA ACG CTG GTG AAA GTA AAA

Asp Ala Glu Asp Gin Leu Gly Ala

HOLE GCT GAA HOLE CAG TTG GGT GCA

55 Arg Val Gly Tyr He Glu Leu Asp

CGA GTG GGT TAC ATC GAA CTG GAT

192116 20

Leu Asn Ser Gly Lys lie Leu Glu

CTC AAC AGC GGT AAG ATC CTT GAG

Ser Phe Arg Pro Glu Glu Arg Ala

5 AGT TTT CGC CCC GAA GAA CGC GCT

Lys Arg Asn Ser Asp Ser Glu Cys

AAA_ CGG AAT AGC GAT TCT GAG TGC

10 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

15

He Glu Ala Leu Asp Lys Tyr Ala

ATC GAA GCT TTG GAT AAA TAC GCG

Cys Asn Cys Val Val Gly Tyr lie

20 TGT AAC TGT GTA GTG GGT TAT ATC

Gyl Glu Arg Cys Gin Tyr Arg Asp

GGT GAA CGC TGT CAA TAC CGT GAT

25 Leu Lys Trp Trp Glu Leu Arg (stop)

CTG AAA TGG TGG GAA TTG CGT TAA

TAGTGAAGATCTGGATCCGTTTAGCGTTTTCCA

ATGATGAGCACTTTTAAAGTTCTGCTATGTGGC

30 GCGGTATTATCCCGTGTTGACGCCGGGCAAGAG

CAACTCGGTCGCCG CATAC

Similarly, the plasmid pUG2101 obtained by the method 8-4-b forms a fused protein with the following primary structure.

With Ser lie Gin His Phe Arg Val

ATG AGT ATT CAA CAT TTC CGT GTC

40 Ala Leu lie Pro Phe Phe Ala Ala

GCC CTT ATT CCC TTT TTT GCG GCA

Phe Cys Leu Pro Val Phe Ala His

TTT TGC CTT CCT GTT TTT GCT CAC

45

Pro Glu Thr Leu Val Lys Val Lys

CCA GAA ACG CTG GTG AAA GTA AAA

Asp Ala Glu Asp Gin Leu Gly Ala

50 HOLE GCT GOA HOLE CAG TTG GGT GCA

Arg Val Gly Tyr He Glu Leu Asp

CGA GTG GGT TAC ATC GAA CTG GAT

55 Leu Asn Ser Gly Lys He Leu Glu

CTC AAC AGC GGT AAG ATC CTT GAG

21 192116

Ser Phe Arg Pro Glu Glu Arg Phe

AGT TTT CGC CCC GAA GAA CGT TTT

Pro With With Ser Thr Phe Lys Vai

5 CCA ATG ATG AGC ACT TTT AAA GTT

Leu Leu Cys Gly Ala Val Leu Ser

CTG CTA TGT GGC GCG GTA TTA TCC

10 Arg Val Asp Ala Gly Gin Glu Gin

CGT GTT GAC GCC GGG CAA GAG CAA

Leu Gly Arg Arg He His Tyr Ser

CTC GGT CGC CGC ATA CAC TAT TCT

15

Gin Asn Asp lie Val Glu Tyr Ser

CAG AT GAC TTG GTT GAG TAC TCA

Pro Val Thr Glu Lys His Leu Thr

20 CCA GTC ACA GAA AAG CAT CTT ACG

Asp Gly With Thr Val Arg Glu Leu

HOLE GGC ATG ACA GTA AGA GAA TTA

25 Cys Ser Ala Ala lie Thr Met Ser

TGC AGT GCT GCC ATA ACC ATG AGT

Asp Asn Thr Ala Ala Asn Leu Leu

HOLE AAC ACT GCG GCC AAC TTA CTT

30

Leu Thr Thr lie Ala Lys Arg Asn

CTG ACA ACG ATC_GCT_AAA_CGG ATH

Ser Asp Ser Glu Cys Pro Leu Ser

35 AGC GAT TCT GAG TGC CCA CTG TCT

His Asp Gly Tyr Cys Leu His Asp

CAC GAT GGC TAT TGT CTG CAC GAC

40 Gly Val Cys With Tyr lie Glu Ala

GGT GTT TGC ATG TAC ATC GAA GCT

Leu Asp Lys Tyr Ala Cys Asn Cys

TTG HOLE AAA TAC GCG TGT AAC TGT

45

Val Val Gly Tyr lie Gly Glu Arg

GTA GTG GGT TAT ATC GGT GAA CGC

Cys Gin Tyr Arg Asp Leu Lys Trp

50 TGT CAA TAC CGT GAT CTG AAA TGG

Trp Glu Leu Arg (stop)

TGG GAA TTG CGT TAA TAGTGAAG AT C

192116 22

TGGATCCGTTTAGCGATCGGAGGACCGAAGG

AGCTAACCGCTTTTTTGCACA

Similarly, the plasmid pUG2701 obtained by the method 8-4-c forms a fused protein with the following primary structure.

5

With Ser lie Gin His Phe Arg Val

ATG AGT ATT CAA CAT TTC CGT GTC

Ala Leu lie Pro Phe Phe Ala Ala

10 GCC CTT ATT CCC TTT TTT GCG GCA

Phe Cys Leu Pro Val Phe Ala His

TTT TGC CTT CCT GTT TTT GCT CAC

15 Pro Glu Thr Leu Val Lys Val Lys

CCA GAA ACG CTG GTG AAA GTA AAA

Asp Ala Glu Asp Gin Leu Gly Ala

HOLE GCT GAA HOLE CAG TTG GGT GCA

20

Arg Val Gly Tyr lie Glu Leu Asp

CGA GTG GGT TAC ATC GAA CTG GAT

Leu Asn Ser Gly Lys He Leu Glu

25 CTC AAC AGC GGT AAG ATC CTT GAG

Ser Phe Arg Pro Glu Glu Arg Phe

AGT TTT CGC CCC GAA GAA CGT TTT

30 Pro With With 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

35

Arg Val Asp Ala Gly Gin Glu Gin

CGT GTT GAC GCC GGG CAA GAG CAA

Leu Gly Arg Arg He His Tyr Ser

40 CTC GGT CGC CGC ATA CAC TAT TCT

Gin Asn Asp He Val Glu Ser Ala

CAG AT GAC TTG GTT GAG TCG_GCT

45 Lys Arg Asn Ser Asp Ser Glu Cys

AAA_CGG AT AGC GAT TCT GAG TGC

Pro Leu Ser His Asp Gly Tyr Cys

CCA CTG TCT CAC GAT GGC TAT TGT

50

Leu His Asp Gly Val Cys Met Tyr

CTG CAC GAC GGT GTT TGC ATG TAC

He Glu Ala Leu Asp Lys Tyr Ala

55 ATC GAA GCT TTG GAT AAA TAC GCG

23 192116

Cys Asn Cys Val Val Gly Tyr lie

TGT AAC TGT GTA GTG GGT TAT ATC

Gly Glu Arg Cys Gin Tyr Arg Asp

5 GGT GAA CGC TGT CAA TAC CGT GAT

Leu Lys Tip Tip Glu Leu Arg (stop)

CTG AAA TGG TGG GAA TTG CGT TAA

10 TAGTGAAGATCTGGATCCGTTTAGCCGACTCAC

CAGT CAC AGAAAAGCATCTTACGGAT

The nucleotide sequences encoding the fused proteins in the plasmids pUG2101, pUG2301 and pUG2701 were analyzed according to the Maxam-Gilbert method.

15 Photo 5 shows the results of the analysis. In photo 5, lanes 1 through 4 show the result obtained with the Mlul-Pstl fragment (224 bp) of pUG2101, lanes 5 through 8 show the result with the Mlul-EcoRI fragment (721 bp) of pUG2101 result obtained, lanes 9-12 show the result obtained with the Mlul-BamHI fragment (452 bp) of p (JG2301, lanes 13-16 show it with the Mlul-Pstl fragment (335 bp) of pUG2701 obtained and lanes 17 to 20 obtained with the Mlul-EcoRI fragment (610 20 bp) of pUG2701 Lanes 1,5,9,13 and 17 show the reaction products for guanide lanes 2, 6, 10, 14 and 18 the reaction products for guanine plus adenine, lanes 3, 7, 11, 15 and 19 the reaction products for thymine plus cytosine and lanes 4, 8, 12, 16 and 20, the reaction products for cytosine. } highlighted section is an adapter.

8-5. System for the expression of fused protein of β-lactamase and β-urogastron linked by Arg-Lys.

8-5-a. Preparation of pUG1102 and pUG1105.

β-urogastron gene was included in the β-lactamase gene of pBR322 at the only Pvul restriction site to obtain vectors for the expression of fused protein of β-lactamase and β-urogastron, as shown in Figure 15.

The plasmid pBR322 was cleaved with Pvul at 37 ° C for 3 hours. Some of the plasmids were checked by electrophoresis with 1 wt% agarose gel to confirm that they are completely cleaved. Adapters D-1-3 and D-3-2 were coupled to the fragment at 12 ° C for 15 hours and the coupled product was then electrophoresed with 1% w / w agarose gel to generate a DNA fragment isolate. Then, the β-urogastrone fragment and the vector were mixed at a molar ratio of about 5: 1 and coupled at 12 ° C for 15 hours. After coupling, the strain HB101 was transformed with the resulting plasmid and the colonies were selected for TcR.

Seventy one TcR transformed colonies were obtained and then checked for their Aβ sensitivity. Plasmid DNA was prepared from 20 APs colonies (28.2%) and then checked for the presence of Mlul restriction site to confirm the uptake of β-urogastrone gene. Five of the 20 plasmids were found to contain the Mlul site of β-urogastron gene. The DNA was cleaved with Hinfl and then electrophoresed with 1.5 wt% agarose gel to check the orientation of the recording. It was found that two of the plasmids, i.e. pUG1102 and pUG1105 contain the gene in the correct orientation.

45 8-5-b. Preparation of pUG1004, pUG 1201 and pUG1301.

The procedure 8-5-a was repeated using Pstl, Hindi and Xmnl instead of Puvl to obtain pUG1004, pUG1201 and pUG1301 as shown in Figures 16.17 and 18, respectively.

9. Confirmation of the expression of β-urogastron.

The expression plasmids thus constructed were used to transform E. coli, HB101 or ECI-2, and the cells were cultured according to the method below, followed by extradie and radioimmunoassay to confirm expression.

9-1. Culture of recombinant microorganisms with β-urogastrone gene and protein extradie.

9-1-a. Expression system using PL promoter.

55 The strain ECI-2 with the expression plasmid pUG103-E and the same strain with the expression plasmid

pUG117-E were each grown at 25 ° C in two flasks, each containing 1 liter of LB culture medium. When the culture in one of the flasks showed an absorbance of 0.3 at 660 nm, the culture was maintained at 42 ° C for 1 hour

192116 24 subject to heat induction. The culture in the other flask was incubated continuously at 25 ° C until the absorbance was 0.4. The cells in each flask were collected, washed with PBS buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM dibasic sodium phosphate and 1.5 mM monobasic sodium phosphate (pH 7.0 »), then resuspended in PBS buffer in 3 vol% of the amount of the original culture and destroyed (for 30 seconds at 100 W, three times) by a sonicator (model 5202, product of Ohtake Works Co., Ltd., Japan) under ice cooling. ultracentrifugation (1 hour at 40,000 g) of the cell debris separated supernatant was dialialized against 0.01 N aqueous acetic acid solution and the dialysate was lyophilized and then subjected to radioimmunoassay (hereinafter referred to as RIA).

10 9-1 -b. System for the expression of fused protein.

The E.coli strain HB101 with plasmids pUG1004,1301,2101, 2303 or 2703 was preincubated at 37 ° C in a culture medium containing 50 µg / ml tetracycline, then diluted to a volume ratio of 1: 100 with the same medium and grown until the absorbance at 660 nm was 0.4. The cells were collected, washed with PBS buffer, then resuspended in PBS buffer in 3% by volume of the original culture and subjected to sonic vibrations (at 100 W for 30 seconds, three times) using the same sonicator as previously mentioned under ice cooling. The supernatant separated from the cell debris by ultracentrifugation (1 hour at 40,000 g) was dialized against 0.01 N aqueous acetic acid solution, lyophilized and then subjected to RIA.

To confirm the accumulation of fused protein in the periplasm, the periplasma fraction was prepared according to S.J. Chan et al. (Chan, S.J. et al., Proc. Natl. Acad. Sci., U.S.A., 78, 5401-5405 (1981)). A portion of the culture was diluted to a volume ratio of 1: 100 with fresh E culture medium (1 liter aqueous solution of 10 g dibasic potassium phosphate, 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 mg L-leucine, 16.85 mg thiamine and 20 mg tetracycline hydrochloride) and then grown at 37 ° C until the absorbance at 660 nm was 0.4. The cells were collected (6000 rpm, 10 min) and washed twice with a mixture of 10 mM tris-HCl (pH 8.0) and 30 mM sodium chloride. The cells (1 g) were resuspended in 80 ml of 20 wt% sucrose-30 mM tris-HSI (pH 8.0) and EDTA was added to the suspension at a concentration of 1 mM. The mixture was run at 180 rpm for 10 minutes. (24 ° C) shaken with a rotary shaker and then centrifuged (13,000 g, 10 minutes) to collect the cells, which were resuspended in 80 ml of distilled water. The suspension was kept in ice for 10 minutes with occasional stirring and then centrifuged (13,000 g, 10 minutes). The supernatant was collected as a periplasmic fraction (0-Sup). The sediment was suspended in a mixture of 10 mM tris-HCl (pH 8.0) and 30 mM sodium chloride and treated with the same sonicator as before to obtain a cytoplasmic fraction (0-Ppt). These samples were subjected to RIA.

9-2. Radioimmunological test.

9-2-a. Establishing the RIA system.

Rabbits were immunized with purified human β-urogastrone as an antigen to obtain antiserum. The β-urogastrone (300 µg) was dissolved in 0.2 ml of distilled water, 1.5 ml of 50% polyvinylpyrrolidone solution was added to the solution and the mixture was stirred at room temperature for 2 hours. Complete Freund's adjuvant (2.0 ml) was added to the mixture to obtain an emulsion, which was injected subcutaneously into the breast portion of three rabbits. After repeating the immunization four times every 2 weeks, 50 µg of the antigen was further injected intravenously, the whole blood collected 3 days later and the serum separated.

45 Next, the following RIA conditions were determined in view of the titration curve for determining the degree of dilution of the antiserum for the test, the incubation time for optimizing the test conditions, the method of separating the bound radiolabeled antigen ( bound) of the free radiolabeled antigen (free), etc.

The dilution solution used was a phosphate buffer (10 mM, pH 7.4) containing 0.5 wt% bovine serum-50 albumin (BSA), 140 mM sodium chloride and 25 mM disodium EDTA. The dilution solution (400 μΙ), 100 μΙ of the sample or standard human β-urogastron and 100 μΙ anti-human β-urogastron serum were mixed. After the mixture was incubated at 4 ° C for 24 hours, 100 µl of a 125 labeled human β-urogastrone solution (approximately 5000 cpm) was added to the mixture. After the mixture was further incubated at 4 ° C for 48 hours, 100 μΙ of a second antibody (anti-55 rabbit γ-globulin goat serum) (20-fold dilution with PBS buffer), 100 μΙ normal rabbit serum (200-fold dilution with PBS buffer) and 900 μ110 mM PBS buffer with 5 wt% polyethylene glycol added to the resulting mixture and incubated for an additional 3 hours at 4 ° C. The culture was maintained at 3000 rpm for 30 minutes. centrifuged, the supernatant was separated and the precipitate was counted. The immunoreactive content as human β-urogastrone in the sample was determined from the standard curve obtained using standard human β-urogastron.

9-2-b. Confirmation of β-urogastrone productivity of the recombinant microorganism.

Table 5 shows the result of RIA for the expression system using XPL promoter.

TABLE 5

Expression plasmid Heat induction Amount produced 10 β-urogastrone (n g / l of culture) pUG103-E yes 450.2 pUG103-E no 3.2 pUG117-E yes 388.4 15 pUG117-E no 3.2

Control (pBR322) no imperceptible

Table 6 shows the result of RIA for fused protein expression systems.

20 TABLE 6

Expression plasmid Amount of β-urogastrone produced (pg / l from culture) 25 - pUG1004 729.6 pUG1301 650.7 pUG2101 31.3 PUG2301 125.2 30 pUG2701 119.2

Table 7 shows the localization of the expressed fused protein.

35 TABLE 7

Expression plasmid Amount of β-urogastrone produced (pg / l from culture) 40 Periplasma fraction Cytoplasma fraction (0-Sup) (0-Ppt) PÜG1004 326.0 4.0 pUG1301 347.4 2.8 45 pUR2101 79.9 10.2 pUG2301 118.8 4.1 pUG2701 65.7 3.6 50 Tables 5 and 6 show that the X-PL promoter system for direct expression of β-urogastrone and the system for expression of the compound as a fused protein both expressed β-urogastrone immunoreactivity in E. coli. Table 7 shows that in the case of fused protein, the expressed β-urogastron immunoreactivity is almost localized in the periplasm.

Claims (18)

192116 26 1. β-urogastron genes with the following nucleotide sequence: 5 'AAT AGC GAT TCT GAG TGC CCA CTG 5 3' TTA TCG CTA AGA CTC ACG GGT GAC TCT CAC GAT GGC TAT TGT CTG CAC AGA GTG CTA CCG ATA ACA GAC GTG A gene according to claim 1, which contains a restriction enzyme recognition site attached to the front and / or back of the gene. A gene according to claim 2, which contains a restriction enzyme recognition site and a start codon at the front of the gene and / or a stop codon and a restriction enzyme recognition site at the back of the gene, wherein the codons and the recognition sites 30 are arranged in the order shown. A gene according to claim 3, having the following nucleotide sequence: -15 -1.1 1 'AAT TCG AAG ATC TGC ATG AAT AGC 3' GC TTC TAG ACG TAC TTA TCG 35 10 20 30 GAT TCT GAG TGC CCA CTG TCT CAC CTA AGA CTC ACG GGT GAC AGA GTG 40 40 50 GAT GGC TAT TGT CTG CAC GAC GGT CTA CCG ATA ACA GAC GTG CTG CCA 60 70 80 45 GTT TGC ATG TAC ATC GAA GCT TTG CAA ACG TAC ATG TAG CTT CGA AAC 90 100 GAT A A A TAC GCG TGT AAC TGT GTA 50 CTA TTT ATG CGC ACA TTG ACA CAT 110 120 GTG GGT TAT ATC GGT GAA CGC TGT CAC CCA ATA TAG CCA CTT GCG ACA 55 27 192 116 130 140 150 CAA TAC CGT GAT CTG AAA TGG TGG GTT ATG GCA CTA GAC TTT ACC ACC 5 160 170 GAA TTG CGT TAA TAG TGA AGA TCT CTT AAC GCA ATT ATC ACT TCT AGA G 3 ' A recombinant plasmid with the gene defined in claim 4. Process for the preparation of the recombinant plasmid according to claim 5, characterized in that the subunit with the nudeotide sequence is: 5 'AAT TCG AAG ATC TGC ATG AAT AGC 3' GC TT C TAG ACG TAC TT A TCG GAT TCT GAG TGC CCA CTG TCT CAC CTA AGA CTC ACG GGT GAC AGA GTG 20 GAT GGC TAT TGT CTG CAC GAC GGT CTA CCG ATA ACA GAC GTG CTG CCA GTT TGC ATG TAC ATC GAA GCT TCG 25 CAA ACG TAC ATG TAG CTT CGA AGC 3 'CTA G 5' 30 and the subunit with the nudeotide sequence: 5 'A GCT TTG GAT A A A TAC GCG TGT AAC CTA TTT ATG CGC ACA AAC TGT GTA GTG GGT TAT ATC GGT 35 TTG ACA CAT CAC CCA ATA TAG CCA GAA CGC TGT CAA TAC CGT GAT CTG CTT GCG ACA GTT ATG GCA CTA GAC 40. A A TGG TGG GAA TTG CGT TAA TAG TTT ACC ACC CTT AAC GCA ATT ATC TGA AGA TCT G 3 'ACT TCT AGA CCT AG 5' 45 record at a suitable recording site of a suitable plasmid vector. A recombinant plasmid, which contains a plasmid vector containing the p-urogastron gene defined in claim 4, wherein in the plasmid vector further at the front of the p-urogastron gene a promoter for controlling the expression of the gene and one linked to the promoter 50 SD order are included. A recombinant plasmid containing a plasmid vector of the sequence of the fourth and subsequent base pairs of the p-urogastron gene defined in claim 4, wherein in the plasmid vector the combination of a promoter, an SD sequence and another gene has been included on the front of the p-urogastron gene. A recombinant plasmid as defined in claim 8, wherein the other gene is a β-lactamase gene. A recombinant plasmid as defined in claim 7 or 8, wherein the promoter λΡι. or lac is UV5. 192116 28 10 CCT AG 5 ' 10 GAC GGT G TT TGC ATG TAC ATC GAA CTG CCA CAA ACG TAC ATG TAG CTT GCT TTG GAT AAA TAC GCG TGT AAC CGA AAC CTA TTT ATG CGC ACA TTG 15 TGT GTA GTG GGT TAT ATC GGT GAA ACA CAT CAC CCA ATA TAG CCA CTT CGC TGT CAA TAC CGT GAT CTG AAA GCG ACA G TT ATG GCA CTA GAC TTT TGG TGG GAA TTG CGT 3 'ACC ACC CTT AAC GCA 5', where other sequences may be present at the front and / or back of this sequence. A recombinant plasmid as defined in claim 7 or 8, wherein the plasmid vector is pBR322. A transformant containing a host cell with a recombinant plasmid capable of expressing the β-urogastron gene defined in claim 1. A transformant as defined in claim 12, wherein the recombinant plasmid is the recombinant plasmid defined in claim 5. A transformant as defined in claim 12, wherein the recombinant plasmid is the recombinant plasmid defined in claim 8. A transformant as defined in claim 12, wherein the host cell is E. coli. 16. A transformant as defined in claim 12, wherein the host cell is transformed with a recombinant plasmid containing a TcR gene and a CI857 gene. A transformant formation method, characterized in that a host cell is transformed with a recombinant plasmid capable of expressing the β-urogastron gene defined in claim 1. 18. A process for the formation of β-urogastron, characterized in that the transformant defined in claim 12 is grown and the expressed β-urogastron is collected. Hereby 23 sheets of drawing