CA1304023C - .beta.-UROGASTRONE GENE, CORRESPONDING RECOMBINANT PLASMIDS, CORRESPONDING TRANSFORMANTS AND PREPARATION THEREOF AND OF .beta.-UROGASTRONE - Google Patents

Abstract

NOVEL .beta.-UROGASTRONE GENE, CORRESPONDING
RECOMBINANT PLASMIDS, CORRESPONDING TRANSFORMANTS
AND PREPARATION THEREOF AND OF .beta.-UROGASTRONE

ABSTRACT OF THE DISCLOSURE:
The present invention provides a novel gene which is suited to the expression of .beta.-urogastrone, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of .beta.-urogastrone.

Description

~3~

NOVEL ~-UROGASTRONE GENE, CORRESPONDING
RECOMBINANT PLASMIDS, CORRESPONDING TRANSFORMANTS
AND PREPARATION THEREOF AND OF ~-UROGASTRONE
_ _ The present invention relates to a novel ~-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of ~-urogastrone.
~-Urogastrone is a polypeptide hormone synthesized in the salivary glands of human, etc.
(see, for example, Heitz et al., Gut, 19, 408-413 (1978)), has a primary structure comprising 53 amino acids in the following sequence (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 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 Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg In the specification, amino acids are repre-sented by the ~ollowing symbols.
Asn: asparagine Ser: serine Asp: aspartic acid Glu: glutamic acid Cys: cysteine Pro: proline Leu: leucine His: histidine .

~3~ 3 Gly: glycine Tyr: tyrosine Val: valine Met: methionine ~le: isoleucine Ala: alanine Lys: lysine Gln: glutamine Arg: arginine Trp: tryptophan Phe: phenylalanine ~ -Urogastrone has physiological activities such as suppression of the secretion of gastric acid and promotion of cell growth (see Elder et al., Gut, 16, 887-893 (1975)) and is therefore useful for treating ulcers and wounds.
Since ~-urogastrone is excreted in small amounts in human urine, the compound is presently prepared from urine by extraction, separation and purification. However, this method involves problems such that large quantities of the compound can not easily be obtained because the compound is a minor component in human urine.
On the other hand, ~uropean Patent Application Publication No.00~6039 discloses an attempt to produce ~-urogastrone by a gene engineering technique with use of a synthetic ~-urogastrone gene. The above publication, however, discloses the synthetic gene having a specific nucleotide sequence but does not teach whether there are other genes which are capable of expressing ~-urogastrone 3~ ;23 by a similar me-thod, IIOL does it mention such a yene oE a particular nucleotide sequence.

A large number o:. nucleotide sequences can code for the amino acid sequence o:E R-urocJastrorle through a yene engineering technique or which yene is most suited to the application of gene engineering techniques. Thus, many experiments and inventive efforts are required to determine the most suitable nucleotide sequence.

The present invention provides a novel B-urogastrone gene which is entirely different from the gene disclosed in the above publica-tion in the nucleotide sequence and which is capable of expressing B-urogastrone through gene engineering techniques.

The present invention also provides a gene which is suited to the expression of ~-urogastrone by gene engineering techniques.

The present invention again provides novel recombinant p].asmids and transformants corresponding to -the novel B-urogastrone gene.

The present invention again provides a process which enables quantity producticn of B-urogastrone with a high purity with r use of the novel gene by gene engineering techniques.

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Preferably, the ~ urogastrone gene has the following nucleotide sequence:

5' A A T A G C G A T T C T G A G T G C C C A C T G
3' T T A ~ 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 & A G T G C T A C C G A T A A C A ~ A C G T 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 & 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 ti: 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' A C C A C C C: T T A A C G C A 5 ' The letters stand for the purine or pyrimidine bases forming the nucleotide sequence. The symbols herein used for bases represent the following: A for adenine, G for guanine, C for cytosine and T *or thymine.

The gene I is entirely novel and unobvious in itself and is obtained by determining the specified nucleotide sequence from a very large number of possible nucleotide sequences.

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The gene I has the following characteristics.
~ rogastrone c~n be expressed ve~y advan-tageously by gene engineering techniques.

(2) The trinucleotide codons constituting the gene I are all acceptable to host cells, especially to Escherichia coli (E.coli) which is easily available with safety consequently assuring a high degree of expression.

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

(4) For the preparation of the gene I, the constituent oligonucleotides can be ligated into blocks and the blocks can be ligated into subunits easily as contemplated, substantially free from undesired ligation thereof.

(5) In expressing ~-urogastrone as a fused protein, means is available by which an unnecessary portion can be easily removed to obtain the desired ~-urogastrone.
When ~-urogastrone is to be expressed actually with use of the gene I, restriction enzyme recognition sites may be provided at the front end and/or the rear end of the gene in view of the ligation with the promotor, Shine-Dalgarno sequence (hereinafter referred to as "SD

~3~2;~

sequencei'), vector, etc. needed ~or the expression.
Further when required, a start codon and/or a stop codon ma~ be provided upstream and downstream of the gene, respectively. The recognition sites, start codon and stop codon are not limited particularly but can be desired ones.
Shown below is an example of gene ha~ing an expanded sequence (hereinafter referred to as "gene II") which includes a restriction enzyme recognition site and a start codon disposed upstream of the gene I and a stop codon and a restriction enz~me recognition site disposed downstream of the gene I, the sites and codons being arranged in the order mentioned, the gene II further including other restriction enz~me recognition sites.
15~ Gene II:
Start codon -15 -1, 1 5' IA A T T¦C G A A ¦G A T C, T G C A T Gl A A T A G C
3' I G C¦ T T C T A Gl A C G T AIC T T A T C G
E Ta Bg(S) Mb GIA T T C T G A G T G C C C A C T G T C T C 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
H~

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

~.3~12~

GTT TGC ATG TAC AT~ GA¦A GCT TTG
CAA ACG TAC ATG TAG ~TT CGA¦AAC
Ta Hd GAT AAA TAL~GCG TGT AAC TGT GTA
CTA TTT ATG ~ ACA TTG ACA CAT
Ml(Th) GT-`G GGT TAT ATC GGT GAA CGC TGT
CAC CCA ATA TAG CCA CTT GCG ACA

CAA TAC CGTIGAT CTG AAA TGG TGG
GTT ATG GCA CTA GiAC TTT ACC ACC

Stop codon 160 ~ 170 G¦A A TTG CGT TAA TAG TGA A¦GA TCT

CTT AA¦C GCA A T T A T C A C T T C T A~A
E Bg(S) Gl 3' ~ CCT A G¦ 5' Ba The symbols representing the restriction enzymes in the above sequence stand for the follol.~ing.
E : EcoRI, Ta: TaqI
Bg: BglII, S : Sau3AI
Mb: MboII, Hf: HinfI
Ba: BamHI, Hd: HindIII
Ml: MluI, Th: ThaI

~L3~ 3 The present invention is not limited to the gene I and gene II but also include other genes which are substantially identical therewith in the nucleotide sequence and which are capable of expressing ~-urogastrone.
In synthetic preparation of the gene I or II, it is advantageous to construct the gene I or II as divided into the front half portion and the rear half portion.
For example, it is possible to prepare a subunit having the front half of the nucleotide sequence of the gene I
or II and another subunit having the rear half of the nucleotide sequence thereof as it is divided approximately at the midportion thereof and to join these two subunits of the gene I or II together into the gene I or II. The subunit having the front half of the nucleotide sequence of the gene II further may have a restriction enzyme recognition site at the rear encl, and another subunit having the rear half of the nucleotide sequence of the gene II may havP a restriction enzyme recognition site at the front end, ànd these subunits are jointed together into the gene II.
Stated more specifically for illustrative purposes, the former subunit can be a subunit A oomprising the front half of the gene II and having a restriction enzyme (BamHI) recognition site provided at its rear end, and the latter subunit can be a subunit B comprising t'ne rear half of the gene II which ha~s a restriction enzyme ~31~

g (HindIII~ recognition site at its front end. These subunits are shown below.
Subunit A:
5' A A TT C G A AG ATC TGC ATG AAT AGC
3' GC TTC TAG ACG TAC TTA TCG
GATTCT GAG TGC CCA CTG TCT CAC
CTAAGA CTC ACG GGT GAC AGA GTG
GAT GGC TAT TGT CTG CA C G A C GGT
CTA CCG ATA ACA GAC GTG CTG CCA
GTT TGC ATG TAC ATC GAA G C T TCG
CAA ACG TAC ATG TAG CTT CGA AGC
3' CTA G S' Subunit B
5' A GCT TTG GAT AAA TAC GCG TGT
3. AAC CTA TTT ATG CGC ACA
A A C TGT GT A GTG GGT TAT ATC GGT
TTG ACA CAT CAC CCA ATA TAG CCA
GAA CGC TGT CA A TAC CGT G AT CTG
CTT GCG ACA GTT ATG GCA CTA GAC
AAA TGG TGG GAA TTG CGT TAA TAG
TTT ACC ACC CTT AAC GCA ATT ATC
TGAA G A TCT G 3' ACTTCT AGA CCT AG 5' The subunits A and B are synthesized, for example, in the following manner. Oligonucleotides having 11, 13 or 15 bases are synthesized (A-ltoA-16, and B-l to B-16, i.e. 32 oligonucleotides). Next, l~ to 60f these oligonucleotides are assembled and ligated into blocks (block 1 to block 7, i.e. 7 blocks).
T'nese oligonucleotides and blocks are shown below.

~L3~23 Block 1:

(A-l) (A-2) (A-3) 5' AATTCGAAGAT CTGCATGAATAGC GATTCTGAGTG 3' 3' GCTTCTAGACGTA CTTATCGCTAA GACTCACGGGTGA 5' tA-16) (A-15) (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) 5' GTTTGCATGTA CATCGAAGCTTCG 3' 3' CGTACATGTAGCT TCGAAGCCTAG S' (A-10) (A-g) 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) 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) ~3~4~23 Next, the blocks 1 to 3 are ligated together into the subunit A, and the blocks 4 to 7 are ligated together into the subunit B.
The present in~ention will be described in greater detail with reference to the accompanying drawings and photos.
Fig. l schematically shows the synthesis of an oligonucleotide by the solid phase process;
Fig. 2 shows a process for ligating oligo-nucleotides A-l to A-16 into a subunit A and introducing the subunit into a plasmid pBR322 derived from E.coli to obtain a recombinant plasmid pUGl;
Fig. 3 shows a similar process for preparing a recombinant plasmid pUG2 by introducing a subunit B
into a plasmid pBP322;
Fig. 4 shows a process for preparing a recombinant plasmid pUG3 from pUGl and pUG2;
Fig. 5 shows the result obtained by analyzing the nucleotide sequence of oligonucleotide A-3 by two-2~ dimensional fractionation by electrophoresis andhomochromatography;
Fig. 6 shows a process for preparing a recombinant plasmid pGH37;
Fig. 7 shows a process for preparing a recombinant plasmid pGH35;

- ~ 3~23 Fig. 8 shows a process Eor preparing a recombinan~ plasmid pEK28;
Fig. 9 shows processes for preparing recombinant plasmids pUG102 to pUG122 and recombinant plasmids pUG103-E and pUG117-R;
Fig. 10 shows processes for preparing xecombinant plasmids pBRH02 and pBRH03;
Fig. 11 shows ~boII restriction map of pUG3 including H fragment (179 b.p.) which contains the present ~-urogastrone gene;
Fig. 12 shows a process for preparing recombinant plasmids pUG2301 to pUG2303;
Fig. 13 shows a process for preparing recombinant plasmids pUG2101 to pUG2105;
Fig. 14 shows a process for preparing recombinant plasmids pUG2701 to pUG2703;
Fig. 15 shows a process for preparing recombinant plasmids pUG1102 and pUG1105;
Fig. 16 shows a process for preparing a recombinant plasmid pUG1004;
Fig. 17 shows a process for preparing a recombinant plasmid pUG1201;
Fig. 18 shows a process for preparing a recombinant plasmid pUG1301; and Photos 1 to 5 are respectively show analytical ~.3~L~:~3 resùlts oE nucleotide sequences of recombinant plasmids obtained in example by the Maxam-Gilbert method.
The procedures themselves for constructing the gene II of the present invention are known. The oligonucleotides for constructing the gene II can be prepared by known processes, for example, by the solid phase process to be described below briefly (see, for example, H. Ito et al., Nucleic Acids Research, 10, 1755-1769 (1982)).
When the solid phase process is resorted ;to, the oligonucleotide is synthesized, as shown in Fig. 1 by successively coupling mononucleotides or dinucleotides with a nucleoside supported on polystyrene resin to obtain a predetermined sequence of nucleotides.
The nucleoside supporting resin can be prepared, for example, with use of a partially crosslinked poly-styrene resin by reacting N-(chloromethyl)-phthalimide with the resin, reacting hydrazine with the product to obtain aminomethylated polystyrene resin, and linking -to the amino group thereo~ a nucleoside having its 5' hydroxyl group free and amino group protected, using succinic acid as a spacer.
On the other hand, various processes are known for preparing mononucleotides or dinucleotides (see, for example, C. Broka et al., Nucleic Acids Research, 8, .3~ 23 5~61-5~71 (1980)). For example, a mononucleotide can be prepared by reacting o-chlorophenylphosphorodichloridate, triazole and a nucleoside having its 5' hydroxyl group protected with a dimethoxytrityl group (DMTr) in the presence o~ triethylamine, then reacting the mono-triazolide obtained with ~-cyanoethanol in the presence of l-methylimidazole as a catalyst, and eluting the reaction product with chloroform-methanol by silica gel column chromatography. This process gives a completely protected mononucleotide.
A dinucleotide can be prepared by treating the completely protected mononucleotide obtained above with benzenesulfonic acid or like acid to give the mono-nucleotide with the 5' hydroxyl group free, which is react with the monotriazolide obtained above, and eluting the reaction product with chloroform-methanol by silica gel column chrorna~o~raphy. This process affords a cotnpletely protected dinucleotide.
The solid phase synthesis of oligonucleotides is conducted advantageously using a DNA synthesizer which is, for example, available as DNA synthesizer of Bachem Inc., U.S.A. The nucleoside supporting resin obtained above is placed into a reaction vessel and washed with dichloromethane-isopropanol, and a solution of zinc bromide in dichloromethane-isopropanol is added 13040;~3 _ 15 ~

to the resin to remove the dimethoxytrityl ~roup 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 dimethyl-formamide to remove the remaining zn2 , thereafter washed with tetrahydrofuran and exposed to nitrogen gas stream ~or several minutes for drying. Separately, the completely protected dinucleotide or mononucleotide is dissolved in pyridine followed by addition of triethyl-amine, and the resulting solution is shaken, then allowed to stand at room temperature for several hours and there-after evaporated under reduced pressure. The resul-ting triethylammonium salt is dissolved in pyridine and azeotropically evaporated several times with use of pyridine for drying. The salt of nucleotide is dissolved in a solution of mesitylenesulfonyl-5-nitrotriazole, (MS~T, condensation reagent) in pyridine. The resulting solution is added to the dried resin and allowed 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 dimethylaminopyridine as catalyst in tetrahydrofuran-pyridine to mask the unreacted hydroxyl group. Finally the resin is washed with pyridine to complete one ~304023 cycle of solid phase synthesis. One cycle extends the nucleotide sequence by one or 2 chain lengths. The above procedure is repeated to couple mononucleotides or dinucleo-tides successively with the resin to the desired length, whereby a completely protected oligo-nucleotide can be obtained as supported on the resin.
To the resulting xesin is added a solution of tetramethylguanidine-2-pyridinealdoximate in pyridine-water, and the mixture is allowed to stand with heating.
The resin is then filtered off and washed with pyridine and ethanol alternately. The washings and filtrate are combined together and concentrated under reduced pressure.
The concentrate is dissolved in an aqueous solution of triethylammonium bicarbonate (TEAB), followed by washing with ether. The aqueous~ solution is subjected to ,~ C~r~e~ ~f'~
Sephadex G-50~column chromatography using a TEAB solution as an eluent. The fractions are collected and the optical density of each fraction is measured at 260 nm.
A fraction including the first eluate peak is concentrated.
The concentrate is purified, for example, by high-speed liquid chromatography until a single peak is obtained.
The oligonucleotide thus obtained still has its 5' end protected by a dimethoxytrityl group, so that the product is treated wi~h an aqueous solution of acetic acid to remove the protective group, followed again by high-speed ~3~4[)23 liquid chromatography or the like for purification until a single peak is obtained.
The desired oligonucleotides are prepared by the process described above and then checked individually for the nucleotide sequence by a two-dimensional fractionating method using electrophoresis and homo-chromatography and (or) the Maxam-Gilbert method and thereafter used for preparing the blocks and subunits.
The two-dimensional fractionating method for checking the nucleotide sequence can be carried out by the procedure of Wu et al. (E. Jay, R. A. Bambara, R.
Padmanabhan and R. Wu, Nucleic Acids Res., 1, 331 (1974)).
To practice this method, the oligonucleotide as lyophilized is dissolved in distilled wa~er to a con-centration of about 0.1 ~g/~l. A portion o this solutionis treated with ~-32P-ATP and T~ polynucleotidekinase to label the 5' end with 32p and then partially digested with snake venom phosphodiesterase. The product is spotted on a cellulose acetate film and subjected to electrophoresis for the ~irst dimensional development to separate the product according to the dif~erence o~
bases. The developed products are then transfered onto a diethylaminoethyl cellulose (DEAE cellulose) plate and subjected to the second dimensional development using a solution o~ partially hydrolysed RNA called a homomixture.

3 ~ 3 _ 18 -(This procedure is termed homochroma-tography). In this way, the oligonucleotide is separated according to the chain length. Subsequently, the nucleotide sequence of the oligonucleotide is read autoradiographically starting with the 5' end.
If it is difficult to check the sequence by this method, the Maxam-Gilbert method is resorted to when required. (A. M. Maxam and W. Gilbert, Proc. Natl.
Acad. Sci., USA, 74, 560 (1977), A. M. Maxam and W.
Gilbert, Methods in Enzymol., Vol 65, p.499, Academic Press 1980).
This method, which is called also a chemical decomposition method, employs a reaction specific to a particular base to cleave the oligonucleotide at the position of the base, and the bands revealed by electro-phoresis serve to read the sequence from the 5' or 3' end. The base-specific reactions are as follows.
Guanine is specifically methylated by dimethyl sulfate.
Guanine and adenine undergo depurination reaction in the presence of an acid. Thymine and cytosine both react with hydrazine in a low concentration of salt, but cytosine only reacts with hydrazine in a high con-centration of salt. After the completion of reactions for the ~our bases, each reaction mixture is reacted with piperidine to displace ring opened base and to ~ 3~ 3 _ 19 -catalyze ~-elimination of both phosphates Erom the sugar, and finally the DNA strand is cleaved at that base.
The resulting reaction mixtures are subjected to poly-acrylamide gel electrophoresis respectively to confirm the nucleotide sequence according to which of the reactions produced each band.
Next, the oligonucleotides are ligated by using a T4 DNA ligase as shown in Fig. 2. For the correct ligation, the 16 oligonucleotides A-l to A-16 cor-responding to the subunit A are ligated as divided intothree sets, i.e. the block 1 comprising A-l, A-2, A-3, A-14, A-15 and A-16, the block 2 comprising A-4, A-5, A-6, A-ll, A-12 and A-13, and the block 3 comprising A-7, A-8, A-9 and A-10 as shown in Fig. 2. By electrophoresis the blocks 1 to 3 having the correct sequences are obtained and are further ligated into the subunit A.
Stated more specifically, some of the 5' ends of the 16 oligonucleotides A-l to A-16 are labeled with 32p with use of ~-32P-ATP and T4 polynucleotidekinase;
and the hydroxyl groups of the remaining 5' ends are phosphorylated with ATP. To form each o~ the three blocks, the oligonucleotid~s are assembled and ligated with use of T4 DNA ligase, and the product is electro-phoresed on polyacrylamide gel to isolate the desired block. The three blocks thus obtained are ligated with ~L3~4~23 _ 20 -use of T4 DNA ligase to produce subunit A. Although a dimer structu~e may be produced in the ligation reation, it is easily cleaved with the restriction enzymes EcoRI
and BamHI to obtain the subunit A. Subsequently, as seen in Fig. 2, a known plasmid vector, pBR322, which is derived from _.coli and readily available, is cleaved with EcoRI and BamHI, and the subunit A is inserted into the vector to obtain a recombinant plasmid pUG 1.
The same procedure as above is followed also for the subunit B. As in the case of the subunit A, the 16 oligonucleotides B-l to B-16 are ligated as divided into ~our sets as seen in Fig. 3, and the blocks are ligated together to produce subunit B. The dimer, if produced, is cleaved with the restriction enzymes HindIII
and BamHI to obtain the subunit B. A plasmid vector, pBR322, is cleaved with HindIII and BamHI, and the subunit B is inserted into the vector to obtain a recombinant plasmid pUG2 as seen in Fig. 3, Further as shown in Fig. 4, pUGl is cleaved with restriction enzymes HindIII and SalI, and a fragment removed from pUG2 with use of the same restriction enzymes is inserted into pUGl to prepare a recombinant plasmid pUG3 havin~ a ~-urogastrone structural gene (gene II).
pUGl, pUG2 and pUG3 are recombinant plasmids which each comprise pBR3~2 and the subunit A which is ....

- 13~23 the front half portion of the ~-urogastrone structural gene, the subunit B which is the rear half of the gene, or the entire structural gene. These recombinant plasmids can be proliferated to large quantities by introducing them into a host, such as the strain HB101 of E.col_ which is known and readily available according to the calcium method as the transformation method (E. Lederberg and S. Cohen, J. ~acteriol., 119, 1072 (1974)).
Whether pUGl, pUG2 and pUG3 are present in the host such a5 the strain HB101 of E.coli can be checked by the ~ollowing methods. After the plasmids are collected by the alkaline extraction method, pUGl and pUG2 are checked or the presence of the BglII recognition site which is not present on the ~ector pBR322. Similarly, pUG2 and pUG3 are checked whether they can b~ cleaved with MluI which is not present on pB~322.
According to the alkaline extraction method E.coli harboring the plasmid is incubated, the cells are then collected, and lysozyme is caused to act thereon to dissolve the cell wall. A mixture of sodium hydroxide and sodium laurylsulfate is used to disrupt the cell and then to denature the DNA, which is then neutralized with sodium acetate buffer. At this time, the chromosomal DNA
remains denatured, but the plasmid, which is an extra-chromosomal DNA, restores the initial double stranded form.

.;.... ...

_ 22 -Plasmids are collected by utilizing these characteristics.
The plasmids are further subjected to density-gradient ultracentrifugation with cesium chloride and ethidiu~
,~ bromide for purif-cation and then passed through a racl~v~
Biogel A~50m column to remove RNA. Thus plasmides can be obtained with a high purity in a large quantity.
In this way, the ~-urogastrone gene of the invention (gene II) can be obtained.
Next, the method of introducing the ~-urogastrone gene into host cells will be described.
The host cells to be used in this invention are not limited particularly and any of those known is usable, for example, those of E.coli, Bacillus, Pseudomonas, yeasts, etc., among which E.coli cells are preferable.
The modes of expressing the ~-urogastrone gene with use of E.coli includes a system for directly expressing ~-urogastrone, and a system wherein it is expressed as a fused prokein with ~-lactamase or other different protein.
For the direct expression of ~-urogastrone gene, it is required to introduce into the recombinant plasmid, upstream o~ the ~-urogastrone gene, a promotor and an SD
sequence. While the promotor is not limited particularly, desirable promotors are those assuring a high degree of expression, such as ~PL which is the left ward promotor ~3 1)4Q~3 of ~ phage,lac UV5 which is present upstream of ~-~alactosidase gene of E.coli, etc. When ~PL is used as the promotor, the SD sequence is not limited parti.cularly, but it is desirable to use the four-base sequence of AGGA. Further wllen lac UV5 is used as the promotor, it is desirable to use the SD sequence which occures downstream of the lac UV5 promotor or the one chemically synthesized.
The system for directly expressing the ~-urogastrone gene will be described with reference to the case wherein ~PL-SD seqùence-~-urogastrone gene is used.
Although ~PL is a powerful promotor (J. Hedgpeth et al., Molecular and General Genetics, 163, 197-203 (1978)), the fully activated ~PL promotor causes lethal effects on the host E.coli cell, so that there is a need to proliferate the cell under the condition free of any lethal action and therea~ter cause the ~PL to function.
On the other hand, CI857 which is a gene withi~ ~ phage is one of the mutated genes of CI repressor which acts on the operator for ~PL. At low temperatures (of up to about 30C), the CI857 repressor binds to the operator to completely inhibit the activity of ~PL as a promotor, consequently permitting proliferation of E.coli.
Therefore, the host cells are allowed to proliferate in this state and thereafter brought to a high temperature ~.3~4~3 _ 24 -(of not lower than 37C), whereby the ~PL is allowed to function. Furthermore, the plasmid vectors, such as pSC101 which is known and readily available, having a stringent replicating mechanism, and those such as 5 pBR322 having a relaxed replicating mechanism are not incompatible with each other but can coexist within the same E.coli cell.
Accordingly it is suitable to construct a recombinant plasmid pGH37 wherein a CI857 gene is incorporated in a tetracycline-resistanct plasmid vector pSC101 (with lac UV5 promotor provided upstream thereof for the efficient expression of CI857) as seen in Fig. 6 and to introduce the recombinant plasmid into E . coli (HB101 strain) to obtain a transformant (ECI-2 strain) for use as a host for the vector for expressing ~-urogastrone under the control of -the ~PL promotor.
According to the present invention, the ~PL-S~
sequence-~-urogastrone gene is introduced, for example, into pBR322 to obtain a ~-urogastrone expressing vector, which is used for transforming the strain ECI-2, whereby a so-called two-plasmid system is provided wherein two useful plasmids coexist in a E. coli cell.
With this system, the CI857 repressor encoded by pGH37 binds to the operator for ~PI promotor on the 25 second plasmid when -the cell is cultured for example at , f!~

` ~.3al~lr3~3 _ 25 -30C, permit-ting the prolifexation of the cell. After the cell is fully proliferated in this state, the temperature is raised for example to ~0C, whereupon the CI857 repressor is dissociated from the operator, permitting the activity of ~PL promotor for -the expression of ~-urogastrone.
Although a similar concept was applied to the e~pression of fibroblast interferon, SV-40 Small t antigen, etc., in these cases a ~ lysogen is used as a host in which the DNA of ~ phage carrying a CI857 gene is introduced into the host chromosome (R. Derynck et al, Nature, 287, 193-197 (19803, C. Derom et al, Gene, 17, 45-54 (1982), K. Kupper et al, Nature, 289, 555-559 (1981)).
With the system of the present invention, however, the CI857 gene is introduced into a different plasmid which is resistant to tetracycline. Accordingly the present system has the advantages that there is no likelihood that the ~ phage introduced into the host chromosome will be induced into proliferation and that the strain can be controlled easily. Of course, the two--plasmid system is used ~or the first time for systems for expressing ~-urogastrone.
According to another system, a portion of any other protein gene such as ~-lactamase gene is ligated to the ~-urogastrone gene to express the ~-urogastrone gene as a fused protein. This method has the advantage that the fused protein is less susceptible to decomposition by the protease within the E.coli to consequQntly aford protection for ~-urogastrone. Another advantage is that the fused protein migrates to and accumulates in the periplasm in the cell of E.coli (S. J. Chan et al, Proc.
~atl. Acad. Sci., U.S.A., 78, 5401-5405 (1981)), is locally present and is therefore easy to separate and purify.
Stated more specifically, a gene coding for two basic amino acids which can provide a cleavage site for taking out ~-urogastrone from the fused protein by cleaving ~ith an enzyme is inserted into the ~-lactamase gene at a suitable restriction enzyme cleaving site, and a ~-urogastrone gene is ligated to the ~-lactamase gene Preferably the sequence of two basic amino acids is -Lys-Arg- or -Arg-Lys-. Examples of enzymes for recognizing the amino acid sequence to cleave ~-urogastrone from the fused protein are ~allikrein, trypsin, etc ~xamples of restriction enzymes for cleaving the ~-lactamase gene are XmnI, HincII, ScaI, PvuI, PstI, BglI, BanI, etc.
The ~-lactamase-~-uro~astrone recombinant plasmid thus prepared can express a fused protein within ~3~2~
_ 27 -E.coli for quantity production. The resulting fused protein is treated with kallikrein or the like, whereby ~-urogastrone can be obtained.
The expression system can be checked by directly analyzing the nucleotide sequence of the gene by the Maxam-Gilbert method, by confirming the insertion oE gene and direction thereof by the mini-preparation or mapping method (H. C. Birnboim e~ al., Nucleic Acids Research, _, 1513-1523 ~1979)), or by radioimmunoassey for ~-urogastrone.
The trans~orman~ of the present invention thus obtained is cultured by the usual method, whereby ~-urogastrone can be collected with a high purity in a large quantity.
The present invention will be described below in greater detail with reference to the following example to which this invention is limited in no way.
Example 1) Preparation of nucleoside supporting resin Various nucleoside supporting resins were prepared by the ~ollowing method.
A quantity of 1 wt.% crosslinked polystyrene resin "S-Xl," ~product oE BIO.RAD Laboratories, U.S.A., 200 to 400 mesh) was mixed with 2.41 g of N-(chloromethyl)-phthalimide, 0.22 ml o~ tri~luoromethanesulEonic acid and ~L3~ 3 50 ml of dichloromethane by stirring at room temperature for 2 hours. Af-ter the completion of reaction, the resin was filtered, washed with dichloromethane, ethanol and methanol in succession, dried under reduced pressure and then refluxed with 50 ml of 5 wt.% solution of hydrazine in ethanol overnight by heating. The resin was filtered and washed with ethanol, dichloromethane and methanol successively and then dried under reduced pressure.
The mixture of aminomethylated polystyrene resin (2.5 g) obtained by the above procedure, 0.75mM of monosuccinic acid ester of 5'-o-dimethoxytritylnucleoside, 1.23mM of dicyclohexylcarbodiimide and lmM of dimethylaminopyridine was allowed to stand overnight at room temperature with addition of 30 ml of dichloromethane. The resin was filtered, washed with dichloromethane, methanol and pyridine successively, then immersed in pyridine-acetic anhydride (90:10 in volume ratio) and allowed to stand at room temperature for 30 minutes. The nucleoside supporting resin obtained was filtered, washed with pyridine and dichloromethane and dried under reduced pressure for use in solid phase synthesis reaction.
2) Synthesis of dinucleotide As an example, synthesis of a completely protected dinucleotide having the base sequence of TA
will be described. Adenosine (13.14 g) having its 5' ~.3~ 3 hydroxyl group protected with a dimethoxytrityl (DMTr) group and the amino group with a benzoyl group and 6.3~ g of triazole were dissolved in anhydrous dioxane.
With ice cooling, 8.35 ml of triethylamine was added to the solution, then 6.86 g of o-chlorophenylphosphoro-dichloridate was added dropwise to the mixture over a period of 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 its volume 3 and 3.6 g of ~-cyanoethanol and 4.8 g of l--methylimidazole were admixed 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 with O.lM aqueous solution of sodiu~l phosphate, dibasic three times and with water twice and therea~ter concentrated under reduced pressure, giving 19.96 g of crude product.
The product was purified by silica gel column chromato-graphy using chloroform-methanol (98:2 in volume ratio) as an eluent. The purifying procedure was repeated to ob-tain 15.12 g of completely pro~ected adenosine mono-nucleotide.
The adenosine mononucleotide (7.81 g) thus obtained was added to a 2 wt.% solution of benzenesulfonic ~L3~ Z~

acid in chloroEorm-methanol (70:30 in volume ratio), and the mixture was stirred with ice cooling for 20 minutes and then neutralized with aqueous solution o~ sodium hydrogen-carbonate. The separated chloroform layer was washed with water and concentrated under reduced pressure, giving 7.11 g of a crude product. The product was subjected to silica gel column chromatograp'ny and eluted with chloroform-methanol (97:3 in volume ratio) to obtain 4,31 g of adenosine mononucleotide having a free 5' hydroxyl group.
Thymidine (1.64 g) having its 5' hydroxy group protected with a dimetho~ytrityl group and 0.95 g of triazole were dissolved in 21 ml of anhydrous dioxane, 1.25 ml of triethylamine was added to the solution, and 0.69 ml of o-chlorophenylphosphorodichloridate was added dropwise to the mixture over a ~eriod of 5 minutes with stirring and ice cooling. The mixture was thereafter stirred at room temperatuxe for 1 hour. The triethylamine hydrochloride resulting from the reaction was filtered Off, and the filtrate was stirred for lO minu~es with 1.1 ml of an aqueous solution of pyridine (lM). To the solution were added a dioxane solution (10 ml) of 1.17 g of the adenosine mononucleotide having the free 5' hydroxyl group and prepared as above and 0.72 ml of l-methylimidazole, and the mixture was stirred at room ~ 3~ 23 temperature for 3 hours. The reac~ion mixture obtained was concen~rated under reduced pressure, the residue was dissolved in ethyl ace-tate, and the solution was washed with an aqueous solution of sodium phosphate, dibasic (O.lM)and then with water and concentrated under reduced pressure, glving 2.39 g of crude product. The product was subjected to silica gel column chromatography and eluted with chloro:Eorm-methanol (98:2 in volume ratio) to obtain 2.39 g of completely protected dinucleotide TA.
In the same method as above various nucleotrides were prepared.
3) Synthesis o~ oligonucleotide The solid phase synthesis of the oligonucleotide A-l, i.e. undecanucleotide AATTCGAAGAT, will be described.
A resin (40 mg) having the nucleoside T
supported thereon and prepared in the above method 1) was placed into a reaction vessel, washed with dichloro-methane-isopropanol (85:15 in volume ratio) three times, and then treated with a solution of zinc bromide (lM) 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 wi~h dichloromethane, then washed with a solution of triethylammonium acetate (0.5M) in dimethylformamide to remove the remaining ~n , further 1~114~2:~

washed with tetrahydrofuran and dried by passing nitrogen gas through the reaction vessel for several minutes.
The dinucleotide GA (50 mg)~ completely protected and prepared as in the above method 2), was dissolved in 1 ml of pyridine, shaken with 1 ml of tri-ethylamine and then allowed to stand at room temperature for several hours. The solution was then evaporated under reduced pressure. The residue was azeotropically evaporated several times with pyridine to convert the nucleotide to a triethylammonium salt. The salt was dissolved in 0.3 ml o~ solution of mesitylene-sulfonyl-5-nitrotriazole (0.3M) in pyridine. The solution was added to the dried resin, followed by reaction at room temperature for 60 minutes. The liquid portion was filtered off from the reaction mixture, and the solid portion was washed with pyridine and then allowed to stand for 5 minutes in a mixture of 0.2 ml of acetic anhydride and 0.8 ml of a solution o~ dimethylamino-pyridine (0.lM) in tetrahydro~uran-pyridine to mask the unreacted hydroxyl group. Finally, the resin was washed with pyridine, whereby one cycle of solid phase synthesis process was completed. One cycle extends the nucleotide chain by 2 base lengths. The same procedure as above was repeated to successi~ely couple the dinucleotides AA, CG, TT and AA with the resulting nucleotide by condensation, ~L3C1 4~3 whereby the completely protected undecanucleotide AATTCGAAGAT was prepared as supported on the resin.
The resin (20 mg) obtained was allowed to stand at 40C for 1 hour with 0.6 ml of a solution of tetra-methylguanidine-2-pyridinealdoximate (0.5M) in pyridine-water (90:10 in volume ratio). The resin was then passed through a pasteur pipette plugged with cotton and thereby filtered off. The resin was washed with pyridine and ethanol alternately. The washings and the filtrate were combined together and concen~rated at 40C under reduced pressure. The residue was dissolved in 2 ml of an aqueous solution of triethylammonium bicarbonate (TEAB, lO~M). The solution was washed with ether three times.
The aqueous phase was applied to a Sephadex G-50 column (2 x 100 cm) and eluted with lOrnM TEAB solution. The fractions were checked for absorbance at 260 nm. The fraction including the first eluate peak was concentrated.
The residue was subjected to high-speed liquid chromato-graphy (pump: Model 6000A, detector: Model 440, products of Waters Associates, U.S.A.) to obtain a purified fraction having a single peak. For the high-spee~
liquid chromatography, the column used was ~-Bondapak~C18 (product o~ Waters Associates, U.S.A.), and acetonitrile~
aqueous solution of triethylammonium acetate (O.lM) was used as an eluent for gradient elution (5 ~ 40 vol.~).

~3~ 23 ~ 3~ -The undecanucleotide thus purified still had lts 5' end protected with dimethoxytrityl group, so that the compound was treated with 80 vol.% aqueous solution of acetic acid for 15 minutes to remove the dimethoxytrityl group and then purified by high-speed liquid chromatography again until a single peak is obtained. The same column as above was used for this purpose, and acetonitrile-aqueous solution of triethylammonium acetate (O lM) was used for gradient elution (5 -~ 25 vol.%).
In the same manner as above, the oligo-nucleotides A-2 to A-16, and B-l to B-16 were synthesized.
Table 1 shows the yield of each oligonucleotide determined with use of 20 mg of the resin resulting from the solid phase synthesis, by cutting off the oligo-lS nucleotide from the resin, fol].owed by removal of theprotective group and purificati.on.
The yield was calculated from the measurement o~ absorbance of the final purified product at 260 nm and the sum of absorbance values for the nucleotide bases ~3g~ 23 Table 1 -A-l, 80~g A-2, 120~g A-3, 90~g A-4, 50~g A-5, 140~g A-6, 70~lg A-7, 80~g A 8, 90~g A-9, lOO~g A-10,90~g A-ll, llO~g A-12; 40~g A-13,50~g A-14,40~g A-15, 60~g A-16, 150~g B-l, 60~g B-2,lOO~g B-3, 50~g B-4, 90~g B-5, lOO~g B-6,90~g B-7, 130~g B-8, lOO~g B-9, llO~g B-10, lOO~g B-ll llO~g B-12, llO~g B-13, 130~g B-14, 60~g B-15, 70~ _B-16, 50~g 4) Checking of the sequence of oligonucleotide bases The sequence was checked in accordance with the two-dimensional fractionation by electrophoresîs and homochromatography of Wu et al. hereinbefore mentioned.
The oligonucleotides A-l to A-16 and B-l to B-16 were each found to have the contemplated nucleotide seq-uence. Fig. 5 shows the result obtained by analyzing the oligonucleotide A-3, in which A-3 was found to have the sequence of GATTCTGAGTG as read from the 5' end.
The nucleotide sequsnce of each oligonucleotide was also checked by the Maxam-Gilbert method stated above.
It was confirmed that the oligonucleotides A-l to A-16 and B-l to B-16 each had the contemplated nucleotide sequence.

~L3~4~3 5) Construction of oligonucleotide blocks and subunits The blocks and subunits were prepared by theprocedure shown in Fig. 2 as described in detai]. below.
First, about 5 ~g of each of oligonucleotides A-l, A--2, A-3, A-14, A~15 and A-16 was dissolved in distilled water (50 ~1~ to obtain a solution having a concentration of about 0.1 ~g/~l. The six kinds of aqueous solutions were placed, each in an amount of 10 ~1 (1 ~g calculated as DN~), into other six Eppendorf tubes individually. A mixture solution (6 ~1) containing 250mM
tris-HCl (pH 7.6~, 50mM magnesium chloride, lOmM spermine and 50mM DTT was placed into each tube, followed by addition of 0.5 ~1 of y-32P-ATP aqueous solution (product of Amersham International Ltd., U.K.), 0.5 ~1 of T4 polynucleotidekinase (product of Takara Shuzo Co., Ltd., Japan) and 13 ~1 of distilled water, to obtain 30 ~1 of mixture. The mix-ture was reacted for 30 minutes at 37C, and further reacted for 30 minutes with addition of 1 ~1 of 30mM ATP aqueous solution. The reac~ion was terminated by heating at 100C for 2 minutes. The reaction mixture was rapidly cooled with ice. The oligonucleotides A-l, A-2, A-3, A-14, A~15 and A-16 thus having the 5' end phosphorylated were placed, each in an amount of 10 ~1, into another single 1.5 ml Eppendorf tube. Into the tube were placed 40 ~1 of 250mM tris-HCl aqueous solution ~3~ 23 ~pH 7.6), 40 ~1 of 50mM magnesium chloride and 35 ~1 of distilled water to obtain a total amount of 175 ~1.
The mixture was heated at 90C for 2 minutes, then gradually cooled to room temperature. With addition of 10 ~1 of 200m~ DTT aqueous solution, 10 ~1 of 20mM ATP
aqueous solution and 5 ~1 ~100 units) of T4 DNA ligase (product of Nippon Gene Co., Ltd., Japan)1 the mixture was reacted overnight at 4C, giving the block 1 o ligated oligonucleotides A-l, A-2, A-3, A-14, A-15 and A-16 was prepared.
The block 2 and block 3 were similarly ormed by the ligation of A-4, A-5, A-6, A-ll, A-12 and A-13 and by the ligation of A-7, A-8, A-9 and A-10.
Ethanol was added to the reaction mixture of the blocks thus prepared in twice the volume thereof, and the mixture was allowed to stand at -80C for 30 minutes to precipitate DNA, followed by electrophoresis on a 12.5 wt.% polyacrylamide gel and autoradiography. This resulted in bands at the positions of 72 b.p. (base pair) and 36 b.p. for the block 1, a band at the position of 36 b.p. for the block 2 and bands at the positions of 48 b.p. and 24 b.p. ~or the block 3. Subsequently, each band was cut out and a mixture of lOmM tris-HCl (pH 7.6) and lOm~ EDTA aqueous solution (tris-EDTA) was added thereto. The mixture was then allowed to stand overnight ~L3~ 23 at room temperature for extraction. The resul-ting mixture was centrifuged, the supernatant was separated, and the supernatant was fully shaken with tris-EDTA saturated phenol and then centrifuged to discard the lower layer.
The same procedure was repeated twice with tris-EDTA
saturated phenol. Finally, the upper layer was passed through a column, 1 cm in diameter and 20 cm in length, packed with ~ephadex G-50 to remove the phenol and acrylamide. The elute was then concentrated to 200 ~1 and thereafter allowed to stand at -80C for 30 minutes with ethaonl in twice the volume of the concentrate to precipitation DNA.
The three blocks obtained were combined together. To the mixture were added 50mM tris-HCl (p~ 7.6), lOmM magnesium chloride, 20mM DTT, lmM ATP
and 5 ~1 (100 unit) of T4 DNA ligase. The resulting mixture was allowed to stand overnight at 4C for ligation. To the mixture was added ethanol in twice the volume thereo~, and the mixture was allowed to stand at -80C ~or 30 minutes to precipitate DNA, ~ollowed by electrophoresis on 8 wt.% polyacrylamide gel and autoradiography, which revealed bands at 96 b.p.
and 192 b.p. Each band was cut out, and tris-EDTA was added thereto, and the mixture was allowed to stand over-night at room temperature for extraction. The mixture ~L3~4~23 was centrifuged to separate the supernatant, -the super-natant was fully shaken with tris-EDT~ saturated phenol, and the lower layer was discarded. With further addl-tion of tris-EDTA saturated phenol, this procedure was repeated twice. The upper layer was passed through a Sephadex G-50 column, the elute was concentrated, and to the concentrate was added ethanol in twice the volume of the concentrate, followed by standing at -80C for 30 minutes to precipitate DN~. The resulting product was cleaved with ~coRI and BamHI to obtain the subunit A.
The same procedure as above was repeated as seen in F'ig. 3 to ligate oligonucleotides B-l, B-2, B-15 and B-16 into the block 4, to ligate oligonucleotides B-3, B-47 B-13 and B-14 into the block 5, to ligate oligonucleotides B-5, B-6, B-ll and B 12 into the block 6, and to ligate oligonucleotides B-7, B-8, B-9 and B-10 into the block 7. The blocks corresponding ~o 26 b.p.
and 52 b.p. were collected, and similarly ligated to give products o 104 b.p. and 208 b.p., which was cleaved with HindIII and BamHI, Thus, the subunit B was obtained 6) Cloning of subunits and analysis of recombinant plasmids With reference to Fig. 2, pBR322 was cleaved with EcoRI and BamHI, and phosphate groups were removed from the 5' ends with alkaline phosphatase (product of Takara Shuzo Co., Ltd., Japan) so as not to restore the original state. Subsequently pBR322 thus cleaved and dephosphorylated and the subunit A were allowed to stand overnight at ~C in a mixture of 50mM tris-HCl (pH 7.6), lOmM magnesium chloride, 20mM DTT and lmM ATP with addition of 5 ~1 of T4 DNA ligase, whereby they were ligated. To the reaction mixture was added ethanol in twice the volume thereof, and the mixture was allowed to stand at -80C for 30 minutes for precipitation.
The mixture was then centrlfuged, the precipitate was dried and dissolved in 100 ~1 of distilled water, whereby a plasmid pUGl was obtained in which the subunit A was incorpora~ed in pB~322.
E.coli strain HB101 was transformed with the plasmld pUGl by the calcium method.
The strain HB101 serving as a host was cultured at 37C in a 50 ml of LB culture medium (1 wt.~ of bactotrypton, 0.5 wt.~ of yeast extract and 0.5 wt.% of sodium chloride). I~hen the absorbance at 610 nm reached 0.25, a 40 ml portion of the culture broth was transferred into a centrifugal tube and centrifuged at 6000 r.p.m.
for 10 minutes at ~C. The supernatant was discarded, the precipitate was suspended in 20 ml of ice-cooled O.lM
magnesium chloride, the suspension was centrifuged under the same condition again, and the supernatant 3l~ 23 was discarded. The precipitate was suspended in 20 ml of ice~cooled solution of O.lM calcium chloride and 0.05M magnesium chloride and ice-cooled for 1 hour.
The suspension was centrif~lged, the supernatant was discarded, and the precipitate was suspended in 2 ml of ice-cooled solution of O.lM calcium chloride and 0.05M
magnesium chloride. To a 200 ~1 portion of the suspension was added 10 ~1 of aqueous solution of pUGl, and the mixture was ice-cooled for 1 hour and then heated in a water bath at ~3.5C for 30 seconds. Subsequently, 2.8 ml of LB culture medium was added to the mixture, followed by incubation at 37C for 1 hour. The culture was then spread over a LB plate containing 50 ~Ig/ml of ampicillin, in an amount of 200 ~l/dish and incubated overnight at 37C. The growing colonies were checked by further transplantation to a LB plate containing 50 ~g/ml of ampicillin and also to a LB plate containing 20 ~g/ml of tetracycline and were incubated overnight at 37C.
The colonies resistant to ampicillin only were separated to obtain a transformed cell.
Plasmids were collected from the cell on a small scale by the alkaline e~traction method and checked for the presence of a BglII cleavage site. One of the cells containing the plasmid having BglII cleavage site was cultured in a large scale to obtain purified plasmid gL~0~ 23 pUGl similarly by the alkaline extraction method.
The nucleotide sequence of the subunit A
incorporated in the resulting pUGl was analyzed on both strands by the Maxam-Gilbert method stated above.
Photos 1 and 2 show the results o-f analysis.
Lanes 1 to 4 axe the result of electrophoresis for EcoRI - SalI fragment, and lanes 5 to 8 are that for 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 show the reaction products for thymine ~ cytosine, and lanes 4 and 8 show the reaction products for cytosine. Photo 2 shows the results achieved by the same specimens as above, in which a region of the higher molecular weight side (corresponding to the upper portion of Photo 1) is enlarged. In this way, the nucleotide sequence of the subunit A was confirmed,, With re~erence to Fig. 3, the plasmid pBR322 was clea~ed with HindIII and BamHI, and the larger fragment was iso~ated by means of electrophoresis and ligated to the subunit B. Thus, a plasmid pUG2 in which subunit B was introduced into pBR322 was obtained similarly as in the case of pUGl. Using the resulting plasmid pUG2, the strain HB101 was transformed, and the colonies resistant to ampicillin only were selected.

~L304~3 - ~3 -Plasmids were collected from the colonies and then checked for the presence of a BglII cleavage site and a MluI cleavage site. The cells containing the plasmid having both sites were selected. One of the selected cells was cultured in a large scale to obtain purified plasmid pUG2. The nucleotide sequence of the subunit B
in pUG2 was analyzed on both strands by the Maxam-Gilbert method.
Photo 3 shows the results of analysis. Lanes 1 to 4 show the result achieved by HindIII-SalI fragment.
Lanes 5 to 8 show the result achieved by the same specimen, in which a region of the higher molecular weight side (corresponding to the upper portion of lane 1 to 4) being shown on an enlarged scale. ~ach lane shows the same corresponding reaction product as in Photo 1. Thus, the nucleotide sequence of the su~unit B was confirmed.
Next with reference to Fig. 4, pUGl was cleaved with HindIII and SalI, and a larger fragment was separated through a Biogel 1.5 m column. pUG2 was cleaved with HindIII and SalI, followed by electrophoresis to obtain a smaller fragment. The two fragments were combined together and treated with T4 DNA ligase for ligation, whereby plasmid pUG3 was obtained wherein the subunits A plus B, i.e. ~-urogastrone gene, was incorporated into pBR322. E. coli strain HB101 was transformed using ~3C~4~2 - ~4 -the plasmid pUG3. The transformant has been deposited under Budapest Treaty on international recognition of deposit with deposition number F~RM BP-543 in Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, since June 22, 19~4.
In the above case also, the cells harboring plasmid pUG3 which was resistant to ampicillin only and had MluI cleavage site were selected. One of the selected cells was cultured in a large scale to obtain purified plasmid pUG3. The nucleotide sequence of the ~-urogastrone gene in pUG3 was analyzed on both strands by the Maxam-Gilbert method.
Photo 4 shows the results of analysis. Lanes 1 to 4 show the result obtained with Bam~ PstI fragment.
Lanes 5 to 8 show the result obtained with the same speciment, in which a region of the higher molecular weight side (corresponding to the upper portion of lanes.
1 to ~) being shown on an enlarged scale. The reaction produc~s of the lanes are the same as the corresponding ones in photo l. The analysis confirmed the nucleotide sequence of the ~-urogastrone gene.

7) Expression vector incorporating~PL promotor The ~PL promotor, left ward promotor of ~ phage, was used for expressing ~-urogastrone as will be described in detail below.

~ 3O4O?~J3 First, preparation of a strain ECI-2 derived from E.coli strain HB101 will be described. ECI-2 served as a host for ~PL expression plasmids. Then described will be the c]oning of a DNA fragment containing ~PL promotor from the DNA of ~CI8S7S7 which is a mutant oE
~ phage, and the construction of expression plasmids from the cloned DNA. Further described will be the expression of ~-urogastrone gene in the host ECI-2 strain by ~PL
promotor.
7-1) Construction of strain ECI-2 The strain ECI-2 is E.coli HB101 harboring a plasmid pGH37 for expression of CI857 gene.
pGH37 was prepared by the process shown in Fig. 6. First, DNA of ~CI857S7 was cleaved with BglII.
15 Then, the cohesive ends at the cleavage site were digested using Sl nuclease. One ~g of DNA of ~CI857S7 cleaved with BglII was reac~ed with 200 units of SI nuclease at 20C
for 30 minutes in 100 ~1 of an aqueous solution (pH 4.5) comprising 200 mM sodium chloride, 30mM sodium acetate and 5mM zinc sulfate. The blunt-ended DNA fragments thus obtained were subjected to 1.0 wt.% agarose gel electro-phoresis ~o isolate therefrom a fragment with 2385 b.p.
having the whole CI857 structural gene. The fragmen-t was inserted into the PvuII cleavage site of plasmids pGL101 to construct a plasmid pGH36 which expresses the Cl857 ~ 3~4~2:~

gene under the control of a promotor, lac UV5.
Subsequently, pGH36 is cleaved with two restriction enzymes, EcoRI and PstI, to obtain a fragment having 1193 b.p., which was inserted into a plasmid pSC101 S between EcoRI and PstI cleavage sites to prepare a plasmid pGH37.
Subsequen~ly, the strain HB101 of E.coli was transformed with pGH37 by the aforementioned calcium method. One of the resulting strains was named ECI-2.
The strain ECI-2 is deposited under Budapest Treaty on international recoginition of deposit with deposition number FERM BP-542 in Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trad,e and Industry, Japan, since June 22, 1984. This strain is resistant to tetracycline, expresses the CI857 gene and permits conjoint presence, through transformation, of other plasmids derived, for example, from pBR322. Accordingly the strain ECI-2 was thereafter used as a host for ~PL expressiOn plasmids. 0 7-2) Cloning of ~PL promotor and preparation of expression plasmids As shown in Fig. 7, pGH35 was constructed first.
DNA of ~CI857S7 was cleaved with EcoRI and SalI to obtain a fragment of 5925 b.p. which includes ~PL promotor and CI857 gene as well as ~PR promotor. The fragment was - ~7 -inserted into plasmid pBR322 between EcoRI and SalI
cleavage sites to construct a plasmid pGH25.
Next, pGH2S was cleaved with BamHI and ligated to pBR322 similarly cleaved with Ba~HI to obtain pGH34.
Subsequently, pGH34 was cleaved with AvaI and BglII and thereafter treated with Sl nuclease into blunt-ended fragment of about 4500 b.p. The fragment was circularized with T~ DNA ligase to constnlct a plasmid pGH35.
Next, pEK28 was constructed as shown in Fig. 8.
Synthetic oligonucleotides C-l-l and C-1-2 as an adapter, which include an SD sequence and have the nucleotide sequence shown below, were ligated to the ~ragment which was obtained by cleaving pGH35 with HpaI. The assembly was further ligated to plasmid pMC1403 cleaved with BamHI
to obtain plasmid pEG2. The pEG2 has two ampicillin-reslstant genes, and expresses ~-galactosidase gene derived from pMC1403 under the control of ~PL promotor, utilizîng a start-codon as well as the SD sequence included in the adapter.
The fragments C-l-l and C-1-2 have the following nucleotide sequence.
SD sequence Start codon C-l-l: 5' A G G A A C A¦G A T C T A T G 3' C-1-2: 3' T C C T T G T ~ A T A C C T A G 5' BglII

,'. ' ' .. - . .... .. .

3L3~41[~23 - ~8 -Miller's method was employed to confinn the expression of ~-galactosidase in the host ECI-2 harboring 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 ~-galactosidase with a synthetic substrate ONPG (o-nitrophenylgalactoside) to liberate a yellow compound o-nitrophenol. Miller's method will be described in greater detail. A 0.1 ml quantity of culture of a bacterium specimen, the absorbance of which has been measured at 610 nm, is mixed with 1.9 ml of assay buffer (0.1 M sodium phosphate, pH 7.0, lmM of magnesium sulfate and 0.1 M ~-mercapto-ethanol) and vigorously shaken for 15 seconds with 0.1 ml of toluene to increase permeability of the bacterium specimen.
The toluene is thereafter evaporated of~ by an aspirator.
With addition of 0.2 ml of ONPG solution (solution of 400 mg of ONPG in 100 ml of assay buffer), the mixture is incubated at 30C until a yellow color develops, whereupon 3.5 ml of 1 M sodium carbonate is added to stop the enzyme reaction. The absorbance of the reaction mixture is measured at 420 nm and 550 nm.
The activity of ~-galactosidase is de~ined by the units in 1 ml of the liquid culture according to the following equation, in which absorbance at 610 nm ~3 [11~23 is calculated as 1Ø
Activity of ~- OD420 - 1.75 x OD
galactosidase = ~ t x v x OD610 t : time of incubation (min~
v : amount of specimen added to the reaction system (0.1 ml) OD610 : absorbance at 610 nm of the specimen The above method, when practiced, revealed the following result. When the ECI-2 strain harboring pEG2 was incubated at 30C, the ~-galactosidase activity was 98 units. However, when the culture was further incubated at ~2C for 1 hour, the ~PL promotor was activated to result in ~-galactosidase activity of 9637 units. This substantiates that the sequence from the ~PL promotor to ~-galactosidase is in the contemplated order.
, Although pEG2 has two BglII cleavage sites, only the BglII site present immediately after the SD
sequence of ~-galactosidase is needed, while the other site is undesirable. Accordingly, the plasmid was cleaved with BamHI and ligated again to remove a fragment having about 770 b.p. The construction of pEK~8 completed which is an expression plasmid with use of ~PL promotor.
7-3) Expression of fused gene of front half of ~-urogastrone and ~-galactosidase Fig. 9 schematically shows the series of ~3~ 3 _ 50 -procedures to be described below.
A plasmid pU5101 was constructed in the following manner which has a fused gene of the front half of ~-urogastrone and a ~-galactosidase. More specifically, S pUGl, which has the front half of ~-urogastrone gene, and pMC1403 having a ~-galactosidase gene were cleaved with BamHI and then ligated to form pUG101. With this plasmid, the front half of ~-urogastrone gene and the ~-galactosidase gene are ligated in the same frame.
Accordingly the plasmid expresses the amino acid sequences of the two as a fused protein.
For the expression with this plasmid under the control of ~PL promotor, pUG101 and pEK28 were each cleaved with BglII.
The cleavage with BglII produces a DNA fragment having a extruding 5' ends in the form of ~ TAcTAG) .
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 kinds o~ deoxribonucleotide.
triphosphates dGTP, dATP, dTTP and dCTP, a blunt end is obtained by filling up with the corresponding nucleotides, that makes the end of ~ TcGTTAG) . If dGTP onl~ is added as a nucleotide component, the end of ( ATCGTAG) is obtained owing to the termination of the reaction.
Next, when Sl nuclease is used to digest ~he remaining single strand, a blunt end is obtained in the form of 9 3~ 3 t ATGC) . Similarly if dGTP and dATP are added in the Klenow reaction, followed by digestion with Sl nuclease, ( TC~T) is obtained. When the Klenow reaction is conducted by addition of dGTP, dATP and dTTP, followed by digestion with Sl nuclease, ~ TAcTATA) is obtained.
Further if only the digestion with Sl nuclease is conducted without efecting the Klenow reaction, ( TA) is obtained~ Thus, when the DNA fragment with the end of~ TAcTAG) is subjected to the Klenow reaction with use of diferent nucleotides and/or to the Sl nuclease reaction, five kinds of blunt ends are obtained which are different from one another by one base pair length.
The Klenow reaction and Sl nuclease reaction were conducted under the follow:ing conditions.
* Klenow reaction:
One ~g of DNA as the substrate was reacted with lmM of eac~ deoxyribonucleotide triphosphate at 12C for 30 minutes in the presence of 1 unit of the enzyme (klenow fragment) and lmM of ATP in 50 ~1 of a reaction medium comprising 40mM of potassium phosphate bufer (pH 7.4), lmM of ~-mercapto-ethanol and 10mM of magnesium chloride.

* Sl nuclease reaction:

~L3~4~3 _ 52 -One ~g of DNA as the substrate and 200 unitsof the enzyme were reacted at 20C for 30 minutes in 100 ~l of a reaction medium comprising 200mM of sodium chloride, 30mM of sodium acetate and 5mM of zinc sulfate (pH 4.5).
pEK28 and pUG101 were each cleaved with BglII
and thereafter subjected to various combinations of the Klenow reaction and Sl nuclease reaction, giving fragments having 5 kinds o~ blunt ends. The two types of these fragments, when combined together, provide 21 combinations which are different from one another in the number and sequence of nucleotides between the SD sequence and the start codon of the fused protein, as listed in Table 2.
In actuality, the DNA fragments thus ob~ained were cleaved with SalI to isolate fragments which contain the gene encoding fused protein of ~ urogastrone front half and ~-galactosidase from pU&101, and fragments containing ~PL promotor from pEK28. These fragments were li~ated by T4 DNA ligase in the combinations shown in Table 2.
Consequently, recombinant plasmids in Table 3 were obtained. The ~-galactosidase activity of the expressed fused proteins was measured by Miller's method.
Table 3 shows that all the plasmids expressed a relatively a high level of ~-galactosidase ac~ivity. Especially pUG103, pUG104 and pUG117 achieved remarkable results.

~1,3~d~3 ,~ ~
t~ l c.) C~ E~
E~ c~ E~
u~ ,V) E~C~ E~ <C
E ,_ E~ o ¢ ~ V oo C~ ,_ O E I E~ o ¢ ~ V ~ ~( ~ E~ c~
C) ¢ ~ C~) ~ ¢ ~ ¢ ~ ¢
E~ V C~ V C~ V V C~ V C~ C~
~ ¢ ¢~ ¢P ~P ¢~ ¢P
a~ V c~ ~ ~) ~ c~
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o~ l C~ C~ _~ E~ ,~ ¢ ,_ ~ E~ ~ E~ ~ ¢ r` ~) o c~7 c~ o ¢ ~ E~ ~ E~
5 E~ E~ ~ ¢ r~ ¢ ~ ¢ ,1 E~ ¢¢ ~ , ¢ V~ V¢ pV ¢ P
O ~ ~ ~ l C~ P C) P
¢ _ ¢ _ ¢ _ ¢
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¢ v¢ v¢ ~¢ c~) l l l ~ ¢l ~ ¢
~n O ¢

o~ ~
a ~:
. _ _ O a~ , ~304 G1~3 . _ _ . _ . _.

E~ l C~
¢ l C~ E~ E~
~ C~ _~C~ _ C~ ~ E-/ --O l C5 ~ E~ o~ ~1 ~o c~ o roC~ E-/ O C~ O E~ ~ ~ c~
O C~ C~ ~ E-l ~ l ¢ ~ ¢ ~
C~ E E~ p ¢ P ~ C~ ¢ P
E~ C¢ P ¢ P~ ¢, ~ ¢ P
~ l l . _ _ . . .
O l CC~
~! I c~ E~
t~ I _~ ~) ,_ C3 ,~ C~ _~c1 C~ ~ c~ co E~ c~ C~ a~S~ , c~) o E~ o C~ ~ E~
J~ C~ E~ ~ C~ ~ ¢ ~ l ¢ ~
ta c~ C~ c~ C~ ~ C~ C~ c3 c3~ E~ ~ ~ ¢ ~ ¢ :~ ¢ ~
,_ C~ C~ ¢~ P C¢~ ~ ~ ¢~ ~
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~ ~ l ~rl tO ~ I ~ C~ _~ C~ _~
~ I c~ C.) r~ ~ ~ E-O ~ C~ o ~ oE~ ~ E-~
.~ l C3 ~ E~~i ¢ ~ ¢
~, ~1 C~ E~ C~ ~C~ C~ C~ C~
o ~ ¢ ~ ¢ o ¢ ~ ¢
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¢ ~ ¢ ¢ ¢
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~3~ 3 _ 55 -Table 3 .
Number of nucleotides between SD sequence Recombinant~-Galactosidase and start co_on plasmid (units) 6 pUG102 1674 7 pUG103 2533 7 pUG107 2260 8 pUG104 2802 8 pUG108 1835 9 pUG105 1018 9 pUG109 1942 pUG106 973 11 pUGllO 1764 11 pUG113 1950 11 pUGll9 1862 12 pUG114 946 12 pUG117 2332 12 pUG120 1374 13 pUG118 2041 13 pUG121 1678 14 pUG122 1814 .

j, .

~L3~

Next, a vector for expression of ~-urogastrone was prepared from pUG103 or pUG117. The plasmid (pUG103 or pUG117) was cleaved with HindIII and PvuII to obtain a fragment of 102 kb containing the region of from ~PL
promotor to the front half of ~-urogastrone gene.
Further pUG2 was cleaved with EcoRI, filled up with Klenow fragment, and cleaved with HindIII to obtain a fragment of 4.1 kb. The two fragments were ligated with T4 DNA ligase, and the strain ECI-2 of E.coli was transformed by the calcium method stated above to obtain a recombinant holding therein the plasmid pUG103-E or pUG117-E for expression of the combination of the front half and rear half of ~-urogastrone gene, i.e. the whole ~-urogastrone gene, under the control of ~PL promotor.
8) Vector for expression of fused protein A ~-lactamase gene on the plasmid pBR322 and a ~-urogastrone gene were ligated to express ~-urogastrone as a fused protein as will be described below.
8-1) Donor o~ ~-lactamase gene pBRH02 is obtained by cleaving pBR322 with AvaI and P wII, followed by the Klenow reaction and ligation by T4 DNA ligase. This plasmid has genes for ampicillin resistance (ApR) and tetracycline resistance (TcR) as markers. pBRH03 is obtained by cleaving pBR325 with AvaI and HindIII, followed by the Klenow reaction ~L3~ 3 and ligation and has Ap and chloramphenicol resistance (Cm ) as markers. Fig. 10 shows these plasmids.
8-2) Donor of ~-urogastrone gene pUG3 prepared as already described w~s cleaved with MboII to obtain 13 kinds of DNA fragments, which were named A to M in the order of size as shown in Fig. 11.
Of these DNA fragments, H fragment was found to be composed of 179 b.p. starting with a nucleotide coding for asparagine at the N-terminus of ~-urogastrone and ending with 16 bases downstream oE the stop codon, the fragmen~ having the whole structural gene of ~-urogastrone.
To isolate the H fragment, the fragments were subjected to 6 wt.% polyacrylamide gel electrophoresis, and the fragment was purified.
8-3) Adaptor For adaptors, the oligonucleotides listed in Table 4 were prepared by the same method as already stated. These adaptors were so designed as to code for the basic amino acid pair of Lys-Arg or Arg-Lys to enzymatically cleave ~-urogastrone from the expressed fused protein.

0~3 Table 4 Adaptor 5' end - - -3' end E-l CGCTAAACGG

E-10. CGTTTG

~.3~ 23 ~ 59 -8-4) System for expression of fused protein of ~-lactamase and ~-urogastrone linked by Lys-Arg A vector for expression of ~-lactamase-~-urogastrone fused protein was so prepared that a restriction enzyme recognition sequence would be generated in the region containing an adaptor.
8-4-a) Construction of pUG2301 to pUG2303 The process shown in Fig. 12 was practiced.
The plasmid pBRH02 was completely cleaved with XmnI at 37C over a period of 3 hours. Subsequently 3 ~g of the vector, about 0.1 ~g of ~-urogastrone fragment of 179 b.p. and about 1 ~g of each of E-l and E-2 (with non-phosphorylated 5' end) serving as an adaptor were ligated in a single step at 12C over a period of 15 hours to obtain plasmids as an expression vector. The strain HB101 was transformed with use of the plasmids by the calcium method.
Of the 499 TcR colonies obtained, 168 colonies (33.7%) were ApS. These colonies were checked by mini-preparation ~or the si~e of plasmid DNA. Thirteenplasmids were about 200 b.p. larger than the vector and considered to have a ~-urogastrone gene inserted therein.
All of them, which had a MluI site, were cleaved with HinfI and checked for the orientation of insertion of the ~-urogastrone gene by 1.5 wt.% agarose gel electrophoresis.

Three of those checked gave fragments of about 1050 b.p.
and about 800 b.p. This indicated that the ~-urogastrone gene was inserted in the same orientation as ~-lactamase.
These three plasmids were named pUG2301 to pUG2303.
8-4-b~ Construction of pUG2101 to pUG2105 The process shown in Fig. 13 was practiced.
The plasmid pBR322 was used as a plasmid vector which has unique PvuI site in the ~-lactamase gene.
According to the process described in 8-4-a), an expression ~ector was constructed using E-l and E-5 for an adaptor.
When the 1626 TcR colonies obtained were checked for Ap sensitivity~ 31 colonies ~1.9%) were Ap . Mini-preparation was conducted for 22 Tc and Ap colonies, and the plasmids were checked for the insertion of ~-urogastrone gene by cleavage with MluI. Twenty plasmids were found to have an MluI site. The orientation was checked by cleaving with HinfI or BamHI. Plasmids pUG2101 to pUG2105 were found to have a ~-urogastrone gene inserted therein in the same orientation as the ~-lactamase gene.
8-4-c) Construction of pUG2701 to pUG2703 Expression plasmids were constructed by the same procedure as in 8-4-a~ using pBR322 as a vector and E-7 and E~8 as an adaptor, as shown in Fig. 14.

~3~ 3 ~ - 61 -Of the 217 TcR colonies obtained, 106 colonies (~8.8%) were ApS. Mini-preparation was conducted for 25 of these colonies. Eight of the plasmids were about 200 b.p. larger than the vector and appeared to have a ~urogastrone gene inserted therein, so that these eight plasmids were cleaved with BamHI and checked ~or the Orientation of the gene. Consequently, three plasmids were found to have the ~-urogastrone gene inserted in the same orientation as ~-lactamase and were named pUG2701 to 2703.
The plasmid pUG2301 obtained by the procedure 8-4-a) above produces a fused protein of a portion of ~-lactamase and ~-urogastrone. Predicted amino acid sequence and the corresponding nucleotide sequence are shown below.

3 ~3~0;~ 3 M et S er I le Gln His P he A r(l V al ATG AGT ATT CAA CAT TTC CGT GTC

Ala Leu I le P ro P he Phe Ala Ala GCC CTT ATT CCC TTT TTT GCG GCA

P he C ys L eu P ro V al P he A la ~-1 is TTT TGC CTT CCT C,TT TTT GCT CAC

P ro G lu T hr L eu V al L ys V al L ys CCA GAA ACG CTG GTG AAA GTA AAA
AsP A la G lu ASP G In Leu G Iy A la GAT GCT GAA GAT CAG TTG GGT GCA

A rs V al GIY TYr I le G lu L eu As~
CGA GTG GGT TAC ATC GAA CTG GAT

Leu Asn S er GIY Lys I le Leu Glu CTC AAC AGC GGT AAG ATC CTT GAG

S er P he A rg P ro G lu Glu Arg Ala AGT TTT CGC CCC GAA GAA CGC GCT
L ys A rg A sn S er ASP Ser G lu CYS
AAA CGG AAT AGC GAT TCT GAG TGC

P ro L eu S er H is AsP G Iy TYr CYs CCA CTG TCT CAC GAT GGC TAT TGT
Leu H is ASP G Iy Val CYs Met TYr CTG CAC GAC GGT GTT TC,C ATG TAC

~L3~ 23 I le Glu Ala Leu As~ Lys TYr Ala ATC GAA GCT TTG GAT AAA TAC GCG
CYS Asn CYS Val Val GIY TYr I le TGT AAC TGT GTA GTG GGT TAT ATC

GYI G lu Ar~ CYS G In TYr Arg ASP
GGT GAA CGC TGT CAA TAC CGT GAT

Leu Lys TrP Tr~ Glu Leu Arg (sto,~) CTG AAA TGG TGG GAA TTG CGT TAA

T A G T G A A G A T C T G G A T C C G T T T A G C G T T T T C C A
A T C, A T G A Gi C A C T T T T A A A G T T C T G C T A T G -r G G C

GCGGTATTATCCCGTGTTGACGCCGGGCAAGAG

CAACTCGGTCGCCGCATAC
Similarly, the plasmid pUG2101 obtained by the procedure 8-4 b) produces a fused protein having the following primary structure.
Met Ser I le Gln His Phe Arg Val ATG AGT ATT CAA CAT TTC CGT GTC

Ala L eu I le Pro Phe P he Ala Ala GCC CTT ATT CCC TTT TTT GCG GCA

P he C YS L eu P ro V al P he A la H is 1-TT TC,C CTT CCT GTT TTT GCT CAC

.3~%3 Pro Glu Thr Leu Val Lys Val L ys CCA GAA ACG CTG GTG AAA GTA AAA
ASP Ala Glu ASP Gln L eu GIY Ala GAT GCT GAA GAT CAG TTG GGT GCA

Ar~ Val GIY TYr I le Glu Leu As~
CGA C,TG GGT TAC ATC GAA CTG GAT

Leu Asn Ser GIY Lys I le Leu Glu CTC AAC AGC GGT AAG ATC C-l-T GAG
Ser Phe Arg Pro G lu G lu Arg Phe AGT TTT CGC CCC GAA GAA CGT TTT

Pro Met Met Ser Thr Phe Lys Val CCA ATG ATG AGC ACT TTT AAA GTT

Leu Leu CYS G Iy A la Val Leu Ser CTG CTA TGT GGC GCG GTA TTA TCC

Arg Val AsP Ala GIY C,ln Giu Gln CGT GTT GAC GCC GGG CAA GAG CAA

Leu GIY Arg Ar~ I le His TYr Ser CTC GGT CGC CGC ATA CAC TAT TCT

Gln Asn AsP I le Val Glu TYr Ser CAG AAT GAC TTG GTT GAG TAC TCA

Pru Val Thr Glu Lys His L eu Thr 1l 3ai~L~23 AsP GIY Met Thr Val Arg Glu l eu GAT (3GC ATG ACA G-rA AGA GAA TTA

CYS Ser Ala Ala I le Thr Met Ser TGC AGT GCT C,CC ATA ACC Al-G AC,l-ASP Asn Thr Ala Ala Asn Leu l eu GAT AAC ACT GCG GCC AAC TTA CTT

Leu Thr Thr I le Ala Lys ArY Asn CTG ACA ACG ATC GCT AAA CGG AAT
Ser ASP Ser Glu CYS Pro Leu Ser AGC GAT TCT GAG TGC CCA C-l-G TCT

H is ASP G Iy TYr CYS Leu H is ASP
CAC . GAT GGC TAT TGT CTG CAC GAC
GIY Val CYS Met TYr I le GIU Ala GGT GTT TGC ATG TAC ATC C,AA GCT

Leu ASP Lys TYr A la CYS Asn CYS
TTG GAT AAA TAC GCG TGT AAC TGT

Val Val GIY TYr I le GIY Glu Arg GTA GTG GGT TAT ATC GGT GAA CGC

CYS Gln TYr Ars AsP Leu Lys TrP
TGT CAA TAC CGT GA T CTGl AAA TGG

Trp Glu Leu Ar~ (stop) TGG GAA T-rG CGT TAA TAGTGAAC,A1-C

AGCTAACCGCTTTTTTGCACA
Similarly, the plasmid pUG2701 obtained by the procedure 8-4-c) produces a fused ~rotein having the following primary structure.

Met Ser I le Gln His Phe Arg Val ATG AGT ATT CAA CAT TTC CGT GTC

Ala Leu I le pro Phe Phe Ala Ala GCC CTT ATT CCC l-TT TTT GCG GCA

Phe CYs Leu Pro Val Phe Ala His TTT TGC CTT CCT GTT TTT GCT CAC

Pro G lu Thr Leu Val LYS Val Lys CCA GAA ACG CTG GTG AAA GTA AAA
ASP A la G lu ASP G In Leu G Iy A la GAT GCT GAA GAT CAG TTG GGT GCA

Ars Val GIY TYr I le GIU Leu ASP
CGA GTG GGT TAC ATC GAA CTG GAT

Leu Asn Ser GIY 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

~3~4~23 Pro Met Met Ser Thr Phe Lys Val CCA ATG ATG AGC ACT TTT AAA GTT

Leu Leu CYS GIY A la Val Leu Ser CTG CTA TGT GGC GCG GTA TTA TCC

Ars Val AsP Ala GIY Gln Glu Gln CGT GTT GAC GCC GGG CAA GA,G CAA
Leu C1IY Arg Arg I le ~lis TYr Ser CTC GGT CGC CGC ATA CAC TAT l-CT
Gln Asn AsP lle Val Glu Ser Ala CAG AAT GAC TTG GTT GAG TCG C,CT

Lys Arg Asn Ser AsP Ser Glu CYS
AAA CGG AAT AGC GAT TCT GAG TGC
PrQ Leu Ser H is ASP GIY TYr CYS
CCA CTG TCT CAC GAT GGC TAT TGT

Leu His ASP GlY Val CYS Met TYr CTG CAG GAC GGT GTT TGC ATG l-AC
Ile Glu Ala Leu AsP Lys TYr Ala ATC GAA GCT TTC, GAT AAA TAC GCG
CYs Asn CYS Val Val GIY TYr Ile TGT AAC TC,T GTA GTG GGT TAT ATC
GIY Glu Arg CYS Gln TYr Ar~ ASP
GGT GAA CGC TGT CAA TAC CGT GAT

~3~23 Leu LYS Trl~ TrP Glu Leu ~r~ (sto~) CTG AAA TGG TGG GAA TTG CGT TAA

TAGTGAAGATCTGGA-rCCGTT-rAGCCGAC'-rCAC

CAGTCACAGA~AAGCATCTTACGGAT

The nucleotide sequences coding for the fused proteins in the plasmids pUG2101, pUG2301 and pUG2701 were analyzed by the Maxam-Gilbert method.
Photo 5 shows the results of analysis.
With reference to Photo 5, lanes 1 to 4 show the result obtained with the MluI-PstI fragment (224 b.p.) of pUG2101, lanes 5 to 8 show the result with the MluI-EcoRI fragment (721 b.p.) of pUG2101, lanes 9 to 12 show that with the MluI-Ba~HI fragment (452 b.p.) of pUG2301, lanes 13 to 16 is that with the MluI-PstI fragment (335 b.p.) of pUG2701, and lane 17 to 20 show that with the MluI-EcoRI fragment (610 b.p.) of 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 plus adenine, lanes 3, 7, 11, 15 and 19 show the reaction products for thymine plus cytosine, and lanes 4, 8, 12, 16 and 20 show the reaction products for cytosine. The portion marked with "}" is an adaptor.

~1 3~23 8-5) System for expression of fused protein oE ~-lactamase and ~-urogastrone linked by Arg-Lys 8-5-a) Preparation of pUG1102 and pUG1105 ~-Urogastrone gene was inserted into the ~-lactamase gene of pBR322 at its unique PvuI restrictionsite to obtain vectors for expression of fused pro-tein of ~-lactamase and ~-urogastrone as shown in Fig. 15.
The plasmid pBR322 was cleaved with PvuI at 37C over a period of 3 hours. Some of the plasmids were checked by 1 wt.% agarose gel electrophoresis to confirm that they had been completely cleaved. The adaptors D-1-3 and D-3-2 were ligated to the fragment at 12C over a period of 15 hours, and the ligated product was thereafter subjected to 1 wt.% agarose gel electro-phoresis to isolate a DNA fragment. Subsequently,~-urogastrone fragment and vector were mixed together in a molar ratio of appro~imately 5:1 and ligated at 12C over a ~eriod of 15 hours. After the ligation, the strain HB101 was transformed with the resulting plasmid, and the colonies were selected with reference to Tc .
Seventy-one TcR transformed colonies were obtained and then checked for Ap sensitivity. Plasmid DNA was prepared from 20 Ap colonies (28.2%) and then checked for the presence of MluI restriction site to ~3~2~

confirm the insertion of ~-urogastrone gene. Five of the 20 plasmids were found to have the MluI site of ~-urogastrone gene. The DNA was cleaved with HinfI and then subjected to 1.5 wt.% agarose gel electrophoresis to check the orientation o insertion. Two of the plasmids, i.e. pU&1102 and pUG1105, were found to have the gene in the proper orientation~
8-5-b) Preparation of pUG1004, pUG1201 and pUG1301 The procedure 8-5-a) was repeated using PstI, HincII and XmnI in place of PvuI to obtain pUG1004, pUG1201 and pUG1301 as shown in Figs. 16, 17 and 18, respectively.
9) Confirmation of expression of ~-urogastrone The expression plasmids thus constructed were used to transform E.coli, HB101 or ECI-2, and the cells were cultured by the following method, followed by extraction and radioimmunoassay to confirm expression.
9-1) Culture of recombinant microorganisms with ~-urogastrone gene and extraction of proteins 9-1-a) Expression system using ~PL promotor The strain ECI-2 harboring expression plasmid pUG103-E and the same strain harboring expression plasmid pUG117-E were each cultured at 25C in two flasks each containing 1 liter of LB culture medium. When the culture in one of the flasks exhibited an absorbance o 0.3 at ~3a9~

660 nm, the culture was subjected to heat induction at 42C for 1 hour. The culture in the other flask was continuously incubated at 25C until the absorbance became 0.4. The cells in each flask was collected, washed with PBS buffer (137mM sodium chloride, 2.7mM
potassium chloride, 8.lmM sodium phosphate, dibasic and 1.5mM sodium phosphate, monobasic (pH 7.0)), then resuspended in PBS buffer in 3 vol.% of the amount of original culture and destroyed (at 100 W for 30 seconds, three times) by a sonicator (Model 5202, product of Ohtake Works Co., Ltd., Japan) with ice cooling. The supernatant separated from the cell debris by ultracentrifugation (at 40000 g for l hour) was dialyzed against 0.01N aqueous solution of acetic acid, and the dialyzate was lyophilized and thereafter subjected to radioimmunoassay (hereinaf~er referred to as "RIA").
9-1-b) System ~or expression of fused protein E. coli strain HB101 harboring plasmids pUG1004, -1301, 2101, 2303 or 2703 was preincubated at 37C in a culture medium containing 50 ~g/ml of tetracycline, then diluted to the volume ratio of 1:100 with the same medium and cultured until the absorbance at 660 nm became 0.4.
The cells were collected, washed wi~h PBS buffer, then resuspended in PBS buffer in 3 vol.% of the amount of the original culture and sonicated (at 100 ~ for 30 seconds, ~L30~

_ 72 -three times) by the same sonicator as above with ice cooling. The supernatant separated from the cell debris by ultracentrifugation (~0000 g for l hour) was dialyzed against O.OlN aqueous solution of acetic acid, lyophilized and then subjected to RIA.
To confirm the accumulation o~ fused protein in the periplasm, the periplasmic fraction was prepared according to S. J. Chan et al. (Chan, S. J. et al., Proc. Natl. Acad. Sci., U.S.A., 78, 5~01-5405 (1981)).
A portion of the culture was diluted to a volume ratio of 1:100 with a fresh E culture medium (1 liter of aqueous solution of 10 g of potassium phosphate, dibasic, 3.5 g of sodium ammonium hydrogenphosphate, 0.2 g of magnesium sulfate heptahydrate, 2 g of citric acid, 2 g of glucose, 0.23 g of L-proline, 39.5 mg of L-leucine, 16.85 mg of thiamine and 20 mg of tetracycline hydrochloride) and then cultured at 37C until the absorbance at 660 nm became 0.4. The cells were collected (6000 r.p.m., 10 minutes) and washed twice with a mixture o~ lOmM
2.0 tris-HCl (pH 8.0) and 30mM sodium chloride. The cells (1 g) were resuspended again in 80 ml o~ 20 wt.% sucrose-30mM tris-HCl (pH 8.0), whereupon EDTA was added to the suspension to a concentration o~ lmM. The mixture was shaken by a rotary shaker at 180 r.p.m. for 10 minutes (24C) and then centrifuged (13000 g, 10 minutes) to ~3~23 collect the cells, which were resuspended in 80 ml of distilled wa~er. The suspension was allowed to stand in ice for about 10 minutes with occasional stirring and then centri~uged (13000 g, lO minutes). The supernatant was collected as a periplasmic fraction (0-Sup).
The pellet was suspended in a mixture of lOmM tris-HCL
(pH 8.0) and 30mM sodium chloride and treated by the same sonicator as above to obtain a cytoplasmic fraction (0-Ppt~. These samples were subjected to RIA.
~-2~ Radioimmunoassay 9-2-a) Establishment of RIA system Rabbits were immunized with purified human ~-urogastrone as an antigen to obtain antiserum. The ~-urogastrone (300 ~g) was dissolved in 0.2 ml of distilled water, 1.5 ml of 50% polyvinylpyrrolidone solution was added to the solution, and the rnixture was stirred for 2 hours at room temperature. Complete Freund's adjuvant (2.0 ml) was added to the mixture to obtain an emulsion, which was subcutaneously injected into the chest portion of three rabbits. After repeating the immunization four ~imes every two weeks, 50 ~g of the antigen was further intravenously injected, the whole blood was collected 3 days -thereafrer, and the serum was separated.
Next, the following RIA conditions were determined in view of the titration curve -Eor determining ~L3~ 23 - 7~ -the dilution degree of the antiserum for the assay, incubation time for optimizing the assay conditions, method of separating the bound radiolabeled antigen (bound) from the free radiolabeled antigen (free), etc.
The diluting solution used was a phosphate buffer (lO~M, pH 7.4) containing 0.5 wt.% of bovine serum albumin (BSA), l~OmM of sodium chloride and 25mM of disodium EDTA. The diluting solution (400 ~1), 100 ~1 of the sample or standard human ~-urogastrone and 100 ~1 of antihuman ~-urogastrone serum were mixed together.
After the rnixture was incubated for 24 hours at ~C, 100 ~1 of 125I-labeled human ~-urogastrone solution (about 5000 cpm) was added to the mixture. After the mixture was further incubated for 48 hours at 4C, 100-~1 of second antibody (anti-rabbit ~-globulin goat serum) (20-fold dilution with PBS buffer), 100 ~1 of normal rabbit serum (200-fold dilution with PBS buffer) and 900 ~1 of lOmM PBS buffer containing 5 wt.% poly-ethylene glycol were added to the resulting mixture, and then further incubated for 3 hours at 4C. The culture was centrifuged for 30 minutes at 3000 r.p.m., the supernatant was separated off, and the precipitate was counted. The content of immunoreactive substance as human ~~urogastrone in the sample was determined from the standard curve obtained with use of standard human ~-urogastrone.

~3~ 3 9 2-b) Confirmation of ,8-urogastrone productivity of recombinant microorganism Table 5 shows the result of RIA conducted for the expression system with use o~ PL promotor.
Table 5 Expression Amount of ~-plasmid Heat induction urogastrone produced (ng/l culture) pUG103-E Yes 450.2 pUG103-E No 3.2 pUG117-E Yes 388.4 pUGl17-E No 3.2 Control NoNot detectable (pBR322) -Table 6 shows the result of RIA conducted for fused protein expression systems.
Table 6 ExpressionAmount of ~-plasmidurogastrone produced (~Q;/l culture) pUG1004 729.6 pUG1301 650.7 pUG2101 31.3 pUG2301 125.2 pUG2701 119.2 23~

Table 7 shows the localization of the expressed fused protein.
Table 7 _ ~Amount of ~-urogastrone produced ~
Expression _ (~g/l culture) plasmid Periplasmic fraction Cytoplasmic fraction (o-Sup) _ _ (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 reveal that the ~PL promotor system for direct expression of ~-urogastrone and the system for expression of the compound as a fused protein both expressed ~-urogastrone imrnunoreactivity in E.coli.
Table 7 reveals that in the case of fused protein, the expressed ~-urogastrone immunoreactivity is almost localized in the periplasm.

Claims (9)

1. A process for preparing .beta.-urogastrone/.beta.-lactamase fusion protein which comprises ligating a .beta.-lactamase gene so as to express the .beta.-urogastrone gene as a fused protein, the process being characterized in that a host cell is transformed with a plasmid containing a gene inserted into a site between a portion of .beta.-lactamase gene and the .beta.-urogastrone gene, the inserted gene being one coding for at least two basic amino acid which can provide a cleavage site for taking out .beta.-urogastrone from the fused protein by cleaving with an enzyme, and then the tranformant is cultured, whereby a fused protein is secreted from the cytoplasm of the cell.

2. A process according to claim 1 wherein the .beta.-urogastrone gene having the following nucleotide sequence:

3. A process according to claim 1 wherein the sequence of the two basic amino acids is -Lys-Arg- or -Arg-Lys-.

4. A process according to claim 1 wherein the portion of .beta.-lactamase gene is a gene coding for the sequence of 63 or more amino acids from the amino terminal of E. coli .beta.-lactamase precursor.

5. A process according to claim 4 wherein the portion of .beta.-lactamase gene is a gene coding for the sequence of 63 to 182 amino acids from the amino terminal of E. coli .beta.-lactamase precursor.

6. A process according to claim 1 wherein the host cell is E. coli.

7. A process for preparing .beta.-urogastrone, comprising producing a fused protein according to the process, as defined in claim 6, wherein the fused protein is collected from the periplasm layer of E. coli and the collected protein is enzymatically cleaved, to yield .beta.-urogastrone.

8. A plasmid having the fused gene as defined in claim 1.

9. A host cell transformed with the plasmid as defined in claim 8.