GB2162851A - beta -urogastrone gene - Google Patents

Abstract

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

Description

SPECIFICATION Novel ss-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of -urogastrone The present invention relates to a novel ss-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of (3-urogastrone.

ss-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 lle Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Giy Tyr lle Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg In the specification, amino acids are represented by the following symbols.

Asn: asparagine Ser: serine Asp: aspartic acid Glu: glutamic acid Cys: cysteine Pro proline Leu: leucine His: histidine Gly: glycine Tyr: tyrosine Val: valine Met: methionine lle: isoleucine Ala: alanine Lys: lysine Gln: glutamine Arg: arginine Trp: tryptophan Phe: phenylalanine ss-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 testing ulcers and wounds.

Since p-urngastrone 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, European Patent Application Publication No. 0046039 discloses an attempt to produce -urogastrone by a gene engineering technique with use of a synthetic ss-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 ss-urogastrone by a similar method, nor does it mention such a gene of a particular nucleotide sequence.

A large number of nucleotide sequences can code for the amino acid sequence of p-urogastrone.

Nevertheless, it is hard to speculate which of such genes is capable of expressing ss-urogastrone through a gene engineering technique or which gene 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.

An object of the present invention is to provide a novel p-urogastrone gene which is entirely different from the gene disclosed in the above publication in the nucleotide sequence and which is capable of expressing ss-urogastrone through gene engineering techniques.

Another object of the present invention is to provide a gene which is suited to the expression of ss- urogastrone by gene engineering techniques.

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

Still another object of the present invention is to provide a process which enables quantity production of ss-urogastrone with a high purity with use of the novel gene by gene engineering techniques.

These and other objects of the present invention will become apparent from the following description.

We have conducted many experiments and found that a gene I of the following nucleotide sequence fulfils the objects of the invention.

Gene 1: 5' AAT AGC GAT TCT GAG TGC CCA CTG 3' -TTA TCG CTA AGA CTC ACG GGT GAC TCT CAC GAT GGC TAT TGT CTG CAC AGA GTG CTA CCG ATA ACA G-AC GTG GAC GGT GTT 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 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 GTT ATG GCA CTA GAC TTT TGG TGG GAA TTG CGT 3' ACC ACC CTT AAC 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 for 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.

The gene I has the following characteristics.

(1) ss-Urogastrone can be expressed very advantageously 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 facilitate ligation with other gene and insertion into the plasmid vector.

(4) For the preparation of the gene I, the constituent oligonucledtides 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 ss-urogastrone as a fused protein, means is available by which an unnecessary portion can be easily removed to obtain the desired ss-urogastrone.

When ss-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 sequence"), vector, etc. needed for the expression. Further when required, a start codon and/or a stop codon may be provided upstream and downstream of the gene, respectively. The recognition sites, stat codon and stop codon are not limited particularly but can be desired ones.

Shown below is an example of gene having an expanded sequence (hereinafter referred to as "gene ll")which includes a restriction enzyme recognition site and a start codon disposed upstream of the gene I and a stop codon and a restriction enzyme 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 enzyme recognition sites.

The symbols representing the restriction enzymes in the above sequence stand for the following.

E: EcoRI, Ta: Taql Bg: Bill, S: Sau3Al Mb: Mboll, Hf: Hinfl Ba: BamHI, Hd: Hindlll M1: M1ul, Th: Thal 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 (3-uro- gastrone.

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 Il further may have a restriction enzyme recognition site at the rear end, and another subunit having the rear half of the nucleotide sequence of the gene II may have a restriction enzyme recognition site at the front end, and these subunits are jointed together into the gene II.

Stated more specifically for illustrative purposes, the former subunit can be a subunit A comprising the front half of the gene Il and having a restriction enzyme (BamHI) recognition site provided at its rear end, and the latter subunit can be a subunit B comprising the rear half of the gene II which has a restriction enzyme (Hindlll) recqgnition site at its front end. These subunits are shown below.

Subunit A: 5' AAT TCG AAG ATC TGC ATG AAT AGC 3' GC TTC TAG ACG TAC TTA TCG GAT TCT GAG TGC CCA CTG TCT CAC CTA AGA CTC ACG GGT GAC AGA GTG GAT 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 3' CTA G5' Subunit B 5' A GCT TTG GAT AAA TAC GCG TGT 3' AAC CTA TTT ATG CGC ACA AAC TGT GTA GTG GGT TAT ATC GGT TTG ACA CAT CAC CCA ATA TAG CCA GAA CGC TGT CAA TAC CGT GAT 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 TGA AGA TCT G 3' ACT TCT 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 (A01 to A-16, and B-1 to B-16, i.e. 32 oligonucieotides).Next, 4 to 6 of these oligonucleotides are assembled and ligated into blocks (block 1 to block 7, e.e. 7 blocks). These oligonucleotides and blocks are shown below.

Block 1: (A-1) (A-2) (1-3) 5' AATTCGAAGAT CTGCATGAATAGC GATTCTGAGTG 3' 3' GCTTCTAGACGTA CTTATCGCTAA GACTCACGGGTGA 5' (A-16) (A-15) (1-14) Block 2: (A-4) (A-5) (A-6) 5' CCCACTGTCTCAC GATGGCTATTG TCTGCACGACGGT 3' 3' CAGAGTGCTAC CGATAACAGACGT GCTGCCACAAA 5' (A-13) (A-12) (A-11) Block 3: (A-7) (A-8) 5' GTTTGCATGTA CATCGAAGCTTCG 3' 3' CGTACATGTAGCT TCGAAGCCTAG 5' (A-10) (A-9) Block 4: (B-1) (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) 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 invention will be described in greater detail with reference to the accompanying drawings and photos.

Figure 1 schematically shows the synthesis of an oligonucleotide by the solid phase process; Figure 2 shows a process for ligating oligo- nucleotides A-1 to A-16 into a subunit A and introducing the subunit into a plasmid pBR322 derived from E.coli to obtain a recombinant plasmid pUG1; Figure 3 shows a similar process for preparing a recombinant plasmid pUG2 by introducing a subunit B into a plasmid pBP322; Figure 4 shows a process 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 process for preparing a recombinant plasmid pGH37; Figure 7 shows a process for preparing a recombinant plasmid pGH35;; Figure 8 shows a process for preparing a recombinant plasmid pEK28; Figure 9 shows processes for preparing recombinant plasmids pUG102 to pUG122 and recombinant plasmids pUG103-E and pUG117-E; Figure 10 shows processes for preparing recombinant plasmids pBRH02 and pBRH03; Figure 11 shows Mboll restriction map of pUG3 including H fragment (179 b.p.) which contains the present ss-urogastrone gene; Figure 12 shows a process for preparing recombinant plasmids pUG2301 to pUG2303; Figure 13 shows a process for preparing recombinant plasmids pUG2101 to pUG2105; Figure 14 shows a process for preparing recombinant plasmids pUG2701 to pUG2703; Figure 15 shows a process for preparing recombinant plasmids pUG1102 and pUG1105;; Figure 16 shows a process for preparing a recombinant plasmid pUG1004; Figure 17 shows a process for preparing a recombinant plasmid pUG1201; Figure 18 shows a process for preparing a recombinant plasmid pUG1301; and Photos 1 to 5 are respectively show analytical results of 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 Figure 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 polystyrene 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 thereof 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, 8a, 5461-5471 (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 of triethylamine, then reacting the monotriazolide obtained with ss-cyanoethanol in the presence of 1-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 mononucleotide 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 chromatography. This process affords a completely 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 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 dimethylformamide to remove the remaining On2+, thereafter washed with tetrahydrofuran and exposed to nitrogen gas stream for several minutes for drying.Separately, the completely protected dinucleotide or mononucleotide is dissolved in pyridine followed by addition of triethylamine, and the resulting solution is shaken, then allowed to stand at room temperature for several hours and thereafter evaporated under reduced pressure. The resulting 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, (MSNT, 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 an hydride using dimethylaminopyridine as catalyst in tetrahydrofuranpyridine to mask the unreacted hydroxyl group. Finally the resin is washed with pyridine to complete one 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 dinucleotides successively with the resin to the desired length, whereby a completely protected oligonucleotide can be obtained as supported on the resin.

To the resulting resin is added a solution of tetramethylguanidine-2-pyridinealdoximate in pyridinewater, 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 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 with an aqueous solution of acetic acid to remove the protective group, followed again by high-speed 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 homochromatography and (or) the Maxam-Gilbert method and thereafter used for preparing the blocks and subunits.

The two-dimensionai 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 water to a con- centration of about 0.1 llg/pi. A portion of this solution is treated with oy-32P-ATP and T4 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 first dimensional development to separate the product according to the difference of bases. The developed products are then transferred onto a diethylaminoethyl cellulose (DEAE cellulose) plate and subjected to the second dimensional development using a solution of partially hydrolysed RNA called a homomixture. (This procedure is termed homochromatography). 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 electrophoresis 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 concentration of salt. After the completion of reactions for the four bases, each reaction mixture is reacted with piperidine to displace ring opened base and to catalyze p-elimination of both phosphates from 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 Figure 2. For the correct ligation, the 16 oligonucleotides A-l to A-16 corresponding to the subunit A are ligated as divided into three 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-li, A-12 and A-13, and the block 3 comprising A-7, A-8, A-9 and A-10 as shown in Figure 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 of the three blocks, the oligonucleotides are assembled and ligated with use of TA DNA ligase, and the product is electrophoresed on polyacrylamide gel to isolate the desired block. The three blocks thus obtained are ligated with use of T4 DNA ligase to produce subunit A.

Although a dimer structure 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 Figure 2, a known plasmid vector, pBR322, which is derived from E.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 four sets as seen in Figure 3, and the blocks are ligated together to produce subunit B. The dimer, if produced, is cleaved with the restriction enzymes Hindlil and BamHI to obtain the subunit B. A plasmid vector, pBR322, is cleaved with Hindlll and BamHI, and the subunit B is inserted into the vector to obtain a recombinant plasmid pUG2 as seen in Figure 3.

Further as shown in Figure 4, pUG1 is cleaved with restriction enzymes Hindlll and Salt, and a fragment removed from pUG2 with use of the same restriction enzymes is inserted into pUG1 to prepare a recombinant plasmid pUG3 having a ss-urogastrone structural gene (gene II).

pUG1, pUG2 and pUG3 are recombinant plasmids which each comprise pBR322 and the subunit A which is the front half portion of the (3-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.coli which is known and readily available according to the calcium method as the transformation method (E. Lederberg and S. Cohen, J.

Bacteriol., 119, 1072 (1974)).

Whether pUG1, pUG2 and pUG3 are present in the host such as the strain HB101 of E.coli can be checked by the following methods. After the plasmids are collected by the alkaline extraction method, pUG1 and pUG2 are checked for the presence of the Bg1ll recognition site which is not present on the vector pBR322. Similarly, pUG2 and pUG3 are checked whether they can be cleaved with Mlul which is not present on pBR322.

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. Plasmids are collected by utilizing these characteristics. The plasmids are further subjected to density-gradient ultracentrifugation with cesium chloride and ethidium bromide for purification and then passed through a Biogel A 50m column to remove RNA. Thus plasmides can be obtained with a high purity in a large quanity.In this way, the ss-urogastrone gene of the invention (gene II) can be obtained.

Next, the method of introducing the (3-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 (3-urogastrone gene with use of E.coli includes a system for directly expressing (3-urogastrone, and a system wherein it is expressed as a fused protein with ss-lactamase or other different protein.

For the direct expression of ss-urogastrone gene, it is required to introduce into the recombinant plasmid, upstream of the ss-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 AP, which is the left ward promotor of Xphage, lac UV5 which is present upstream of ss-galactosidase gene of Ecoll, etc. When AP, is used as the promotor, the SD sequence is not limited particularly, but it is desirable to use the four-base sequence of AGGA. Further when lac UV5 is used as the promotor, it is desirable to use the SD sequence which occurs downstream of the lac UV5 promotor or the one chemically synthesized.

The system for directly expressing the ss-urogastrone gene will be described with reference to the case wherein AP,-SD sequence-ss-urogastrone gene is used.

Although AP, is a powerful promotor (J. Hedgpeth et al., Molecular and General Genetics, 163, 197-203 (1978)), the fully activated AP, 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 thereafter cause the AP, to function. On the other hand, Cl857 which is a gene within A phage is one of the mutated genes of Cl repressor which acts on the operator for XPL. At low temperatures (of up to about 30"C), the Cm857 repres sor binds to the operator to completely inhibit the activity of XP, 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 (of not lower than 37"C), whereby the AP, 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 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 Cm857 gene is incorporated in a tetracycline-resistanct plasmid vector pSC101 (with lac UV5 promotor provided upstream thereof for the efficient expression of Cm857) as seen in Figure 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 ss-urogastrone under the control of the AP, promotor According to the present invention, the AP,-SD sequence-ss-urogastrone gene is introduced, for example, into pBR322 to obtain a ss-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 Ecoli cell.

With this system, the Cm857 repressor encoded by pGH37 binds to the operator for AP, promotor on the second plasmid when the cell is cultured for example at 30"C, permitting the proliferation of the cell.

After the cell is fully proliferated in this state, the temperature is raised for example to 40"C, whereupon the Cl857 repressor is dissociated from the operator, permitting the activity of AP, promotor for the expression of (3-urogastrone.

Although a similar concept was applied to the expression of fibroblast interferon, SV-40 Small t antigen, etc., in these cases a A lysogen is used as a host in which the DNA of A phage carrying a Cm857 gene is introduced into the host chromosome (R. Derynck et al, Nature, 287, 193-197 (1980), C. Derom et al, Gene, 17, 45-54 (1982), K. Kipper et al, Nature 289, 555-559 (1981)).

With the system of the present invention, however, the Cl857 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 X phase 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 for the first time for systems for expressing (3-urogastrone.

According to another system, a portion of any other protein gene such as ss-lactamase gene is ligated to the ss-urogastrone gene to express the ss-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 consequently afford protection for (3-urogastrone. Another advantage is that the fused protein migrates to and accumulates in the periplasm in the cell of Ecoli (S. J. Chan et al, Proc. Natl. Acad. Sci., USA., 78, 5401-5404 (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 ss-urogastrone from the fused protein by cleaving with an enzyme is inserted into the ss-lacta- mase gene at a suitable restriction enzyme cleaving site, and a ss-urogastrone gene is ligated to the ss- 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 ss-urogastrone from the fused protein are kallikrein, trypsin, etc. Examples of restriction enzymes for cleaving the ss-lactamase gene are Xmnl, Hincll, Scal, Pvul, Pstl, Bgll, Banl, etc.

The p-lactamase-p-urogastrone recombinant plasmid thus prepared can express a fused protein within E.coli for quantity production. The resulting fused protein is treated with kallikrein or the like, whereby ss- 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 of gene and direction thereof by the mini-preparation or mapping method (H. C. Birnboim et al., Nucleic Acids Research, 7, 1513-1523 (1979)), or by radioimmunoassey for (3-urogastrone.

The transformant of the present invention thus obtained is cultured by the usual method, whereby ss- 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 following method.

A quantity of 1 wt.% crosslinked polystyrene resin "S-X1," (product of BIO.RAD Laboratories, U.S.A., 200 to 400 mesh) was mixed with 2.41 g of N-(chloromethyl)- phthalimide, 0.22 ml of trifluoromethanesulfonic acid and 50 ml of dichloromethane by stirring at room temperature for 2 hours. After 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 polysty rene resin (2.5 g) obtained by the above procedure, 0.75mM of monosuccinic acid ester of 5'-o-dimethoxytritylnucleoside, 1.23mM of dicyclohexylcarbodiimide and 1mM 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 pyridineacetic 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' hydroxyl group protected with a dimethoxytrityl (DMTr) group and the amino group with a benzoyl group and 6.34 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- chlorophenylphosphorodichloridate 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, and 3.6 g of ss-cyanoethanol and 4.8 g of 1-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 0.lM aqueous solution of sodium phosphate, dibasic three times and with water twice and thereafter concentrated under reduced pressure, giving 19.96 g of crude product. The product was purified by silica gel column chromatography using chloroform-methanol (98:2 in volume ratio) as an eluent. The purifying procedure was repeated to obtain 15.12 g of completely protected adenosine mono-nucleotide.

The adenosine mononucleotide (7.81 g) thus obtained was added to a 2 wt.% solution of benzenesulfonic acid in chloroform-methanol (70:30 in volume ratio), and the mixture was stirred with ice cooling for 20 minutes and then neutralized with aqueous solution of 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 chromatography 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 dimethoxytrityl 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 period of 5 minutes with stirring and ice cooling. The mixture was thereafter stirred at room temperature for 1 hour. The triethylamine hydrochloride resulting from the reaction was filtered off, and the filtrate was stirred for 10 minutes with 1.1 ml of an aqueous solution of pyridine (1M). 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 1-methylimidazole, and the mixture was stirred at room temperature for 3 hours.The reaction mixture obtained was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, and the solution was washed with an aqueous solution of sodium phosphate, dibasic (0.1band then with water and concentrated under reduced pressure, giving 2.39 g of crude product. The product was subjected to silica gel column chromatography and eluted with chloroform-methanol (98:2 in volume ratio) to obtain 2.39 g of completely protected dinucieotide TA.

In the same method as above various nucleotrides were prepared.

3) Synthesis of oligonucleotide The solid phase synthesis of the oligonucleotide A-1, 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 (1 M) 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 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 of 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 of dimethylamino-pyridine (0.lem) in tetrahydrofuran-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 successively couple the dinucleotides AA, CG, TT and AA with the resulting nucleotide by condensation, whereby the completely protected undecanucleotide AATTCGAAGAT was prepared as supported on the resin.

The resin (20 mg) obtained was allowed to stand at 40 C for 1 hour with 0.6 ml of a solution of tetramethylguanidine-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 concentrated at 40 C under reduced pressure. The residue was dissolved in 2 ml of an aqueous solution of triethylammonium bicarbonate (TEAB, 10mM). 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 10mM 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 chromatography (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-speed liquid chromatography, the column used was ll-Bondapak C18 (product of Waters Associates, U.S.A.), and acetonitrile-aqueous solution of triethylammonium acetate (0.1M) was used as an eluent for gradient elution (5 # 40 vol.%). The undecanucleotide thus purified still had its 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 (0.1M) was used for gradient elution (5 # 25 vol.%).

In the same manner as above, the oligo-nucleotides A-2 to A-16, and B-1 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- nucleotide from the resin, followed by removal of the protective group and purification.

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

TABLE 1 A-1, 80 g A-2, 120 > g A-3, 90g A-4, 501lg A-5, 140 g A-6, 70 g A-7, 80 g A-8, 90 g A-9, 100A9 A-10, 90g A-11 110A9 A-12, 40 g A-13, 50 g A-14, 40 g A-15, 60 g A-16, 150 g B-1, 60 g B-2, 100 g B-3, 50 g B-4, 90 g B-5 100 g B-6, 90 g B-7, 130 g B-8, 100 g B-9, 110 g B-10, 100 g Cell, 110 g B-12, 110 g B-13, 130 g B-14, 60,LLg B-15, 70 g B-16, 50pLg 4) Checking of the sequence of oligonucleotide bases The sequence was checked in accordance with the two-dimensional fractionation by electrophoresis and homochromatography of Wu et al. hereinbefore mentioned.

The oligonucleotides A-1 to A-16 and B-1 to B-16 were each found to have the contemplated nucleotide sequence. Figure 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 sequence of each oligonucleotide was also checked by the Maxam-Gilbert method stated above.

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

5) Construction of oligonucleotide blocks and subunits The blocks and subunits were prepared by the procedure shown in Figure 2 as described in detail below.

First, about 5 g of each of oligonucleotides A-1, A-2, A-3, A-14, A-15 and A-16 was dissolved in distilled water (50 l) 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 A (I g calculated as DNA), into other six Eppendorf tubes individually. A mixture solution (6 pI) containing 250mM tris-HCI (pH 7.6), 50mM magnesium chloride, 10mM spermine and 50mM DTT was placed into each tube, followed by addition of 0.5 l of y-32P-ATP aqueous solution (product of Amersham International Ltd., U.K.), 0.5 l of T4 polynucleotidekinase (product of Takara Shuzo Co., Ltd., Japan) and 13 l of distilled water, to obtain 30 pl of mixture. The mixture was reacted for 30 minutes at 37 C and further reacted for 30 minutes with addition of 1 l of 30mM ATP aqueous solution. The reaction was terminated by heating at 100 C 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 FI, into another single 1.5 ml Eppendorf tube. Into the tube were placed 40 l of 250mM tris-HCI aqueous solution (pH 7.6), 40 ul of 50mM magnesium chloride and 35 u1 of distilled water to obtain a total amount of 175 iii. The mixture was heated at 90"C for 2 minutes, then gradually cooled to room temperature.With addition of 10 u1 of 200mM DTT aqueous solution, 10 ;il of 20mM ATP aqueous solution and 5 ul (100 units) ofT4 DNA ligase (product of Nippon Gene Co., Ltd., Japan), the mixture was reacted overnight at 40C, giving the block 1 of 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 formed by the ligation of A-4, A-5, A-6, A-11, 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 -80 C 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. for the block 3. Subsequently, each band was cut out and a mixture of 10mM tris-HCI (pH 7.6) and 10mM EDTA aqueous solution (tris-EDTA) was added thereto. The mixture was then allowed to stand overnight at room temperature for extraction.The resulting 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 Sephadex G-50 to remove the phenol and acrylamide. The elute was then concentrated to 200 l and thereafter allowed to stand at -80"C for 30 minutes with ethanol in twice the volume of the concentrate to precipitation DNA.

The three blocks obtained were combined together. To the mixture were added 50mM tris-HCI (pH 7.6), 10mM magnesium chloride, 20mM DTT, 1mM ATP and 5 al (100 unit) of T4 DNA ligase. The resulting mixture was allowed to stand overnight at 4"C for ligation. To the mixture was added ethanol in twice the volume thereof, 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, 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 was centrifuged to separate the su -pernatant, the supernatant was fully shaken with tris-EDTA saturated phenol, and the lower layer was discarded. With further addition 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 -80"C for 30 minutes to precipitate DNA. The resulting product was cleaved with EcoRI and BamHI to obtain the subunit A.

The same procedure as above was repeated as seen in Figure 3 to ligate oligonucleotides B-1, B-2, B15 and B-16 into the block 4, to ligate oligonucleotides B-3, B-4, B-13 and B-14 into the block 5, to ligate oligonucleotides B-5, B-6, B-11 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 to 26 b.p. and 52 b.p. were collected, and similarly ligated to give products of 104 b.p. and 208 b.p., which was cleaved with Hindlll and BamHI. Thus, the subunit B was obtained.

6) Cloning of subunits and analysis of recombinant plasmids With reference to Figure 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 4"C in a mixture of 50mM tris-HCI (pH 7.6), 10mM magnesium chloride, 20mM DTT and 1 mM ATP with addition of 5 pl 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 -80"C for 30 minutes for precipitation.The mixture was then centrifuged, the precipitate was dried and dissolved in 100 pl of distilled water, whereby a plasmid pUG1 was obtained in which the subunit A was incorporated in pBR322.

Ecoli strain HB101 was transformed with the plasmid pUGI by the calcium method.

The strain HB101 serving as a host was cultured at 37"C 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). When 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 4"C. The supernatant was discarded, the precipitate was suspended in 20 ml of ice-cooled 0.1M magnesium chloride, the suspension was centrifuged under the same condition again, and the supernatant was discarded. The precipitate was suspended in 20 ml of icecooled solution of 0.lM calcium chloride and 0.05M magnesium chloride and ice-cooled for 1 hour. The suspension was centrifuged, the supernatant was discarded, and the precipitate was suspended in 2 ml of ice-cooled solution of 0.lM calcium chloride and 0.05M magnesium chloride. To a 200 AI portion of the suspension was added 10 ul of aqueous solution of pUG1, and the mixture was ice-cooled for 1 hour and then heated in a water bath at 43.5"C for 30 seconds. Subsequently, 2.8 ml of LB culture medium was added to the mixture, followed by incubation at 37"C for 1 hour.The culture was then spread over a LB plate containing 50 g/ml of ampicillin, in an amount of 200 'LI/dish and incubated overnight at 37"C. The growing colonies were checked by further transplantation to a LB plate containing 501lg/ml of ampicillin and also to a LB plate containing 20 Wg/ml of tetracycline and were incubated overnight at 37"C. 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 extraction method and checked for the presence of a Bgill cleavage site. One of the cells containing the plasmid having Bglll cleavage site was cultured in a large scale to obtain purified plasmid pUG1 similarly by the alkaline extraction method.

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

Photos 1 and 2 show the results of analysis. Lanes 1 to 4 are the result of electrophoresis for EcoRI Sail fragment, and lanes 5 to 8 are that for BamHI - Pstl 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 reference to Figure 3, the plasmid pBR322 was cleaved with Hindlll and BamHI, and the larger fragment was isolated by means of electrophoresis and tigated to the subunit B. Thus, a plasmid pUG2 in which subunit B was introduced into pBR322 was obtained similarly as in the case of pUG1. Using the resulting plasmid pUG2, the strain HB101 was transformed, and the colonies resistant to ampicillin only.

were selected. Plasmids were collected from the colonies and then checked for the presence of a 89111 cleavage site and a M1ul 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 Hindlll-Sail 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. Each lane shows the same corresponding reaction product as in Photo 1. Thus, the nucleotide sequence of the subunit B was confirmed.

Next with reference to Figure 4, pUG1 was cleaved with Hindlll and Salt, and a larger fragment was separated through a Biogel 1.5 m column. pUG2 was cleaved with Hindlll and Sail, 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. (3-uro- gastrone gene, was incorporated into pBR322. E. coli strain HB101 was transformed using the plasmid pUG3.The transformant has been deposited under Budapest Treaty on international recognition of deposit with deposition number FERM BP-543 in Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, since June 22, 1984.

In the above case also, the cells harboring plasmid pUG3 which was resistant to ampicillin only and had M1ul 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 (3-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 BamHI-Pstl fragment.

Lanes 5 to 8 show the result obtained with the same speciment, in which a region of the higher molequ- lar weight side (corresponding to the upper portion of lanes 1 to 4) being shown on an enlarged scale.

The reaction products of the lanes are the same as the corresponding ones in photo 1. The analysis confirmed the nucleotide sequence of the ss-urogastrone gene.

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

First preparation of a strain ECI-2 derived from Ecollstrain HB101 will be described. ECI-2 served as a host for #PL expression plasmids. Then described will be the cloning of a DNA fragment containing #PL promotor from the DNA of AC1857S7 which is a mutant of X phage, and the construction of expression plasmids from the cloned DNA. Further described will be the expression of ss-urogastrone gene in the ost ECI-2 strain by APL promotor.

7-1) Construction of strain ECI-2 The strain ECI-2 is E.coli HB101 harboring a plasmid pGH37 for expression of C1857 gene.

pGH37 was prepared by the process shown iri Figure 6. First, DNA of AC1857S7 was cleaved with BG1#.

Then, the cohesive ends of the cleavage site were digested using S1 nuclease. One g of DNA of XC185787 cleaved with Bg ill was reached with 200 units of SI nuclease at 20"C for 30 minutes in 100 pl 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 electrophoresis to isolate therefrom a fragment with 2385 b.p. having the whole Cl857 structural gene. The frag ment was inserted into the Pvull cleavage site of plasmids pGL101 to construct a plasmid pGH36 which expresses the C1857 gene under the control of a promotor, lac UV5.Subsequently, pGH36 is cleaved with two restriction enzymes, EcoRI and Pstl, to obtain a fragment having 1193 b.p., which was inserted into a plasmid pSC101 between EcoRI and Pstl cleavage sites to prepare a plasmid pGH37.

Subsequently, 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 recognition of deposit with deposition number FERM BP-542 in Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, since June 22, 1984. This strain is resistant to tetracycline, expresses the Cl857 gene and permits conjqint presence, through transformation, of other plasmids derived, for example, from pBR322.

Accordingly the strain ECI-2 was thereafter used as a host of #PL expression plasmids.

7-2) Cloning of XPL promotor and preparation of expression plasmids As shown in Figure 7, pGH35 was constructed first. DNA of #C1857S7 was cleaved with EcoRI and Sal I to obtain a fragment of 5925 b.p. which includes XPL promotor and C1857 gene as well as XPR promotor.

The fragment.was inserted into plasmid pBR322 between EcoRI and Sail cleavage sites to construct a plasmid pGH25.

Next, pGH25 was cleaved with BamHI and ligated to pBR322 similarly cleaved with BamHI to obtain pGH34.

Subsequently, pGH34 was cleaved with Aval and Bgill and thereafter treated with S1 nuclease into blunt-ended fragment of about 4500 b.p. The fragment was circularized with T4 DNA ligase to construct a plasmid pGH35.

Next, pEK28 was constructed as shown in Figure 8. Synthetic oligonucleotides C-1-1 and C-1-2 as an adapter, which include an SD sequence and have the nucleotide sequence shown below, were ligated to the fragment which was obtained by cleaving pGH35 with Hpal. The assembly was further ligated to plasmid pMC1403 cleaved with BamHI to obtain plasmid pEG2. The pEG2 has two ampicillin- resistant genes, and expresses ss-galactosidase gene derived from pMC1403 under the control of #PL promotor, utilizing a start codon as well as the SD sequence included in the adapter.

The fragments C-1-1 and C-1-2 have the following nucleotide sequence.

SD sequence Start codon

Miller's method was employed to confirm the expression of ss-galactosidase in the host ECI-2 harboring the plasmid pEG2 (Milier, J. (1972) "Experiments in Molecular Genetics" New York, Cold Spring Harbor Laboratory pp352-355). This method is based on the reaction of ss-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, 1mM of magnesium sulfate and 0.1 M (3-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 off 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 300C until a yellow color develops, whereupon 0.5 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 ss-galactosidase is defined by the units in 1 ml of the liquid culture according to the following equation, in which absorbance at 610 nm is calculated as 1.0.

Activity of ss- - OD420 - 1.75 x OD550 galactosidase t x v x OD610 x 1000 (units) 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 30 C, the ss-galactosidase activity was 98 units. However, when the culture was further incubated at 42 C for 1 hour, the EPL promotor was activated to result in ss-galactosidase activity of 9637 units. This substantiates that the sequence from the AP, promotor to ss-galactosidase is in the contemplated order.

Although pEG2 has two Bgill cleavage sites, only the Bgill site present immediately after the SD sequence of ss-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 pEK28 completed which is an expression piasmid with'use of AP, promotor.

7-3) Expression of fused gene of front half of ss-urogastrone and ss-galactosidase.

Figure 9 schematically shows the series of procedures to be described below.

A plasmid pUG101 was constructed in the following manner which has a fused gene of the front half of ss-urogastrone and a ss-galactosidase. More specifically, pUG1, which has the frohnt half of ss-urogastrone gene, and pMC1403 having a ss-galactosidase gene were cleaved with BamHI and then ligated to form pUG101. With this plasmid, the front half of ss-urogastrone gene and the ss-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 AP, promotor, pUG101 and pEK28 were each cleaved with Bg111.

The cleavage with ssg1# produces a DNA fragment having a extruding 5' ends in the form of

# ... A #. ... TCTAG 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 of deoxribonucleotide triphosphates dGTP, dATP, dTTP and DCTP, a blunt end is obtained by filling up with the corresponding nucleotides, that makes the end of

# ... AGATC #.

... TCTAG If dGTP only is added as a nucleotide component, the end of

# ... AG # ... TCTAG is obtained owing to the termination of the reaction. Next, when S1 nuclease is used to digest the remaining single strand, a blunt end is obtained in the form of

# ... AG # ... TC # Similarly if dGTP and dATP are added in the Klenow reaction, followed by digestion with S1 nuclease,

AGA TCT is obtained.When the Klenow reaction is conducted by addition of dGTP, dATP and dTTP, followed by digestion with S1 nuclease,

# ... AGAT # ... TCTA is obtained. Further if only the digestion with S1 nuclease is conducted without effecting the Klenow reaction,

...Ass is obtained. Thus, when the DNA fragment with the end of

# ... A # ... TCTAG is subjected to the Klenow reaction with use of different nucleotides and/or to the S1 nuclease reaction, five kinds of blunt ends are obtained which are different from one another by one base pair length.

The Klenow reaction and S1 nuclease reaction were conducted under the following conditions.

*Klenow reaction: One g of DNA as the substrate was reacted with 1mM of each deoxyribonucleotide triphosphate at 12 C for 30 minutes in the presence of 1 unit of the enzyme (klenow fragment) and 1mM of ATP in 50 l of a reaction medium comprising 40mM of potassium phosphate buffer (pH 7.4). 1mM of ss-mercaptoethanol and iOmM of magnesium chloride.

*S1 nuclease reaction: One g of DNA as the substrate and 200 units of the enzyme were reacted at 20"C 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 Bgill and thereafter subjected to various combinations of the Klenow reaction and S1 nuclease reaction, giving fragments having 5 kinds of 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 obtained were cleaved with Sail to isolate fragments which contain the gene encoding fused protein of ss-urogastrone front half and ss-galactosidase from pUG101, and fragments containing kP, promotor from pEK28. These fragments were ligated by T4 DNA ligase in the combinations shown in Table 2.

Consequently, recombinant plasmids in Table 3 were obtained. The ss-galactosidass 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 ss-galactosidase activity. Especially pUG103, pUG104 and pUG117 achieved remarkable results.

TABLE 2 Nucleotide sequence upstream of start codon (A TG) ATCTGC- GATCTGC -ACA -ACAATCTGC- -ACAGATCTGC (pUG105) (pUG016) -ACAG -ACAGGATCTGC (pUG110) Nucleotide sequence downstream of SD -ACAGA -ACAGAATCTGC- -ACAGAGATCTGCsequence (AGGA) (pUG113) (pUG114) -ACAGAT -ACAGATATCTGC- -ACAGATGATCTGC (pUG117) (pUG118) -ACAGATC -ACAGATCATCTGC- -ACAGATCGATCTGC (pUG121) (pUG122) Nucleotide sequence upstream of start codon (A TGJ TGC- CTGC- TCTGC -ACA -ACATGC- -ACACTGC- -ACATCTGC (pUG102) (pUG103) (pUG104) -ACAG -ACAGTGC- -ACAGCTGC- -ACAGTCTGC (pUG107) (pUG108) (pUG109) Nucleotide sequence downstream of SD -ACAGA ACAGATGC- -ACAGACTGC- sequence (AGGA) (pUG111) (pUG112) -ACAGAT -ACAGATTGC- - -ACAGATTCTGC (pUG11S) (pUG116) -ACAGATC- -ACAGATCCTGC- -ACAGATCTCTGC (pUG119) (pUG120) TABLE 3 Number of nucleotides between SD sequence Recombinant ss-Galactosidese and start codon plasmid (units) 6 pUG102 1674 7 pUG103 2533 7 pUG107 2260 8 pUG104 2802 8 pUG108 1835 9 pUG1OS 1018 9 pUG109 1942 10 pUG106 973 11 pUG110 1764 11 pUG113 1950 11 pUG119 1862 12 pUG114 946 12 pUG117 2332 12 pUG120 1374 13 pUG118 2041 13 pUG121 1678 14 pUG122 1814 Next, a vector for expression of ss-urogastrone was prepared from pUG103 or pUG117. The plasmid (pUG103 or pUG117) was cleaved with Hindlll and Pvull to obtain a fragment of 1.2 kb containing the region of from XP, promotor to the front half of ss-urogastrone gene. Further pUG2 was cleaved with EcoR1, filled up with Klenow fragment, and cleaved with Hindill to obtain a fragment of 4.1 kb.The two fragments were ligated with T4 DNA ligase, and the strain ECl-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 ss-urogastrone gene, i.e. the whole ss- urogastrone gene, under the control of AP, promotor.

8) Vector for expression of fused protein A ss-lactamase gene on the plasmid pBR322 and a ss-urogastrone gene were ligated to express (3-uro- gastrone as a fused protein as will be described below.

8-1) Donor of ss-lactamase gene pBRH02 is obtained by cleaving pBR322 with Aval and Pvull, 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 Aval and Hindlll, followed by the Klenow reaction and ligation and has ApR and chloramphenicol resistance (CmR) as markers. Figure 10 shows these plasmids.

8-2) Donor of ss-urogastrone gene pUG3 prepared as already described was cleaved with Mboll to obtain 13 kinds of DNA fragments, which were named A to M in the order of size as shown in Figure 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 Nterminus of ss-urogastrone and ending with 16 bases downstream of the stop codon, the fragment having the whole structural gene of (3-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 ss-urogastrone from the expressed fused protein.

TABLE 4 Adaptor 5' end 3' end D-1-3 CCGTAAG D-1-4 TTACGG D-2-1 CGTAAG D-2-2 TTACG D-3-2 TTACGGAT D-4-2 TTACGTGCA E-1 CGCTAAACGG E-2 CGTTTAGCG E-3 GACAAACGG E-4 CGTTTGTC E-5 CGTTTAGCGAT E-6 CGTTTGTCTGCA E-7 CGGCTAAACGG E-8 CGTTTAGCCG E-9 CAAACGG E-10 CGTTTG 8-4) System for expression of fused protein of ss-lactamase and ss-urogastrone linked by Lys-Arg A vector for expression of ss-lactamase-ss- 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 Figure 12 was practiced.

The plasmid pBRHO2 was completely cleaved with Xmnl at 37 C over a period of 3 hours. Subsequently 3 g of the vector, about 0.1 g of ss-urogastrone fragment of 179 b.p. and about 1 g of each of E-1 and E-2 (with non-phosphorylated 5' end) serving as an adaptor were ligated in a single step at 12 C 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 Ape. These colonies were checked by minipreparation for the size of plasmid DNA. Thirteen plasmids were about 200 b.p. larger than the vector and considered to have a ss-urogastrone gene inserted therein. All of them, which had a M1ul site, were cleaved with Hinfl and checked for the orientation of insertion of the ss-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 ss-urogastrone gene was inserted in the same orientation as ss-lactamase.

These three plasmids were named pUG2301 to pUG2303.

8-4-b) Construction of pUG2101 to pUG2105 The process shown in Figure 13 was practiced.

The plasmid pBR322 was used as a plasmid vector which has unique Pvul site in the ss-lactamase gene.

According to the process described in 8-4-a), an expression vector was constructed using E-1 and E-5 for an adaptor.

When the 1626 TcR colonies obtained were checked for Ap sensitivity, 31 colonies (1.9%) were Ape.

Mini- preparation was conducted for 22 TcR and Aps colonies, and the plasmids were checked for the insertion of ss-urogastrone gene by cleavage with M1ul. Twenty plasmids were found to have an Mlul site. The orientation was checked by cleaving with Hinfl or BamHI. Plasmids pUG2101 to pUG2105 were found to have a ss-urogastrone gene inserted therein in the same orientation as the ss-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 Figure 14.

Of the 217 TcR colonies obtained, 106 colonies (48.8%) were Ape. 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 ss-urogastrone gene inserted therein, so that these eight plasmids were cleaved with Bam-Hl and checked for the orientation of the gene, Consequently, three plasmids were found to have the ss-urogastrone gene inserted in the same orientation as ss-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 ss-lactamase and (3-urogastrone. Predicted amino acid sequence and the corresponding nucleotide sequence are shown below.

Met Ser Ile Gln 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 Phe Cys Leu Pro Val Phe Ala His TTT TGC CTT CCT GTT TTT GCT CAC Pro Glu Thr Leu Val Lys Val Lys CCA GAA ACG CTG GTG AAA GTA AAA Asp Ala Glu Asp Gln Leu Gly Ala GAT GCT GAA GAT CAG TTG GGT GCA Arg Val Gly Tyr lie Glu Leu Asp CGA GTG GGT TAC ATC GAA CTG GAT Leu Asn Ser Gly Lys Ile Leu Glu CTC AAC AGC GGT AAG ATC CTT GAG Ser Phe Arg Pro Glu Glu Arg Ala AGT TTT CGC CCC GAA GAA CGC GCT Lys Arg Asn Ser Asp Ser Glu Cys AAA CGG AAT AGC GAT TCT GAG TGC Pro Leu Ser His Asp Gly Tyr Cys CCA CTG TCT CAC GAT GGC TAT TGT Leu His Asp Gly Val Cys Met Tyr CTG CAC GAC GGT GTT TGC ATG TAC Ile Glu Aa Leu Asp Lys Tyr Ala ATC GAA GCT TTG GAT AAA TAC GCG Cys Asn Cys Val Val Gly Tyr Ile TGT AAC TGT GTA GTG GGT TAT ATC Gyl Glu Arg Cys Gln Tyr Arg Asp GGT GAA CGC TGT CAA TAC CGT GAT Leu Lys Trp Trp Glu Leu Arg (stop) CTG AAA TOO TGG GAA TTG CGT TAA TAGTGAAGATCTGGATCCGTTTAGCGTTTTCCA ATGATGAGCACTTTTAAAGTTCTGCTATGTGGC GCG GTATTATCCCGTGTTGACGCCG G G CAAGAG CAACTCGGTCGCCGCATAC Similarly, the plasmid pUG2101 obtained by the procedure 8-4-b) produces a fused protein having the following primary structure.

Met Ser lie Gln 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 Phe Cys Leu Pro Val Phe Ala His TTT TGC CCT CCT GTT TTT GCT CAC Pro Glu Thr Leu Val Lys Val Lys CCA GAA ACG CTG GTG AAA GTA AAA Asp Ala Glu Asp Gln Leu Gly Ala GAT GCT GAA GAT CAG TTG GGT GCA Arg Val Gly Tyr Ile Glu Leu Asp CGA GTG GGT TAC ATC GAA CTG GAT Leu Asn Ser Gly Lys Ile 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 Pro Met Met Ser Thr Phe Lys Val CCA ATG ATG AGC ACT TTT AAA GTT Leu Leu Cys Gly Ala Val Leu Ser CTG CTA TGT GGC GCG GTA TTA TCC Arg Val Asp Ala Gly Gln Glu Gln CGT GTT GAC GCC GGG CAA GAG CAA Leu Gly Arg Arg Ile His Tyr Ser CTC GGT CGC CGC ATA CAC TAT TCT Gln Asn Asp Ile Val Glu Tyr Ser CAG AAT GAC TTG GTT GAG TAC TCA Pro Val Thr Glu Lys His Leu Thr CCA GTC ACA GAA AAG CAT CTT ACG Asp Gly Met Thr Val Arg Glu Leu GAT GGC ATG ACA GTA AGA GAA TTA Cys Ser Ala Ala lie Thr Met Ser TGC AGT GCT GCC ATA ACC ATG AGT Asp Asn Thr Ala Ala Asn Leu Leu GAT AAC ACT GCG GCC AAC TTA CTT Leu Thr Thr Ile Ala Lys Arg Asn CTG ACA ACG ATC GCT AAA CGG AAT Ser Asp Ser Glu Cys Pro Leu Ser AGC GAT TCT GAG TGC CCA CTG TCT His Asp Gly Tyr Cys Leu His Asp CAC GAT GGC TAT TGT CTG CAC GAC Gly Val Cys Met Tyr Ile Glu Ala GGT GTT TGC ATG TAC ATC GAA GCT Leu Asp Lys Tyr Ala Cys Asn Cys TTG GAT AAA TAC GCG TGT AAC TGT Val Val Gly Tyr lie Gly Glu Arg GTA GTG GGT TAT ATC GGT GAA CGC Cys Gln Tyr Arg Asp Leu Lys Trp TGT CAA TAC CGT GAT CTG AAA TGG Trp Glu Leu Arg (stop) TGG GAA TTG CGT TAA TAGTGAAGATC TGGATCCGTTTAGCGATCGGAGGACCGAAGG AGCTAACCGCTTTTTTGCACA Similarly, the plasmid pUG2701 obtained by the procedure 8-4-c) produces a fused protein having the following primary structure.

Met Ser Ile Gln His Phe Arg Val ATG AGT ATT CAA CAT TTC CGT GTC Ala Leu Ile 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 Pro Glu Thr Leu Val Lys Val Lys CCA GAA ACG CTG GTG AAA GTA AAA Asp Ala Glu Asp Gln Leu Gly Ala GAT GCT GAA GAT CAG TTG GGT GCA Arg Vai Gly Tyr Ile Glu Leu Asp CGA GTG GGT TAC ATC GAA CTG GAT Leu Asn Ser Gly Lys Ile 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 Pro Met Met Ser Thr Phe Lys Val CCA ATG ATG AGC ACT TTT AAA GTT Leu Leu Cys Gly Ala Val Leu Ser CTG CTA TGT GGC GCG GTA TTA TCC Arg Val Asp Ala Gly Gln Glu Gln CGT GTT GAC GCC GGG CAA GAG CAA Leu Gly Arg Arg Ile His Tyr Ser CTC GGT CGC CGC ATA CAC TAT TCT Gln Asn Asp Ile Val Glu Ser Ala CAG AAT GAC TTG OU GAG TCG GCT Lys Arg Asn Ser Asp Ser Glu Cys AAA CGG AAT AGC GAT TCT GAG TGC Pro Leu Ser His Asp Gly Tyr Cys CCA CTG TCT CAC GAT GGC TAT TGT Leu His Asp Gly Val Cys Met Tyr CTG CAC GAC GGT GTT TGC ATG TAC Ile Glu Ala Leu Asp Lys Tyr Ala ATC GAA GCT TTG GAT AAA TAC GCG Cys Asn Cys Val Val Gly Tyr lie TGT AAC TGT GTA GTG GGT TAT ATC Gly Glu Arg Cys Gln Tyr Arg Asp GGT GAA CGC TGT CAA TAC CGT GAT Leu Lys Trp Trp Glu Leu Arg (stop) CTG AAA TGG TGG GAA TTG ' CGT TAA TAGTGAAGATCTGGATCCGTTTAGCCGACTCAC CAGTCACAGAAAAGCATCTTACGGAT The nucleotide 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 Mlul-Pstl fragment (224 b.p.) of pUG2101, lanes 5 to 8 show the result with the M1ul- EcoRi fragment (721 b.p.) of pUG2101, lanes 9 to 12 show that with the M1ul-BamHI fragment (452 b.p.) of pUG2301, lanes 13 to 16 is that with the M1ul-Pstl fragment (335 b.p.) of pUG2701, and lane 17 to 20 show that with the M1ul-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, 8-5) System for expression of fused protein of ss-lactamase and ss-urogastrone linked by Arg-Lys 8-5-a) Preparation of pUG1102 and pUG1105 ss-Urogastrone gene was inserted into the ss- lactamase gene of pBR322 at its unique Pvul restriction site to obtain vectors for expression of fused protein of ss-lactamase and ss-urogastrone as shown in Figure 15.

The plasmid pBR322 was cleaved with Pvui at 37 C 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 12 C 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, ss-urogastrone fragment and vector were mixed together in a molar ratio of approximately 5:1 and ligated at 12 C over a period of 15 hours. After the ligation, the strain HB101 was transformed with the resulting plasmid, and the colonies were selected with reference to TcR.

Seventy-one TcR transformed colonies were obtained and then checked for Ap sensitivity. Plasm it DNA was prepared from 20 Aps colonies (28.2%) and then checked for the presence of M1ul restriction site to confirm the insertion of ss-urogastrone gene.Five of the 20 plasmids were found to have the M1ul site of -urogastrone gene. The DNA was cleaved with Hinfl and then subjected to 1.5 wt.% agarose gel electro- phoresis to check the orientation of insertion. Two of the plasmids, i.e. pUG1102 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 Pstl, Hincil and Xmnl in place of Pvul to obtain pUG1004, pUG1201 and pUG1301 as shown in Figures 16, 17 and 18, respectively.

9) Confirmation of expression of ss-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 ss-urogastrone gene and extraction of proteins 9-1-a) Expression system using XP, promotor The strain ECI-2 harboring expression plasmid pUG103-E and the same strain harboring expression plasmid pUG117-E were each cultured at 25"C in two flasks each containing 1 liter of LB culture medium.

When the culture in one of the flasks exhibited an absorbance of 0.3 at 660 nm, the culture was subjected to heat induction at 42"C for 1 hour. The culture in the other flask was continuously incubated at 25"C until the absorbance became 0.4. The cells in each flask was collected, washed with PBS buffer (137mM sodium chloride, 2.7mM potassium chloride, 8.1mM 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 1 hour) was dialyzed against 0.01N aqueous solution of acetic acid, and the dialyzate was lyophilized and thereafter subjected to radioimmunoassay (hereinafter referred to as "RIA").

9-1-b) System for expression of fused protein E. coli strain HB101 harboring plasmids pUG1004, 1301, 2101, 2303 or 2703 was preincubated at 370C in a culture medium containing 50 Ag/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 with PBS buffer, then resuspended in PBS buffer in 3 vol.% of the amount of the original culture and sonicated (at 100 W for 30 seconds, three times) by the same sonicator as above with ice cooling. The supernatant separated from the cell debris by ultracentrifugation (40000 g for 1 hour) was dialyzed against 0.01 N aqueous solution of acetic acid, lyophilized and then subjected to RIA.

To confirm the accumulation of 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, 5401-5405 (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 37"C 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 of 1OmM tris-HCI (pH 8.0) and 30mM sodium chloride. The cells (1 g) were resuspended again in 80 ml of 20 wt.% sucrose-30mM tris-HCI (pH 8.0), whereupon EDTA was added to the suspension to a concentration of 1mM. The mixture was shaken by a rotary shaker at 180 r.p.m. for 10 minutes (24"C) and then centrifuged (13000 g, 10 minutes) to collect the cells, which were resuspended in 80 ml of distilled water. The suspension was allowed to stand in ice for about 10 minutes with occasionai stirring and then centrifuged (13000 g, 10 minutes).The supernatant was collected as a periplasmic fraction (O-Sup). The pellet was suspended in a mixture of 1OmM tris-HCL (pH 8.0) and 30mM sodium chloride and treated by the same sonicator as above to obtain a cytoplasmic fraction (O-Ppt).

These samples were subjected to RIA.

9-2) Radioimmunoassay 9-2-a) Establishment of RIA system Rabbits were immunized with purified human ss-urogastrone as an antigen to obtain antiserum. The ss- urogastrone (300 pg) 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 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 times every two weeks, 50 Ag of the antigen was further intravenously injected, the whole blood was collected 3 days thereafter, and the serum was separated.

Next, the following RIA conditions were determined in view of the titration curve for determining 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 (1OmM, pH 7A) containing 0.5 wt.% of bovine-serum albumin (BSA), 140mM of sodium chloride and 25mM of disodium EDTA.The diluting solution (400 l), 100 l of the sample or standard human ss-urogastrone and 100 lli of antihuman ,3-urogastrone serum were mixed together. After the mixture was incubated for 24 hours at 4 C, 100 'LI of 251-labeled human ,3- urogastrone solution (about 5000 cpm) was added to the mixture.After the mixture was further incubated for 48 hours at 4 C, 1 00-'Ll of second antibody (anti-rabbit -globulin goat serum) (20-fold dilution with PBS buffer), 100 l of normal rabbit serum (200-fold dilution with PBS buffer) and 900 l of 10mM PBS buffer containing 5 wt.% polyethylene glycol were added to the resulting mixture, and then further incubated for 3 hours at 4 C. 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 (3-urogastrone.

9-2-b) Confirmation of ss-urogastrone productivity of recombinant microorganism Table 5 shows the result of RIA conducted for the expression system with use of AP, promotor.

TABLE 5 Expression Amount of (3- plasmid Heat induction urogastrone produced (ngll culture) pUG103-E Yes 450.2 pUG103-E No 3.2 pUG117-E Yes 388.4 pUG117-E No 3.2 Control No Not detectable (pBR322) Table 6 shows the result of RIA conducted for fused protein expression systems.

TABLE 6 Expression Amount of ss- plasmid urogastrone produced ( gll culture) pUG1004 729.6 pUG1301 650.7 pUG2101 31.3 pUG2301 125.2 pUG2701 119.2 Table 7 shows the localization of the expressed fused protein.

TABLE 7 Amount of ss-urogastrone produced Expression ( gll 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 XP, promotor system for direct expression of ss-urogastrone and the system for expression of the compound as a fused protein both expressed ss-urogastrone immunoreactivity in E.coli. Table 7 reveals that in the case of fused protein, the expressed ss-urogastrone immunoreactivity is almost localized in the periplasm.

Claims (24)

1. A novel ss-urogastron gene having the following nucleotide sequence: 5' AAT AGC GAT TCT GAG TGC CCA CTG 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 GAC GGT GTT 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 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 GTT ATG GCA CTA GAC TTT TGG TGG GAA TTG CGT 3' ACC ACC CTT AAC GCA 5'

2. A subunit of the gene defined in claim 1, the subunit having the front half of the nucleotide sequence in claim 1 and divided approximately at the midportion thereof.

3. A subunit of the gene defined in claim 1, the subunit having the rear half of the nucleotide sequence in claim 1 and divided approximately at the midportion thereof.

4. A gene as defined in claim 1 which has a restriction enzyme recognition site attached to each of the front end and/or the rear end of the gene.

5. A gene as defined in claim 4 which has a restriction enzyme recognition site and a start codon provided upstream of the gene and/or a stop codon and a restriction enzyme recognition site provided downstream of the gene, the codons and the recognition sites being arranged in the order mentioned.

6. A gene as defined in claim 5 which has the following nucleotide sequence: -15 -1, 1 5' AAT TCG AAG ATC TGC ATG AAT AGC 3' GC TTC TAG ACG TAC TTA TOG 10 20 30 GAT TCT GAG TGC CCA CTG TCT CAC CTA AGA CTC ACG GGT GAC AGA GTG 40 50 GAT GGC TAT TGT CTG CAC GAC GGT CTA CCG ATA ACA GAC GTG CTG CCA 60 70 80 GTT TGC ATG TAC ATC GAA GCT TTG CAA ACG TAC ATG TAG CTT CGA AAC 90 100 GAT AAA TAC GCG TGT AAC TGT GTA 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 130 140 150 CAA TAC CGT GAT CTG AAA TOG TGG GTT ATG GCA CTA GAC TTT ACC ACC 160 170 GAA TTG CGT TAA TAG TGA AGA TCT CTT AAC GCA ATT ATC ACT TCT AGA G 3' CCT AG 5'

7.A subunit of the gene defined in claim 6, the subunit having the front half of the nucleotide sequence defined in claim 6 and divided approximately at the mid- portion thereof, the subunit further having a restriction enzyme recognition site at the rear end thereof.

8. A subunit as defined in claim 7 having the following nucleotide sequence: 5' AAT TCG AAG ATC TGC ATG AAT AGC 3' GO TTC TAG ACG TAC TTA TCG GAT TCT GAG TGC CCA CTG TCT CAC CTA AGA CTC ACG GGT GAC AGA GTG GAT 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 3' CTA G5'

9. A subunit of the gene defined in claim 6, the subunit having the rear half of the nucleotide sequence defined in claim 6 and divided approximately at the midportion thereof, the subunit further having a restriction enzyme recognition site at the front end thereof.

10. A subunit as defined in claim 9 having the following nucleotide sequence: 5' A GCT TTG GAT AAA TAC GCG TGT AAC CTA TT.T ATG CGC ACA AAC TGT GTA GTG GGT TAT ATC GGT TTG ACA CAT CAC CCA ATA TAG CCA GAA CGC TGT CAA TAC CGT GAT 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 TGA AGA TCT G 3' ACT TCT AGA CCT AG 5'

11. A recombinant plasmid having the gene defined in claim 6.

12. A process for preparing the recombinant plasmid as defined in claim 11 comprising inserting the subunit defined in claim 8 and the subunit defined in claim 10 into an appropriate insertion site of an appropriate plasmid vector.

13. A recombinant plasmid comprising a plasmid vector having inserted therein the ss-urogastrone gene defined in claim 6, the plasmid vector further having inserted therein upstream of the ss-urogastrone gene a promotor for controlling the expression of the gene and an SD sequence joined to the promotor.

14. A recombinant plasmid comprising a plasmid vector having the sequence of the fourth and following pairs of bases of the ss-urogastrone gene defined in claim 6, the plasmid vector having the combination of a promotor, an SD sequence and another gene inserted therein upstream of the ss-urogastrone gene.

15. A recombinant plasmid as defined in claim 14 wherein said another gene is a ss-lactamase gene.

16. A recombinant plasmid as defined in claim 13 or 14 wherein the promotor is AP, or lac UV5.

17. A recombinant plasmid as defined in claim 13 or 14 wherein the plasmid vector is pBR322.

18. A transformant comprising a host cell having a recombinant plasmid capable of expression of the (3-urogastrone gene defined in claim 1.

19. A transformant as defined in claim 18 wherein the recombinant plasmid is the one defined in claim 13.

20. A transformant as defined in claim 18 wherein the recombinant plasmid is the one defined in claim 14.

21. A transformant as defined in claim 18 wherein the host cell is E.coli.

22. A transformant as defined in claim 18 wherein the host cell is transformed with a recombinant plasmid having a TcR gene and a C1857 gene.

23. A process for producing a transformant by transforming a host cell with a recombinant plasmid capable of expressing the ss-urogastrone gene defined in claim 1.

24. A process for producing ss-urogastrone characterized by culturing the transformant defined in claim 18 and collecting the expressed (3-urogastrone.