AU599003B2 - Novel beta-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of beta-urogastrone - Google Patents

Description

PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Form Short Title: Int. Ci Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: lPrjnrify: Rs'acl~ Art: TO BE COMPLETED BY APPLICANT Nchn3 of Applicant Address of Applicant: Actu,:iIlnventor; EARTH CHEMICAL COMPANY, LIMITED.

3218/12, Sakoshi, Ako-shi, Hyogo-ken,

JAPAN.

Address tor Service: CLEMENT HACK CO.F 140 wi32iam Street, Melbourn~e, Vic. 3000.

Australia.

Complete Specification for the invention entitled: NOVEL j3-UROGASTRONE GENE, CORRESPONDING RECOMBINANT PLASMIDS, CORRESPONDING TRANSFORMANTS AND PREPARATION THEREOF AND OF O-UROGASTiRONE.

The following statement Is a full description of this invention, Including the best method of perfoeming it known to me.,- PF/CPlF/2/80 9 1A.

NOVEL B-UROGASTRONE GENE, CORRESPONDING RECOMBINANT PLASMIDS, CORRESPONDING TRANSFORMANTS AND PREPARATION THEREOF AND OF B-UROGASTRONE The present invention relates to a novel B-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of B-urogastrone.

B-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 i 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 lie Gly Glu Arg Cys Gin 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 i.

-D*I

-2- Gly: glycine Tyr: tyrosine Val: valine Met: methionine lie: isoleucine Ala: alanine Lys: lysine Gin: glutamine Arg: arginine Trp: tryptophan Phe: phenylalanine B-3rogastrone 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 0-urogastrone is excreted in small amounts in human urine, the compound is presently prepared from urine by extraction, separation and purification. However, this met.od 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 P-urogastrone by a gene engineering technique with use of o a synthetic P-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 P-urogastrone L 3 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 B-urogastrone.

Nevertheless, it is hard to speculate which of such genes is capable of expressing B-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 B-urogastrone gene which is entirely different from the gene disclosed in the above publication in the nucleotide sequence and which is capable of expressing B-urogastrone through gene engineering techniques.

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

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

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

ri i u; 4- 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 Gene I: AAT A G 3' TT A T C TC T CA AGA G T GA C G G CT G C C 0 G CT T T C GA A A TGT G T AC A CA C G C T G G C G A C T GG T G ACC A C the invention.

G AT C T A G AT C T A G TT C A A G AT C T A CTA G TG C A C C A A G TT G A A C TT G A C T T A

AT

AT

TA

T A

AT

TA

AT

C G G C C G G C T G C AC G

TGT

AC A

TAC

AT G G C G C G C

GCG

CGC

AT C

TAG

GAT

CT A 3' C C A CCA G GT

CTG

GAC

AT C

TAG

TGT

AC A G GT C CA CT G

GAC

T G

AC

A C T G

AA

TT

A C T G

AA

T T

AA

TT

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.

L. I L- Y ili The gene I has the following characteristics.

B-Urogastrone can be expressed very advantageously by gene engineering techniques.

The trinucleotide codond 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.

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.

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.

In expressing 0-urogastrone as a fused protein, means is available by which an unnecessary portion can be easily removed to obtain the desired B-urogastrone.

When P-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 6sequence"), 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, start 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 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 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.

Gene II: Start codon 1 JA A T TC G A AIG A T C T G C AT G A A T AG C 3' GC TT TTC AG ACG TAC TTA T CG E Ta Bg(S) Mb 20 GA 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 AIG A C T C AC G GG T G A C A G A GT G Hf GAT G G C TAT T GT CT G C A C G A C G GT CTA CC G ATA A CA G A C GT G CT G CCA -7 70 G TT TOG CA TG T AC ATIC OA'A GC.T TTG C AA ACOG TAGC A TG T AG CITT C GAl AA C Ta Hcl G AT A AA TAG GC TOT AA C C TA T TT ATG AG AGA TT G Ml (Th) 100 TOGT GT A A CA C AT 110 0 T G 0 0 T CGAGCC GA 120 TAT ATCGGOT OAA A TA TAO G CCA CGTT CGOG T GT GGCG A CA 150 TOO TOGG A CC AGCC 130 G A A OT T 140 TAG CGOTI0T GTG AAA ATO GG CA G TA GAGC TT T

S

GLAA T TOG C T T A A 7

C

E

Stop codon 160 37 COGT TA TA G TOGA G CA A TT A TC A CT AIGA T CT TGCT A-G]A Bg(S) C C T Ba The symbols representing the restriction enzymes in the above sequence stand for the following.

E :EcoRI, Bg: BelII, Mb- tMboII, Ba: BainHI, Ml: MlIIi, Ta: TaqI S 3cu3AI Hf: Ilif I Hd: HinduI Th: ThaI 8 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 0-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 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 II 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 L 9 (HindIII) recognition site at its front end. These subunits are shown below.

Subunit A: AAT T C G A AG AT C T G C AT G AAT 3' G C TT C T A G AC G T A C TT A

GAT

CT A

GAT

CT A GT T C A A C T

GA

G C C G G C C G

GAG

CTC

TAT

AT A AT G TA C T G C

TGC

A C G T G T AC A

TAC

ATG

C C A CCA G G T CT G

GAC

AT C

TAG

C T G G A C

GAC

GT G G A A C T T T CT AG A G A C GAC CT G G C T C GA A G C TC G CA C GT G G T C C A CCA TC G AG C CTA G Subunit B i-- i A G CT T TG GAT AAA TAC G C G T GT 3' A A C C T A T T T A T G C G C A C A AAC T G T G T A G T G G GT T A T ATC G TT G ACA CAT CAC CCA ATA TAG C GAA C G C T GT CAA TAGC GT G AT C CTT GC G ACA GTT AT G G CA CTA G AAA T G G TGG GAA TT G C GT TAA T TTT A C C A C C CTT A A C G C A AT T A T GA A G A T C T G 3' ACT TCT AGA CCT AG The subunits A and B are synthesized, for example, in the following manner. Oligonucleotides having 11, 13 or 15 bases are synthesized (A-1 to A-16, and B-1 to B-16, i.e. 32 oligonucleotides). Next, 4 to 6 of these oligonucleotides are assembled and ligated into blocks (block 1 to block 7, i.e. 7 blocks).

These oligonucleotides and blocks are shown below.

G T C A T G A C

AG

T C L Block 1:

AA

TT

GAGAT~ C

T

GTAAG GTTGGG3 (A-16) (A-15) (A-14) Block 2: (A-6) GCCACTGTCTCAC GATGGCTATTG TCTQCACGACGGT 31 3' CAGAGTGCTAC CCATAACAGACGT GCTGCCACAAA (A-13) (A-12) (A-1l) Block 3: (A-8) GTTTCCATGTA CATGGAAGCTTLJCG 31 3' CGTACATGTAGCT TCGAAGCCTAG (A-9) Block 4; (B-2) AGGTTTGGATA AATACGCGTGTAAICT 3' 31 AAGCTATTTATGC GCACATTGACACA (B-16) Block (B-4) 5' GTGTAGTGGGT TATATCGGT.GAACGC 3' 31 TCACCGAATATAG CCACTTGCGACAG (B-14) (B-13) Block 6: (B-6) TGTCAATACCG TGATCTGAAATGGTG 3' 3' TTATGGCAGTAGA CTTTACCAGGCTT (B-12) (B-1l) Block 7; GGAATTGCGTT AATAGTO.AGATCTG 3' 3f AACGCAATTATGA C]TTCTAGACOThG (B-9) i S- 11

I

Next, the blocks 1 vo 3 are ligated together jinto 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.

Fig. 1 schematically shows the synthesis of an oligonucleotide by the solid phase process; Fig. 2 shows a process for ligating oligonucleotides 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 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 preparlog a recombinant plasmid pUG3 from pUGI and pUG2; Fig. 5 shows the result obtained by analyzing the nucleotide sequence of oligonucleotide A-3 by twodimensional fractionation by electrophoresis and homochromatography; Fig. 6 shows a process for preparing a recombinant plasmid pGH37; Fig. 7 shows a process for preparing a recombinant plasmid L -12 Fig. 8 shows a process for preparing a recombinant plasmid pEK28; Fig. 9 shows processes for preparing recombinant plasmids pUG102 to pUG122 and recombinant plasmids pUG103-E and pUG117o-E; Fig. 10 shows processes for preparing recombinant plasmids pBRH02 and pBRHO3; Fig. 11 shows MboII restriction map of pUG3 including H fragment (179 which contains the present B-urogastrone gene; Fig. 12 shows a process for preparing recombinant plasmids pUG2301 to pUG2303; Fig. 13 shows a process for preparing recombinant plasmids pCG2101 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 1 L.i i.

~i r 13 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, 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 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 nuc.leoside having its hydroxyl group free and Amino group protected, using succinic acid as a spacer.

On the other 'iand, various processes are known for preparing mononucleotides or dinucleotides (see, for example, C. Broka et al., Nucleic Acids Research, 8, 14 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 B-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 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 oligonucleotide 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 l to the resin to remove the dimethoxytrityl group at the 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- 2+ formamide to remove the remaining Zn 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 (MSNT, condensation reagent) in pyridine. The resulting solution is added to the dried resin and allowed to stand i 20 at room temperature. The liquid portion of the reaction I mixture is removed, and the resin portion is washed with pyridine and then reacted with acetic anhydride using dimethylaminopyridine as catalyst in tetrahydrofuranpyridine to mask the unreacted hydroxyl group. Finally the resin is washed with pyridine to complete one

I---I

-16 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 R 17 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-dimensional fractionating method for checking the nucleotide sequence can be carried out by the procedure of Wu et al. 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 concentration of about 0.1 vg/pl. A portion of this solution is treated with y- P-ATP and T 4 polynucleotidekinase to 32 label the 5' end with P and then partially digested with snake venom phosphodiesterase. The product is o 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 transfered onto a diethylaminoethyl cellulose (DEAE cellulose) plate and subjected to the second dimensional development using a solutio of partially hydrolysed RNA called a I-omomixture.

i I-L'~YUI 18 (This procedure is termed homochromatography). In this way, the oligonucleotide is separated according to the chain length. Subsequently, the nucleotide sequence of the oligcnucleotide 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. 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 -19catalyze B-elimination of both phosphates from the sugar, and finally the DNA strand is cleaved at that base.

ThJe resulting reaction mixtures are subjected to polyacrylamide 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 T DNA ligase as shown in Fig. 2. For the correct ligation, the 16 oligonucleotides A-1 to A-16 corresponding to the subunit A are ligated as divided into three sets, i.e. the block 1 comprising A-1, A-2, A-3, A-14, A-15 and A-16, the block 2 comprising A-4, A-6, A-11, 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 co 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-i to A-16 are labeled with 32P with use of y- 32 P-ATP and T 4 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 T 4 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 T 4 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 Fig. 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 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 io obtain a recombinant plasmid pUG2 as seen in Fig. 3.

Further as shown in Fig. 4, pUGI is cleaved with restriction enzymes HindII and Sail, 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 p-urogastrone structural gene (gene II).

pUGl, pUG2 and pUG3 are recombinant plasmids which each comprise pBR322 and the subunit A which is -21 the front half portion of the P-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 Lederberg and S. Cohen, J. Bacteriol., 119, 1072 (1974) Whether pUJGl, 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, pUGl and pUG2 are checked for the presence of the BglII recognition site which is not present on the vector pBR322. Similarly, pUG2 and pUG3 are checked whether they can be cleaved with MluI 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 extrachromosomal DNA, rectores the initial double stranded form.

I, Lc-- 22- 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 quantity.

In this way, the P-urogastrone gene of the invention (gene II) can be obtained.

Next, the method of introducing the purogastrone 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.

S 15 The modes of expressing the 8-urogastrone gene with use of E.coli includes a system for directly expressing B-urogastrone, and a system wherein it is expressed as a fused protein with B-lactamase or other different protein.

For the direct expression of B-urogastrone gene, it is required to introduce into the recombinant plasmid, upstream of the P-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 XPL which is the left ward promotor 23 of X phage, lac UV5 which is present upstream of B-galactosidase gene of E.coli, etc. When XPL 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 occures downstream of the lac UV5 promotor or the one chemically synthesized.

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

Although XPL is a powerful promotor Hedgpeth et al., Molecular and General Genetics, 163, 197-203 (1978)), the fully activated APL 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 XPL to function.

On the other hand, CI857 hich is a gene within A phage is one of the mutated getnas of CI repressor which acts on the operator for XP At low temperatures (of up to about 30°C), the 01857 repressor binds to the operator to completely inhibit the activity of APL 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 1 i j II i_ LL-L--

~L.L

IY ii 24 (of not lower than 37°C), whereby the XPL is allowed to function. Furthermore, the plasmid vectors, such as pSCIO1 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 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 P-urogastrone under the control of the XPL promotor.

According to the present invention, the XFP.-SD sequence-B-urogastrone gene is introduced, for example, into pBR322 to obtain a B-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 XPL promotor on the second plasmid when the cell is cultured for example at -3-i r i 25 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 C1857 repressor is dissociated from the operator, permitting the activity of XP L promotor for the expression of B-urogastrone.

Although a similar concept was applied to the expression of fibroblast interferon, SV-40 Small t antigen, etc., in these cases a X lysogen is used as a host in which the DNA of X phage carrying a C1857 gene is introduced into the host chromosome Derynck et al, Nature, 287, 193-197 (1980), 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 X 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 for the first time for systems for expressing 0-urogastrone.

According to another system, a portion of any other protein gene such as a-lactamase gene is ligated to d i 26 the B-urogastrone gene to express the B-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 g-urogastrone. Another advantage is that the fused protein migrates to and accumulates in the periplasm in the cell of E.coli J. Chan et al, Proc.

Natl. Acad. Sci., 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 B-urogastrone from the fused protein by cleaving with an enzyme is inserted into the P-lactamase gene at a suitable restriction enzyme cleaving site, and a 0-urogastrone gene is ligated to the 0-lactamase gene.

Preferably the sequence of two basic amino acids is -Lys-Arg- or -Arg-Lys-. Examples of enzymes K<o for recognizing the amino acid sequence to cleave 0-urogastrone from the fused protein are kallikrein, trypsin, etc. Examples of restriction enzymes for cleaving the B-lactamase gene are XmnI, HincII, Scal, Pvul, PstI, BglI, BanI, etc< The B-lactamase-P-urogastrone recombinant plasmid thus prepared can express a fused protein within 27- E.coli for quantity production. The resulting fused protein is treated with kallikrein or the like, whereby 0-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 C. Birnboim et al., Nucleic Acids Research, 7, 1513-1523 (1979)), or by radioimmunoassey for B-urogastrone.

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

i 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 (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

M

28 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 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 ImM 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 -29 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 8-cyanoethanol and 4.8 g 0 0 of 1-methylimidazole were admixed with the concentrate 0 by stirring at room temperature for 3 hours. The reaction mixture was corcentrated under reduced pressure.

S 15 The residue was dissolved in ethyl acetate, washed with 0.1M 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 a obtain 15.12 g of completely protected adenosine mononucleotide.

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-methazol (97:3 in volume ratio) to obtain 4.31 g of adenosine mononucleotide having a free hydroxyl group.

Thymidine (1.64 g) having its 5' hydroxy group protected with a dimethoxytrityl group and 0.95 g of S°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-chlorophenylphoaphorodichloridate was added dropwise to the mixture over a period of 5 minutes with stir.ing and ice cooling. The mixture was thereafter stirred at room temperature for 1 hour. The triethylamine S. hydrochloride resulting from the reaction was filtered off, and the filtrate was stirred for 10 minutes with F 1.1 ml of an aqueous solution of pyridine To the solution were added a dioxan,, solution (10 ml) of 1.17 g of the adenosine mononucleotide having the free hydroxyl group and prepared as above and 0.72 ml of 1-methylimidazole, and the mixture was stirred at room LL r II~- 31 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 (O.1M)and 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 dinucleotide TA.

In the same method as above various nucleotrides were prepared.

3) Synthesis of 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 dichloromethane-isopropanol (85:15 in volume ratio) three times, and then treated with a solution of zinc bromide (1M) in dichloromethane-isopropanol to remove the dimethoxytrityl group at the 5' position. This procedure was repeated several times until the color of the solution disappeared.

The resin was washed with dchloromethane, then washed with a solution of triethylammonium acetate (0.5M) in dimethylformamide to remove the remaining Zn 2 further II_~ _I II 32 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 was dissolved in 1 ml of pyridine, shaken with 1 ml of triethylamine 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- (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 dimethylaminopyridine (0,1M) 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, -33 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 tetramethylguaaidine-2-pyridinealdoximate (0.5M) in pyridinewater (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 pyricine and i ethanol alternately. The washings and the filtrate were i 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, 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 Wacers Associates, to obtain a purified fraction having a single peak. For the high-speed liquid chromatography, the column used was k-Bondtpak C18 (product of Waters Associates, and acetonitrileaqueous solution of triethylammonium acetate (0.1M) was used as an sluent for gradient elution (5 4 40 vol.%).

34 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 oligonucleotides 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 oligonucleotide 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.

35 Table 1.

A-i, 8Opg A-2, lL Opg A-3, 90i'g A-4, 140p~g A-6, 70PCY A-7, 80pig A-8, A-13, 50p~g A-14, 4OPg A-15, 60pg A-16, B-i, 60pg B-2, 100jig B-3 Y 50pg B-4, i00pg B-6, 90p4g B-7, 130pg 1B-8, lO0pg B-9, ilOvig B-10, i0pg B-il liOjag B-12, liOpg B-13, i30pg 13-14, 60vtg B-15, 70pg B-16, 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-i to B-16 were each found to have the contemplated nucleotide sequence. 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 sequence of each oligonucleotide was also checked by the 1ax-a-Gilbert method stated above.

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

L- -36 Construction of oligonucleotide blocks and subunits The blocks and subunits were prepared by the procedure shown in Fig. 2 as described in detail below.

First, about 5 pg of each of oligonucleotides A-l, A-2, A-3, A-14, A-15 and A-16 was dissolved in distilled water (50 pl) to obtain a solution having a concentration of about 0.1 pg/pl. The six kinds of aqueous solutions were placed, each in an amount of 10 pl (1 pg calculated as DNA), into other six Eppendorf tubes individually. A mixture solution (6 ul) containing 250mM tris-HCl (pH 50mM magnesium chloride, 10mM spermine and 50mM DTT was placed into each tube, followed by o addition of 0.5 ul of Y- 3 2 P-ATP aqueous solution (product of Amersham International Ltd., 0.5 1l of T 4 polynucleotidekinase (product of Takara Shuzo Co., Ltd., Japan) and 13 pl of distilled water, to obtain 30 pl of mixture. The mixture was reacted for 30 minutes at 37 0

C,

and further reacted for 30 minutes with addition of 1 Pu 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 pl, into another single 1.5 ml Eppendorf tube. Into the tube were placed 40 il of 250mM tris-HC1 aqueous solution 1 37 (pH 40 il of 50mM magnesium chloride and 35 il of distilled water to obtain a total amount of 175 pl.

The mixture was heated at 90°C for 2 minutes, then gradually cooled to room temperature. With addition of 10 pl of 200mM DTT aqueous solution, 10 p1 of 20mM ATP aqueous solution and 5 pl (100 units) of T 4 DNA ligase (product of Nippon Gene Co., Ltd., Japan), the mixture was reacted overnight at 4*C, giving the block 1 of ligated oligonucleotides A-1, 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 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 electrophor(,;sis 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 &nd bands at the positions of a. 48 b.p, and 24 b.p. for the block 3. Subsequently, each band was cat ott" and a mixture of 10mM tris-HCl (pH 7.6) and 10mM EDTA aqueous solution (tris-EDTA) was added thereto. The mixture was then allowed to stand overnight L I -ii 38 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 pl and thereafter allowed to stand at -80 C 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-HC1 (pH lOmM magnesium chloride, 20mM DTT, ImMt ATP and 5 pl (100 unit) of T 4 DNA ligase. The resulting mixture was allowed to stand overnight at 4 0 C for ligation. To the mixture was added ethanol in twic.

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-EOTA was added thereto, and the mixture was allowed to stand overnight at room temperature for extraction. The mixture 39was centrifuged to separate the supernatant, 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 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 Fig. 3 to ligate oligonucleotides B-1, B-2, 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 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 which was cleaved with HindllI and BamHl. 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 i_ -Y t- i 40 Takara Shuzo Co., Ltd., Japan) so as not to restore the original state. Subsequently pBR322 thus cleaved and dephosphorylated and, the subinit A were allowed to stand overnight at 4°C in a mixture of 50mM triA-HCl (pH 7.6), 10mM magnesium chloride, 20mM DTT and ImM ATP with addition of 5 4p of T 4 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 precipita,tion.

The mixture was then centrifuged, the precipitate was dried and dilisolved in 100 pl of distilled water, whereby a plasmid pUG1 was obtained in which the subvit A was incorporated in pBR322.

E.coli strain HB101 was transformed with the plasmid pUG1 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 Ssodium chloride). When the absorbanc- at 610 run 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. 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 L- 41was discarded. The precipitate was suspended in 20 ml of ice-cooled solution of 0.1M calcium chloride and i 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 0.1M calcium chloride and 0.05M magnesium chloride. To a 200 pl portion of the suspension was added 10 pl of aqueous solution of pUG1, and the mixture was ice-cooled for 1 hour and then heated in a water bath at 43.5 0 C for 30 seconds. Subsequently, 2.8 ml of LB culture medium was added to the mixture, followed by incubation at 37 0 C for 1 hour. The culture was then spread over a LB plate containing 50 pg/ml of ampicillin, in an amount of 200 pl/dish and incubated overnight at 37 0 C. The growing colonies were checked by further transplantation to a LB plate containing 50 pg/ml of ampicillin and also to a LB plate containing 20 pg/ml of tetracycline and were incubated overnight at 37 0

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 BglII cleavage site. One of the cells containing the plasmid having BglII cleavage site was cultured in a large scale to obtain purified plasmid 42 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 Sall fragment, anA 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 reactionu products for guanine adenine, lanes 3 and 7 sho 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 Fig. 3, the plasmid pBR322 was cleaved with HindIII and BamHI, and the larger I 20 fragment was isolated 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 pUG1. Using the resdlting plasmid pUG2, the strain HB101 was transformed, and the colonies resistant to ampicillin only were selected.

43 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. 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 Fig. 4, pUG1 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 T 4 DNA ligase for ligation, whereby plasmid pUG3 was obtained wherein the subunits A plus B, i.e. B-urogastrone gene, was incorporated into pBR322 E. coli strain HB101 was transformed using -44 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 Mlul 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 Burogastrone 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-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 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 P-trogastrone gene.

7) ExpressiLc~ vector incorporatingP L promotor The XPL promotor, left ward promotor of A phage, was used for expressing B-urogastrone as will be described in detail below.

45 First, preparation of a strain ECI-2 derived from E.coli strain HB101 will be described. ECI-2 served as a host for XPL expression plasmid. Then described will be the cloning of a DNA fragment containing XPL promotor from the DNA of XCI857S 7 which is a mutant of X phage (Sanger et al, J. Mol. Biol. 1982 162 729-733), and the construction of expression plasmids from the cloned DNA. Further described will be the expression of g-urogastrone gene in the host ECI-2 strain by XPL promotor.

7-1) Construction of strain ECI-2 The strain ECT-2 is E.coli HB101 harboring a o"0oo plasmid pGH37 for expression of CI857 gene.

pGH37 was prepared by the process shown in Fig.

o 6. First, DNA of XCI857S 7 was cleaved with BglII. Then, .o the cohesive ends at the cleavage site were digested using S1 nuclease. One g of DNA of ACI857S7 cleaved with BglII was react-d with 200 units of SI nuclease at 20 0 C for minutes in 100 pl of an aqueous solution (pH comprising 200 mM sodium chloride, 30 inM sodium acetate and mM zinc sulfate. The blunt-ended DNA fragments thus obtainrd were subjected to 1.0 wt.% agarose gel electrophoresis to isolate therefrom a fragment with 2385 b.p.

o having the whole CI857 structural gene. The fragment was inserted into the PvuII cleavage site of plasmids pGL101 (Thummel et al, J.Virol. 1981 37 683-697) to construct a plasmid pGH36 which expresses the CI857 -46- Hgene under the control of a promotor, lac Subsequently, pGH36 is cleaved with two restriction enzymes, EcoRI and PstI, to obtain a fragment having 1193 which was inserted into a plasmid pSC101 between EcoRI and PstI 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 strt,.ns 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 Trade and Industry, Japan, since June 22, 1984. This strain is resistant to tetracycline, expresses the CI857 gene and permit3 conjoint presence, through transformation, of other plasmids derived, for Sexample, from pBR322. Accordingly the strain ECI-2 was thereafter used as a host for XP L expression plasmids.

7-2) Cloning of XPL promotor and preparation of expression plasmids As shown in Fig. 7, pGH35 was constructed first.

DNA of CI857S7 was cleaved with EcoRI and Sail to obtain a fragment of 5925 b.p. which includes XPL promotor and CI857 gene as well as XPR promotor. The fragment was 47 inserted into plasmid pBR322 between EcoRI and Sail cleavage sites to construct a plasmid Next, pGH25 was cleaved with BamrI and ligated to pBR322 similarly cleaved with BamHI to obtain pGH34.

Subsequently, pGH34 was cleaved with Aval and BglII and thereafter treated with Sl nuclease into bluntended fragment of about 4500 b.p. The fragment was circularized with T DNA ligase to construct a plasmid Next, pEK28 was constructed as shown in Fig. 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 cleavirg pGH35 with Hpal. The assembly (C a-ScM~w~n M lC'3- i CIIce&aeA was further liga d to plasmid pMC1403 7Zclav&d with BamHI to obtain plasmic pEG2. The pEG2 has two ampicillinresistant genes, and expresses B-galactosidase gene derived from pMC1403 under the control of XPL promotor, utilizing a start codon as well as the SD sequence included in the adapter.

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

SD sequence Start codon Z' C-l-1: 5' A G G A A C A G A T C T X T G 3' C-l-2: 3' T C C T T G T C T A GA T A C C T A G SBglI

A

-48 Miller's method was employed to confirm the expression of B-galactosidase in the host ECI-2 harboring the plasmid pEG2 (Miller, J. (1972) "Experiments in Molecular Genetics" New York, Cold Spring Harbor Laboratory pp35 2 -355). This method is based on the reaction of B-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 B-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 30°C 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 P-galactosidase is defined by the units in 1 ml of the liquid culture according to the following equation, in which ,absorbance at 610 nm T.i- m h M ii. .11.in i" a 49 is calculated as Activity of B- OD 4 2 0 1.75 x OD 5 5 0 galactosidase x 0 x OD x 1000 (units) t x v x 610 t time of incubation (min) v amount of specimen added to the reaction system (0.1 ml)

OD

6 1 0 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 0 C, the g-galactosidase activity was 98 units. However, when the culture was further incubated at 42°C for 1 hour, the XPL promotor was activated to result in B-galactosidase activity of 9637 units. This substantiates that the sequence from the XPL promotor to P-galactosidase is in the contemplated order.

Although pEG2 has two BglII cleavage sites, only the Bgl!I site present immediately after the SD sequence of P-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 plasmid with use of XPL pr-' .tor.

7-3j Expression of fused gene of front half of Burogastrone and B-galactosidase Fig. 9 schematically shows the series of I. i i~W 50 procedures to be described below.

A plasmid pUG101 was constructed in the following manner which has a fused gene of the front half of -urogastrone and a B-galactosidase. More specifically, pUG1, which has the front half of B-urogastrone gene, and pMC1403 having a O-galactosidase gene were cleaved with BamHI and then ligated to form pUG101. With this plasmid, the front half of B-urogastrone gene and the p-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 XPL 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 TCTA ;However, when the DNA fragment is reacted with a large Ifragment 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, AGATC f dGTP only is added that makes the end ofAG) If dGTP only is added

AG

as a nucleotide component, the end of TCTAG is obtained owing to the termination of the reaction.

Next, when S1 nuelease is used to digest the remaining single strand, a blunt end is obtained in the form of 51 TC) Similarly if dGTP and dATP are added in the Klenow reaction, followed by digestion with S1 nuclease, AGA is obtained. When the Klenow reaction is ""Tc5 conducted by addition of dGTP, dATP and dTTP, followed by digestion with Sl nuclease,K TCTA is obtained.

Further if only the digestion with Sl nuclease is conducted without effecting the Klenow reaction, A' is obtained. Thus, when the DNA fragment with v A h the end of TCTAG is subjected to the Klenow

TCA

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 Sl nuclease reaction were conducted under the following conditions.

Klenow reaction: One pg of DNA as the substrate was reacted with ImM of each deoxyribonucleotide triphosphate at 12°C for 30 minutes in the presence of 1 unit of thr enzyme (klenow fragment) and lmM of ATP in 50 Pl of a reaction medium comprising 40mM of potassium phosphate buffer (pH ImM of 0-mercapto-ethanol and 10mM of magnesium chloride.

S1 nuclease reaction; -L ii i l ii:iii-~L--iii'l'~'-i I 52 One pg of DNA as the substrate and 200 units of the enzyme were reacted at 20'C for 30 minutes in 100 pl of a reaction medium comprising 200mM of sodium chloride, 30mM of sodium acetate and 5mM of zinc sulfate (pH pEK28 and pUG101 were each cleaved with BglII 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 S. sequence and the start codon of the fused protein, as listed in Table 2.

In actuality, the DNA fragments thus oEtained were cleaved with Sall to isolate fragments which contain the gene encoding fused prote-.n of -urogastrone front half and B-galactosidase from pUG101, and fragments containing XPL promotor from pEK28. These fragments were ligated by T 4 DNA ligase in the co mbinations shown in Table 2, Consequently, recombinant plasmids ble 3 were obtained. The B-galactosidase activity the expressed fused proteins was measured by Millet's method, Table 3 shows that all the plasmids expressed a relatively a high level of $-galactosidase activity. Especially pUG103, pUG104 and pUG117 achieved remarkable results.

j Y -e Table 2 Nucleotide sequence upstream of start codon (ATG) ATCTGC- GATCTGC 4 1 -ACA -ACAATCTGC- -ACAGATCTGC (PUG1O6) Nucleotide sequence downstream of SD sequence (AGGA) -ACAG -ACAGGATCTGC -ACAGA -ACAGAATCTGC -ACAGAGATGTGC (pUG114) -ACAGAT -ACAGATATCTGC- -ACAGATGATCTGC- (pUG118) -ACAGATC -ACAGATCATCTGC (PUGl21)

-ACAGATCGATCTGC-

(pUG122) 7- Table 2 (continued) 1 Nucle~otide sequen-a upstream of start codon (ATG) TGC CTGC- TCTGC-___ -ACA -AGATG"'- (pUG1CZ)-"

-ACACTGC-

(TnUG103) -ACATCTGC (pUG1O4) iiucleotide sequence downstream of SD sequence (AGGA) -AGAG -ACAGTGC- -ACAGCTGC- I-ACAGTCTGC- (pUG1O7) ___p108f jPUGlO9) -ACAGA -ACAGATGnC- -ACAGACTGC- (IpUG1l2) -ACAGAT -ACAGATTGC- ACAGATTCTGC I (pUG116) -ACAGATC -ACAGATCCTGC (-PUG119)

-ACLAGATCTCTGC-

55 Table 3 Number of nucleotides between SD sequence and start codon 6 7 7 8 8 9 9 11 11 11 12 12 13 13 14 Recombinant -Galactosidase plasinid pUG102 pUGi 03 pUG107 pUG104 pUGlOS pUG105 pUG109 pUG106 pUGilO pUG113 pUG119 pUG 114 pUG1 17 pUG120 pUG1 18 pUG121 pUG122 (units) 1674 2533 2260 2802 1835 1018 1942 973 1764 1950 1862 946 2332 1374 2041 1678 1814 a 56 Next, a vector for expression of B-urogastrone was prepared from pUG103 or pUG117. The plasmid (pUG103 or pUG117) was cleaved with HindIII and PvuII to obtain a fragment of 1.2 kb containing the region of from XPL promotor to the front half of B-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 w'.h T 4 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 P-urogastrone gene, i.e. the whole P-urogastrone gene, under the control of XPL promotor.

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

8-1) Donor of P-lactamase gene pBRH02 is obtained by cleaving pBR322 with Aval and PvuII, followed by the Klenow reaction and ligation by T 4 DNA ligase. This plasmid has genes for ampicillin resistance (Ap

R

and tetracycline resistance

R

(Tc as markers. pBRH03 is obtained by cleaving pBR325 with Aval and HindIII, followed by the Klenow reaction t 57 and ligation and has Ap R and chloramphenicol resistance (Cm

R

as markers. Fig. 10 shows these plasmids.

8-2) Donor of B-urogastrone gene pUG3 prepared as already described was 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 B-urogastrone and ending with 16 bases downstream of the stop codon, the fragment having the whole structural gene of B-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 B-urogastrone from the expressed fused protein.

.58 Table 4 -Adaptor 5' end 3' end 00 D-1-3 D-1-4 D-2-1 D-2-2 D-3-2 E- 1 E-3 E -4 E-6 E- 7 E-8 E-9 CCGT AAG

TTACGG

CGTAAG

TTACG

TTACGGAT

TTACGTGCA

CGCTAAACCG

CGTTTAGCG

GACAAACGG

CGTTTGTC

COTTTAGCGAT

CGTTTGTCTGGA

CGGCTAAACGG

CGTTTAGCCG

CAAACGG

CGTTT G 0 (-0 I~ 59 8-4) System for expression of fused protein of B-lactamase and B-urogastrone linked by Lys-Arg A vector for expression of B-lactamase-0urogastrone 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 37 0 C over a period of 3 hours. Subsequently 3 pg of the vector, about 0.1 pg of B-urogastrone fragment of 179 b.p. and about 1 pg of each of E-l and E-2 (with nonphosphorylated 5' end) serving as an adaptor were ligated in a single step at 12 0 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 Tc R colonies obtained, 168 colonies were Ap

S

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 P-urogastrone gene inserted therein.

All of them, which had a Mlul site, were cleaved with Hinfl and checked for the orientation of insertion of the B-urogastrone gene by 1.5 wt.% agarose gel electrophoresis.

60 Three of those checked gave fragments of about 1050 b.p.

and about 800 b.p. This indicated that the 0-urogastrone gene was inserted in the same orientation as B-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 Pvul site in the B-lactamase gene.

According to the process described in an expression vector was constructed using E-l and E-5 for an adaptor.

When the 1626 TcR colonies obtained were checked for Ap sensitivity, 31 colonies were Ap

S

Minipreparation was conducted for 22 TcR and Ap S colonies, and the plasmids were checked for the insertion of B-urogastrone gene by cleavage with Mlul. Twenty plasmids were found to have an Mlul site. The orientation was checked by cleaving with Hinfl or BamHI. PLasmids pUG2101 to pUG2105 were foun,. to have a B-urogastrone gene inserted therein in the same orientation as the P-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.

I_ r, i i- -C I- U-l 'i IIIU-LI-.

61 Of the 217 TcR colonies obtained, 106 colonies were Ap

S

Mini-preparation was conducted for of these colonies. Eight of the plasmids were about 200 b.p. larger than the vector and appeared to have a P-urogastrone gene inserted therein, so that these eight plasmids were cleaved with BamHI and checked for the orientation of the gene. Consequently, three plasmids were found to have the P-urogastrone gene inserted in the same orientation as B-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 P-lactamase and P-urogastrone. Predicted amino acid sequence and the corresponding nucleotide sequence are shown below.

62 Met Ser I le Gin His Phe Arg Val ATG AGT ATT CAA CAT TTC CGT GTC Ala Leu I le Pro Phe Phe Ala Ala GCC COTT ATT COCC TTT TTT GCG GOA Phe Cys Leu Pro Val Phe Ala His TTT TGC OTT OCT GTT TTT GOT CAC Pro Gli Thr Leu Val Lys Val Lys CCA GAA ACG OTG GTG AAA GTA AAA Asp Ala Glu Asp Gin Leu GIY Ala GAT GOT GAA GAT CAG TTG GGT GCA Arg Val GIY Tyr I le Glu Leu Asp CGA GTG GGT TAO ATC GAA CTG GAT Leu Asn Ser Gly Lys I e Lou GuI OTO AAC AGO GGT AAG ATO COTT GAG Ser Phe A rg PF ro Glu GIu A rg Ala AGT TTT OGO 000 GAA GAA OGO GOT Lys Arg Asn Ser Asp Ser Glu Cys AAA CGG AAT AGO GAT TOT GAG TGC Pro Lou Ser 1-1is Asp Gly TYr Oys CCA GTG TOT CAO GAT GGC TAT TGT Leou Hi is Asp Gly Val Cys Met Tyr CTG CAG GAC G GT GTT TGO ATG T A C I L I le

ATO

Cys

TGT

Gvl

GGT

L eu

CG

Glu

GAA

Asn

AAO

Glu

GAA

L ys

AAA

Ala

GOT

CYS

TGT

Arg

OGC

T rp

TGG

63 L eu

TTG

Val

GTA

CYS

TGT

TrP

TGG

GAT

Val GT G G In

CAA

G W

GAA

L

AAA

Giy

GGT

Tyr

TAO

L eti T T G Tyr

TAO

Tyr

TAT

A rg

OGT

A rg

OGT

Ala

GOG

Ile

ATO

Asp

GAT

stop)

TAA

TAGTGAA

ATGATGA

QAT OTGQAT 0 GTTTAGOGTTTT CCA G CA OTTTTAAAGTT OTG CTATO T-GG C G OGGTATI'ATC00 GTGTT'GA 0GO OGGGOAAGAG

OAACTCOGTOGOOGOATAC

Similarly, the plastuid pUG2l01 obtained by the procedure 8-4-b) Droduces a fused protein having the following primary structu7.e, met

ATG

Ala

GC

Phe

ITTT

Ser

AGQT

L eu

OTT

Cys

TGO

Ilie

ATT

I Ie AlT L eu

OTT

G In

CAA

Pro 000 P ro

OCT

HIS

OAT

Phe

TTT

ValI

GTT

Phe

ITO

P he

ITTT

Phe

TTT

Arg 0 GT A la

GOG

Ala

GOCT

Val

GTO

Ala

GOA

H is

CG

64 P ro

OGA

ASP

GAT

A rg

OGA

L eU

OTO

Ser

AGT

Pro

OCA

L eu

OTG

A rg O GT L eu

OTO

G In

GAG

pro

OCA

Glu

GAA

Ala

GCT

Val

QOTG

Asn

AAC

Phe

TTT

Met

ATO

L eu

OTA

Val

OTT

Gly As~

AAT

GTO

Thr

AG

Glu

GAA

Gly

GT

8cr

AGO

A rg

OGO

Met

ATO

Gys

TGT

Asp

GAO

A rg

OGO

*Asp

GAO

Thr

ACA

L eu

OTO

Asp

GAT

T yr

TAO

Gly

GOQT

P ro 000 8cr

AGO

GIY

GGG,

Ala

GCO

Arg

GO

Ile TT G GI GA A Val

GTG

G In

CG

Ile

ATO

LYvs

AAG

Oiu

OAA

Thr

ACT

AlIa

GOG

Giv

GGG

IT e

ATA

Val

OT

LYvS AA G Lyvs

AAA

L eti

ITOF

Giu

GAA

Ile

ATO

Giu

OAA

Phe TT T Val

GTA

G In

CAA

Hi1s

GAO

GI u G AG His

OAT

Val

GTA

GI y

GOT

L eu

COTO-

L eu O TT A rg

CGT

L YS

AAA

L eu

TTA

G ILI G AG Tyr

TAT

Tyr

TAO

L et

OTT

Lyvs

AAA

Alia

GA

Asp OA T Ohl

GAG

P he TT T V/alI

OTT

S ep

TO

G In

CAA

Ser

TOT

8cr I

TOA

Thr AO0G Asp G ly Met Thr Val A rg G lu L Eu GAT GGC ATG ACA GTA AGA GAA TTA Cys Ser Ala Ala Ile Thr Met 8cr TGO AGT GOT GOC AlA O ATG AGT Asp Asn T hr Ala Ala Asn L et L eu GAT AAO ACT GOG GOC AAO TTA OTT L eu Thr Thr I I e Ala LYS Arg A sn OTO ACA ACG A TC GOT AAA NIGG AAT 8cr Asp S er C-1I u Qys Pro L eu 8cr AGO GAT TOT GAG TGC OCA OTG TOT His Asp GWY Tyr Cys Loeu H is Asp GAO GAl GGC TAT TGT OTG CAC GAO GIy Val Cys Met Tyr Iloe G lu AlIa GGT GTT IGO AIG TAO ATO GAA GOT Leu Asp Kvs Tyr Ala CYS Asn ys TTG GA' 'A A TAO GOG TOT AAO TOT Vai Val Qly Tyr Ile Oiv G u Arg GIA GTG GOT TAT ATO GGT QAA 000 Cys Gin Tyr A rg Asp Loeu LYs Trp TGT CAA TAO 001 GAT O T G AAA TG Trp G Wu Loll Arg stop) TO O AA TT G 001 TAA TAGTGAAOATO 66 TGGATCOGTTTAGCGATOGu-GAGGAOOGAAGG AG OTAA C CGCOTTTTTG CACA Similarly, the plasmid pUG27Ol obtained by the procedure 8-4-c) produces a fused protein having the following primary structure.

Met

ATG

A la aGO Phe

TTT

P ro

OCA

ASP

GAT

Ar'9

CGA

L eu

OTO

Ser AG T Ser

AGT

L eu

OTT

CYS

TGC

Gilu

GAA

Ala

GOT

Val

GTG

Asn

AAC

Phe

TTT

Ile AlT I le AT T L eu.

OTT

Thr

AOG

Giu QA A GlY

GGT

Ser

AGO

Ang

CGG

G In

CAA

Pro

CCC

P ro

COCT

L eu

CTG

Asp

GAT

Tyr

TAO

GIy

GGT

Pro

COO

1- is

CAT

Phle

T-T

Val

GTT

Val

GTG

G In

GAG

Ile

ATO

L ys

AAG

G lu

GAA

P he

TTC

Phe T TT Phe

TTT

Lyvs

AAA

L eu TT G G!u G AA Ilie

ATO

Gilu

GAA

A rg C C,,T Ala GaCG Ala

GOT

Val

GTA

Gly

GGT

L eu CT G L eu

OTT

Arg O 0,T Vai GT C Ala

GCA

H i S

CAC

Lyvs

AAA

Ala

GCA

Asp

GAT

Giu G A G Phle

TTT

67 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 TCO Arg Val Asp Ala Gly Gin G1( Gin CGT GTT GAC GCC GGG CAA GAG CAA Leu Giy Arg Arg I le His Tyr Ser CTC GGT CGC CGC ATA CAC TAT TCT G In Asn Asp I le Vat Glu Ser Ala CAG AAT GAC TTG GTT GAG CG GOT Lys Arg Asn Ser Asp Ser Glu Cys AAA OGG AAT AGC GAT TCT GAG TGC Pro Leu Ser His Asp GIY Tyr Cys CCA CTG TCT GAC GAT GGC TAT TGT Leu H is Asp Gly Val Cys Met Tyr CTG CAC GAC GGT GTT TGC ATG TAOC I le Glu Ala Leu Asp Lys Tyr Ala ATC GAA GCT TTG GAT AAA TAC GCG Cys Asn Cys Val Val Gly Tyr I le TGT AAC TGT GTA GTG GGT TAT ATC Gly GIu A rg Cys Gin Tyr A r Asp GGT GAA CGC TGT CAA TAO CGT GAT L -68 Leu Lys Trp Trp G lu Leu Arg (stop) CTG AAA TGG TGG GAA TTG CGT TAA

TAGTGAAGATCTGGATCCGTTTAGCCGAC-CAC

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 MluI-PstI fragment (224 of pUG2101, lanes 5 to 8 show the result with the MluI- EcoRI fragment (721 of pUG2101, lanes 9 to 12 show that with the MluI-BamHI fragment (452 of pUG2301, lanes 13 to 16 is that with the MluI-PstI fragment (335 of pUG2701, and lane 17 to 20 show that with Stne MluI-EcoRI fragment (610 of pUG2701. Lanes 1, j| 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, lares 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.

69 System for expression of fused protein of B-lactamase and B-urogastrone linked by Arg-Lys Preparation of pUG1102 and pUG1105 B-Urogastrone gene was inserted into the Blactamase gene of pBR322 at its unique Pvul restriction site to obtain vectors for expression of fused protein of 0-lactamase and B-urogastrone as shown in Fig. The plasmid pBR322 was cleaved with Pvul 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 electrophoresis to isolate a DNA fragment. Subsequently, SB-rogastrone fragment and vector were mixed together i in a molar ratio of approximately 5:1 and ligated at S12°C over a period of 15 hours. After the ligation, I the strain HB101 was transformed with the resulting 20 plasmid, and the colonies were selected with reference to Tc Seventy-one Tc R transformed colonies were obtained and then checked for Ap sensitivity. Plasmid DNA was prepared from 20 Ap S colonies and then checked for the presence of Mlul restriction site to ~iyl-i-i 1L I-ii-~il i. confirm the insertion of B-urogastrone gene. Five of the 20 plasmids were found to have the Mlul site of B-urogastrone gene. The DNA was cleaved with Hinfl and then subjected to 1.5 wt.% agarose gel electrophoresis 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.

Preparation of pUG1004, pUCl201 and pUG1301 The procedure 8-5-a) was repeated using PstI, HincIl and XmnI in place of Pvul to obtain pUG1004, pUG1201 and pUG1301 as shown in Figs. 16, 17 and 18, respectively.

9) Confirmation of expression of B-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 SBP-urogastrone gene and extraction of proteins 9-1-a) Expression system using XPL promotor iThe strain ECI-2 harboring expression plasmid pUG103-E and the same strain harboring expression plasmid pUG117-E were each cultured at 25 0 C in two flasks each containing 1 liter of LB culture medium. When the culture S' 25 in one of thz flasks exhibited an absorbance of 0.3 at 71 660 run, the culture was subjected to heat induction at 42°C for 1 hour. The culture in the other flask was continuously incubated at 25 0 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 sodium phosphate, monobasic (pH 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 37°C in a culture medium containing 50 pg/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, a- 72 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 0.01N 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., 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 0 C until the absorbance at 660 nm became 0.4. The cells were collected (6000 r.p.m., minutes) and washed twice with a mixture of tris-HCl (pH 8.0) and 30mM sodium chloride. The cells S(1 g) were resuspended again in 80 ml of 20 wt.% sucrosetris-HCl (pH whereupon EDTA was added to the suspension to a concentration of ImM. The mixture was shaken by a rotary shaker at 180 r.p.m. for 10 minutes S 25 (24 0 C) and then centrifuged (13000 g, 10 minutes) to i _1 73 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 occasional stirring and then centrifuged (13000 g, 10 minutes). The supernatant was collected as a periplasmic fraction (0-Sup).

The pellet was suspended in a mixture of 10mM 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.

9-2) Radioimmunoassay 9-2-a) Establishment of RIA system Rabbits were immunized with purified human B-urogastrone as an antigen to obtain antiserum. The 8urogastrone (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 ml) was added to the mixture to obtain an emulsion, 1 which was subcutaneously injected into the chest portion o 20 of three rabbits. After repeating the immunization four times every two weeks, 50 pg 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 for determining -74 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 (10mM, pH 7.4) containing 0.5 wt.% of bovine serun albumin (BSA), 140mM of sodium chloride and 25mM of disodium EDTA. The diluting solution (400 PI), 100 pl of the sample or standard human P-urogastrone and 100 pl of antihuman B-urogastrone serum were mixed together.

After the mixture was incubated for 24 hours at 4''C, 125 100 PI of I-labeled human P-urogastrone solution (about 5000 cpm) was added to the mixture. After the mixture was further incubated for 48 hours at 4UC, 100-pl of second antibody (anti-rabbit y-globulin goat serum) (20-fold dilution with PBS buffer), 100 pl of normal rabbit serum (200-fold dilution with PBS buffer) and '00 pl of 10nmM PBS buffer containing 5 wt.% polyethylene glycol were added to the resulting mixture, and thei further incubated for 3 hours at 4 0 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 B-urogastrone in the sample was determined from the standard curve obtained with use of standard human 75 9-2-b) Confirmation of P-urogastrone productivity of recombinant microorganism Table 5 shows the result of RIA conducted for the expression system with use of XP promotor.

Table Expres sion plasmid pLIGlO3-E pUGl03-E pUG1l7-E pUG117-E Control (pBR322) Heat induction Yes No Yes Amo'int of urogastrone produced (ng/l culture) 450.2 3.2 388. 4 3.2 Not detectable Table 6 shows the result of RIA conducted for fused protein expression systems.

Table 6 Expression plasmid pUG 1004 pUGl301 ptJG210l pUG2 301 DUG2701 Amount of urogastrone produced (lag/l culture) 729 .6 650 .7 31.3 125.2 119.2 76 Table 7 shows the localization of the expressed fused protein.

Table 7 Amount of 0-urogastrone produced Expression (pg/l culture) plasmid Periplasmic fraction Cytoplasmic fraction (O-Sup) (0-Ppt) pUG1004 326.0 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 XPL promotor system for direct expression of B-urogastrone and the system for expression of the compound as a fused protein both expressed B-urogastrone immunoreactivity in E.coli.

Table 7 reveals that in the case of fused protein, the expressed B-urogastrone immunoreactivity is almost localized in the periplasm.

Claims (23)

1. A novel 0-urogastrone gene having the following nucleotide sequence: AAT A G 0 G AT T C T G A G T G C C C A C T G 3' TTA TC G CT A A G A CTC AC G G GT G AC TCT CAC GAT G G C TAT T GT CTG C AC AGA GT G CTA C CG A T A A C A GAC GT G GAC G GT GT T T G C AT G TA C ATC GAA CTG C C A C A A AC G T AC AT G TAG C T T G CT T T G AT AAA T AC G C G TGT AAC CG A A A C CT A T T T AT G C G C AC A T T TGT G T A GT G G GT TAT A T C GGT GAA ACA CAT C A C CCA ATA TAG C A TT C G C T G T CA A T A C GT GAT C T G A AA G G A C A GTT AT G G CA C T A G A C TTT S T G G T G G GA A T T G C GT 3' A C C A C C T T A A C G C A

2, A subunit of the gene defined in claim 1, the subunit having the front half of the uciJbctide 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.

A gene as defined in claim 4 which has a 78 restriction enzyme recognition site and a start codon provided upstreatm of the gene and/or a stop codon and a restriction enzyme recognition site provided downstreatm of the gene, the codons and the recognition sites being arranged in the order mentioned.

6. A gene as defined nucleotide sequence: in claim 5 which has the following AAT G AT CTA T C G AAG G C TTC T CT GAG AGA CTC G G C TAT C CG ATA AT C TAG 20 T G C A C G T GT ACA T A -1, G C C G 1 AT G TA C AAT TTA T C T AG A A G C T C G C AC GT G C C A CTG G G T G A CT G CA C GAC GT G A T T A T T A A 70 TAC AT C A T G TAG GAC G CTG C C T G C A ACG T T G A C A C T G GAA G CT CTT CGA 100 AAC T GT TTG ACA T T A A 4 e i; GAT AAA C T A TTT 110 G T G G GT CAC CCA G C G T C G C A G T CA G T A CTA CAT 120 GT G AA CA C T T 130 TAT A T C AT A TAG 140 C AA T A CGT GTT TATG G CA 160 GAA TTG C GT C TT A C G CA G A T CT G A A A C T A GA TTT 170 T AA TAG T GA ATT AT C ACT C G C T GT G C G ACA 150 TGG T G G AC C A C C AGA T C T TC T AGA G 3' CCT AG L r 79

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 follwing nucleotide sequence: A A T TC G A A G A T C T G C AT G A A T AG C 3' G C T T C T A G AC G T A C T T A TC G GAT TCT GAG T G C CCA CT G T CT CAC CTA AGA CTC ACG GGT GAC AGA GTG 0 GAT G G C TAT T GT CTG CAC GAC GGT CT A CCG ATA ACA GAC GT G CTG G C A GTT T G C ATG TAC ATC GAA G CT TC G CAA AC G TAC ATG TAG CTT C GA AG C 3' C T A G

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.

A subunit as defined in claim 9 having the following nucleotLde sequence: A Gr T T G A T A A A T AC G C G T G T AA C CT A TTT AT G C G C A CA AAC TGT GTA GTG G GT TAT ATC GGT T G A C A C AT C AC CCA ATA TAG C C A i., 80 GAA CG GC TGT CAA TAC CGT GAT CTG CTT G C G ACA GTT ATG G CA CTA GAC AAA T GG TG G GAA TTG C GT T AA TAG TTT ACC ACC CTT AAC G C A ATT ATC T GA A G A T C T G 3' ACT TCT AGA CCT AG

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 000000 plasmid vector. a

*13, A recombinant plasmid comprising a plasmid vector having inserted therein the B-urogastrone gene defined in claim 6, the plasmid vector further having inserted therein upstream of the p-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 8-urogastrone gene defined in claim 6, the plasmid vector having the combination of a promotor, an SD sequence and a 8-lactamase gene inserted therein upstream of the 0-urogastrone gene, so that 8-urogastrone is expressed as a fused protein with the B-lactamase.

A recombinant plasmid as defined in claim 13 >or 14 wherein the promotor is XPL or lac \w t t 81

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

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

18. A transformant as defined in claim 17 wherein the recombinant plasmid is the one defined in claim 13.

19, A transformant as defined in claim 17 wherein the recombinant plasmid is the one defined in claim 14. 0

20. A transformant as defined in claim 17 o wherein the host cell is E. coli, 0°

21. A transformant as defined in claim 17 tell wherein the host cell is transformed with two recombinant plasmids, having a Tc gene and a CI857 gene respectively.

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

23. A process for producing 0-urogastrone characterized by culturing the transformant defined in claim 17 and collecting the expressed B-urogastrone. DATED this 19th day of May 1989 EARTH CHEMICAL CO LTD By Their Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia