FR2566799A1 - B-UROGASTRONE GENE AND SUBUNITS, PLASMIDS AND CORRESPONDING TRANSFORMANTS AND THEIR PREPARATION, PROCESS FOR THE PRODUCTION OF B-UROGASTRONE - Google Patents

Abstract

THE PRESENT INVENTION CONCERNS A NEW GENE OF B-UROGASTRONE AS WELL AS ITS SUB-UNITS, THE RECOMBINANT PLASMIDS CONTAINING THEM, AS WELL AS THE PROCESS FOR MANUFACTURING THESE PLASMIDS, THE TRANSFORMERS INCLUDING HOT CELLS HAVING THESE PLASMIDS. THAN THE PROCESS FOR OBTAINING THESE TRANSFORMERS, A PROCESS FOR CULTURING THESE TRANSFORMERS FOR THE PRODUCTION OF B-UROGASTRONE.

Description

The present invention relates to a novel 8-urogastrone gene, the

  corresponding recombinant plasmids, the corresponding transformants

  and their preparation as well as that of 13-urogastrone.

  B-urogastrone is a polypeptide hormone synthesized in the salivary glands of humans, etc. (cf, eg, Heitz et al., Gut, 19, 408-413 (1978)), it has a primary structure consisting of of 53 amino acids in the following sequence (see H. Gregory et al., Int. 3. Peptide Protein

Res., 9, 107-118 (1977)).

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

  In the description, the amino acids are represented by the

  following symbols: Asn: Asparagine Set'r. serine Asp: aspartic acid Glu: glutamic acid Cys: cysteine Pro: proline Leu: leucine His: histidine Gly: glycine Tyr: tyrosine Val: valine Met: methionine: isoleucine Ala: alanine LVs: lysine Gln: glutamine Arg: arginine Trp: tryptophan

Ph: phenylalanine.

  The [3-urogastrone has physiological activities such as suppression of gastric acid secretion and promotion of cell growth (see Elder et al., Gut, 16, 887-893 (1975)) and is therefore useful for treat the

- ulcers and wounds.

  Since 13-urogastrone is excreted at low levels in human urine, the compound is presently prepared from the urine by extraction, separation and purification. However, this process poses problems such that it is difficult to obtain large quantities

  of the compound because the compound is a minor component of human urine.

  On the other hand, European Patent Application Publication No. 0046039 discloses a test for producing r3-urogastrone by a technique

  genetic engineering with use of a synthetic 13-urogastrone gene.

  However, the above publication describes the synthetic gene having a specific nucleotide sequence but does not say whether there are other genes that are capable of expressing 13-urogastrone by an analogous method,

  nor does it mention such a gene of a particular nucleotide sequence.

  A large number of nucleotide sequences can encode the amino acid sequence of 13-urogastrone. However, it is difficult to know which of these genes is capable of expressing 13-urogastrone by

  a genetic engineering technique or gene that is best suited for the application

  cation of genetic engineering techniques. Thus, many experiments and many inventive efforts are required to determine the sequence

nucleotide the most appropriate.

  An object of the invention is to provide a novel 13-urogastrone gene which is entirely different from the gene described in the above publication in the nucleotide sequence and which is capable of expressing 13urogastrone.

  by genetic engineering techniques (or engineering).

  Another object of the invention is to provide a gene which is appropriate

  the expression of [3-urogastrone by genetic engineering techniques.

  Another object of the invention is to provide new plasmids

  recombinants and transformants corresponding to the new 13urogastrone gene.

  Another object of the invention is to provide a process which allows the production of 13-urogastrone with high purity.

  with use of the new gene by genetic engineering techniques.

  These and other objects of the invention will appear from

the following description.

  We conducted many experiments and found that a gene 1

  of the following nucleotide sequence meets the objects of the invention.

GOne 1: -

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

  3 'T T T T C G C T A A G A C T C A C G G G T G-A C

  T C T C A C G A T T G G C T A T G T: C T G C A C

  A G G T G C T A C C G AT A A C A G C G T G

  G A C G G T G T T T G C AT G TA C A T C G. A A

  CTG C C A C AA A C G T A C AT G T A G CTT

  G C T T T G G AT AAA T A C G C G T G T AAC

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

  T G T G G A T T G T G G A T G G A G

  ACA C A T C A C C AT AT G -CCA CT T

  C G C T G T C AA T-A C C G A T T C T G AA A

GCG ACA GTT ATG AT C TA GAG TT

T GG T G G AA T T G CGT 3 '

A C C A C C C T T AA C G C A 5 '

  The letters represent the purine or pyrimidine bases forming the nucleotide sequence. The symbols used here for the bases represent

  the following: Adenine, G guanine, C cytosine and T thymine.

  The gene I is entirely new and non-obvious in itself and

  is obtained by determining the nucleotide sequence specified from a -

  very large number of possible nucleotide sequences. - -

  The gene I has the following characteristics: (1) The 13-urogastrone can be expressed very advantageously by:

genetic engineering.

  (2) The trinucleotide codons that make up gene I are all acceptable -

  for host cells, in particular Escherichia coli (E. coli) that can be

  easily obtain, thus ensuring a high degree of expression.

  (3) Specific restriction enzyme recognition sites may be provided in the gene and at both ends, and sites may be

  be manipulated as desired to facilitate ligation with another -

  gene and insertion into the plasmid vector ..

  (4) For the preparation of gene 1, the constituent oligonucleotides can be bound in blocks and the blocks can be easily linked into subunits

  as contemplated, substantially without unwanted ligation.

  (5) By expressing B-urogastrone as fused protlein, there is a means by which an unnecessary fraction can be easily removed

  to obtain the desired B-urogastrone.

  When B-urogastrone is to be effectively expressed using gene I, restriction enzyme recognition sites can be provided at the forward end and / or back end of the gene for ligation with the promoter. , the sequence of Shine-Dalgarno (called

  below 'SD sequence'), the vector, etc., necessary for the expression.

  In addition, if necessary, a start codon and / or a codon may be provided.

  stopping upstream and downstream of the gene, respectively. The recognition sites.

  the start codon and the stop codon are not limited in particular but

  can be those who are desired.

  An example of a gene having an enlarged sequence (hereinafter referred to as "gene II") is given below which comprises a restriction enzyme recognition site and a start codon arranged upstream of the I gene and a stop codon and a restriction enzyme site disposed downstream of the gene 1, the sites and codons being arranged in the order mentioned, the gene II further including other

  sites of restriction enzyme restriction.

Gene II: Starting codon

-15 - -1, 1

  'AA T TCG A G A T C T G C A T IM A -A T A G C

  3 | Cl T T C iA C G T AIC T T A. STC G E Ta Bg (S) Mb

-. 20 30

  GA T T C T G T G T G T C C ACT C T C A C C

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

Hf

50

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

  C T A C C G A T A A C A G A C G T G-C T G CC A

  C bO o U U E9 Uc, oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo = H

t1. ó. _ <tW N a .....

O.Cc.4) HE-4 ó C <o, -

  1E. F4H cnCCD U Oqvo r-c E-CD 'Livt) ó -'8ó Ek z a-4 C) c. E4O '<CDE <H

<O Hó <CDH <H

HH OR '<EH <) C DO c tR.oZZH'

OD F <óó '

E-44: ## EQU1 ##

uacatO -

  o'o 4 <HE '<H' <UoOH CD JEr --- j CD L <HH 'a) OD ciCD u

OR '<H-400' <H H '<L O)

  The invention is not limited to gene I and gene II but also includes other genes that are substantially identical in the gene.

  nucleotide sequence and which are capable of expressing B-urogastrone.

  In the synthetic preparation of gene I or 11, it is advantageous to construct the gene I or II as divided into the front half and back half. Thus, it is possible to prepare one subunit before the first half of the nucleotide sequence of gene I or II and another subunit having the back half of its nucleotide sequence as it is divided approximately to its half. and to join these two subunits of the gene I or II together in the gene I or 11. The subunit having the front half of the nucleotide sequence of the gene II may further have a restriction enzyme recognition site at the same time. rear end, and another subunit having the back half of the gene II nucleotide sequence may have a restriction enzyme recognition site at the end

  before, and these subunits are joined together in gene II.

  In other words, more specifically for purposes of precision, the first subunit may be a subunit A comprising the front half of the II gene and having a restriction enzyme recognition site (BamHI) provided at its rear end. and the last subunit may be a B subunit comprising the back half of the gene II which has a restriction enzyme recognition site (HindIII) at its forward end. These

  subunits are presented below.

Subunit A:

  'AT A T T C G A AG AT C T G AT G AT AT G C

  3 'G C TT C T A G A C G T A C TT AT CG

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

  CT A AG A CT C ACG G G T G A C A G A GT G

  GAT G G C T A T T G-T CT G C A C G A C G GT

  CT A C C G AT A A C A GA C G T G CT G C C A

  G T TT G C A T G T.A C A T C G A A G C T T C G

  CAA A C G T A C A T G T A G C T A CG A AG C

3 '

CTA G 5 '

25667 = 99

7 -

Sub-unit B:

  'A G C T T T G G AT A A T A C G C G T G T

  3 'A AC C T AT T T T G C G C A C A

  A A C T G T G TA G T G G T T T TT TT C GG T

  T T G A C A C AT C A C CC AT A T A T: G C C A

  G AA C G C T GT C Y AA TA C C G T G AT C T G

  CT T G C G A CA T T G G C A C T A G A-C

  A A T T G G T G G A T T TG C-G T T A A G

  T T A C C A C T T A A C C C A A - T T C

TGA AGA T CT G 3 '

ACT T C T AG A C C T A'G 5 '

: - i - '

  Subunits A and B are synthesized, for example, in the following manner.

  Oligonucleotides having 11, 13 or 15 bases (A-1 to A-16, and B-1 to B-16, ie 32 oligonucleotides) are synthesized. Then, 4 to 6 of these oligonucleotides are assembled and bound in blocks (block 1 to block 7, ie 7 blocks). These oligonucleotides and blocks are shown below: cc CJ% P

E-i-i -

C, Or-4 C.) Ln ói- '

-D E4f- i E (f- -r ---- -

  ## STR5 ## wherein R is a hydrogen atom, a hydrogen atom, a hydrogen atom, a hydrogen atom or a hydrogen atom ## STR5 ## wherein: ## STR2 ## wherein ## STR5 ## Ha ó-) EkU) Eó O CD <ó.EO óFó E-4i'ó E

<3'H1C3 0 C'HE-% 0 <C4FIH '

## STR1 ## 4

## STR1 ##

  F4-C: ## STR2 ## ## STR1 ##

  7-4--7 --- dr-And-C Cr-I -cr-4--

  -C.) Eqóó '-CI. ## STR5 ## wherein Eq 4 has the same formula as the one of the formula ## STR1 ## where ## STR2 ## is shown in FIG. - ó Eq- -CD V)

  <ÓEqéC) C) r ^ óEqHC) CJ87E9óunEóSCDUr-

  Then, blocks 1 to 3 are bonded to subunit A, and blocks 4 to 7 are bonded to subunit B. The invention will be described in more detail with reference to the drawings.

and attached photos.

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

  Fig. 2 represents a process for binding oligonucleotides A-

  1 to A-16 into a subunit A and introducing the subunit into an E. coli derived plasmid pBR322 to obtain a recombinant plasmid pUG1; Fig. 3 shows an analogous method for preparing a recombinant plasmid pUG2 by introducing a B subunit into a plasmid pBP322; Fig. 4 shows a process for preparing a recombinant plasmid pUG3 from pUG1 and pUG2; Fig. 5 shows the result obtained by analyzing the nucleotide sequence of oligonucleotide A-3 by two-dimensional fractionation by electrophoresis and homochromatography; Fig. 6 shows a method for preparing a recombinant plasmid pGH37; Fig. 7 shows a process for preparing a recombinant plasmid pGH35; Fig. 8 shows a process for preparing a recombinant plasmid pEK28; Fig. 9 shows methods for preparing the recombinant plasmids pUG102 to pUG122 and the recombinant plasmids pUG103-E and pUG117-E;

  Fig. 10 shows methods for preparing recombinant plasmids

  pBRH02 and pBRH03; Fig. 11 shows the MbolI restriction map of pUG3 including fragment H (179 bp) which contains the present β-urogastrone gene; Fig. 12 shows a method for preparing the recombinant plasmids pUG2301 to pUG2303; Fig. 13 shows a method for preparing the recombinant plasmids pUG2101 to pUG2105; Fig. 14 shows a method for preparing the recombinant plasmids pUG2701 to pUG2703; Fig. 15 shows a method for preparing the recombinant plasmids pUG1102 and pUG1105; Fig. 16 shows a method for preparing a recombinant plasmid pUG1004; Fig. 17 shows a method for preparing a recombinant plasmid pUG1201; Fig. 18 shows a method for preparing a recombinant plasmid pUG1301; and Photos 1 to 5 respectively show the analytical results of nucleotide sequences of recombinant plasmids obtained in the example

by the method of Maxam-Gilbert.

  The methods themselves for constructing the gene II of the invention are known. Oligonucleotides can be prepared to construct gene II by known methods, e.g. ex. by the solid phase method which will be briefly described below (see, for example, H. Ito et al., Nucleic Acids Research,

, 1755-1769 (1982)).

  When the solid phase process is used, the oligomer is synthesized

  nucleotide as shown in FIG. 1 by successively coupling mono-

  nucleotides or dinucleotides with a nucleoside on a resin support

  of polystyrene to obtain a predetermined sequence of nucleotides.

  The nucleoside carrier resin can be prepared, for example, using a partially crosslinked polystyrene resin by reacting N- (chloromethyl) phthalimide with the resin, reacting

  hydrazine with the product to obtain an amino polystyrene resin

  methylated, and linking to its amino group a nucleoside having its free 5 'hydroxyl group and the protected amino group, using succinic acid

as a spacer.

  On the other hand, various methods are known for preparing mononucleotides or dinucleotides (see, for example, C. Broka et al., Nucleic Acids Research, 8, 5461-5471 (1980)). Thus, a mononucleotide can be prepared by reacting o-chlorophenylphosphorodichloridate, triazole and

  a nucleoside having its 5 'hydroxyl group protected with a dimethoxy group

  trityl (DMTr) in the presence of triethylamine and then reacting the mono-

  triazolide obtained with B13-cyanoethanol in the presence of 1-methylimidazole

  as a catalyst, and eluting the reaction product with chloroform-

  1,1 = methanol by column chromatography on silica gel, This method gives a completely protected mononucleotide: A dinucleotide can be prepared by treating the completely protected mononucleotide obtained above with benzenesulfonic acid or a similar acid to give the mononucleotide with the 5 'free hydroxyl group, which is reacted with the monotriazolide obtained above, and

  the product of the reaction with chloroform-methanol by chiro-

  Silica gel columnography. This process gives a completely protected dinucleotide:

  Solid phase synthesis of oligonucleotides is conducted beforehand.

  happily using a DNA synthesizer that is., p. eg, available as the DNA synthesizer from Bachem Inc., USA. The nucleoside-supporting resin obtained above is put in a reaction vessel and washed with dichloromethane-isopropanol, and a solution of zinc bromide in dichloromethane isopropanol is added to the resin to remove the dimethoxytrityl group at the 5 'position. This process is repeated several times until the color of the solution disappears. We wash the resin with

  dichloromethane-isopropanol and then with a solution of triethyl acetate

  ammonium in dimethylformamide (DMF) to remove the remaining Zn 2+, then washed with tetrahydrofuran (THF) and exposed to a stream of nitrogen gas for several minutes for drying-Separately, the dinucleotide or mononucleotide is dissolved completely protected in pyridine and then triethylamine is added, and the resulting solution is stirred and then allowed to stand at room temperature (RT) for several hours.

  hours and then evaporated at reduced pressure. The trieth / y-salt is dissolved

  resulting triethylammonium in pyridine and evaporated in a

  azeotropic several times using pyridine for drying purposes. -

  The nucleotide salt is dissolved in a solution of mesitylenesulfonyl

  -Nitrotriazole (MSNT, condensation reagent) in pyridine. The resulting solution is added to the dried resin and allowed to stand at RT. The liquid fraction is removed from the reaction mixture, and the resin fraction is washed with pyridine and reacted with acetic anhydride. using dimethylaminopyridine as a catalyst in THF-pyridine to mask the unreacted hydroxyl group. Finally we wash the resin

  with pyridine to complete a solid phase synthesis cycle.

i 2

  ULin cycle extends the nucleotide sequence by one or two chain lengths.

  The above method is repeated to couple mononucleotides or dinucleotides sequentially with the resin to the desired length, whereby a fully protected oligonucleotide can be obtained.

supported on the resin.

  To the resulting resin is added a solution of tetramethylguanidine-

  2-pyridinealdoximate in pyridine-water, and the mixture is allowed to stand on heating. The resin is then filtered off and washed with pyridine and ethanol alternately. The washings are combined with the filtrate and concentrated under reduced pressure. The concentrate is dissolved in an aqueous solution of triethylammonium bicarbonate (TEAB) and then washed with ether. The aqueous solution is subjected to chromatography

  on Sephadex G-50 column using TEAB solution as 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 phase chromatography to a single peak. The oligonucleotide thus obtained still has its 5 'end protected by a dimethoxytrityl group, so that the product is treated with an aqueous acetic acid solution to remove the protecting group, followed again by high-level liquid chromatography. speed or a similar process for purification

until one peak.

  The desired oligonucleotides are prepared by the method described above.

  above, and then the nucleotide sequence is individually verified by a two-dimensional fractionation method using electrophoresis and homochromatography and (or) the Maxam-Gilbert method and then

  uses them to prepare blocks and subunits.

  The two-dimensional fractionation method for checking the nucleotide sequence can be conducted by the method of Wu et al. (E. Jay, R.

  A. Bambara, R. Padmanabhan and R. Wu, Nucleic Acids Res. 1, 331 (1974)).

  To practice this method, the oligonucleotide as lyophilized is dissolved in distilled water at a concentration of about 0.1 μg / liter. We

  treats a fraction of this solution with 32 P-ATP and T4 polynucleotide.

  kinase to label the 5 'end with P and then are patiently digested with phosphodiesterase of snake venom. The product is distributed in drops on a cellulose acetate film and subjected to electrophoresis for the first dimensional development to separate the product according to the base difference. The developed products are then transferred to a plate of diethylaminoethyl cellulose (DEAE cellulose) and. they are subjected to the second dimensional development using a partially hydrolysed RNA solution called a homomix. (This process

  is called homochromatography). In this way, we separate the oligonucleotide

  cleotide according to the length of the chain. Then we read the nucleotide sequence

  of the oligonucleotide by autoradiography starting from the 5 'end.

  If it is difficult to verify the sequence by this method, the Maxam-Gilbert process is used if necessary. (A.M. Maxam and W. Gilbert, Proc Natl Acad Sci, UJSA, 74, 560 (1977), A.M. Maxam and W. Gilbert, Methods in

  Enzymol., Vol. 65, p 499, Academic Press 1980).

  This process, which is also called a chemical decomposition process, employs a specific reaction to a particular base to cleave

  the oligonucleotide at the base position, and the electro-revealed bands

  phoresis are used to read the sequence from the 5 'or 3' end. The reactions

  Specific bases are as follows. Guanine is specifically

  methylated with dimethylsulfate. Guanine and adenine undergo a depurination reaction in the presence of an acid. Thymine and cytosine both react with hydrazine at a low salt concentration, but cytosine only reacts with hydrazine at a high salt concentration. After the

  At the end of the reactions for the four bases, each reaction mixture is reacted.

  With the aid of piperidine, it is possible to displace the open-core base and to catalyze the removal of the two phosphates from the sugar, and finally the DNA strand is cleaved to this base. The reaction mixtures obtained are subjected to polyacrylamide gel electrophoresis respectively to confirm the nucleotide sequence according to that of the reactions which

produces each band.

  Next, the oligonucleotides are ligated using a T4 DNA ligase as seen in Figure 2. For correct ligation, the 16 A-1 oligonucleotides to A-16 corresponding to the A subunit are divided into three

  sets, namely block 1 comprising A-1, A-2, A-3, A-14, A-15 and A-

  16, block 2 comprising A-4, A-5, A-6, A-11, A-12 and A-13, and block 3 comprising A-7, A-8, A-9 and A-10 as we see it in fig. 2. By electrophoresis blocks 1 to 3 having the correct sequences are obtained and are further bound to subunit A. In other words, more specifically, some of the 5 'ends of the 16 nucleotides A-1 to A are labeled. With 32 p using 32 P-ATP and T4 polynucleotide kinase, and the hydroxyl groups of the remaining 5 'ends are phosphorylated with ATP. To form each of the three blocks, the oligonucleotides were assembled and ligated using T4 DNA ligase, and the product was electrophoresed on polyacrylamide gel to isolate the desired block. The three blocks thus obtained are ligated using T4 DNA ligase to produce the A subunit. Although a dimer structure can be produced in the iigation reaction, it cleaves easily with the restriction enzymes EcoRI and BamHI to obtain the subunit A. Then, as seen in fig. 2, a known plasmid vector, pBR322, which is derived from E, is cleaved. coli and readily available, with EcoRI and BamHI, and inserting the A subunit into the vector to obtain a plasmid

recombinant pUG 1.

  The same procedure as above is also followed for the sub-

  as in the case of subunit A, the 16 oligonucleotides B-

  1 to B-16 are linked divided into 4 sets as seen in fig. 3, and the blocks are ligated together to produce the B subunit. The dimer, if produced, is cleaved with the HindIII and BamHI restriction enzymes to obtain the B subunit. A plasmid vector, pBR322, is cleaved. with HindIII and BamHI, and the B subunit is inserted into the vector to obtain a plasmid

  recombinant pULG2 as seen in FIG. 3.

  In addition, as can be seen in FIG. 4, pUG1 was cleaved with HindIII and SaII restriction enzymes, and a fragment removed from pUG2 was inserted using the same restriction enzymes in pUG1 to prepare a recombinant plasmid pUG3 having a B-urogastrone structural gene.

(gene II).

  pUG1, pUG2 and pUG3 are recombinant plasmids which each comprise pBR322 and subunit A which is the front half of the B-urogastrone structural gene, subunit B which is the back half of the gene, or the gene of entire structure. These recombinant plasmids can be proliferated in large quantities by introducing them into a host, such as E. coli strain HB101. coli which is known and readily available according to the method

-2566799

  calcium as a transformation process (E. Lederberg and S. Cohen, 3.

  Bacteriol., 119, 1072 (1974)): It can be verified whether pUG1, pUG2 and pUG3 are present in the host like E. coli strain HB101. by the following methods. After having collected the plasmids by the alkaline extraction method, the presence of the BglI recognition site which is not present on the pBR322 vector is verified in pUG1 and pUG2. Similarly, we check if pUG2 and pUG3 can be

  cleaved with MluI which is not present on pBR 32-2.

  According to the alkaline extraction method, the E. coli harboring the plasmid is incubated, the cells are then collected, and the lysozyme is activated to dissolve the cell wall. A mixture of sodium hydroxide and sodium lauryl sulphate is used to break the cell and denature the DNA, which is then neutralized with a sodium acetate buffer. At the moment,

  the chromosomal DNA remains denatured, but the plasmid, which is extra-DNA

  chromosome, restores the initial two-stranded form. Plasmids are collected using these characteristics. The plasmids are then subjected to

  density gradient ultracentrifugation with cesium chloride and

  ethidium bromide for purification and then passed through a 50m Biogel column to remove the RNA. It is thus possible to obtain plasmids with high purity in large quantities. In this way,

  the β-urogastrone gene of the invention (gene II) can be obtained. -

  Next, the method of introducing the 13-uro-gene will be described.

  gastrone in the host cells. The host cells to be used in the invention are not particularly limited and any of those known, e.g. ex. those of E. coli, Bacillus, Pseudomonas, yeast, etc., among which E. coli cells are preferred. coli .: The expression modes of the 13-urogastrone gene using E. coli include a system for directly expressing 13-urogastrone, and a system where it is expressed as a protein fused with

  β-lactamase or another different protein.

  For the direct expression of the 13-urogastrone gene, it is necessary

  to introduce in the piasmde ;; nt e; the end of the R-Qir-qast.rnnp.

  a promoter and an SD sequence. While the promoter is not particularly limited, the desirable promoters are those that provide a high level of expression, such as AP which is the promoter of the left-hand L-part of the phage ?, Lac UV5 that is present upstream of the gene. Bgalactosidase of E. coli, etc. When using. As a promoter, the SD sequence is not particularly limited, but it is desirable to use the 4-base sequence of AGGA. In addition, when using UV5 as a promoter, it is desirable to use the SD sequence which is downstream of the promoter.

  UV5 lake or one that is chemically synthesized.

  The system for directly expressing the β-urogastrone gene will be described with reference to the case where the β-sequence-SD-13 urogastrone gene is used. Although APL is a potent promoter - (J Hedgpeth et al., Molecular and General Genetics, 163, 197-203 (1978)), the fully activated PL promoter produces lethal effects on the E host cell. .. coli, so that it is necessary to proliferate the cell in conditions devoid of any lethal action and then operate the 3L. On the other hand, C1857 which

The

  is a gene in phage X is one of the mutated CI repressor genes that acts on the operator for PL. At low temperature (up to about 30 C), the CI857 repressor binds to the operator to completely inhibit the activity.

  of P as promoter, therefore allowing the proliferation of E.

  The coli. The host cells are thus allowed to proliferate in this state and then brought to a high temperature (not lower than 37 C), whereby the XPL can function. In addition, plasmid vectors, such as pSC101 that is known and readily available, have a strict replication mechanism, and those such as pBR322 that have a relaxed replication mechanism are not incompatible with each other but may coexist

in the same cell of E. coli.

  It is therefore appropriate to construct a recombinant plasmid pGH37

  a CI857 gene is incorporated into a tetra-resistant plasmid vector

  cyclin pSC101 (with the lac UV5 promoter provided upstream for efficient expression of IC857) as seen in FIG. 6 and to introduce the recombinant plasmid into E. coli (strain H8101) to obtain a transformant (strain

  ECI-2) for application as a host for the vector to express B-uro-

* gastrone. under the control of the PL promoter According to the invention, the PL-sequence SD-B-urogastrone gene, p. eg, in pBR322 to obtain a vector expressing β-urogastrone; it is used to transform the strain ECI-2, thanks to which it is provided that it is used to transform the strain ECI-2, thanks to what it is provided what is called a system with two plasmids o two useful plasmids coexist

in a cell of E. coli.

  In this system, the C1857 repressor encoded by pGH37 binds to the operator for the PL promoter on the second plasmid when the cell is cultured. ex. at 30 C, allowing proliferation of the cell. When the cell has fully proliferated in this state, the temperature p is raised; ex. at 40 ° C., after which the CI857 repressor is dissociated from the operator,

  enabling the activity of the promoter; P for the expression of Burogastrone.

  Although a similar concept is applied to the expression of fibroblast interferon, SV-40 Small t antigen, etc., in these cases a lysogen is used as the host in which the phage DNA is introduced. carrying a CI857 gene in the host chromosome (R. Derynck et al., Nature, 287, 193-197 (1980),

  C. Derom et al., Gene, 17, 45-54 (1982), K. Kpper et al., Nature, 289, 555-

559 (1981).

  However, with the system of the invention, the Cl857 gene is introduced.

  in a different plasmid that is resistant to tetracycline. The advantages of the present system are therefore that it is unlikely that the phage Xintroduced into the host chromosome is induced to proliferate and that the strain can be easily controlled. Naturally, the two-plasmid system

  is used for the first time for systems to express 1urogastrone.

  According to another system, a fraction of any other protein gene such as the β-lactamase gene is linked to the Burogastrone gene to express the 3-urogastrone gene as a fused protein. The advantage of this method is that the fused protein is less exposed to decomposition by the protease in E. therefore provide protection for 8-urogastrone. Another advantage is that the fused protein migrates to the cytoplasm of the E. coli cell. coli

  and accumulates there (S.J. Chan et al., Proc Natl Acad Sci., USAS 78, 5401-

  5405 (1981)), is locally present and therefore easy to separate and purify.

  That is, more specifically, a gene encoding two basic amino acids that can provide a cleavage site for removing burogastrone from the fusion protein by enzyme cleavage is inserted into the β-lactamase gene at an appropriate site. enzyme cleavage

  restriction, and a 13-urogastrone gene is linked to the Blactamase gene.

  Preferably the sequence of two basic amino acids is -Lys-Arg- or -Arg-Lys-. Examples of enzymes for recognizing the amino acid sequence for cleaving β-urogastrone from the fused protein include kallikrein, trypsin, etc. Examples of restriction enzymes for cleaving the β-lactamase gene include XmnI, HincI], ScaI, PvuI, PstI, BglI, BanI, etc. The recombinant plasmid 13-lactamase-B-urogastrone thus prepared can express a fused protein in E. coli for quantity production. The resulting fusion protein is treated with kallikrein

  or a similar substance, whereby B-urogastrone can be obtained.

  The expression system can be verified by directly analyzing the nucleotide sequence of the gene by the method of MAxam-Gilbert, in

  confirming the insertion of the gene and its direction by the process of minimizing

  preparation or establishment of a map (H.C. Birnboim et al., Nucleic

  Acids Research, 7, 1513-1523 (1979)), or by radioimmunoassay for B-

urogastrone.

  The transformant of the invention thus obtained is cultured by the usual method, whereby B-urogastrone can be collected with purity.

high in large quantity.

  The invention will be described below in more detail with reference

  to the following example to which the invention is not limited in any way.

EXAMPLE

  1) Preparation of a resin supporting a nucleoside

  Various nucleoside-supporting resins are prepared by the following method. A certain amount of cross-linked polystyrene resin "S-XI" was mixed at 1% by weight (product of BIO-RAD Laboratories, USA, 0.074 to 0.043 mm mesh size) with 2.41 g of N (chloromethyl) phthalimide, 0.22 ml of trifluoromethanesulfonic acid and 50 ml of dichloromethane with stirring at RT for 2 h. After the end of the reaction, the resin is filtered, washed successively with dichloromethane, ethanol and magnesium, dried under reduced pressure and then refluxed with 50 ml of% solution. weight of hydrazine in ethanol overnight while heating. The resin is filtered and washed with ethanol, dichloromethane and methanol successively and then dried under reduced pressure. Let's rest

2: 566 799

  overnight at RT the resin mixture, aminylated polystyrene (2.5 g) obtained by the above method, 0.75 mM 5'-o-dimethoxytritylnucloside monosuccinic acid ester, 1.23 mM dicyclohexylcarbodiimide and

  1 mM dimethylaminopyridine with addition of 30 ml of dichloromethane.

  The resin is filtered off, washed with dichloromethane, methanol and methanol. of-

  pyridine successively, then it is immersed in the pyridine-acetic anhydride (90:10 in volume ratio) and allowed to stand at -TA for minutes. The resulting nucleoside-supporting resin is filtered, washed with pyridine and dichloromethane and dried at reduced pressure

  for application in a solid phase synthesis reaction.

2) Dinucleotide synthesis.

  By way of example, the synthesis of a complete dinucleotide will be described.

  protected with the base sequence of TA. Adenosine (13.14 g) having a 5 'hydroxyl group protected with a dimethyloxytrityl group (DMTr) and a benzoyl group (6.34 g) are dissolved in anhydrous dioxane. triazole. While cooling with ice, 8.35 ml of triethylamine is added to the solution, then 6.86 g of o-chlorophenylphosphorodichloridate are added dropwise to the mixture over a period of 10 minutes.

  minutes, and the resulting mixture is stirred at RT for 2.5 hours.

  The triethylamine hydrochloride formed is separated by filtration, the filtrate is concentrated to approximately 2/3 of its volume, and 3.6 g of 13-cyanoethanol and 4.8 g of 1-methylimidazole are mixed with stirring while stirring.

  at RT for 3 h. The reaction mixture is concentrated under reduced pressure.

  The residue is dissolved in ethyl acetate (EA), washed with a 0.1M aqueous solution of sodium phosphate, dibasic 3 times and with water.

  2 times and then concentrated under reduced pressure giving 19.96 g of crude product.

  The product is purified by column chromatography on silica gel in:

  using chloroform-methanol (98: 2 in volume ratio) as eluent.

  The purification process is repeated to obtain 15.12 g of monounsine adenosine.

  nucleotide compiLtinmerit proL.:é.

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

in 5 'gross.

  Thymidine (1.64 g) having a hydroxy group protected with a dimethoxytrityl group and 0.95 g of triazole in 21 ml of anhydrous dioxane is dissolved, 1.25 ml of triethylamine are added to the solution, and 0.69 ml of o-chlorophenylphosphorodichloridate are added dropwise to the mixture over a period of 5 minutes while stirring and cooling with ice. The mixture is then stirred at RT for 1 h. The triethylamine hydrochloride resulting from the reaction is filtered off and the filtrate is stirred for 10 minutes with 1.1 ml of an aqueous solution of pyridine (1M). To the solution is added a solution in dioxane (10 ml) of 1.17 g of the adenosine mononucleotide having the hydroxyl group in free 5 'and prepared as above and with 0.72 ml of 1-methylimidazole, and the mixture is stirred at RT for 3 h. The reaction mixture obtained is concentrated under reduced pressure, the residue is dissolved in the EA, and the solution is washed with an aqueous solution of sodium phosphate, dibasic (0.1 M), then with water and concentrated to room temperature. reduced pressure, giving 2.39 g of crude product. The product was subjected to column chromatography on silica gel (CCGS) and eluted with chloroform-methanol (98: 2 to volume ratio) to obtain 2.39 g of fully protected TA dinucleotide.

  In the same way as above, various nucleotides are prepared.

3) Oligonucleotide synthesis.

  The solid phase synthesis of oligonucleotide A-1 is described.

  that is to say the undecanucleotide AATTCGAAGAT.

  A resin (40 mg) supporting the nucleoside T and prepared according to process 1) above is washed in a reaction vessel, washed with dichloromethane-isopropanol (85:15 in volume ratio) 3 times, then

  treated with a solution of zinc bromide (1M) in dichloromethane-

  isopropanol to remove the dimethoxytrityl group at the 5 'position. This process is repeated several times until the color of the solution disappears. The resin is washed with dichloromethane and then washed with a solution of triethylammonium acetate (0.5M) in DMF to remove the remaining Zn2 +, further washed with THF and dried by passing nitrogen

  gaseous in the reaction vessel for several minutes.

  The dinucleotide GA (50 mg), dissolved in 1 ml of pyridine, is

  Protected and prepared as in Method 2) above, stirred with 1 ml of triethylamine and allowed to stand at RT for several hours. The reduced pressure solution is then evaporated. The residue is azeotropically evaporated several times with pyridine to convert the nucleotide to a triethylammonium salt. The salt is dissolved in 0.3 ml

  of mesitylene-sulfonyl-5-nitritrotriazole solution (0.3 M) in pyridine.

  The solution is added to the dried resin and then reacted at RT for minutes. The liquid fraction is filtered off from the reaction mixture, and the solid fraction is 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 (0.1 M) in THF-pyridine to mask the unreacted hydroxyl group. Finally, the resin is washed with pyridine, whereby a solid phase synthesis process cycle is completed. One cycle extends the nucleotide chain of base lengths. The same procedure as above is repeated for successively coupling the dinucleotides AA, CG, TT and AA with the resulting nucleotide by condensation, whereby the completely protected undecucleotide is prepared.

  AATTCGAAGAT supported on the resin.

  The resulting resin (20 mg) was allowed to stand at 40 ° C. for 1 hour with 0.6 ml of a solution of tetramethylguanidine-2-pyridinaldiximate (0.5 M) in pyridine-water (90:10 in volume ratio). ). The resin is then passed through a Pasteur pipette plugged with cotton and thus filtered off. The resin is washed with pyridine and ethanol alternately. The washing waters and the filtrate are combined and concentrated at 40 ° C. under reduced pressure. The residue is dissolved in 2 ml of an aqueous solution of triethylammonium bicarbonate (TEAB, 10 mM). The solution is washed 3 times with ether. The aqueous phase is applied in a Sephadex G-50 column (2 x 100 cm) and eluted with 10 mM TEAB solution. The absorption of the fractions at 260 nm is checked. The fraction including the first eluate peak is concentrated. The residue was subjected to high-speed liquid chromatography (Model 6000A pump, Model 440 detector, Waters Associates products, USA) to obtain a purified fraction with a single peak. For high-speed liquid chromatography, the

  column is [-Bondapak C18 (product of Waters Associates, USA)

  Uni), and an aqueous solution of acetonitrile of triethyl acetate

  ammonium (0.1 M) as eluent for gradient elution (5 - 40% vol).

  The purified undecanucleotide always has its 5 'end protected with a dimethoxytrityl group, so that the compound is treated with an aqueous solution at 80% vol. of acetic acid for 15 minutes to remove the dimethoxytrityl group and then purified again by high speed liquid chromatography until a single peak. The same column as above is used for this purpose, and an aqueous acetonitrile solution of triethylammonium acetate (0.1 M) is used for gradient elution.

(5 -> 25% vol.).

  In the same way as above, the oligonucleotides are synthesized

A-2 to A-16, and B-1 to B-16.

  Table 1 shows the yield of each oligonucleotide determined using 20 mg of the resin resulting from solid phase synthesis, separating the oligonucleotide from the resin, and then removing the group.

protecting and purifying.

  The yield is calculated from the measurement of the absorption of the final purified product at 260 nm and the sum of the absorption values for

the bases of nucleotides.

TABLE 1

  A-1, 80g A-2, 120g A-3, 90g A-4, 50g A-5, 140g A-6, 70lg A-7, 80g A-8, 90g A-9, 1001g A-10, 90g A-11-, 11Og A-12, 40gg A-13, 50ug A-14, 40g A-15, 60g A-16, 150g Bl, 60gg B-2, 100g B-3, 50gg B-4, 90g B-5, 100g B-6, 90g B-7, 130g B-8, 010g B-9, O110g B-10, 100g B10-10G B-12, 11Oig B-13, 130gg B-14, 60g B-15 , 70g B-16, 50g

  4) Verification of the Oligonucleotide Base Sequence.

  We verify the sequence according to the two-dimensional fractionation by

  electrophoresis and homochromatography of Wu et al.-mentioned above.

  Each of the oligonucleotides A-1-to-A-16 and B-1 to B-16 is found to have the nucleotide sequence envisioned. Fig. 5 shows the result obtained by analyzing oligonucleotide A-3, where it is found that A-3 has the sequence

  GATTCTGAGTG read from the 5 'end.

  The nucleotide sequence of each oligonucleotide is also checked.

  cleotide by the method of Maxam-Gilbert discussed above. -

  It is confirmed that oligonucleotides A-1 to A-16 and B-1 to B-16

  each have the nucleotide sequence envisaged.

  ) Construction of Oligonucleotide Blocks and Subunits. Blocks and subunits are prepared by the method shown in

  fig. 2. as described in detail below.

  First, about 5 g of each of the oligonucleotides are dissolved

  A-1, A-2, A-3, A-14, A-15 and A-16 in distilled water (50 VI) to obtain -

  a solution having a concentration of about 0.1 μg / l. The 6 types of aqueous solution, each in an amount of 10 l (1 μg calculated in DNA), are placed in 6 other Eppendorf tubes individually. A mixture solution (6 L1) containing tris-HCl is placed in each tube. 250 mM (pH 7.6), 50 mM magnesium chloride, 10 mM spermine and 50 mM DTT followed by addition of 0.5 μl aqueous solution of β-3P-ATP (product of Amersham International Ltd). , UK), 0.5 μl of T4 polynucleotide kinase (product of Takara Shuzo Co., Ltd., Japan) and 13 μl of distilled water to obtain 30.1. mixture. We

  The mixture is reacted for 30 minutes at 37 ° C and is further reacted.

  for 30 minutes with the addition of 1 μl of 30 mM aqueous ATP solution.

  The reaction is terminated by heating at 100 ° C. for 2 minutes. The reaction mixture is rapidly cooled with ice. Oligonucleotides A-1, A-2, A-3, A-14, A-15 and A-16 thus have the 5 'end. phosphorylated, each in an amount of 10-11, in another single 1.5-ml Eppendorf tuk. 40 μl of aqueous solution of 250 mM tris-HCl (pH 7.6), 40 μl of 50 mM magnesium chloride and 35 μl of distilled water are placed in the tube to obtain a total quantity of 175 μl. The mixture is heated at 90 ° C. for 2 minutes and is then gradually cooled to RT. With the addition of 10 μl of aqueous solution of 200 mM DTT, 10 μl of 20 mM aqueous ATP solution and 5 μl (100 units) of T4 DNA ligase (product of Nippon Gene Co., Ltd., Japan),

  the mixture is reacted overnight at 4 ° C. giving the oligonucleotide block 1

  bound A-1, A-2, A-3, A-14, A-15 and A-16.

  Similarly, block 2 and block 3 are formed by the ligation of A-4, A-

  5, A-6, A-11, A-12 and A-13 and by ligation of A-7, A-8, A-9 and A-10. Ethanol was added to the reaction mixture of the blocks thus prepared at 2 times their volume, and the mixture was allowed to stand at -80 ° C. for 30 minutes to precipitate the DNA, and then electrophoresis was carried out on a polyacrylamide gel at 12 ° C. , 5% by weight and autoradiography. This gives bands in the positions of 72 bps (base pairs) and 36 bps for the block 2 and bands at the 48 bps and 24 bps positions for the block 3. Then, each band is debossed and a mixture is added. 10 mM tris-HCl (pH 7.6) and a 10 mM aqueous solution of EDTA (tris-EDTA). The mixture is then left to stand overnight for the purposes of extraction. The resulting mixture is centrifuged, the supernatant is separated, and the supernatant is thoroughly shaken with tris-EDTA-saturated phenol and centrifuged to discard the bottom layer. The same process is repeated twice with phenol saturated with tris-EDTA. Finally, the top layer is passed through a column 1 cm in diameter and 20 cm long filled with Sephadex G-50 to remove phenol and acrylamide. The eluate is then concentrated at 200 ° C. and then left to stand at -80 ° C. for 30 minutes with ethanol in twice.

  the volume of the concentrate to precipitate the DNA.

  We combine the 3 Obtained blocks. To the mixture was added mM tris-HCl (pH 7.6), 10 mM magnesium chloride, 20 mM DTT, 1 mM ATP and 5 μl of T4 DNA ligase. The resulting mixture is allowed to stand overnight at 40C for ligation. To the mixture is added ethanol at 2 times its volume, and the mixture is allowed to stand at -80 ° C. for 30 minutes.

  to precipitate the DNA, followed by electrophoresis on a poly-

  8% by weight acrylamide and autoradiography, which reveals 96 bp and 192 bp bands. We decite each band, and we add tris-EDTA,

  and allowed to stand overnight at RT for extraction.

  The mixture is centrifuged to separate the supernatant, the supernatant is thoroughly shaken with tris-EDTA saturated phenol, and the bottom layer is discarded. By adding more saturated phenol with tris-EDTA, this process is repeated twice. The top layer is passed through a column

'66799

  of Sephadex G-5, the eluate is concentrated and the concentrate is added to the ethanol concentrate at 2 times, and then allowed to stand at -80 ° C for 30 minutes to precipitate the DNA. The product obtained is cleaved with EcoRI and BamHI to obtain the A subunit. The same procedure as above is repeated as shown in FIG. 3 to bind oligonucleotides B-1, 8-2, B-15 and 8-16 to block 4, to link oligonucleotides B-3, B-4, B-13 and B-14 to block 5, to bind

  oligonucleotides B-5, B-6, B-11 and B-12 in block 6, and to bind the oligonucleotides

  cleotides B-7, B-8, B-9 and B-10 in block 7. The blocks corresponding to 26 bp and 52 bp are collected and similarly bound to give products of 104 bp

  and 208 bp, which is cleaved with HindIII and BamHI. Thus, we obtain the sub-

unit B.

  6) Cloning of subunits and analysis of recombinant plasmids.

  With reference to FIG. 2, pBR322 was cleaved with EcoRI and BamHI, and the 5'-end phosphate groups were removed with alkaline phosphatase (a product of Takara Shuzo Co., Ltd., Japan) so as not to restore the original state. Then left overnight at 4 C pBR322 thus cleaved and dephosphorylated and the A subunit in a mixture of 50 mM tris-HCl (pH 7.6), 10 mM magnesium chloride, 20 mM DTT and 1 mM ATP with addition of 5 μl of T4 DNA ligase, whereby they are bound. To the reaction mixture was added ethanol at 2 times its volume, and the mixture was allowed to stand at -80 ° C for 30 minutes for precipitation. The mixture is then centrifuged, the precipitate is dried and dissolved in 100 ml of distilled water, whereby a plasmid pUG1 is obtained where subunit A is incorporated.

in pBR322.

  We transform E. col! strain HB101 with the plasmid pUG1 by the

calcium process.

  Host strain HB101 was cultured at 37 ° C. in 50 ml of LB culture medium (1% by weight of bactotrypton, 0.5% by weight of yeast extract and 0.5% by weight of sodium chloride. ). When the absorption at 610 nm reaches 0.25, a 40 ml fraction of the culture broth is transferred to a centrifugation tube and centrifuged at 6000 rpm for 10 minutes at 4 ° C. The supernatant is discarded, the precipitate suspended in 20 ml of 0.1 M magnesium chloride cooled with centrifuged ice

  the suspension again under the same conditions, and discard the supernatant.

  Z6 The precipitate is suspended in 20 ml ice-cold solution of 0.1 M calcium chloride and 0.05 M magnesium chloride and cooled with ice for 1 h. The suspension is centrifuged, the supernatant is discarded, and the precipitate is suspended in 2 ml of ice-cold solution of 0.1 M calcium chloride and 0.05 M magnesium chloride. To a 200 LA fraction of the suspension 10 μl of aqueous solution of pUG1 is added, and the mixture is cooled with ice for 1 h and then heated in a water bath at 43.5 ° C. for 30 s. 2.8 ml of LB culture medium are then added to the mixture, followed by incubation at 37 ° C. for 1 hour. The culture is then extended on an LB plate containing 50 μg / ml of ampicillin in an amount of 200 μl / dish and incubated overnight at 37 ° C. The growing colonies are checked by further transplanting to an LB plate. containing 50 μg / ml of ampicillin and also to a LB plate containing 20 μg / ml of tetracycline and incubated overnight at 37 ° C. Resistant colonies are separated.

  with ampicillin alone to obtain a transformed cell.

  Small cell pakir plasmids are collected by the alkaline extraction method and the presence of a BglII cleavage site is verified. One of the cells containing the plasmid having the large BGILI cleavage site is cultured to obtain the purified plasmid pUG1.

  likewise by the alkaline extraction process.

  The nucleotide sequence of the subtype is analyzed on both strands.

  unit A incorporated into the resulting pUG1 by the Maxam-Gilbert process

explained above.

  Photos 1 and 2 show the results of the analysis. Paths 1 to 4 are the result of electrophoresis for the EcoRI-SalI fragment, and lanes 5 to 8 are for the BamHI-PstI fragment. Paths 1 and 5 show the reaction products for the. guanine, lanes 2 and 6, the reaction products for guanine + adenine, lanes 3 and 7 show the reaction products for thminin + cytosine, and lanes 4 and 8 show the reaction products for cytosine. Photo 2 shows the results obtained by the same specimens as above, where a region on the higher molecular weight side (corresponding to the upper part of the photo)

  1) is enlarged. In this way, the nucleotide sequence of the sub-

unit A is confirmed.

-2566799

  Referring to FIG. 3, plasmid pBR322 is cleaved with HindIII and BamHI, and the larger fragment is isolated by electrophoresis and bound to B subunit. Thus, a plasmid pUG2-o is obtained. Subunit B is introduced. in pBR322 as in the case of pUG1. Using the resulting pUG2 plasmid, the HB101 strain was transformed, and the ampicillin-resistant colonies alone were selected. The plasmids are collected from the colonies and then the presence of a BglII cleavage site and a MluI cleavage site is verified. Plasmid-containing cells having both

  sites. One of the selected cells is grown on a large scale to obtain -

  the purified plasmid pUG2. The nucleotide sequence of the sub-

  unit B in pUG2 on both strands by the Maxam-Gilbert process.

  Photo 3 shows the results of the analysis. Paths 1 to 4 show the result obtained by the HindIII-SalI fragment. Paths 5 to 8 show the result obtained by the same specimen, where a region on the higher molecular weight side (corresponding to the upper part of paths 1 to 4) is shown on an enlarged scale. Each path shows the same corresponding reaction product as in photo 1. Thus, the nucleotide sequence of subunit B is confirmed., Then referring to FIG. 4, pUG1 was cleaved with HindIII and SalI, and a larger fragment was separated through a 1.5 m Biogel column. PUG2 was cleaved with HindIII and SalI, and then electrophoresed to obtain a smaller fragment. The two fragments are combined and treated with T4 DNA ligase for ligation, whereby we obtain

  the plasmid pUG3 o the subunits A plus B, that is to say the gene of 13B-

  urogastrone, are incorporated in pBR322. E. coli strain HB101 is transformed using plasmid pUG3. The transformant was deposited under the terms of the Budapest Treaty on the international recognition of the deposit with the deposit NO0 FERM BP-543 at the Fermentation Research Institute,

  Agency of Industrial Science and Technology, Ministry of. international trade

  and Industry, Japan, 22 June 1984.

  In the above case also, cells harboring plasmid pUG3 which is ampicillin-resistant alone and has a MluI cleavage site are selected. One of the selected cells is grown on a large scale to obtain the purified plasmid pUG3. The nucleotide sequence of the B3-urogastrone gene in pUG3 was analyzed on both strands by the Maxam-Gilbert method. Photo 4 shows the result of the analysis. Paths 1 to 4 show the result obtained with the BamHI-PstI fragment. Paths 5 to 8 show the result obtained with the same specimen, or a region on the higher molecular weight side (corresponding to the upper part of paths 1 to 4) is shown on an enlarged scale. The reaction products of the paths (or paths) are the same as those which correspond in the photo

  1. The analysis confirms the nucleotide sequence of the Burogastrone gene.

  7) Expression vector incorporating the AP L promoter The PL promoter, promoter of the left phage, L is used to express Burogastrone as will be described in detail below. First, the preparation of an ECI-2 strain derived from the strain of E. coli is described. coli HB101. ECI-2 serves as a host for i5 P- expression plasmids. Cloning of a DNA fragment containing the P promoter from phage mutant ACI857S7 DNA and the construction of expression plasmids from DNA is then described. clone. We describe

  in addition, the expression of the B-urogastrone gene in the strain ECI-

2 host by promoter P

  7-1) Construction of strain ECI-2.

  The strain ECI-2 is E. coli HB101 harboring a plasmid pGH37 for

the expression of the CI857 gene.

  PGH37 is prepared by the method shown in FIG. 6. First, the ICI857S7 DNA is cleaved with BglII. The cohesive ends are then digested at the cleavage site using S1 nuclease. 11 μg of BglI-cleaved C6I857S7 DNA is reacted with 200 units of C nuclease for 30 minutes in 100 μl of an aqueous solution (pH 4.5) comprising 200 mM sodium chloride. 30mM sodium acetate and 5mM zinc sulfate. The blunt-ended DNA fragments thus obtained are subjected to 1.0% by weight agarose gel electrophoresis to isolate a 2385 bp fragment having the entire structural gene CI857. The fragment is inserted into the PvuII cleavage site of plasmids pGL101 to construct a plasmid pGH36 which expresses the C1857 gene under the control of a promoter, lac UV5. Next, pGH36 was cleaved with two restriction enzymes, EcoRI and PstI, to obtain a fragment having 1193 bp, which was inserted into a plasmid pSC101 between the EcoRI and PstI cleavage sites to prepare

a plasmid pGH37.

  Then, strain HB101 of E. coli with pGH37 by the calcium method mentioned above, - One of the strains obtained is named ECI-2. The strain ECI-2 is deposited under the Treaty of Budapest on

  international recognition of the deposit with the deposit N FERM-BP-

  542, Fermentation Research Institute, Ministry of International Trade and Industry, Japan, June 22, 1984. This strain is resistant to tetracycline, expresses the gene CI857, and allows for the joint presence, by transformation. , others

  derived plasmids, p. eg, from pBR322. We then use the strain ECI-

  2 as a host for A P L expression plasmids

  7-2) Cloning of the 1 P promoter and preparation of expression plasmids.

The

  As seen in fig. 7, pGH35 is built first.

  The ACI857S7 DNA was cleaved with EcoRI and SalI to obtain a 5925 bp fragment which comprises the P promoter and the IC857 gene as well as the L promoter> P. The fragment in the plasmid pBR322 was inserted between the R-cells.

  EcoRI and SalI cleavage sites to construct a pGH25 plasmid.

  PGH25 was then cleaved with BamHI and cleaved pBR322 was similarly

with BamHI to obtain pGH34.

  Then, pGH34 was cleaved with AvaI and BglII and treated with S1 nuclease to give a blunt-ended fragment of about 4500 bp. The fragment is annealed with T4 DNA ligase to construct

a plasmid pGH35.

  PEK28 is then constructed as shown in FIG. 8. The synthetic oligonucleotides C-1-1 and C-1-2 are ligated as an adapter, which comprises an SD sequence, and which have the nucleotide sequence shown below,

  to the fragment that is obtained by cleaving pGH35 with HpaI. The assembly is still

  Bam HI cleaved plasmid pMC1403 to obtain plasmid pEG2.

  PEG2 has two ampicillin-resistant genes, and expresses the β-galactosidase gene derived from pMC1403 under the control of the promoter.

  using a start codon as well as the SD sequence included in the adapter.

  Fragments C-1-1 and C-1-2 have the following nucleotide sequence: Sequence SD start codon

  C-1-1: 5 'A G G A A C A G A T C T A T G 3'

  C-1-2: 3 'T C C T T G T C T A G A T A C C T A G 5'

  BglII The Miller method is used to confirm the expression of Bgalactosidase in the host ECI-2 harboring plasmid pEG2 (Miller, 3. (1972) "Experiments in Molecular Genetics" New York, Cold Spring Harbor Laboratory pp. 352- 355). This process is based on the reaction of Bgalactosidase with a synthetic ONPG substrate (o-nitrophenylgalactoside) to release a

  o-nitrophenol yellow compound. The Miller process will be described in more detail.

  An amount of 0.1 ml of culture of a species of bacteria, the absorption of which was measured at 610 nm, is mixed with 1.9 ml of assay buffer (0.1 M sodium phosphate, pH 7, 0.1mM magnesium sulfate and 0.1M mercaptoethanol) and stirred vigorously for 15 sec with 0.1ml toluene to increase the permeability of the bacterial species. Toluene is then removed

  by evaporation by means of a vacuum cleaner. With addition of 0.2 ml of solution -

  ONPG (solution of 400 mg of ONPG in 100 ml of assay buffer), the mixture is incubated at 30 ° C until a yellow color appears, after which 0.5 ml of 1 M sodium to stop the enzymatic reaction. The absorption of the reaction mixture is measured at 42.degree. im and

550 nm.

  The activity of β-galactosidase by units in 1 ml of the liquid culture according to the following equation, where the absorption at 610 nm is calculated as 1.0. Activity of - OD 420 - 1.75 x OD550 - 1 x 1000 galactosidase D) 4050X10 (units txvx DO610 (unit.9 t: incubation time (minutes) v: amount of specimen added to the reaction system (0.1 ml )

  DD610 absorption at 610 nm of the specimen.

  The above method, one practiced, reveals the following result.

  When the ECI-2 strain harboring gEP2 is incubated at 30.C, the activity of.

  13-galactosidase is 98 units. However, when the culture is still incubated at 42ZC for 1 h. activating the promoter = PL to result in β-galactosidase activity of 9637 units. This gives evidence that the 13-galactosidase i'PL promoter sequence is in

the order envisaged.

  Although pEG2 has two BglIL cleavage sites, only the BglII site present immediately after the SD sequence of β-galactosidase is needed, while the other site is undesirable. The plasmid is thus cleaved with BamHI and ligated again to remove a fragment having about 770 Pb. The construction of pEK28, which is an expression plasmid, is completed using a promoter; P L 7-3) Expression of the fused gene of the front half of 13-urogastrone

and 13-galactosidase.

  Fig. 9 schematically shows the series of methods to be described below. A plasmid pUG101 having a fused gene of half is constructed

  before S-urogastrone and 13-galactosidase as follows.

  More specifically, pugt, which is half-forward of the 13-urogastrone gene, and pMC1403 having a 13-galactosidase gene with BamHI are cleaved and ligated to form pUG101. With this plasmid, the front half of the B3-urogastrone gene and the B3-galactosidase gene are linked in the same frame. The plasmid therefore expresses the amino acid sequences of both: in the form of a fused protein; For expression with this plasmid under the control of the promoter

  PL pUG101 and pEK28 are each cleaved with BglII.

  Cleavage with BglII produces a DNA fragment having extrudate ends in the form of (:: A G)

\ .TCTAG).

  However, when the DNA fragment is reacted with a large fragment of DNA polymerase I of E. coli (Klenow fragment), in the presence of the 4 types of deoxyribonucleotide triphosphates dGTP, dATP, dTTP and dCTP, a blunt end is obtained by supplementing with the corresponding nucleotides giving an end. A AGATC: AT TCTAGff If we add dGTP only as a nucleotide component, nn contains the end of f.AG

È ... TCTAG)

  given the end of the reaction. Then, when using Si1 nuclease to digest the remaining single strand, a "blunt" end in the form of / AG is obtained. Even if dGTP and dATP are added in the Klenow reaction, then if digests with S1 nuclease, we obtain. AGA 'TCT) When the Klenow reaction is conducted by addition of dGTP. dATP and dTTP, then by digestion with S1 nuclease, we obtain AGAT \

È ... TCTA)

  Furthermore, if digestion with S1 nuclease alone is carried out without performing the Klenow reaction, the result is that when the DNA fragment is subjected to the (TCTA) end.

TCTAGJ

  the Klenow reaction using different nucleotides and / or the reaction of S1 nuclease gives five types of blunt ends

  which differ from one another by a length of a base pair.

  The Klenow reaction and the S1 nuclease reaction are conducted under the following conditions: Klenow reaction: 1 μg of DNA is reacted as substrate with 1 mM of each deoxyribonucleotide triphosphate at -12 ° C. for 30 minutes in the presence of a unit of the enzyme (Klenow fragment) and 1 mM ATP in 50 μl of a reaction medium comprising 40 mM potassium phosphate buffer

  (pH 7.4), 1 mM β-mercaptoethanol and 10 mM magnesium chloride.

* Reaction of S1 nuclease

  1 μg of DNA is reacted as substrate and 200 units of the enzyme at 20 ° C. for 30 minutes in 100 μl of a reaction medium comprising mM sodium chloride, 30 mM sodium abate and 5 mM sulphate

zinc (pH 4.5).

  pEK28 and pUG101 are both cleaved with BglII and then subjected to various combinations of the Klenow reaction and the reaction of the

  S1 nuclease, leaving fragments with 5 types of blunt ends.

  The two types of these fragments, when combined, provide 21 combinations which differ from each other in the number and sequence of nucleotides between the SD sequence and the start codon of the fused protein, such as

this is listed in Table 2.

  In fact, the DNA fragments thus obtained are cleaved with SalI to isolate fragments which contain the gene encoding the fused protein of the forward half of B-urogastrone and 13-galactosidase from

  pUG101, and fragments containing the? P promoter from pEK28.

  These fragments are bound by T4 DNA ligase in the combinations shown

in Table 2.

  Therefore, the recombinant plasmids are obtained in Table 3. The β-galactosidase activity of the fused proteins expressed by the Miller method is measured. Table 3 shows that all plasmids express a relatively high level of β-galactosidase activity. In particular

  pUG103, pUG104 and pUG117 achieve remarkable results.

Table 2

  Nucleotide sequence upstream of the start codon (ATG)

ATCTGC- GATCTGC-

-ACA -ACAATCTGC- -ACAGATCTGC-

p1. (pUG105) (UG106)

-ACAG -ACAGGATCTGC-

Nucleotide sequence.

  downstream of the sequence SD -ACAGA -ACAGAATCTGC- -ACAGAGATCTGC- <aL

<AGGA)

(, (pUGl13) (pUG114)

  -ACAGAT -ACAGATATCTGC- -ACAGATGATCTGC-

_______; (pUG117) (pUGll8)

  -ACAGATC -ACAGATCATCTGC- -ACAGATCGATCTGC-

  ___ ___ ,. ___ ___-_ _ (pUG121) (pUG122) Nt o o Table 2 (continued)

  ...., .. DTD: Nucleotide sequence upstream of the start codon (ATG)

TGC-CTGC- TCTGC-

-ACA -ACATGC- -ACACTGC- -ACATCTGC-

(pUG102 pUG3) (pUG104)

  -ACAG -ACAGTGC- -ACAGCTGC- -ACAGTCTGC-

  Nucleotide sequence in (pUG107) (pUG108) (pUG109) ', downstream of SD sequence

. -ACAGA '-ACAGATGC- -ACAGACTGC-:

(AGGA)

Z 'i: (pUG111) (pUG112)

-ACAGAT -ACAGATTGC- -ACAGATTCTGC-

(pUG115) _, .: (pUGl16)

  : -ACAGATC - -ACAGATCCTGC- -ACAGATCTCTGC-

', _;,. : '' '' ',:' P. '' ',' '

  i____ ____ ____ ____, ___ __, i (pUG1 9); (PUGl2)

*: ,, ',. . .. ',' \:

,,,,,,,,, s lu i, ',' 'o,

"'' '' Z

Table 3

  Number of nucleotides between the SD sequence and the recombinant eGalactosidase plasmid-codon (unitDs) 6 pUG102 1674 7 pUG103 2533 7 pUG107 2260 8 pUG104 2802 8 pUG108 1835 9 pUG105 1018 9 pUG109 1942 pUG106 973 11 pUGl1lO 1764 11 pUGl13 1950 11 * pUGl PUG114 946 12 pUGI17 2332 12 pUG112 1374 13 pUG118 2041 13 pUG121 1678 14 pUGl22 1814 Next, a vector is prepared for the expression of 13-urogastrone from pUG103 or pUG117. Dn cleaves the plasmid (pUG103 or pUG117) with HindII and PvuI] to obtain a 1.2 kb fragment containing the region from the P L promoter to the forward half of the 2-urogastrone gene. Then pUG2 was cleaved with EcoRI, complete with Klenow fragment, and cleaved with Hindill to obtain a 4.1 kb fragment. The two fragments are ligated with T4 DNA ligase, and E. coli strain ECI-2 is transformed. coli by the calcium method set forth above to obtain a recombinant containing the plasmid pUG103-E or pUG117-E for the expression of the combination of the front half and the back half of the B-urogastrone gene, that is, the entire B-urogastrone gene, under the control of the AP promoter

  8) Vector for the expression of the fused protein.

  A β-lactamase gene is linked to the plasmid pBR322-and a B3urogastrone gene to express S-urogastrone as a protein

  merged as will be described below.

  8-1) B-lactamase gene donor.

  PBRH02 was obtained by cleaving pBR322 with Aval and PvuII, followed by Klenow reaction and ligation with T4 DNA ligase. This plasmid has genes for ampicillin resistance (ApR) and tetracycline resistance (TcR) as markers. PBRHO3 is obtained by cleaving pBR325 with Aval and HindIII, followed by Klenow reaction and ligation.

  and he has Ap R and chloramphenicol resistance (CmR) as markers.

Fig. 10 shows these plasmids.

  8-2) 3-urogastrone gene donor.

  PUG3 prepared as previously described with MboII was cleaved to obtain 13 types of DNA fragments, which are referred to as A to M in size order as shown in FIG. 11. On these DNA fragments, it is found that fragment H is composed of 179 bp starting from a nucleotide coding for asparagine at the N-terminus of 13-urogastrone and ending with 16 bases downstream db stop codon, the fragment having all the structural gene of the i-urogastrone. -iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii The fragments are electrophoresed on 6% by weight polyacrylamide gel.

and the fragment is purified.

8-3) Adapter.

  For the adapters, the oligonucleotides listed in Table 4 are prepared by the same method as that already discussed. These adapters are designed to encode the basic amino acid pair of Lys-Arg or Arg-Lys to enzymatically cleave B-urogastrone from the

expressed fusion protein.

2S66799

Table 4

  Adapter End 5 '. 3 'end

D-1-3 CCGTAAG -

D-1-4 TTACGG: -

D-2-1 CGTAAG

D-2-2 TTACG

D-3-2 TTACGGAT

D-4-2 TTACGTGCA:

E-1 CGCTAAACGG

E-2 CGTTTAGCG

E-3 GACAAACGG

E-4 'CGTTTGTC

E-5 CGTTTAGCGAT

E-6 CGTTTGTCTGCA

E-7 CGGCTAAACGG

E-8 CGTTTAGCCG:

E-9 CAAACGG ...

E-10 CGTTTG :: -

  8-4) System for expression of fused 13-lactamase protein and

  of 8-urogastrone linked by Lys-Arg.

  A vector for the expression of the fused Blactamase-B-urogastrone protein is prepared so that a recognition sequence

  restriction enzyme is generated in the region containing an adapter.

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

  The process shown in FIG. 12.

  Plasmid pBRH02 was completely cleaved with XmnI at 37 C over a period of 3 h. 3 μg of the vector is then ligated, approximately 0.1 μg of the fragment

  B-urogastrone of 179 bp and about 1 μg each of E-1 and E-2 (with the

  5 'unphosphorylated mite) serving as a single step adapter at 12 C

  during a period of 15 hours to obtain plasmids in vector form.

  expression. The strain HB101 is transformed using plasmids

by the calcium process.

  Of the 499 TcR colonies, 168 colonies (33.7%) are ApS. In these colonies, the size of the plasmid DNA is checked by mini-preparation. Thirteen plasmids are about 200 bp larger than the vector and considered to have a B-urogastrone gene inserted therein. All, which have a MluI site, are cleaved with HinfI and verified for the orientation of insertion of the 13-urogastrone gene by 1.5% by weight agarose gel electrophoresis. Three of those verified gave fragments of about 1050 bp and about 800 bp. This indicates that the β-urogastrone gene is inserted in the same orientation as β-lactamase. These three plasmids

are called pUG2301 to pUG2303.

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

  The process shown in FIG. 13.

  Plasmid pBR322 is used as a plasmid vector that has a unique PvuI site in the β-lactamase gene. Selects the process described in Figure 1, in which:

adapter.

  When verifying in the 1626 TcR colonies obtained the sensitivity Ap, 31 colonies (1.9%) are Ap S. a mini-preparation for 22 colonies Tc R and ApS, and the plasmids were checked for the insertion of the β-urogastrone gene by cleavage with MluI. Z0 plasmids are found to have a Mlu site. Orientation is checked by cleaving with Hinf or BamHI. There is a Mlul site. The orientation is checked with HinfI or BarnilL. It is found that the plasmids pUG2101 to pUG2105 have a B-urogastrone gene which

  is inserted in the same orientation as the β-lactamase gene.

  8-4-c) Construction of pUG2701 to pUG2703 Expression plasmids were constructed by the same method as in 8-4-a) using pBR322 as vector and E-7 and E-8 as an adapter,

as seen in fig. 14.

  Of the 217 TcR colonies, 106 colonies (48.8%) are ApS. A mini-preparation is carried out for 25 of these colonies. Eight of the plasmids are about pb larger than the vector and appear to have an inserted Burogastrone gene, so that these 8 plasmids are cleaved and checked for orientation.

  of the gene. Therefore, it is found that 3 plasmids have the B-gene

  urogastrone inserted in the same orientation as β-lactamase and are

referred to as pUG2701 to 2703.

  The plasmid pUG2301 obtained by the process 8-4-a) above produced

  a fused protein of a β-lactamase and β-urogastrone fraction.

  The predicted amino acid sequence and the corresponding nucleotide sequence

  are presented below.

Met Ser I the Gin His Phe Arg Val

ATG AGT AT OAA CAT TTC CGT GTC

Ala Leu I Pro Phe Phe Ala Ala

GCC CTT ATT CCC TTT TTT GOG GCA

Phe Cys Pro Leu Val Phe Ala His

TT TGC CTT OCT GTT TT GCT CAD

Pro Glu Thr Leu Val Lys Val Lys

CCA GAA ACG CTG GTG AAA GTA AAA

Asp.Ala Asp Glin Glin Leu Gly Ala

GAT GCT GAAT GAT CAG TTG GGT GCA

Arg Val GIy Tyr [Glu Leu Asp

CGA GTG GGT ATC TAC GAA CTG GAT

Leu Asn Ser Gly Lys I Leu Glu

CTC AAC AGC GGT AAG ATC 'CTT GAG

Ser Phe Arg Pro Glu Glu Arg Ala

AGT TT CGC CCC GAA GAA CGC GOT

Arg Asn Ser Asp Lys Ser Glu Cys

AAA CGG AGC AAT GAT TCT GAG TGC

Pro Leu Ser His Asp Giy Tyr Cys

CCA CTG TCT CAC-GAT GGC TAT TGT

Leu His ASP Gly Val Cys Met Tyr

CTG. CAC GAC GG T GTT TGC ATG TAC

I leGlu Ala Leu Asp LYs Tyr Ala:

ATC GAA TTG GCT GAT AAA TAC GCG

CYs Asn Cys Valley Val GIY Tyr: I the

TGT AAC TGT GTG GTG GGT TAT ATC-O

GYI Glu Arg Gln Cys Gln Tyr Asp Asp

GGT GAA CGC TGT CAA TAC CGT GA-T

  Leu Lys Trp Trp Glu Leu Arg (stop)

CTG AAA TGG TGG GAA TTG CGT TAA;

TAGTGAAGATCTGGATCCGTTTAGCGTTTTCCA

ATGATGAGCACTTTTAAAGTTCTGCTA-TGTGGC

  GCGGTATTATCCCGTGTTGACGCCQGGGCAAGAG -

CAACTCGGTCGCCGCATAC

  Similarly, the plasmid pUG2101 obtained by the method 8-4-b) produces a fused protein having the primary structure:

next: -

Met Ser Gln His Phe Arg, Val

ATG AGT CAA ATT CAT-TTC CGT = GTC

  : '-: -i Ala Leu I the Pro Phe Phe a Aa: Ala

GCC CTT ATT CCC -TTT TT GCG GCA

Phe Cys Pro Leu Val Phe Ala His

TT-TGC CT COT GTT TT GCT CAC

Pro Glu Thr Leu Val Lys Val Lys

CCA GAA ACG CTG GTG AAA GTA AAA

Asp Ala Glu Asp- Gin Leu Gly Ala

GAT GCT GAAT GAT CAG TTG GGT GCA

Arg Val Gly Tyr I leGlu Leu Asp

CGA G-TG GGT ATC-GAA TAC CTG GAT

Leu Asn Ser Gly Lys binds Leu Glu

CTC AAC AGC GGT AAG ATC CTT GAG

Ser.Phe Arg Pro Glu Glu Arg Phe

AGT TT CGC CCC GAA GAA CGT TT

Pro Met Met Ser Thr Phe Lys Val

CCA ATG ATG AGC ACT TTT AAA GTT

Leu Leu Cys Gly Leu Val Leu Ser

CTG CTA TGT-GGC GCG GTA TTA TBI

Arg Ala GIy Gin Glu Gin

CGT GTT GAC GCC GGG CAA GAG CAA

Leu Gly Arg Arg I leHis Tyr, Ser

CTC GGT CGC CG-C ATA CAC TAT TCT

Gin Asn Asp I the Val Glu Tyr Ser

CAG AAT TTG GAC GTT GAG TAC TCA

Pro Thr Thru Glu LYS His Leu Thr

CCA GTC ACA GAA AAG CAT CTT ACG

Asp Gly Met Thr Val Arg Glu Leu

GAT GGC ATG ACA GTA AGA GAA TTA

Cys Ser Ala Ala I Thr Met Ser

TGC AGT GCT GCC ATA ACC ATG AGT

Asp Asn Thr Ala As Leu Leu

GAT AAC ACT GCG GCC AAC TTA CTT

Leu Thr Thr I theAla Lys Arg Asn

CTG ACA ACG ATC GCT AAA CGG AAT

Ser Asp Ser Glu Leu Ser Cys Pro

AGC GAT TCT GAG TGC CCA CTG TCT

His Asp Gly Tyr Cys Leu His ASP

ACC GAT GGC TAT TGT CTG CAC GAC

Gly Val Cys Met Tyr I leGlu Ala

GGT GTT TGC ATG ATC TAC ATC GAA GCT

Leu Asp Lys Tyr Ala Cys Asn Cys

TTG GAT AAA TAC GCG-TGT AAC TGT

Val Val Gly Tyr the Gly Glu Arg

GTA GTG ATC GGT TAT GGT GAA CGC

Cys Gln Tyr Arg Asp Leu Lys Trp

TGT OAA CGT TAC GAT CTG AAA TGG

Trp Glu Leu Arg (stop)

TGG GAAT TTG CGT TAA TAGTGAAGATC

TGGATCCGTTTAGCGATCGGAGGACCGAAGG

AGCTAACCGCTTTTTTGCACA

  Similarly, the plasmid pUG2701 obtained by the method 8-4-c) produces a fused protein having the following primary structure: Met Ser I Gin His Phe Arg Arg

ATG AGT ATT CAT CAA TTC CGT GTI

Ala Leu I Pro Phe Phe Ala Ala

CCG CTT ATT CCC TTT TTT GCG GCA

Phe CYS Pro Leu Val-Phe Ala His

TT TGC CTT CCT GTT TT GCT CAC

Pro Glu Thr Leu Val Lys Val Lys

CCA GAA ACG CTG GTG AAA GTA AAA

Asp Ala Glu ASP In Leu GIY Ala

GAT GCT GAAT GAT CAG TTG GGT GCA

Arg Val Giy Tyr I the Glu -Leu Asp

CGA GTG GGT ATC TAC GAA CTG GAT

Leu Asn Ser Gly Lys I Leu-Glu

CTC AAC AGC GGT AAG ATC CTT GAG

Ser Phe Arg Pro.Glu Glu Arg Phe

AGT TT CGC C0CC GAA GAA CGT TT

Pro Met Met Ser Thr Phe LLYS Val

CCA ATG ATG AGOC ACT TT AAA GTT

Leu Leu Cys Gly Leu Val Leu Ser

CTG CTA TGT GGC GCG GTA TTA TCCO-

  Arg Asp Asp Ala Gly Gin Glu - Gin- -

CGT GTT GAC GCC GGG CAA GAG -CAA

Leu Gly Arg Arg the F HisTyr-Ser,

CTC GGT CGC CGC ATA CAC TAT TCT

Gin Asn AsP Iie Val Glu Ser- Ala

CAG AAT TTG GAC GTT GAG TCG GOT

Lys Arg Asn Ser Ser Asp Glu-Cys

AAA CGG AGC AAT GAT TCT GAG TGC

Pro Leu Ser His Asp Gly: Tyr Cys

CCA CTG TCT CGAC GAT GGC TAT TGTLeu His Asp Gly -Vai Cys Met -TYr-

CTG CAC GAC GG T GTT: TGC ATG TAC

I leGlu Ala Leu Asp Lys -Tyr Ala

ATO GAA GCT TTG GAT- AAA TAC GCG

Cys Asn Cys Val Val Gly Tyr I

TGT AAC TGT GTG GTG GGT ATC ATC

Gly Glu-Arg Cys Gin Tyr Arg Aspv,

GGT GAA CGC TGT CAA TAC CGT GATI

  Leu Lys Trp Trp Glu - Leu Arg (arr4t)

CTG AAA TGG TGG GAA TTG. CGT TAA

TAGTGAAGATCTGGATOCCGTTTAGCCGACTCAC

CAGTCACAGAAAAGCATCTTACGGAT

  The nucleotide sequences coding for the fused proteins in pEG2101, pUG2301 and pUG2701 are analyzed by the method

of Maxam-Gilbert.

  Photo 5 shows the results of the analysis.

  - If one fades to the picture 5, the paths 1 to 4 show the result obtained with the MluI-PstI fragment (224 pb) of pUG2101, the ways 5 to 8 show the result with the MluI-EcoRI fragment (721 pb ) of pUG2101, the paths 9 to 12 show the result with the MluI-BamHI fragment (452 bp) of pUG2301, the paths 13 to 16 the result with the MluI-PstI fragment (335 bp) of pUG2701, and the paths 17 to Show the result with the Mlul-EcoRI fragment (610 bp) of pUG2701. Paths 1, 5, 9, 13 and 17 show the reaction products for guanine, paths 2, 6, 10, 14 and 18 show the reaction products for guanine plus adenine, routes 3, 7, 11, 15 and 19 show the reaction products for thymine plus cytosine, and the pathways 4, 8, 12, 16 and 20 show the reaction products for

  cytosine. The fraction marked with "3" is an adapter.

  8-5) System for expression of fused β-lactamase protein and

3-urogastrone linked by Arg-Lys.

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

  The 13-urogastrone gene is inserted into the 13-lactamase gene of PBR322 at its sit. pvuI restriction vector to obtain vectors for expression of fused B-lactamase and Burogastrone protein

shown in fig. 15.

  Plasmid pBR322 was cleaved with Pvu I at 37 C over a period of 3 h. Some of the plasmids were checked by agarose gel electrophoresis at 1% by weight to confirm that they had been completely cleaved. Adapters D-1-3 and D-3-2 are ligated to fragment at 12 for a period of h, and the bound product is subjected to 1% by weight agarose gel electrophoresis to isolate a fragment of DNA. Then, the B13-urogastrone fragment and the vector are mixed in a molar ratio of about 5: 1 and ligated at 12 ° C. for a period of 15 hours. After ligation, strain HB101 is transformed with the resulting plasmid, and colonies are selected.

referring to Tc.

  71 colonies transformed with TcL are obtained and their Ap sensitivity is verified. The plasmid DNA is prepared from 20 Ap S colonies (28.2%) and then the presence of the MluI restriction site is verified to confirm the insertion. of the B-urogastrone gene. It is found that 5 out of 20 plasmids have the MluI site of the 13-urogastrone gene. The DNA was cleaved with HinfI and then subjected to 1.5% by weight agarose gel electrophoresis to verify the orientation of the insertion. It is found that two of the plasmids, at

  know pUG1102 and pUG1105, have the gene in the correct orientation.

  8-5-b) Preparation of pUG1004, pUG1201 and pUG1301.

  Process 8-5-a) is repeated using PstI, HincII and XmnI instead of PvuI to obtain pUG1004, pUG1201 and pUG1301 as seen

  in figs. 16, 17 and 18, respectively.

  9) Confirmation of the expression of 8-urogastrone.

  Expression plasmids thus constructed are used to transform E. coli, HB101 or ECI-2, and the cells are cultured by the following method,

  followed by extraction and radioimmunoassay to confirm the expression.

  9-1) Culture of recombinant microorganisms with the Burogastrone gene

and protein extraction.

  9-1-a) Expression system using the PL promoter

  The EC-2 strain harboring the expression plasmid pUG103 was cultured.

  E and the same strain abbritant expression plasmid pUG117-E at 25 C in two flasks each containing 1 liter of LB culture medium. When the culture in one of the flasks has an absorption of 0.3 to 600 nm, the culture is subjected to induction of heat at 42 C for 1 h. The culture of the other bottle is continuously incubated at 25 ° C until the absorption becomes 0.4. Cells from each vial were collected, washed with STP buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM dibasic sodium phosphate, and sodium phosphate).

2566? 99

  1.5 mM monobasic sodium (pH 7.0), then resuspended in STP buffer in 3% vol. of the original culture amount and destroyed (at 100 W for 30 sec, 3 times) by an ultrasonic device (Model 5202, product of Ohtake Works Co., Ltd., 3apon) under cooling with ice . The supernatant separated from the cellular extract is dialysed by ultracentrifugation (at 40000 g for 1 h) against a 0.01 N aqueous solution of acetic acid, and the dialysate is lyophilized and then subjected to radioimmunoassay (called

below "RIA").

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

  The strain of E is preincubated. coli HB101 harboring the plasmids pUG1004, 1301, 2101, 2303 or 2703 at 370C in a culture medium containing Eg / ml of tetracycline, then diluted to volume ratio of 1: 100 with the same medium and cultivated until the absorption at 660 nm becomes 0.4. Cells were collected, washed with STP buffer, then resuspended in STP buffer in 3% v / v of the original culture amount and treated with ultrasound (at 100 W for 30 sec, 3 sec. times)

  same ultra-sound device as above cooling with ice.

  The supernatant separated from the cell debris was dialysed by ultracentrifugation (40000 g for 1 h) against a 0.01 N aqueous solution of acetic acid,

  it is lyophilized then submitted to RIA.

  To confirm the accumulation of fused protein in the cytoplasm, the cytoplasmic fraction is prepared according to S.J. Chan et al., Proc. Natl. Acad. Sci. USA, 78, 5401-5405 (1981)). A fraction of the culture is diluted to a volume ratio of 1: 100 with fresh culture medium E (1 liter of aqueous solution of 10 g of dibasic potassium phosphate, 3.5 g of sodium-ammonium , 2 g of magnesium sulphate 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 hydrochloride of tetracycline) then grown at 370C until the absorption at 660 nm becomes 0.4. We collect the

  cells (6000 rpm, 10 minutes) and washed twice with a mixture of

  10 mM HCl (pH 8.0) and 30 mM sodium chloride. The cells (1 g) are resuspended in 80 ml of 20% by weight tris-HCl 30 mM sucrose (pH 8.0), whereupon EDTA is added to the suspension to a concentration of 1 ml. mM. The mixture was shaken with a rotary shaker at 180 rpm for 10 minutes (Z4-L) and then resuspended (13000 g, 10 minutes) to collect the cells, which was resuspended in 80 ml of distilled water. The suspension is allowed to sit in ice for about

  minutes stirring occasionally and then centrifuged (13000 g, 10 minutes).

  The supernatant is collected as a cytoplasmic fraction (O-

  Sup). The pellet is suspended in a mixture of 10 mM Tris-HCl (pH 8.0) and 30 mM sodium chloride and treated by the same apparatus.

  with ultrasound as above to obtain a cytoplasmic fraction (O-Ppt).

These samples are submitted to RIA.

9-2) Radioimmunoassay.

  9-2-a) Establishment of the RIA system.

  Rabbits are immunized with purified human B-urogastrone as an antigen to obtain the antiserum. The 13-orogastrone is dissolved

  (300 μg) in 0.2 ml of distilled water, 1.5 ml of solution is added to the solution.

  50% polyvinylpyrrolidone, and the mixture is stirred for 2 hours at RT.

  Freund's complete adjuvant (2.0 ml) was added to the mixture to obtain an emulsion, which was injected subcutaneously into the rib cage of three rabbits. After repeated immunization 4 times every 2 weeks, 50 t of the antigen is injected intravenously.

  whole blood 3 days later, and the serum is separated.

  The following RIA conditions are then determined for the titration curve to determine the degree of dilution of the antiserum for the assay, the incubation time to optimize the dosing conditions, the method of separating the radiolabeled antigen. bound (bound) with free radiolabeled antigen (free), etc. The diluent solution used is a phosphate buffer (10 mM, pH 7.4) containing 0.5% by weight of beef serum albumin (BSA), 140 mM sodium chloride and 25 mM disodium EDTA. The diluent solution (400 μl), 100 μl of the sample or standard human B-urogastrone and 100 μl of anti-human urogastrone serum are mixed. After incubation for 24 h at 4 ° C., 100 μl of 125I-labeled 13-urogastrone solution (approximately 5000 cpm) was added to the mixture. After again incubating the mixture for 48 h at 4 ° C, add to the mixture

* obtained 100 μl of second antibody (goat serum

  rabbit) (dilution at 20 ° with STP buffer), 100 μl of normal rabbit serum (200-fold dilution with STP buffer) and 900 μl of 5% by weight glycol containing buffer, and then the polyethylene glycol is further incubated. at 5% by weight, and incubated for 3 h at 40C. The culture is centrifuged for 30 minutes at 3000 rpm, the supernatant is separated, and the precipitate is counted. The content of the immunoreactive substance in human S-urogastrone form in the sample is determined from the standard curve obtained using standard human β-urogastrone. 9-2-b) Confirmation of the B-urogastrone Productivity of the Recombinant Microorganism. Table 5 shows the result of the RIA conducted for the expression system using the P L promoter.

TABLE 5

  Plasmid Induction Amount - -

  Urogastrone heat expression produced (ng / fl of culture) pUG103-E yes 450.2 pUG103-E No 3.2 pUG117-E Yes 388.4 pUG117-E No 3.2 Control - no. Not detectable (pBR322). Table 6 shows the result of the RIA conducted for the systems

  expression of fused proteins.

TABLE 6

Plasmid Quantity of -

  urogastrone expression expression produced (MS / 1 culture) pUG1004 729.6 pUG1301 650.7 pUG2101 31.3 pUG2301 125.2 pUG2701 119.2

  Table 7 shows the location of the expressed fused protein.

TABLE 7

  Amount of -urogastrone produced Plasmid (vg / 1 culture) expressive Periplasmic fraction Cytoplasmic fraction (O-Sup) (O-Ppt) pUG1004 326.0 4.0 pUG1301 347.4 2.8 pUG2101 79.9 10.2 pUG2301 118.8 '4.1

pUG2701 65.7 3.6.

  Tables 5 and 6 reveal that the P promoter system for L and the direct expression of B-urogastrone and the system for expression

  of the compound in the form of fused protein both express the immuno-

  reactivity of B-urogastrone in E. coli. Table 7 reveals that in the case of the fused protein, the immunoreactivity of the expressed B-urogastrone

  is almost entirely localized in the cytoplasm.

Claims (20)

1. A novel 13-urogastrone gene having the following nucleotide sequence: AA T-AGCGATTCTG; AGT G-CCCA CTG 3'T TA TCGCTAAGACTCACGGGTGAC T CT CACGA T-GGCTA TG TG: TCTGCACAGAGTGCTACCGAT AACAG AA AG C- GTGG AC GGTGTTTGCATG TAC ATCG AA C TG CCACAAACGTACA: T G_TAGCTTGCTTT GG ATAAATAC GC GT GTAACC GA AACCTATTTATGCG: CA: C AT T GC TTGTGT AG TGGGTT AT ATCGGTG AA TO CA CATCACCCAATA TACG CCA CTT C GC TGTCAATA CC G: TGATCTGAAAGCG AC AGTTAT GG C A-C T- AGACTTTTGGTGGGAAGGCG T 3 ':: ACCACCCTTAACGCA 5'   2. The subunit of the gene defined in claim 1, the subunit having the front half of the nucleotide sequence of claim 1 and divided about halfway.   3. The subunit of the gene defined in claim 1, wherein the subunit has the back half of the nucleotide sequence of claim 1 and is divided approximately to half thereof. 4. A gene as defined in claim 1 which has a restriction enzyme recognition site attached to each of the front and / or rear ends of the gene.   A gene as defined in claim 4 which has a restriction enzyme recognition site and a start codon provided upstream of the gene and / or   a stop codon and a restriction enzyme recognition site provide   o. cn 4a a (<H CDE C 0-4 <ó E 03 ó C) CD ca Lf 'o cU ZEqOC) OE ^ ó E9 in n 1 <E-Ic ó0 (3E-óo E- ó oom, C E4s < ## EQU1 ## where: ## STR1 ## ## STR2 ## % n O <H E'.03 03Q- <0 E-4 <E- -' this-   o. at). E-4 E- '' ó C) <E4 E-ó E-E'ó Oc  n G @ (0 (30 (E- 'd' ó - <E-   ## EQU1 ## "ci -, 4 (30 E-i cC'E OO,: a cc- 'ó30 E <CD -4' <E-4O <OE. d0 H <EE4 4 Cu E-4 '-' "- * E- < c e. 4.E-o a u 300- '<- tD 'c g ,.   -. ## EQU3 ## -f <00 O O4 E ó Sn CD ó - c-   ## STR5 ##   OAIO. E-E-4 '* H <E -' <E '(' <E- 'H H-   CI) w '<' <- '' E- 'CE-4' <aE-E "<E '' E- CD IDL   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 to its half, the subunit further having   a restriction enzyme recognition site & its rear end.   The subunit as defined in claim 7 having the following nucleotide sequence:   'A AT T CG A AT G AT G AT AGC AT AGC   3 'G C TT C T A G A C G AT C TT AT CG   G A T T C T G A G T G C C C C C T C T C AC   CT A A G A CT A C G G G T GAC AG A GTG   GAT GGC T AT T GT C T G C AC GA C GGT   CTA CC G A T A A C A G AC GTG CTG C CA   GTT T G C A T G T A C A T C GA T G CT T CG   C AA A C G T A C A T G TAG CTT C G A -A GC 3 ' CTA G 5 '   The subunit of the gene defined in claim 6, the subunit having the back half of the nucleotide sequence defined in claim 6 and divided approximately to its half, the subunit further having   a restriction enzyme recognition site at its front end.   The subunit as defined in claim 9 having the following sequence of nicletotides:   'A G CT T T G G AT A A T A C G C G T GT A C C T A T T T T T C G C A C A   AAC T G T G TA GT G G G T AT AT C G GT   T G A C A C AT C AC CC A AT A T A G C CA   GAA C G C T G T C A T T C C G T G AT CTG   CT TG C G AC G T T G G C A C T A G T C   A AA T G G T GG G Y TT TT G -CGT T A A TAG   TTT A C C AC C C T T AA CG CA AT AT C T G A A G T C T G 3 ' ACT T C T A G C C T AG 5 '   11. Recombinant plasmid having the gene defined in claim 6.   A process for preparing the recombinant plasmid as defined in claim 11 wherein the subunit defined in claim 8 and the subunit defined in claim 10 are inserted into a suitable insertion site of a vector. appropriate plasmid. 13. Recombinant plasmid comprising a plasmid vector where is inserted the Burogastrone gene defined in claim 6, with further insertion into the plasmid vector upstream of the B-urogastrone gene a promoter   to control gene expression and an SD sequence attached to the promoter.   A recombinant plasmid comprising a plasmid vector having the sequence of the fourth base pair, and the following, of the Burogastrone gene defined in claim 6, the plasmid vector having the combination of a promoter, an SD sequence and a another gene inserted into it upstream of the B-urogastrone gene.   15. The recombinant plasmid as defined in claim 14, wherein said   Another gene is a β-lactamase gene.   16. Recombinant plasmid as defined in claims 13 and 14 o the promoter is FPL or lac UV5.   17. Recombinant plasmid as defined in claims 13 and 14 the plasmid vector is pBR322.   A transformant comprising a host cell having a recombinant plasmid capable of expressing the β-urogastrone gene defined in claim 1.   19. The transformant as defined in claim 18, wherein the plasmid recom-   is the plasmid defined in claim 13. 59 2566799   20. Transformant as defined in claim -18 o the plasmid recom-   kicking is. that defined in claim 1 4.   21. Transformant as defined in claim 18 o-a host cell is E. coli.   22. The transformant as defined in claim 18, wherein the host cell is transformed with a recombinant plasmid having a TR gene and a CI857 gene.   23. A process for producing a transformant by transforming a host cell with a recombinant plasmid capable of expressing the urogastr gene. one defined in claim 1.   24. A process for producing β-urogastrone, characterized in that the transformant defined in claim 18 is cultivated and in which one collects the expressed B-urogastrone. -