FR2508928A1 - 27-DESAMIDOSECRETINE, PREPARATION THEREOF BY RECOMBINANT DNA TECHNIQUE, AS WELL AS TRANSFORMED ESCHERICHIA COLI STRAIN, POLYDESOXYRIBONUCLEOTIDES WITH TWO CHAINS AND PLASMIDS USED IN THIS PREPARATION - Google Patents

Abstract

27-Deamidosecretin, which is defined by a specific amino-acid sequence, is described. The preparation of the protein by recombinant DNA technology is likewise described, using a transformed E. coli strain, double-stranded polydeoxyribonucleotides and plasmids for the preparation.

Description

27-deamidosecretin, preparation thereof by the recombinant DNA technique, as well as transformed Escherichia coli strain, two-chain polydeoxyribonucleotides and plasmids used in this preparation.

 The invention relates to 27-deamidosecretin (U27n will sometimes be omitted hereinafter) and its preparation. More particularly, the invention relates to a process for the preparation of deamidosecretin by a genetic engineering technique using a structural gene suitable for desamidosecretin and chemically synthesized.

 Secretin is a compound known as one of the gastrointestinal hormones. It is known that it has physiological activities such as an action promoting the secretion of water and bicarbonates by the pancreas and is practically used for a test of pancreatic function or as a therapeutic agent for the pancreas. duodenal ulcer.

 In the remainder of the present description, reference will be made to the following publications which, for the sake of brevity, will be designated by reference numerals 1) to 24) of the following list.

References 1) Acta Chemica Scandinavica, 15, 1790, (1961) 2) a) Chemical industry, 1966, 1757,
(b) Journal of the American Chemical Society, 89,
6753 (1967); 3) Proceedings of the National Academy of Sciences
the United States of America, 75, 3737, (1978) 4) a) Science, 198, 1056, (1977),
b) Proceedings of the National Academy cf Sciences
of the United States of America, 75, 5765, (1978),
(c) Nature, 281, 18, (1979),
d) Biochemistry, 1980, 6096, (1980) 5) Science, 203, 614, (1979); 6) a) Nucleic Acids Research, 8, 5491, (1980),
b) Nucleic Acids Research, 8, 5193, (1980),
c) Tetrahedron Letters, 21, 4159, (1980)
d) Nucleic Acids Research, 8, 2331, (1980);
7) a) The Journal of Biological Chemistry (J. Biol.Chem.),
241, 2014, (1966),
b) Nucleic Acids Research, 1, 1665, (1974);
8) Nucleic Acids Research, 8, 5753, (1980);
9) Chemical and Pharmaceutical Bulletin, 26, 2396, (1978) 10) Genetic Engineering, 1978, 17, (1978) 11) Guidelineafor Recombinant DNA Experiment, published in
August 1979 12) The Japanese Journal of Pharmacology, 21, 325, (1971) 13) a) Chemische Berichte, 105, 2508, (1972),
b) Chemische Berichte, 105, 2515, (1972) 14) Tetrahedron Letters, 1978, 2727, (1978); 15) Proceedings of the National Academy of Sciences of the
United States of America, 73, 3900, (1977) 16) Gene, 2, 95, (1977) 17) Proceedings of the National Academy of Sciences of the
United States of America, 76, 106, (1979); 18) The operon, 31 (1980) (& it by JH Miller, WS Resni
koff, published by Cold Spring Harbor Laboratory); 19) Proceedings of the National Academy of Sciences
of the United States of America, 74, 1507, (1977); 20) Additional volumes of nProtein, nucleic acid & BR <
enzymes, last volume p. 19, (1973) 21) Methods in Enzyioiaogy, 68, 265, (1979) 22) Microbiological Reviews, 44, 1-50, (1980) 23) Nature, 224, 768, (1969) 24) Nature, 217, 1110 , (1968).

It is recalled that the secretin is a polypeptS.de which has the structural formula
5 10
His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser
15 20
Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu
Leu-Gln-Gly-Leu-Val-NH2
In order to obtain this secretin, methods have been proposed such as the extraction of a secretin of natural origin (typically porcine secretin), but it has also been found that the amino acid sequence of bovine secretin is identical to that of porcine secretin) and chemical synthesis. However, each of these processes has drawbacks with regard to cost, speed, yield, etc. In addition, according to the prior process in which the secretin is extracted from the small intestine of pigs and purified, it has been found that a difficulty in that the multitude of gastrointestinal hormones (motilin, VIP, CCKPZ, GIP, GLI etc.) is opposed to precise biological or radioimmunological assay of secretin.

 Furthermore, recent advances in the so-called genetic engineering technique using a synthetic gene have been so notable that it is now beginning to commercially make various physiologically active polypeptides by this technique. Therefore, if the secretin could be prepared by genetic engineering, it would be possible to discover indications for solving the problems mentioned above and which arise in the previous processes.

 it is generally established that it is possible to prepare a polypeptide by a genetic engineering technique by using a synthetic gene by steps which include the chemical synthesis of a structural gene, the insertion of the gene into a suitable plasmid serving as a vector, the transformation of a suitable host and the preparation and recovery of a desired polypeptide, by culture of the transformant, but it is not necessarily possible to know in advance with certainty if it can, by this method, to obtain a particular polypeptide having a desired physiological activity.

 If this problem is considered in the case of secretin, since the C-terminus of the secretin polypeptide is a valine amide, as indicated above, the end of the DNA of the structural gene expressing the The polypeptide must necessarily comprise the codon corresponding to valine, and, therefore, the resulting polypeptide is considered to carry valine as endpoint C. With this reasoning, it is doubtful whether the polypeptide obtained has the physiological activity of secretin.

 The invention has been confirmed by confirming that a structural gene expressing a secretin derivative whose end C is valine in a non-amide form, thus the disamidine dosecretin, can be synthesized chemically, which can be inserting this gene into a suitable plasmid serving as a vector to form a chimeric plasmid, it is possible to transform with the chimeric plasmid an appropriate hSte cell as well as to prepare and recover the deamidosecretin by cultivating the transformant, that the deamidosecretin obtained has a similar activity to that of secretin and, furthermore, that the lactose operon expression system can be used elsewhere in the preparation of deamidosecretin and that the yield of deamidosecretin can be greatly increased by using this system expression.

Thus, the 27-deamidosecretin according to the invention constitutes a polypeptide represented by the amino acid sequence
His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser Arg Leu-Arg-Asp-SerAl α-Arg-Leu-Gln Arg -Leu-
Leu-Gln-Gly-Leu-Val-CH
wherein Val-OH indicates that the C-terminus of the polypeptide comprising valine is a carboxylic acid.

The invention also proposes a process for preparing 27-deamidosecretin comprising the following steps
(1) chemically synthesizing a structural gene suitable for the deamidosecretin, corresponding to the polypeptide in which the amino acid of the C-terminus is the line,
(2) inserting this gene into a vector plasmid capable of proliferating in a host cell to form a chimeric plasmid capable of proliferating in the cell,
(3) transforming the host cell with the chimeric plasmid,
(4) The resulting transformant is cultured and the deaminosecretin formed is recovered.

Another preferred method for the preparation of 27-deamidosecretin according to the invention comprises the following steps
(1) chemically synthesizing a structural gene suitable for the desamidosecretin, corresponding to the polypeptide in which the amino acid of the C-terminus of the secretin is valine,
(2) there is provided a vector plasmid capable of utilizing the lactose operon and also capable of proliferating in a predetermined hte cell,
(3) inserting the structural gene into the vector plasma to obtain a chimeric plasmid capable of proliferating in the cell,
(4) transforming the host cell with the chimera plasma,
(5) the resulting transformant is cultivated and the de-amidosecretin formed is recovered.

The process for the preparation of 27-deamidosecretin according to the invention comprises, in its broadest aspect, the following steps
(1) there is provided a chimeric plasmid which comprises a fragment of a structural gene suitable for a deamidosecretin corresponding to a polypeptide in which the amino acid of the C-terminus is valine, the chimeric plasmid being capable of proliferating in a cell predetermined host and being capable of expressing the appropriate structural gene to a deamidosecretin in the host cell,
(2) the hut cell is transformed with the chimeric plasmid,
(3) the transformant obtained is cultivated and the deamidosecretin formed is recovered.

 A typical example of host cells for use in the transformation method defined above is Escherichia coli, belonging to the genus Escherichia, and the lactose operon is derived from Escherichia coli.

Thus, the preferred method of preparing the 27-deamidosecretin according to the invention can also be defined as a process which consists in cultivating a microorganism producing deamidosecretin belonging to the species
Escherichia coli, to which was incorporated a plasmid containing the deamidosecretin structural gene and to recover the desamidosecretin formed when using lactose loperon in Escherichia coli.

 In another aspect, the invention also relates to an Escherichia coli culture to be used in the practice of the invention, this Escherichia coli having been transformed so as to have a phenotype such that the strain produces, when the cultivates 27-deamidosecretin. The invention thus proposes an Escherichia coli characterized by the fact that it has been transformed by a plasmid which has been incorporated or in which has been inserted a structurally synthesized structural gene of the desamidosecretin corresponding to the polypeptide of which the amino acid of the C-terminus is valine, which plasmid is capable of utilizing the lactose operon and also capable of proliferating in a predetermined high cell.

 In another aspect of the invention, there is provided a two-membered polydeoxyribonucleotide which comprises the structural gene expressing 27-deamidosecretin.

 In yet another aspect, there is provided a plasmid which comprises the structural gene expressing 27-deamidosecretin.

 According to the invention as summarized above, it is thus possible to avoid the problems of extraction and purification encountered in the case of natural secretin extraction, which was used as the only commercially feasible means. when the extract of the recombinant gene according to the invention is used as raw material.

 According to the method of the invention, it is possible to modify one or more kinds of amino acids constituting a peptide, so that it becomes possible to study the correlations between the structures and the activities of the peptide hormones. In other words, there is provided a method of easily obtaining any desired derivative of a physiologically active peptide substituted with an amino acid.

It can be said that the formation of a polypeptide derivative by gene manipulation is the best method, since the formation of a high molecular weight polypeptide derivative by chemical synthesis is very difficult according to the methods currently available.

 Furthermore, by using the hSte-vector system, as in the preferred method of making deamidosecretin mentioned above, it has become possible to prepare a deacidosecretin having a secretin activity which corresponds to about 3 × 10 4 molecules per date cell. , what we can call a practically applicable performance.

On the other hand, based on the fused protein described below, there is a yield of 2.85 × 10 5 molecules / cell, a value which suggests that the preparation of deamidosecretin with high yield is rendered possible by direct expression by means of the chimeric plasmid according to the invention in bacteria, without destructive action (proteolytic) exerted by proteases.

 There are also polypeptides other than secretin which have a C-terminus in amide form and the invention suggests the preparation of deamidated derivatives of these other polypeptides as well as the expression of their physiological activity.

1 Desamidosecretin
The 27-deamidosecretin according to the invention is a polypeptide represented by the amino acid sequence (I) below
His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser Arg-Leu-Arg-Asp-Ser-Aa-Arg-Leu-Gln-Arg-
Leu-Gln-Gly-Leu-Val-CH .t (I)
In formula (I), His and other symbols are accepted symbols for amino acids such as lthistidine etc.

 The polypeptide is different from secretin in that C-terminal valine is not in the form of amide (Val-NH 2) but in carboxylic form (Val-OH).

 The deamidosecretin, which has a physiological activity similar to that of secretin, for example an action promoting the secretion of pancreatic juice, can be used as a physiologically active polypeptide similar to secretin. On the other hand, it is also possible to use the deamidosecretin after having it amide on the carboxylic moiety of the C-terminus which is secretin. The amidation can be carried out by a purely chemical process, an enzymatic process or an in vivo biological method.

2. Preparation of deamidosecretin
Although deamidosecretin can be obtained by modifying the C-terminus of secretin, it is preferably prepared by genetic engineering techniques according to the invention by chemically synthesizing a structural gene of this polypeptide, preparing a plasmid so that the polypeptide can further expressed, by transforming a host cell with the plasmid, by preparing the desired peptide by culturing the transformant and recovering the deamidosecretin.

1) Structure gene
(1) Constitution of the gene
The basic sequence of DNA that constitutes the structural gene of secretin is unknown.

Therefore, among certain codons which designate the amino acids constituting the peptide, those for the synthesis of DNA are chosen to satisfy those which fulfill the following conditions:
(i) it must be determined in such a way that the region enriched in fundamental pairs A - T does not follow consecutively the region enriched in fundamental pairs
GC;
(ii) it must also be determined in such a way that each synthetic fragment as described hereinafter does not comprise any undesirable complementary fundamental sequence, in intramolecular or intermolecular position.

It is desirable, also, to easily select the transformed strain, to make the structural gene in such a way that it contains one or more basic restriction enzyme recognition sequences. In the case of deamidosecretin, it is preferable that the gene comprises fundamental Hinf recognition sequences.
I and Hae he.

From this point of view, typical examples of codons for amino acids in the structural gene of the samidosecretin are:
Amino acid Codon
His CAC
Ser TCT, TCA
Asp GAT
Gly CGT
Thr ACT, ACC
Phe TTC
Glu GAA
Leu TTG, CTC, CTA,
CTG, TTA, CTT,
Arg CGT, CGC
Ala GCA
Gln CAA, CAG
Val GTT
Consequently, a preferred embodiment of the structural gene of the deamidosecretin according to the invention comprises a fundamental sequence such as that indicated hereinafter in the experimental examples and on the structural scheme given at the end of the present description (on this scheme, the fundamental sequence is of course the fragment which goes from CAC, corresponding to His, to
GTT, corresponding to 'Val'.

In other words, the subject of the invention is, in one aspect, a two-ring polydeoxynucleotide comprising a structural gene which expresses 27-deamidosecretin as indicated above and a preferred embodiment of the structural gene which comprises a basic sequence such as the one shown below
His Asp Asp Gly Thr Phe Thr
CAC - TCA - GAT - GGT - ACT - TTC - ACC
GTG - AGT - CTA - CCA - TGA - AAG - TGG
Ser Glu Leu Ser Arg Arg Arg
TCA - GAA - CTA - TCT - CGT - CTA - CGT -
AGT - CTT - GAT - AGA - GCA - GAT - GCA -
Asp Ser Ala Arg Leu Gln Arg
GAT - TCA - GCA - CGC - CTC - CAG - CGC
CTA - AGT - CGT - GCG - GAG - GTC - GCG
Leu Leu Gln Leu Val
TTG - CTG - CAA - GGT - CTC - GTT
AAFC - GAC - GTT - CCA - GAG - CAA
The mode of expression of such a structural gene is described in detail for example in published Japanese Patent Application No. 92696/1979. In the case where pBR 322 is used as the plasmid in which it is a question of incorporating the gene and when the deamidosecretin is to be expressed in the form of protein fused with its lactamase operon, the point at which the gene must be incorporated is advantageously in the operon, the restriction enzyme recognition point Pst I. In other words, the ATG codon of methionine, which is the point to be attacked by the cyanogen bromide, is provided on the side of the end St of the structure gene while one or more stop codons are provided on the 3 'end. Then, for synchronization 3) with the frame that starts with the initial codon of the lactamase gene, the gene is applied to the gene, on the 5 'ATG, randomly selected bases in the number of 2 + 3n (n = 0, 1, 2 ...) and, in addition, a fundamental Pst I recognition sequence to form sticky ends at both ends.In general, structural genes are designed and synt 4) in a state where both "sticky" ends are disengaged, but if desired, both ends may be truncated, so that it is necessary to provide two or more randomly selected bases in other outer portions of the fundamental recognition sequence, to increase the hydrolysis efficiency of the restriction enzyme.

The optional pairs of bases to predict from the
S 'of AGT are 3m pairs, (3m + 1) pairs or (3m + 2) pairs, m being an integer, ie O or 1 or more.

These considerations having thus been weighed, a preferred embodiment of the de-amidosecretin gene suitable for use in the invention is that which is indicated hereinafter in the experimental examples.
In other words, a preferential embodiment of the deamidosecretin gene has the structure indicated below.
Met
ACCTGCAGCC - ATG
TGGACGTCGG - TAC
His Asp Asp Gly Thr Phe Thr
CAC - TCA - GAT - CGT - ACT - TTC - ACC
GTG - AGT - CTA - CCA - TGA - AAG - TGC
Ser Glu Leu Ser Arg Arg Arg
TCA - GAA - CTA - TCT - CGT - CTA - CGT
AGT - CTT - GAT - AGA - GCA - GAT - GCA
Asp Ser Ala Arg Leu Gln Arg
GAT - TCA - GCA - CGC - CTC - CAG - CGC
CTA - AGT - CGT - GCG - GAG - STC - GSG
Leu Leu Gln Leu Val
TTG - CTG - CAL - CGT - CTC - GTT AC - GAC - GTT - OCA - GAG - CAA -
END FIN
TGA - TAG - GGCTGCTGGT
ACT - ATC - CCGACGTCCA
(2) Summary
For the synthesis of the gene designed as described above, each of the two + and - catnos can be divided into fragments. These fragments can be chemically synthesized and then the respective fragments are joined together.

It is best to divide each cat into about 16 fragments each of which has 9 to 16 bases, so that 6 to 7 bases will overlap.

 As the synthesis method of each fragment, mention may be made of the ester method 5), the triester method), the solid phase method 4), the liquid phase procodate or the enzyme method 7). Given the duration of the synthesis, yield and purification, the triester solid phase is the most suitable.

 For the details of the synthesis, reference is made to the cited literature and the experimental examples described hereinafter.

(3) Purification
When synthesizing an oligonucleotide, separation and purification of the final product generally becomes more difficult as the ring lengthens. In particular, in the solid phase synthetic process, appropriately protected oligonucleotide blocks are periodically condensed and, therefore, the purification can not easily be carried out by conventional techniques such as gel filtration, electrophoresis of gel, ion exchange column, high speed liquid chromatography etc.

 In the reverse phase column, the retention time differs greatly depending on whether or not the oligonucleotide has a lipophilic protecting group. Therefore, when using the final oligonucleotide block-stable blocking stage with a stable protecting group under the Elimination of other protecting groups, and subsequent removal of the other protecting groups, can provide a mixture of oligonucleotides having only the stable protecting groups in the desired end product.

By taking advantage of the lipophilic nature of the protecting group, the desired final product can be separated from the mixture of the unaltered species by means of a reverse phase column, after which the protecting group is removed to obtain the desired oligonucleotide.

 According to this method, it is possible to separate and purify in good yield the oligonucleotide thus synthesized and which is mixed with the unaltered species.

(4) Phosphorylation and correlation
The synthetic fragments thus prepared are sequentially coordinated with one another by means of a DNA ligase. To prepare the synthetic fragments as enzyme substrates it is necessary to phosphorylate the 5'-hydroxyl groups of the fragments.

 For this purpose, a polynucleotide kinase is generally used, but chemical phosphorylation is also possible. Although the coordination of fragments is generally done by means of DNA ligase, it is also possible to use the method in which the phosphoric acid groups of the 5 'ends are activated by suitable means (for example imidazolylation). and we coordinate them chemically, the catney of the opposite side serving as a model9).

2) Preparation of a vector comprising the lactose operon
In the preferred method for preparing 27-desamidosecretin according to the invention, it is possible to use various plasmids containing all or part of the lactose operon derived from DNA of E. coli chromosomes.

coli and which are able to proliferate in E. coli. The preparation of these plasmids can be carried out according to standard methods well known in the field of molecular biology. The DNA containing the lactose operon can be drawn directly from the E chromosome. coli, but various transduction bacteriophages containing all or part of the lactose operon (eg Pld1, F'-lac, X Odplac, # h80dlac, #plac, etc.) are obtained and, consequently, a necessary part lactose operon can advantageously be derived from these bacteriophages.In addition, in order for the plasmid to be able to proliferate in E. coli, it is necessary to coordinate the necessary part of the above lactose operon with another plasmid from E. coli (e.g. pBR322, p9C101, XdV1) to form a single plasmid vector.

In one example of the invention, transduction bacteriophage (plac523) is used as a lactose operon containing DNA. The DNA of # plac5 can be titrated for example from E. coli PK1512, which is a lysogenic bacterium, according to a conventional method20). This plac5 has the lactose-lactone region which runs from the middle of the "i" gene to the middle of the "y" gene and, advantageously, it contains no other E coli gene than the lactose operon. ), so that it is preferable in the invention. coli, pBR322, which is chosen because it is one of the most easily accessible plasmids, is used to determine the entire fundamental sequence and has ampicillic marker genes. and resistant to tetracycline. In addition, in the coordination of these genes, each gene (hplac5 and pBR322) is treated with the restriction enzymes ECORI and
HindIII, the 3.8 Mb (mega-dalton) fragment (15) is removed for # pl5 and the larger fragment (e) for pBR322 and ligated together to form the desired plasmid vector.

This vector is called pRE in the invention.

 The lactose operon derived from the chromosome dtE was selected. coli for the expression of the desired deamidosecretin, for example because a foreign gene can be inserted into the "z" gene of the lactose operon at the recognition point of the restriction enzyme EcoRI, to express it under the form of a fusion protein with β-galactosidase4a)) 17) because a large amount of protein can be prepared, production can be induced when a suitable microorganism is used and can be easily recover the product stably in the form of a substantially pure fused protein.

Thus, it is desirable that the vector prepared in the invention has only one restriction endonuclease recognition point EcoRI and, to this end, DNA fragments obtained by cleaving are used. Pl5 and pBR322 respectively with EcoRI and HindIII, as described above, these being in turn chained together. 3) Chimeric plasmid
(1) Preparation
In an appropriate position of the vector, provided for a foreign gene to be expressed as indicated above, is incorporated the aforesaid deamidosecretin structure gene.

The incorporation operation itself can be carried out by a method well known in the field of molecular biology. For details of the process used, reference should be made to the following experimental examples.

 According to one embodiment of the invention, pBR322 is used as the vector plasmid and the gene is incorporated at the PstI recognition point thereof to form a chimeric plasmid. In the invention, this chimeric plasmid is called pMG.

The main reasons for choosing pBR322 as the vector plasmid are that it is one of the most easily accessible plasma, that the entire base sequence is determined and that it has ampicillin-resistant marrow genes and resistant to tetracycline. The plasmid pBR322 is deposited at the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Maryland 20852, USA under ATCC37017.The main reasons for choosing the PstI site as the site of incorporation of the gene are that the lactamase operon can be used as is, that the the transformant can easily be found because the ampicillin resistance (Apr) is changed to aicillin sensitivity (Ap5) and there is only one PstI point in pBR322.

 In the preferred process for the preparation of 27-desanidosecretin, described above, according to another embodiment of the invention, the vector plasmid pRE1, which will be described in more detail below, is incorporated in the invention. EcoRI recognition point thereof, a gene containing the deamidosecretin structural gene to obtain a chimeric plasmid. In the invention, this plasmid is known as pLS.

 In this preferred embodiment of the invention, also, the gene containing the above structural gene, that is to say the gene which has the fundamental sequence represented by the scheme given in fine, is preferable. . In order to incorporate at pRE1 that gene which has PstI restriction enzyme recognition points at both ends, it is necessary to convert the PstI restriction enzyme recognition points to EcoRI.

(2) Buying agent
Thus, in the preferred method of preparing deamidosecretin according to the invention, a two-chain DNA for the packaging agent between the gene containing the structural gene and pRE1 is needed. In other words, the two-stranded DNA is required to have two restriction enzyme recognition points, PstI and EcoRI, the base pairs between the two restriction enzyme recognition points being 3n + 1 (n = 1). , 2, 3 ...) so as to be synchronized with the reading frame for translation and which starts at the initial codon of the p-galactosidase gene (the base pair types being randomly selected, from the moment when do not appear a nonsense codon). However, it is necessary that the portion intended for the linking agent ultimately has the above function and it is possible to obtain the enchanment agent by the method by which the structural gene is synthesized. -above. In one embodiment of the invention which is an example of such a synthetic process, the linking agent is obtained by applying a method such as that described hereinafter.

First, a single-chain DNA, as indicated below, having PstI and EcoRi restriction enzyme recognition points is made: 5 '1 2 3 4 S 6 7 8 9 10 11 12 13 15 16 17 18 19 20 3 '

<tb> C <SEP> T <SEP> G <SEP> A <SEP> A <SEP> T <SEP> T <SEP> C <SEP> A <SEP> G <SEP> C <SEP> T <SEP > C <SEP> T <SEP> G <SEP> C <SEP> A <SEP> G <SEP> A <SEP> G
<tb><SEP> Eco <SEP> RI <SEP> Pst <SEP> I
<tb> and in which 1, 2, 9 & 12, 19, 20 can be randomly selected as long as they fulfill the following conditions.

Condition (1): 1 and 10, 2 and 9, 11 and 20, and 12
and 19 are so many pairs of
its complementary one of the water-
be
Condition (2): The sequence 9, 10, 11 is not a
codon called nonsense.

 The so-called single chaste DNA (referred to as the pre-encapsulation agent in the invention), since it is complementary in itself, takes a long two-chain DNA structure with a number of faults. (non-spaced cuts formed on one of the two DNA channels). Accordingly, two-chain DNA can be derived without spacing by DNA-ligase action. The double-stranded DNA thus prepared is a double-stranded poly-DNA having alternately the recognition points of the Pst I and Eco RI restriction zymes, ie recurrent base sequences according to 1 to 20 above.

 Thus, by treating the two-stranded DNA with the Pst I restriction endonuclease, a Pst I-Eco RI-Pst I linking agent having cohesive ends can be obtained.

In general, a linker having two restriction enzyme recognition points has various uses and the goals in the prior art have been achieved using two kinds of oligomers. The idea mentioned here makes it possible to achieve this by using a single type of oligomer according to the method described above and the DNA with a single chain can be represented as follows:

<tb> n ... X'2X'1 <SEP><SEP><SEP> Point <SEP> Enzyme Recognition <SEP> X1X2 ... YnYm
<tb><SEP> ... <SEP> Y2 <SEP><SEP> Y1 <SEP><SEP><SEP><SEP> Enzyme recognition <SEP><SEP><SEP><SEP>Y'2 ... Y'm <SEP>
<tb><SEP> of <SEP> restriction <SEP> B
<Tb>
X and X ', Y and Y' being all desired bases (A, G, C, T) complementary to each other (n and m = 0, 1, 2 ...)
In the case of the invention, A is EcoRI and B is
Pst I and n + m = 3p + 1 (p = 1, 2), p being advantageously 1 or 2.

(3) Determination of direction
To determine the direction of the 27-deamidosecretin gene incorporated into the plasmid, it is possible to cleave the specific point contained in the structural gene with an enzyme (Hae II) recognizing this specific point, to cleave another point in a certain position outside the gene of structure and analyze the size of the fragment obtained.

4) Transformation.

(1) Cell hate
A typical example of the host cell to be transformed using a chimeric plasmid incorporating the desamidosecretin structural gene as described above, such as the above pMG, is a strain of E. coli. coli Ki2C6OO (FERM BP-115, deposited at the Institude of Fermentation Research,
Agency of Industrial Science and Technology, Japan). The strain of E. coli K12C600 is described in the literature24) and its bacteriological properties are also described therein.

 Another example of the chimeric plasmid in which the desamidosecretin structural gene as mentioned above is incorporated is pLS, for example pLS 58, to be used in the preferred method for preparing deamidosecretin according to the invention. A typical example of the host cell to be transformed using pLS 58 is the strain of E. coli XA35 derived from the known strain of E. coli. coli K1222) and having the properties indicated below, the other properties being no different from those of strain K 12.

[Smr, Lac- (i3-, z-)]
The transformation with the chimeric plasmid in which is incorporated the deamidosecretin structure gene according to the invention is possible in all strains of E. coli. cola,
However, in order to recover deamidosecretin as β-galactosidase fused protein, it is preferable to use a defective β-galactosidase gene strain to avoid the presence of normal β-galactosidase mixed in the protein. In general, the formation of protein is regulated by the repressor gene (gene) of the lactose operon. Therefore, when a wild-type B. coli is used, the induced production of the fused protein is possible with an inducer. for example IPTG (isopropyl-thiogalactoside); when a strain with a highly temperature sensitive gene is used, the fused protein can be formed by raising the temperature; when using a gene-deficient strain, the fused protein can still be obtained18)
In the preferred method for preparing the desamidosecretin according to the invention, the strain of E.

coli XA35 which is deficient in genes of ss-galactosidase and gene.

It should also be noted that the transformation with the plasmid in which the DMSA gene is incorporated is not limited to E. coli as a host, but that a suitable host can also be selected in a broader spectrum of dia- midosecretin. species of bacteria, if one chooses a suitable vector, in a manner well known in the field of molecular biology. A typical example of such a host cell can be found in the aforementioned Japanese patent application
N 92696/1979.

(2) Transformation
The transformation operation itself may be carried out according to standard techniques well known in the field of molecular biology. For the details of the foregoing, reference should be made to the experimental examples described hereinafter.

(3) Transforming
A typical example of transformants is that obtained by transforming K12C600 by pMG. It is called, according to the invention, Kl2C6O0 (pMG103).

The transformant K12C600 (pMG103) has the following genetic properties:
[m, r, F, lacY, Leu, Thr, tonA, supE, recBC i
Another example of the transformants is that obtained by transforming the strain d1E with pLS 58. coli
XA35 for use in the preferred method of preparing desanidosecretin according to the invention as described above. It will be called here E. coli XA35 (pLS58).

The E. coli transformant strain XA35 (pLS 58) differs from E. coli strain XA35 by the following properties, as explained in the experimental examples described below:
[Apr, Lac +] 5) Preparation of deamidosecretin
The deamidosecretin can be prepared by culturing the bacterium thus transformed according to a conventional method
For details on the process, refer to the experimental examples below.

3. Experimental examples
Example 1
Design of the deamidosecretin gene 1) A gene having the base sequence consisting of a combination of blocks A, B and C is provided as shown in the final scheme. These blocks respectively include the fragments below
Fragment Block
AS "1 - S-4, S-6
B S-5, S-7 - S-10, S-12
C S-11, S-13 - S116 2) The design process is described below
(1) Codon selection
The codons are selected as shown in the diagram.

 (2) The ATG codon of methionine is added to the N-terminus, so that the synthesized polypeptide is cleaved at this point by the chemical treatment (+ CNBr).

 (3) At the C-terminus, two translation termination codons (TAG or TGA) are added, so that no superfluous peptide is formed.

 (4) To synchronize with the framework that begins at the initial codon of the lactamase gene, two base pairs are added upstream of the methionine, gen- erally 2 + 3n (n = 0, 1, 2, 3 3, ....).

 (5) Any desired number of pairs of bases are added immediately before the Pst I point downstream, if appropriate for the synthesis of each fragment.

 (6) Finally, two base pairs chosen at will are added at both ends to increase the hydrolysis efficiency of the Pst I restriction enzyme.

The gene designed as above contains the point
Hinf I and Hae II point in the 27-desamidosecretin structure gene.

Chemical synthesis of the fragment 1) Synthesis
The synthesis of the fragments is carried out according to the solid phase method described in the literature (6a). However, the isolation and purification of the synthetic fragments is carried out according to the improved method described hereinafter.

The synthesis yields of the respective fragments are shown in the following table
Fragment Basic Sequence Chain Length Yield
S-1 ACCTGCAGCATGChC 16 33
S-2 GCTGCAGGT 9 52
S-3 TCAGATGGTACTT 13 56
S-4 ATCTGAGTGCATG 13 42
S-5 TCACCTCAGAACTAT 15 43
S-6 GAGGTGAAAGTACC 14 47
S-7 CTCGTCTACGTGATT 15 38
S-8 AGACGAGATAGTTCT 15 27
S ~ 9 CAGCACGCCTOCAGC 15 32
S-10 OGTGCTGAATCACGT 15 43
S-11 GCTTGCTGCAAGGT 14 22
S-12 AGCAAGCGCTGGAGG 15 44
S113 CTCGTTTGATAGG 13 47
5-14 AAACGAGACCTTGC 14 42
S-15 as S-2
S-16 ACCTGCAGCCCTATC 15 32 2) Purification
To 50 mg of the resin which has undergone solid phase synthesis, 0.5 mole of d-picolinic-aldoxime-tetramethylguanidine and 100 to 200 l of a mixture of dioxane and water (1: 1) 14) and the mixture is allowed to stand for several hours at room temperature. 2 ml of concentrated aqueous ammonia are then added to the mixture and the mixture is left to stand overnight at 55 ° C. with hermetic sealing. The mixture obtained is filtered to separate the resin and the filtrate is concentrated and subjected to gel filtration. Elution is effected with 50 mmol of TEAB buffer (pH 7.5) and the eluted fractions are collected in the empty volume. Concentrated and subjected to C-18 reverse phase high pressure liquid chromatography. (Waters: "Radial Pack
A ", diameter 8 cm × 10 cm) to accomplish the eluvium in a 0.01 M ethylenediamine diacetate buffer (pH 7.8) with a concentration gradient of acetonitrile from 10% to 32% at a flow rate The eluted fractions are collected in 11 to 12 minutes during this process, and oligonucleotides lacking a trityl group are eluted as the injection peak. 1 ml of 80% acetic acid is added, the mixture is allowed to stand at room temperature for 15 minutes. The tritanol is removed by extraction, the aqueous layer is concentrated and applied again to the phase column. In the same conditions as above, the elution is carried out with an acetonitrile concentration gradient of 0 to 20% and the fractions eluted in 12 to 13 minutes are collected.

phosphorylation
Each fragment (30 g) is dissolved in 30 mmol of Tris-HCl buffer (pH 7.5) and 60 μCi (19.8 μmol) of 32 P / ATP and 21 μL (9 units) are added to the solution. T4 polynucleotide kinase to make a total of 50 μl, after which it is incubated at 37 ° C for 20 minutes. Then, 10 equivalents were added to the ATP and T4 polynucleotide kinase fragment (9 units) and incubated at 37 ° C. for 20 minutes. The reaction is stopped by heating at 100 ° C. for 2 minutes and the product is purified by gel filtration. Each fragment is confirmed by 20% gel electrophoresis.

Coordination of fragments
Each of 0.05A260 of Si, S-2, S-3 is dissolved,
S-4 and S-6 in a buffer (20 mmol of Tris-HCl pH 7.5, 10 mmol of MgCl2, 10 mmol of DTT (dithiothreitol), 0.2 mmol dlATP (adenosine triphosphate) to form a solution containing a total of 30 L. 1 μl (150 units) of
T4-DNA ligase and the solution is allowed to stand overnight at 10 ° C.

The reaction is confirmed by electrophoresis on 8% polyacrylamide gel. Similarly, the blocks are synthesized
B and C.

 The reaction mixtures of Block A and Block B are mixed and, after adding 3 μl of 0.2 mM ATP and 1 μl (150 units) of T4-DNA ligase, the mixture is allowed to stand overnight at 10 ° C. After the reaction has been confirmed by 8% polyacrylamide gel electrophoresis, the reaction mixture of Block C is added and the reaction is allowed to proceed similarly overnight. The progress of the reaction is confirmed by electrophoresis on 5% polyacrylamide gel.

 The mixture is heated at 68 ° C. for 15 minutes and then cooled to room temperature. 15 l of 0.5 M NaCl and 80 units of the restriction enzyme Pst I are then added and the mixture is allowed to stand overnight at 37 ° C.

The reaction is stopped by heating at 90 ° C. for one minute and the mixture is subjected to separation by electrophoresis on 5% polyacrylamide gel. The longest cat-length band is separated and transferred to 1% low-melting agarose gel electrophoresis to extract the resulting band.

Cloning
Plasmid pBR 322 (4 μg) is added to a mixture (total amount: 50 l) of 100 mmol of Tris-HCl buffer, 5 mmol of MgCl 2 and 50 mmol of NaCl and the reaction is carried out using 6 units of Pst I restriction enzyme at 37 C for 2 hours. Then, the reaction is stopped by heating at 68 ° C. for 15 minutes. The above synthetic gene is added to the reaction mixture and the coordination reaction is conducted in a similar manner using T4 DNA-ligase.

Using 5 μl of the resulting mixture, which is equivalent to 0.68 g of pBR322 DNA, transformation of the E. coli K12C600 according to the Kuschner method10) in a physical confinement facility
P-311).

 The transformed strain was selected on an L tray (1% "Bactotripton11, 0.5X" Bactoyeast "extract, 0.5% NaCl, 1.5%" 1Bactoagar ") containing 10 μg / ml tetracycline (Tc and 500 transformed strains were examined for ampicillin resistance and 45 Tcr / Aps transformed strains were isolated. They will be referred to herein as C600 (pMG 101) to C600 (pMG 145).

 Of the transformed Tcr / Aps strains, 8 strains (C600 (pMG 101) to C600 (pMG 108)) were randomly selected and the plasmid DNA was isolated by equilibrium sedimentation through a cesium chloride density gradient. containing ethidium bromide. The plasmid AJN thus isolated will be called here pMG 101 ta pMG 108.

Confirmation of direction
5 μg of the plasmid are dissolved in a mixture (30 μl) of 10 mmol of Tris-HCl buffer, 66 mmol of MgCl 2, 6 mmol of ss-mercaptoethanol and 60 mmol of NaCl and the 30 units of the restriction enzyme are added. Hae II1 to the solution, after which it is heated at 37 ° C. for one hour. 375 bp fragments were extracted by electrophoresis on a 1.5% low melting agarose gel. The extracted fragments were similarly hydrolyzed using 18 units of the restriction enzyme Hae II and the product was hydrolyzed to 191 bp and 176 bp, determined by 5% polyacrylamide gel electrophoresis, and evaluated as a plasmid having the correct direction.

Identification of the fused protein with minicells
A minicell-producing E. coli (F-, thr +, ara +, leu +, azis, tonAs, mina, minB, gal +, # -, sfrr, malA, xyl, mt1, thi, sup) was transformed with pMG 103 and pBR 322 according to Kuschner's method) to obtain a transformant of
Tc / Aps.

 The transformant is precultivated in 20 ml of Davis minimal culture medium containing 0.5% Casamino acid, 20 μg / ml thymine, 2 μg / ml thiamine and 10 g / ml tetracycline at 37 ° C. for 16 hours. and, by inoculating 500 ml of the same medium (but without tetracycline) with 10 ml of the preculture, the culture is continued up to OD 620 = 0.6 to 0.8.

The culture broth was centrifuged by means of a Hitachi refrigerated centrifuge (model RPR-9 at 3000 rpm) for 12 minutes to remove host cells and then to 8500 centrifugation. rpm for 25 minutes to obtain minicell pellets. These are washed by suspending them in 25 ml of "To" buffer (1 mole of Tris-HCl, pH 7.3, MgSO4.7H2O 150 mg, 1 ml of 1% gelatin, 1 mole of CaCl 2, 0.25 ml / l H2O). The suspension was centrifuged at 10,000 rpm (in an RPR-20 rotor) for 15 minutes, and the obtained minicellule precipitates were suspended in 1 ml of Tl buffer.

The resulting slurry was subjected to gradient gradient centrifugation at a density of 10% -15% -20% (at 4000 rpm, in a RPRS-4 rotor, for 25 minutes) to recover minicells. The suspension of minicells is then suspended in 20 ml of T1 buffer and similar operations are repeated.

The minicellules are suspended in 1 ml of Davis minimal culture medium containing a final concentration of 50 g / ml each of 18 amino acids (alanine, valine, isoleucine, proline, phenylalanine, tryptophan, glucine, serine, threonine, cystine, tyrosine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, arginine, histidine), 20 μg / ml of thymine and 2 μg / ml of thiamine and heated with stirring at 37 ° C. for 12 minutes. After adding to the suspension 20 μCi of (35S) methionine (NEN, New
27
England Nuclear: 1260.1 Ci / mmol) and 5 Cl of (H) -leucine (NEN: 115.2 Ci / mmol), the mixture is further heated for 10 minutes.Then, immediately, the same buffer containing 50 mmol of NaN3, 100 mg / ml of methionine and 100 g / ml of leucine. The centrifugation is then carried out at 3000 rpm for 20 minutes to recover minicellules which are resuspended in 20 μl of sample buffer (6 μl H 2 O, 1.25 moles of Tris-HCl (pH 6.8) 1 ml, SDS (sodium dodecyl sulphate) 0.4 g, glycerol 2 ml, 2-mercaptoethanol 1 ml, BPB (bromophenol blue) 4mg) and heated at 100 ° C. for 3 minutes. 18 μl of this suspension are then subjected to electrophoretic separation on
SDS-polyacrylamide.

 In the cells of E. coli K12C600 (pBR 322) (FERM-P 6017), β-lactamase was synthesized and a line corresponding to a molecular weight of approximately 29 kilodaltons appeared on the electrophoresis gel, but this was not detected. approximately 29 kilodalton protein in E. coli K12C600 (pMG 103). Instead, a polypeptide of about 24 kilodaltons appears as new line. It is visibly the gene formed by coding with pMG 103 and is related to the disappearance of the β-lactamase line. PMG 103 has a structure in which a part of the β-lactamase gene of pBR 322 (coding of 182 amino acids in starting from the amine end of the signal peptide of the ss-lactamase structural protein is coordinated downstream (for transcription) with the 27-deamidosecretin gene (encoding 27 amino acids) and the gene form must be detected as a fused protein ss-lactamase fragment (182 amino acids) 27-deamidosecretin (27 amino acids) The calculated molecular weight of the fused product is 23.4 kilodestons and therefore the newly emerging line in E. coli extracts can be considered K12600 (pMG 1023 represents this fused protein.

Purification of the protein
We submit the E. coli K12C600 (pMG 103) to a culture stirred in 3 liters of Luria broth at 37 ° C. and centrifuged when the concentration of microorganisms reached in
The resulting pellets are washed with 10 mmol of Tris-ElCl buffer (pH 8.0) and 1 mmol of phenylmethylsulfonyl fluoride and centrifuged again. The pellets are resuspended in the same buffer.

The suspension is treated with EDTA at a final concentration of 10 mmol at 0 ° C for 5 minutes and then treated with egg white lysozyme at a final concentration of 0.1 mg / ml at 0 ° C for 30 minutes. to perform spheroplast formation lysis. The lysate obtained was treated with a Kubota Insonator 200 Mn ultrasonic wave generator at the maximum flow rate (200 W) for 10 minutes to completely destroy the bacterial cells, followed by centrifugal separation (using A Hitachi refrigerated centrifuge, model PR52, with RPR20-2 rotor, at 18,000 rpm, for 30 minutes, at OC) to remove cell debris. To the supernatant liquid (110 ml), magnesium at a final concentration of 50 mmol, then the mixture is subjected to centrifugation (using the above rotor at 8000 rpm for 10 minutes at 0 ° C.) To the supernatant liquid is added 400 DNase I and 5 mg of RNase I to perform the treatment at 4 ° C for one hour and then the proteins and peptides were plated with 80K saturated ammonium sulfate.

The precipitates obtained by centrifugation (using an RPR 20-2 rotor at 8000 rpm for 10 minutes) are dissolved
OC) in 10 mmol Tris-HCl (pH 8.0), desalted with the same buffer and centrifuged (under the conditions above).

Acetone is then added to the final supernatant at a final concentration of 75% and the precipitate formed with 1.0 g of cyanogen bromide in 20 ml of 80% formic acid is treated overnight. After evaporation, the residue is extracted with 40 ml of isopropanol and then with an equal volume of methanol. Then the evaporation is again carried out and the residue is dissolved in 0.1 N acetic acid. After removing the insoluble fraction by centrifugation (3000 to 4000 g, for 5 minutes), the filtration is carried out on a column of gel "Sephadex G-25" (manufactured by Pharmacia
Co.), fractions having molecular weights of 1,000 to 10,000 are collected and lyophilized.

Biological dosage
The lyophilized sample is subjected to a biological assay method 12) in which the sample is dissolved in a total volume of 2 ml of isotonic sodium chloride solution; five portions of 0.25 ml each are injected into the groin of five rats; I am assured at intervals of 10 minutes the quantities of secreted secretive juice flowing from an artificial pancreatic tube; The increase in amount which represents the biological activity of the secretins is determined. As a standard secretin, the same rats were injected intravenously with "secrepan" (Eisai, Japan) at 0.25 U. / kg and 0.50 U / kg and the amounts of secreted pancreatic juice were measured to determine the correspondence with the Eisai unit (practically equal to the Crick Harper Raper unit) .Therefore, samples taken from E. coli K12C600 (pMG 103) caused an increase of 2Q2% +76 (mean + # SD) of secreted pancreatic juice, 10 minutes after injection, and 180% - 37.6, 20 minutes after injection. On the other hand, the "Secrepan" causes an increase of 162% + 28 and 206% * 58 respectively after 10 to 20 minutes in the case of injection of 0.25 U / kg, respectively. 159.8% # 83.7 and 250.8 + 216.5, 10 and 20 minutes after the injection of 0.5 U / kg.

These values, resulting from a test "t" give a significant difference at the rate of p <0.05. On the other hand, when one tries by the same method the sample which one obtained starting from the extracts of the bacterial cells taken from the clone containing only pBR 322 and free from the gene of deamidosecretin, one does not maintain any increase of the quantity of secreted pancreatic juice, This indicates that a substance with a biological activity similar to secretin is forming in E. coli cells. coli K12 C600 (pMG 103) while the formation of such a substance in E. coli cells is not maintained. coli K12 C600 (pBR 322). Carrying increased amounts of secreted pancreatic juice from samples obtained from E. coli K12 C600 (pMG 103) in a straight line obtained by plotting on a semi-paper logarithmic the increased amounts of secreted pancreatic juice and on the abscissa the units Secrepan (logarithmic scale), one can determine, by the extrapolation method (provided that the linear relation is carried out) that the secretin unit is from 0.17 to 0.24 unit Eisai (# 0.17 to 0.24 unit Crick Harper Raper) / 0.25 mlXkg rat. When calculated taking into account the weight of the rats used, this value is calibrated between 0.051 and 0.072 U / O, 25 ml / rat, the total activity being from 0.408 to 0.576 U for the total quantity of 2 ml. It is known that the secretin of the highest purity has a specific activity of 16,000 CHR units / mg, the values of 0.408 to 0.576 U corresponding to 25.5 to 36 ng of secretin. This corresponds to 7.15 to 10.1 pmol of secretin molecules, indicating that a deacidosecretin whose activity corresponds to 4.3 to 6.1 x 1012 secretin molecules has been biosynthesized. By preparing deamidoscretin with about 3 x 1012 E. coli K 12 C600 (PMG 103) as a raw material, at least 1.4 to 2.0 molecules of secretin equivalent per bacterial cell are recovered.

Radioimmunoassay
The sample is diluted with 50 mmol of Tris-HCl (pH 8.0) / 0.1% of serum albumin of beef. The radioimmunoassay of the diluted sample is carried out using the "Secretin Kit Kaiichi" manufactured by Daiichi Radioisotope
Research Institude, Japan, according to the prescribed method to confirm its activity.

Example 2
The steps leading to the coordination reaction between the fragments are conducted in a manner similar to Example 1.

Preparation of # plac5 DNA and pBR322 DNA
The A # plac5 DNA is extracted from the lysogenic bacterium E.

coli PK 1512 (IFO 14149) and the preparation operations are carried out according to the method of Oshima20).

 The pBR322 DNA is extracted from E. coli K-12 C600 (pBR 322) (FERM-P 6017) and the preparation is carried out according to the method of M. Kahn et al.21).

Preparation of pREl
Plasmid DNA (10 μg) was added to a mixture (total amount: 20 μl) of 100 mmol pH 7.5 Tris-HCl buffer, 7 mmol MgCl 2 and 50 mmol NaCl and the reaction was run using units of the restriction enzyme EcoRI and 10 units of the restriction enzyme Hind III at 37 ° C. for 2 hours. Then, by electrophoresis on a 1% agarose gel, 3.8 megadaltons of DNA fragments are purified.
1 g of pBR322 DNA is added to a mixture (total amount 10 l) similar to the above mixture and the reaction is carried out using 1 unit of the restriction enzyme
EcoRI and 1 unit of restriction enzyme Hind III at 37 C for 2 hours. Then, by 1% agarose gel electrophoresis, 2.6 megadaltons of DNA fragments were purified.

 The obtained fragments are added to a mixture (total amount: 10 l) of 50 mmol of Tris-HCl buffer pH 7.8, 10 mmol of MgCl 2, 20 mmol of DTT and 1 mmol of ATP and the reaction is carried out using 30 units of T4 DNA ligase at 14 C for 24 hours.

 Using the reaction mixture, the transformation of the E. coli strain is carried out. coli X 35 according to the method of Kuschner10) in a physical confinement plant p-111).

We select the transformed strain on a plateau
L (1% "Bactotrypton", 0.5% "Bactoyeast" extract, 0.5% NaCl, 1.5% "Bactoagar") containing Ap (20 g / ml) and reapplied strains transformed on an EMB-lake plateau (2.25% "Bacto-EMB" broth, 1.5% "Bactoagar"). In addition, the strains converted to Las (red colony) are selected. Between these strains, 5 strains were chosen at random to prepare a plasmid which was analyzed with several kinds of restriction enzymes and it was found that the four strains were formed of a 3.8 megadalton fragment of
Aplac5ADN coordinates to a @co RI-Hind ITI fragment of pBR 322
Such strains are called an E. coli X (pRE 1).

In other words, E. XA transforming coli (pRE 1) is different from the strain of E. coli. coli XA 35 aforesaid by the following properties
[Apr, Lake +]
Web Agent
The synthesis of a fragment having the fundamental sequence CTGAATTCACTCTGCAGAG is carried out. The synthesis and purification are carried out according to the methods of synthesis and purification of the respective structural genes described above (yield 40%). In the invention, this single-stranded DNA is referred to as the pre-linking agent.

 The pre-cation agent (15 μg) is incubated with 20 units of T4-polynucleotide kinase at 37 ° C. for 40 minutes in a mixture (total amount: 30 μl) of 50 mmol of Tris-HCl buffer pH 7.5. Zmol of MgCl2, 0.1 mmol of spermidine, 0.1 mmol of EDTA, 10 mmol of DTT and 0.2 mmol of ATP. The reaction is stopped by heating at 60 ° C. for 2 minutes.

After allowing the mixture to stand at room temperature for one hour, 300 units, 11 μl of T4-DNA ligase were added and the reaction was carried out at 14 ° C overnight. By heating at 60 ° C. for 2 minutes, the reaction is stopped.

The mixture is allowed to stand at room temperature for 1 hour and 20 units of the restriction enzyme are added.
Pst I to conduct the reaction for 5 hours. By heating at 60 ° C. for 2 minutes, the reaction is stopped.

15 g of the packaging agent are thus obtained.
Pst I-Eco RI-Pst I.

Preparation of the EcoRI-EcoRI structural gene
The Pst-I-EcoRI-PstI linking agent prepared as above (4 g) and the PstI structural gene are added.
Pst-I mentioned above in Example 1 (0.6 μg) to a mixture (total amount: 35 l) of 100 mmol of Tris-HCl buffer pH 7.5, 7 mmol of MgCl 2 and 50 mmol of NaCl and the reaction was conducted using 300 units of T4 DNA ligase at 14 C for 24 hours.

 The reaction is then stopped by heating at 68 ° C. for 10 minutes, and the mixture is then gradually cooled.

 To the resulting mixture was added the Eco RI restriction enzyme (100 units) to conduct the reaction at 37 ° C for 5 hours, and then purified the DNA fragment at 120 base pairs by 5% polyacrylamide gel electrophoresis.

Preparation of pLS and cloning
0.25 μg of pRE1 is added to a mixture (total amount of 4 μl) of 100 mmol of Tris-HCl buffer pH 7.5, 7 mmol of MgCl 2 and 50 mmol of NaCl, and the reaction is carried out using a the Eco RI restriction enzyme at 37 ° C. for 1 hour.

The plasmid pRE 1 thus prepared (cleaved with EcoRI) (0.25 μg) and 0.02 g of the structural gene EcoRI are added.
EcoRI to a mixture (total amount: 10 l) of 50 mmol of Tris-HCl buffer pH 7.8, 10 mmol of MgCl 2, 20 mmol of DTT and 1 mmol of ATP and the reaction was carried out using 30 units of T4 DNA ligase at 14 C for 24 hours.

Using the resulting mixture, transformation of the strain dEE is carried out. coli XA35 according to the method of Kuschner10) in a physical confinement installation
P-311)
The transformed strain is selected on a platelet L containing Ap (20 μg / ml) (as described above). The transformed strains are reapplied on the EMB-lac plate (as indicated above) for a new selection of the Lac strain. Among these strains, 200 strains were selected to prepare plasmid DNAs whose structure was analyzed by means of Pst I or Hae II restriction enzyme to confirm that 11 strains had the above structural gene.

Determination of direction
The following experiment is conducted for each plasmid of the 11 strains having the structural gene.

 The plasmid (20 g) is added to a mixture (total amount: 20 l) of 10 mmol of Tris-HCl buffer pH 7.5, 66 mmol of MgCl 2 and 60 ml of NaCl, and 20 more units are added to the solution. the restriction enzyme Hae 11 is then conducted incubation at 37 C for 2 hours. From the resulting mixture, fragments of about 1 megadalton and about 0.7 megadalton fragments were extracted by electrophoresis on 1% agarose gel. The respective fragments were cleaved with the restriction enzyme EcoRI according to the above method and subjected to 15% polyacrylamide gel electrophoresis. The plasmid in which electrophoresis of the 40 base pair DNA fragments was obtained. of the approximately 1 megadalton fragment and the 80 base pair DNA fragment is derived from the approximately 0.7 megadalton fragment was evaluated as the plasmid in which the structural gene was inserted in the correct direction.

 By these experiments, it is found that 5 plasmid strains, out of the 11 strains that have the structural gene, have the correct direction. One of these strains is randomly chosen and is called E. coli XA35 (pLS58).

Identification and purification of the fused protein
We submit the E. coli XA35 (pLS58) to a culture stirred in 1 liter of "Luria" broth containing 20 g / ml of ampicillin at 37 C until the bacterial cell count reaches 5 x 108 cells / ml. The bacterial cells are then collected by centrifugation and they are suspended in a mixture (total amount: 100 ml) of 10 mmol of Tris-HCl buffer (pH 7.5) / 10 mmol of MgCl 2 and the suspension is treated with of a Kubota "Insonator 200 M" ultrasound generator for 30 minutes to destroy the bacterial cells.

 The thus obtained solution (2 l) is subjected to 7.5% SDS polyacrylamide gel electrophoresis and a line of a protein of 120,000 daltons in size is recognized. Such lines are not recognized in the similarly extracted extract of the bacterium without plasmid (strain E.

coli XA35). This protein is therefore considered to be derived from plasmid pLS58.

 It is also confirmed that the protein is practically the same size as ss-galactosidase (19) derived from E. coli, which is not in contradiction with the size (117,873 daltons) of the fused protein of deamidosis. - Cretin and ss-galactosidase.

 It is therefore concluded that this protein at about 120,000 daltons is the fused protein and is purified therefrom.

 The extract obtained by ultrasonic destruction is centrifuged and the precipitates are recovered. The fused protein is recovered essentially in the precipitates. The precipitates are suspended in a mixture (total amount: 100 ml) of 10 mmol of pH 7.5 Tris-HCl buffer and 1 mmol of MgCl 2 and subjected to centrifugation followed by recovery of the precipitates. This operation is repeated twice more in order to eliminate the water-soluble bodies.

The precipitates obtained are dissolved in a mixture (total amount: 100 ml) of 20 mmol of Tris-HCl buffer pH 7.5, 1 mmol of MgCl 2, 10 mmol of NaCl and 7 moles of urea and applied to a column ( 5 cm in diameter x 3 cm) of cellulose
DEAE balanced with the same buffer.

 After washing with 20 ml of the same buffer, the column is washed again with 100 ml of a similar buffer in which the concentration of NaCl is brought to 50 mmol. The fused protein is then eluted with 150 ml of a similar buffer in which the concentration of NaCl is brought to 150 mmol.

 To the eluate obtained, ss-mercaptoethanol is added to the final concentration of 1% and the mixture is dialyzed three times with respect to 5 liters of water. The dialysate is subjected to centrifugation to recover the fused protein as precipitates.

The fused protein obtained in a line virtually unique to polyacrylamide gel electrophoresis
SDS at a rate of about 28 mg per 1 liter of culture broth, as calculated from the absorption at 280 nm.

It is calculated that this value corresponds to about 280,000 molecules / cell.

 The purified fused protein was dissolved in 2 ml of 70% formic acid and treated with 100 mg of cyanogen bromide for 18 hours. After evaporation, the residue is extracted with 5 ml of isopropanol and then with 5 ml of methanol, this extraction being followed by freeze-drying.

Biological dosage
The freeze-dried sample thus obtained is assayed by a biological assay method) in which the freeze-dried sample is dissolved in a total volume of 25 ml of isotonic sodium chloride solution; 5 μl portions are injected intravenously into the groin of each rat; the quantities of pancreatic juice secreted and stole from an artificial pancreatic tube are measured at intervals of 5 minutes and the increment is determined as the biological activity of the secretin. As a standard secretin, the same rats are injected beforehand. intravenously, "Secrepant" (Eisai, Japan) at 1 unit, 2 units and 4 units / rat, and the amounts of secreted pancreatic juice are measured to determine the correspondence with the unit.
Eisai (practically equal to the Crick Harper Raper unit).

 The result is that samples taken from E. coli XA35 (pLS58) show an increase in the secretion of pancreatic juice. On the other hand, when one tries by the same method the sample which one obtains starting from the extracts of bacterial cells taken from the clone containing only pRE1 and free from the gene of deamidosecretin, one does not maintain an increase of the secretion of sucks pancreatic. This indicates that a substance having a biological activity similar to that of secretin is produced in E. coli cells.

coli XA35 (pLS58), whereas the production of such a substance does not take place in E. coli cells. coli XA35 (pRE1).

By carrying increased amounts of pancreatic juice secreted by pulling samples from E. coli
XA35 (pLS580 on a straight line obtained by plotting the increased amounts of secreted pancreatic juice on the ordinate on a semilogarithmic paper and the "Secrepan" units (logarithmic scale) on the abscissa, it can be determined by interpolation that the unit secretin is 3 Eisai units / 50 μl / rat, which represents about 1 sse Eisai units as 25 ml of isotonic sodium chloride solution containing the lyophilized sample.Since it is known that the most secretin pure present) 3) a specific activity of 16,000 units CHR / mg, 1,500 units correspond to about 26 nmol of secretin, which indicates that it has biosynthesized a desamidosecretin whose activity corresponds to 1.6 x 1016 secretin molecules . When deamidosecretin is prepared using as raw material 5 x 10 11 cells of E. coli XA35 (pLS58), 3x104 molecules of secretin equivalent per bacterial cell are recovered at moinx.

 From this result, it can be concluded that the synthetic 27-desamidosecretin gene is expressed by the action of the lactose operon adjuvant of E. coli in the cells of E. coli XA35 (pLS58) and that its translational product, 27-desamidosecretin, has an activity similar to that of secretin.

Radioimmunoassay
The sample is diluted with 50 mmol of Tris-HCl (pH 8.0) / 0.1% of serum albumin of beef. The radioimmunoassay of the diluted sample is conducted using the "Secretinkit Daiichi", manufactured by Daiichi Radioisotope Research Institute, Japan, according to the prescribed method to confirm its activity.

4. Diagram of the gene
The following diagram shows the basic sequence of the gene comprising the structural gene 27-des-samidosecretin separately in blocks A, B and C while a is an optional base pair, b the point Pst I, c a optional base pair, an initial codon, e the Hinf I point and f the Hae II point.

Diagram of the gene

<SEP> Block <SEP> A
<tb><SEP> Word <SEP> I @@ s <SEP><SEP> Asp <SEP> Gly <SEP> Thr <SEP> Phe
<tb><SEP> S <SEP> - <SEP> 1 <SEP> S <SEP> - <SEP> 3
<tb> A <SEP> C <SEP> C <SEP> T <SEP> G <SEP> C <SEP> A <SEP> G <SEP> C <SEP> C <SEP> A <SEP> T <SEP > GC <SEP> A <SEP> CT <SEP> C <SEP> AG <SEP> A <SEP> TG <SEP> G <SEP> TA <SEP> C <SEP> TT
<tb> T <SEP> G <SEP> G <SEP> A <SEP> C <SEP> G <SEP> T <SEP> C <SEP> G <SEP> G <SEP> T <SEP> A <SEP > CG <SEP> T <SEP> GA <SEP> G <SEP> TC <SEP> T <SEP> AC <SEP> C <SEP> AT <SEP> G <SEP> AA <SEP> A <SEP > GT <SEP> G <SEP> GA <SEP> G
<tb><SEP> a <SEP> b <SEP> c <SEP> d
<tb><SEP> S <SEP> - <SEP> 2 <SEP> S <SEP> - <SEP> 4 <SEP> S <SEP> - <SEP> 6
<tb><SEP> Block <SEP> B
<tb> Phe <SEP> Thr <SEP> Ser <SEP> Gln <SEP> Leu <SEP> Ser <SEA> Arg <SEP> Leu <SEA> Arg <SEA> Asp <SEA> Ser <SEA> Ala <SEP > Arg <SEP> Leu <SEP> Gn <SEP> Arg
<tb><SEP> S <SEP> - <SEP> 5 <SEP> S <SEP> - <SEP> 7 <SEP> S <SEP> - <SEP> 9
<tb> T <SEP> CA <SEP> C <SEP> CT <SEP> C <SEP> AG <SEP> A <SEP> AC <SEP> T <SEP> AT <SEP> C <SEP> TC <SEP> G <SEP> TC <SEP> T <SEP> AC <SEP> G <SEP> TG <SEP> A <SEP> TT <SEP> C <SEP> AG <SEP> C <SEP> AC <SEP > G <SEP> CC <SEP> T <SEP> CC <SEP> A <SEP> GC
<tb><SEP> TC <SEP> T <SEP> TG <SEP> A <SEP> TA <SEP> G <SEP> AG <SEP> C <SEP> AG <SEP> A <SEP> TG <SEP> C <SEP> AC <SEP> T <SEP> AA <SEP> G <SEP> TC <SEP> G <SEP> TG <SEP> C <SEP> GG <SEP> A <SEP> GG <SE> T <SEP> CG <SEP> C <SEP> GA <SEP> A <SEP> CG <SEP> A
<tb><SEP> e <SEP> f
<tb><SEP> S <SEP> - <SEP> 8 <SEP> S <SEP> - <SEP> 10 <SEP> S <SEP> - <SEP> 12
<tb><SEP> Block <SEP> C
<tb> Arg <SEP> Leu <SEP> Leu <SEP> Gln <SEP> Gly <SEP> Leu <SEP> Val <SEP> End <SEP> End
<tb><SEP> S <SEP> - <SEP> 11 <SEP> S <SEP> - <SEP> 13 <SEP> S <SEP> - <SEP> 15
<tb><SEP> G <SEP> CT <SEP> T <SEP> GC <SEP> T <SEP> GC <SEP> A <SEP> AG <SEP> G <SEP> TC <SEP> T <SEP> CG <SEP> T <SEP> TT <SEP> G <SEP> AT <SEP> AT <SEP> GG <SEP> G <SEP> C <SEP> T <SEP> G <SEP> C <SEP> A <SEP> G <SEP> G <SEP> T
<tb><SEP> CG <SEP> T <SEP> TC <SEP> C <SEP> AG <SEP> A <SEP> GC <SEP> A <SEP> AA <SEP> C <SE> TA <SEP> T <SEP> CC <SEP> C <SEP> G <SEP> A <SEP> C <SEP> G <SEP> T <SEP> C <SEP> C <SEP> A
<tb><SEP> b <SEP> a
<tb><SEP> S <SEP> - <SEP> 14 <SEP> S <SEP> - <SEP> 16
<tb> 5.Deposit of microorganisms
E. coli K12C600, E. coli XA35 and its transformant
E. coli XA35 (pRE1) with plasmid Prel were deposited, pursuant to the Budapest Treaty on the International Recognition of Deposition and Microorganisms for the Purposes of the Fermentation Research Instance, Aaency of Patent Procedure
Industrial Science Technology (FERM), 1-3, Higashi l-cho, Yatabemachi, Tsukuba-gun, Ibaraki-ken, Japan, as FERM BP-115, dated June 9, 1981, FERM BP-116, on January 7, 1982, and FERM BP-117, dated Jan. 7, 1983. Plasmid pBR322 is deposited at FERM as a transformant of E. coli. coli K12C600 with pBR322, namely under the designation E. coli Kl2C600 (pBR322), under the number
FERM P-6017, dated June 9, 1981.

E. coli K12C600 (pMG 103), which is the transformant obtained with the chimeric plasmid pMG, was deposited at the American Type Culture Collection (ATCC), 12301, Parklawn Drive,
Rockville, Maryland 20852, USA, under N ATCC 39042, dated January 26, 1982.

E. coli PK 1512, which contains the lysogenic bacterium # plac5, was deposited at ItInstitude for Fermentation, Jus-
Hommachi 2-17-85, Yodogawa-ku, Osaka-Shi, Japan, under the subway IFO N 14149, dated February 1, 1982.

 E. coli XA (pLS58), which is the transformant obtained with the chimeric plasmid, was deposited under the Budapest Treaty at ATCC under ATCC No. 39 040 dated January 11, 1982.

Claims (34)

R E V E N D I C A T IO N S  1 - 27-deamidosecretin, constituting a polypeptide represented by the amino acid sequence  His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu  Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln  Arg-leu-Leu-Gln-Gly-Leu-Val-OH in which Val-OH indicates that the C-terminus of the polypeptide, formed of valine, is a carboxylic acid.  2 - Process for the preparation of 27-deamidosecretin, characterized by the following steps  (1) chemically synthesizing a gene of suitable structure for the deamidosecretin, corresponding to the polypeptide in which the amino acid of the C-terminus is valine,  (2) inserting this gene into a vector plasmid capable of proliferating in a hood cell to form a chimeric plasmid capable of proliferating in the cell,  (3) transforming the host cell with the chimeric plasmid,  (4) The resulting transformant is cultivated and the deaminosecretin is recovered.  3 - Process according to claim 2, characterized in that the hood cell is an Escherichia coli.  4 - Process according to claim 3, characterized in that the E. coli is E. coli K12C600, FERM BP-11.  5 - Process according to claim 3, characterized in that the vector plasmid is pBR322.  6 - Process according to claim 5, characterized in that the chimeric plasmid is p which is incorporated the structural gene at the Pst I recognition point of pBR322.  7 - Process for the preparation of 27-deamidosecretin characterized by the following steps  (1) chemically synthesizing a gene of suitable structure for the deamidosecretin corresponding to the poly peptide in which the amino acid of the C-terminus of the secretin is valine,  (2) there is provided a vector plasmid capable of using the lactose operon and also capable of proliferating in a pre-seeded host cell,  (3) inserting the structural gene into the vector plasmid to obtain a chimeric plasmid capable of proliferating in the cell,  (4) transforming the host cell with the chimeric plasmid,  (5) the resulting transformant is cultivated and the de-amidosecretin formed is recovered.  8 - Process according to claim 7, characterized in that the host cell is an Escherichia coli.  9 - Process according to claim 8, characterized in that the E. coli is E. coli XA35, FERM BP-116, deficient strain of β-galactosidase gene and deficient also in gene "i".  10 - Process according to claim 8, characterized in that the lactose operon is drawn from E. coli.  II - Process according to claim 10, characterized in that the vector plasmid contains all or part of the lactose operon derived from the DNA of E. coli chromosomes. coli and that it is able to proliferate in E. coli.  12 - Process according to claim 1, characterized in that the vector plasmid is derived from a transduction bacteriophage containing all or part of the lactose operon and a plasmid of E. coli.  13 - Process according to claim 12, characterized in that the transduction bacteriophage is pdld, F'-lac, t80dplac, Ah80dlac or #plac.  14 - Process according to claim 12, characterized in that the plasmid taken from E. coli is pBR 322, pSC 101 or #dv1.  15 - Process according to claim 13, characterized in that the bacteriophage is XplacS,  16 - Process according to claim 14, characterized in that the plasmid is pBR 322.  17 - Method according to one of claims 15 and lô, characterized in that the vector plasmid is pRE which comprises 3.8 Md of fragment # plac5, chained to the largest fragments obtained by digestion of pBR322 with EcoRI and Hind 1II.  18 - Process according to claim 17, characterized in that the chimeric plasmid is pLS which is an insertion product of the structural gene mentioned in pRE at its point of recognition of EcoRI.  19 - Escherichia coli transformed with a plasmid of which is incorporated a structurally synthesized deamidosecretin structural gene corresponding to the polypeptide whose amino acid of the C-terminus is valine and capable of using the lactose operon and also to proliferate in a predetermined hood cell.  An E. coli according to claim 19, which is E. coli XA35 (pLS58), ATCC 39040.  Two-chain polydeoxyribonucleotide comprising a structural gene express 27-deamidosecretin.  22) A two-chain polydeoxyrubenucleotide according to claim 21, characterized in that the structural gene has the basic sequence:  His Asp Asp Gly Thr Phe Thr  CAC - TCA - GAT - GGT - ACT - TTC - ACC  GTG - AGT - CTA - CCA - TGA - AAG - TGG  Ser Glu Leu Ser Arg Arg Arg  TCA - GAA - CTA - TCT - CGT - CTA - CGT  AGT - CTT - GAT - AGA - GCA - GAT - GCA  Asp Ser Ala Arg Leu Gln Arg  GAT - TCA - GCA - CGC - CTC - CAG - CGC  CTA - AGT - CGT - GCG - GAG - GTC - GCG  Leu Leu Gln Leu Val  TTG - CTG - CAA - GGT - CTC - GTT  AAFC - GAC - GTT - CCA - GAG - CAA.  23 - A two-chain polydeoxyribonucleotide according to one of claims 21 and 22, characterized in that the structural gene contains a codon designating the methionine at the 5t end and one or more codons used to designate the translational stop at the end 3t.  24 - A polyoleoxyribonucleotide with two rings according to claim 23, characterized in that the end 5t of the methionine codon and the end 38 of the translational stop codon have 3m base pairs, (3m + 1) base pairs or (31 + 2) base pairs (where m is 0 or an integer of at least 1) of randomly selected bases.  A two-membered polydeoxyribonucleotide according to claim 24, characterized in that it comprises a fundamental recognition sequence of any desired restriction at both ends and has at least two pairs out of the resulting ends. base, den to give dull ends chosen randomly sieved.  26 - A two-chain polydeoxyribonucleotide according to claim 21, characterized in that it has the following structure:  Met  ACCTGCAGCC - ATG  TGGACGCGG - TAC  His Asp Asp Gly Thr Phe Thr  CAC - TCA - GAT - GGT - ACT - TTC - ACC  GTG - AGT - CTA - CCA - TGA - AAG - TGG  Ser Glu Leu Ser Arg Arg Arg  TCA - GAA - CTA - TCT - CGT - CTA - CGT  AGT - CTT - GAT - AGA - GCA - GAT - GCA  Asp Ser Ala Arg Leu Gln Arg  GAT - TCA - GCA - CGC - CTC - CAG - CGC  CTA - AGT - CGT - GCG - GAG - GTC - GCG  Leu Leu Gln Leu Val  TTG - CTG - CAA - GGT - CTC - GTT  AAFC - GAC - GTT - CCA - GAG - ss  END FIN  TGA - TAG - GGCTGCAGGT  ACT - ATC - CCGACGTCCA  Plasmid comprising a structural gene which expresses 27-desamidosecretin.  28 - Plasmid according to claim 27, characterized in that it comes from pBR322.  29 - Process for the preparation of 27-deamidosecretionic acid characterized by the following steps t  (1) there is provided a chimeric plasmid which comprises a fragment of a structural gene suitable for a desamidosecretin corresponding to a polypeptide in which the amino acid of the C-terminus is valine, the plasmid chimer being capable of proliferating in a a predefined host cell capable of expressing the appropriate structural gene for a deamidosecretin in the host cell,  (2) transforming the host cell with the chimeric plasmid,  (3) the transformant obtained is cultivated and the deamidosecretin formed is recovered.  30. The method according to claim 29, wherein the chimeric plasmid is capable of utilizing the lactose operon.  31 - Process according to claim 30, characterized in that the chimeric plasmid is obtained by inserting the structural gene into a vector plasmid capable of using the lactose operon and also capable of proliferating in the host cell.  32 - Method according to claim 31, characterized in that the vector plasmid contains all or part of the lactose operon derived from the DNA of E chromosomes. coli and that it is able to proliferate in E. coli.  33 - Method according to claim 32, characterized in that the vector plasmid is derived from a transduction bacteriophage containing all or part of the lactose operon and a plasmid taken from E. coli.  34 - Method according to claim 33, characterized in that the transduction bacteriophage is pldl '-lac, 80dplac, h80dplac or J plac.  35 - Method according to claim 33, characterized in that the plasmid dgE. coli is pBR322 pSC101 or dvl.  36 - Process according to claim 34, characterized in that the bacteriophage is # plac5.  37. The method according to claim 35, characterized in that the plasmid is pBR322.  38 - Process according to claims 36 and 37, characterized in that the vector plasmid is pRE, which comprises 3.8 Md of the fragment of # plac5 chained to the largest fragments obtained by digestion of pBR322 with EcoRI and HindIII.  39. Method according to claim 38, characterized in that the chimeric plasmid is pLS, which is an insertion product of the structural gene into pRE at its EcoRI recognition point.