JP2744553B2 - Human calcitonin polymerized protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a human calcitonin polymerized protein for producing human calcitonin, and a DNA encoding the protein.

[0002]

2. Description of the Related Art Calcitonin has the effect of lowering the calcium ion concentration in serum and is useful as a therapeutic agent for osteoporosis, bone page disease, hypercalcemia, etc. (Bioindustry, Bioindustry, 6, 778 pages, (1
989)). Calcitonin is composed of 32 amino acids, C
This is a peptide hormone in which the carboxyl group of the terminal Pro residue is amidated. Conventionally, humans, salmon, eels,
Known from chickens, pigs, cows, sheep, and rats, there is a mutual difference in the amino acid composition among animal species when comparing their molecular structures. When calcitonin derived from a foreign animal other than human is used as the above-mentioned therapeutic agent, it may show antigenicity as a foreign protein, and there is a risk of causing anaphylactic shock when administered again to a patient. Therefore, human form of calcitonin used as a therapeutic agent is preferred. Calcitonin is produced in the thyroid gland of mammals (Takuo Fujita, Hajime Orimo: Calcitonin, Life Science Publishing, (1984)), but its production is very small, so natural human calcitonin is collected in large amounts from tissues. It is impossible.

[0003] As a method for overcoming this point and obtaining a considerable amount of human calcitonin, (1) production by a chemical synthesis method (R. Neher et al: Helvetica Chimica Acta. 51, 1900,
(1986)), (2) Production of recombinant human calcitonin by genetic manipulation (JP-A-58-501121, JP-A-58-203953)
And JP-A No. 1-10999).

[0004] In particular, the method (2) makes it possible to collect a large amount of the target substance and reduce the production cost.

On the other hand, in the field of genetic engineering, a method for dramatically increasing the yield of the same peptide has been pioneered.

[0006] That is, when a peptide hormone is usually produced by a genetic engineering technique, a host transformed by inserting one gene encoding the amino acid sequence into a suitable expression vector and using the same. A method has been adopted in which the target substance is expressed by culturing to collect the target substance. In this method, in the case of a peptide hormone having a small molecular weight, a host was transformed with an expression vector constructed so as to obtain a fusion protein fused with another protein in order to express the peptide hormone in a stable state and expressed. A method for obtaining a target peptide from a fusion protein is usually used. The increase in the production amount of the target peptide can be attained by selecting a host-vector system capable of expressing efficiently, selecting appropriate culture conditions for increasing the amount of the transformed host, or obtaining It depends on how efficiently the target peptide is cleaved from the obtained fusion protein.

However, if the ratio of the target peptide to the molecular weight in the obtained fusion protein is small, the target peptide cannot be obtained in high yield.

Therefore, in order to increase the ratio of the target peptide in the fusion protein, a gene in which a plurality of genes encoding the target peptide are linked (hereinafter referred to as "tandem repeat type gene"). ) Is constructed, and a method for expressing and collecting a large amount of a target peptide by culturing a transformed host using the expression vector has been reported (Shen: Proc. Natl. Acad. Scie. USA
81, 4627, (1984)). By this method, the expression vector
It is possible to express a target peptide in an amount corresponding to the number of genes encoding the target peptide inserted into the gene, compared to the case where a conventional expression vector having one gene inserted is used. As a result, it has become possible to secure several times the production volume.

[0009] Even when human calcitonin is produced by genetic engineering, the expression vector into which the above-mentioned tandem-repeat type gene is inserted and the host is transformed can produce a much larger amount than before. Human calcitonin can be produced.

The Met residue at position 8 of human calcitonin is replaced with Val
Regarding human calcitonin converted to a residue, there is a report that the human calcitonin was expressed in a tandem repeat type gene (I.Iv
anovet al: Biotechnol. Appl. Biochem. 11, 401, (198
9)). However, to date, tandem recombinant human calcitonin having the same amino acid sequence as the natural type has
There has been no example of successful production of human calcitonin using an expression vector into which a repeat-type gene has been inserted.

[0011]

That is, (1) calcitonin is amidated at the carboxyl group of the Pro residue of the C-terminal amino acid, and this acid amide is indispensable for the expression of the physiological activity of calcitonin. When a peptide having a C-terminal amide is obtained by genetic manipulation, the C-terminal carboxyl group must be artificially subjected to amidation after the peptide chain is synthesized. It must be produced as a human calcitonin precursor having a unique structure. Specifically, a peptide containing one unit of a human calcitonin precursor in which one or more amino acids are bonded to the C-terminal Pro residue of human calcitonin (hereinafter, referred to as “precursor peptide”) may be finally obtained. In addition, it is necessary to design a tandem repeat type gene. (2) The protein translated by the tandem repeat type gene can be obtained as a macromolecule protein having a repeating sequence of amino acids. It is necessary to design a tandem-repeat-type gene by subjecting this to a hydrolysis treatment with a protease to finally obtain the above-mentioned one unit precursor peptide. Is
It is necessary to select one that does not cleave human calcitonin itself. (3) The tandem repeat type gene is obtained by linking a plurality of genes encoding the precursor peptide. Inevitably, the technique used in normal genetic manipulation must be adopted.
There were problems such as the need to satisfy (1) and (2).

[0012]

Means for Solving the Problems The present inventors have constructed a tandem repeat type gene which solves the above problems and completed the present invention.

That is, the present invention provides (1) a protein encoded by a tandem repeat type gene, which is represented by the general formula (I): (N) -α-δ-X- [δ-β -Δ-X] _n -δ-γ- (C) (I) (wherein X is a peptide consisting of the amino acid sequence of amino acids 1 to 33 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing) , Α, β and γ are [(i) to
(Iii) one or two or more identical or different amino acid residues [-i-
A restriction enzyme recognition sequence exists in the DNA sequence encoding δ, and a restriction enzyme recognition sequence exists in the DNA sequence encoding δ-γ- in the formula (ii). The sequence is different from the recognition sequence shown in (i), and (iii) the DNA sequence coding for β in the formula is a DNA sequence coding for -α-δ and δ-γ- in the formula, These were ligated after cutting with the corresponding restriction enzymes. ], Δ is Arg residue or Glu residue, n is 1
(N) indicates the N-terminal, and (C) indicates the C-terminal. And (2) a DNA encoding the human calcitonin polymerized protein described in (1) above.

One embodiment of the present invention provides a tandem lipid
A protein encoded by the G-type gene, which is represented by the general formula (I): (N) -α-δ-X- [δ-β-δ-X] _n -δ-γ- (C) (I (Wherein, X is a peptide consisting of the amino acid sequence of amino acids 1 to 33 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, α, β and γ are the following [(i) to
(Iii) one or two or more identical or different amino acid residues [-i-
A restriction enzyme recognition sequence exists in the DNA sequence encoding δ, and a restriction enzyme recognition sequence exists in the DNA sequence encoding δ-γ- in the formula (ii). The sequence is different from the recognition sequence shown in (i), and (iii) the DNA sequence coding for β in the formula is a DNA sequence coding for -α-δ and δ-γ- in the formula, These were ligated after cutting with the corresponding restriction enzymes. ], Δ is Arg residue or Glu residue, n is 1
(N) indicates the N-terminal, and (C) indicates the C-terminal. ) Is a human calcitonin polymerized protein represented by

Human calcitonin is the amino acid sequence of amino acids 1 to 32 of the amino acid sequence shown in SEQ ID NO: 1 in which the carboxyl group of the C-terminal Pro residue is amidated. However, when produced by a microorganism such as Escherichia coli, the C-terminal amidation reaction is not performed in the cells. Thus, it is necessary to produce human calcitonin as a precursor obtained by adding one or more amino acids to the C-terminus, and then perform amidation. In order to obtain this precursor, the above-mentioned tandem repeat type gene is used.

The cleavage site of the protease for excision of one unit of human calcitonin precursor from the polymerized protein expressed by using the tandem repeat type gene is limited to those not present in the amino acid sequence of human calcitonin. Is done. In the present invention, an Arg residue or a Glu residue is preferable (δ in the general formula (I)), and the corresponding protein
As zero, for the Arg residues, clostripain, trypsin, mouse submandibular gland Protea -. Ze, thrombin, kallikrein, plasmin, trypsin-like enzyme of cathepsin B ₁ or microorganisms ( `The Enzymes' ed by PDBaye
r, AcademicPress, New York, 3, 721, (1971))
For the lu residue, V8 protease or sea urchin hatching enzyme is used, but clostripain or trypsin or V8 protease is preferably used.

The amino acid adjacent to the C-terminal Pro residue in the amino acid sequence of human calcitonin (amino acid number 33 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing)
For the amidation with carboxypeptidase Y, a Leu residue, an Ile residue, a Val residue, a Phe residue or an Ala residue is used, and a Leu residue is preferably used. On the other hand, a Gly residue is suitable for amidation with peptidylglycine α-amidating monooxygenase.

In view of the above, the present inventors have developed a peptide (X-δ in the general formula (I)) consisting of amino acids 1 to 34 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Was selected as one unit of precursor peptide excised from the polymerized protein.

Further, in order to cut out one unit of the precursor peptide from the polymerized protein, an Ar terminal is added to the N-terminal side of the amino acid sequence of the precursor peptide in the amino acid sequence of the polymerized protein.
g or Glu residues must be added adjacent to each other.

To construct a tandem repeat type gene for expressing a gene encoding a peptide having an Arg residue or a Glu residue adjacent to the N-terminal side of the above-mentioned precursor peptide to express a polymerized protein. In order to obtain one unit, the upstream region or the downstream region including a region encoding an Arg residue or a Glu residue in the gene (-α-δ or δ-γ-
It is necessary to allow a recognition sequence by a restriction enzyme to be present in the region encoding the As the recognition sequence by the restriction enzyme selected here, those having different sequences in the upstream region and the downstream region and generating the same protruding end are preferable.
Bgl II or a combination of SalI and AvaI is good.
In addition, blunt ends can be used instead of protruding ends. In addition, T4 does not generate the same overhanging ends.
Any material can be used as long as it can be blunted with a polymerase or the like.

The DNA sequence encoding β in the general formula (I) is obtained by digesting the DNA sequences encoding -α-δ and δ-γ- in the formula with the corresponding restriction enzymes. It is a concatenation. In this case, when the obtained tandem repeat type gene is translated from the upstream, it is preferable that the above-mentioned restriction enzyme recognition sequence is present so that the reading frame to amino acids does not shift. If the reading frame is shifted, it is possible to match the reading frame after connection by using a site direct mutagenesis method or the like.

Another aspect of the present invention relates to a DNA sequence encoding a human calcitonin polymerized protein. Such a DNA sequence includes a DNA encoding the general formula (I).
Includes all sequences. As a specific example, of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, a DNA sequence shown in nucleotide numbers 1 to 234 (a DNA sequence encoding the peptide of n = 1 in the general formula (I)) ), But the present invention is not limited to this.

Hereinafter, a method for constructing a tandem repeat type gene will be described. A gene which is a basic gene for constructing a tandem repeat type gene (in the general formula (I), a DNA sequence encoding the amino acid sequence when n = 0):
Hereinafter, it is referred to as “basic gene I”. ) Can be obtained by genetic engineering using cDNA or genomic DNA prepared by extracting mRNA from an organ, or a chemical synthesis method. The chemical synthesis method is most suitable.

When the DNA sequence is designed by the chemical synthesis method, the frequency of codon usage of the host to be expressed and
Secondary structure must be considered. Of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, the basic gene I when expressed in Escherichia coli at nucleotide numbers 1 to 120
1 shows an example of the DNA sequence of the present invention. In this DNA sequence, in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, the amino acid residue represented by amino acid number 33 is a Leu residue, and δ in the general formula (I) is an Arg residue. BamHI and BamHI were constructed in the upstream and downstream regions of the gene in order to construct a tandem repeat type gene.
A cleavage site by Bgl II is provided. Further, a translation stop codon is provided downstream of the basic gene I (nucleotide numbers 121 to 130 in the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing), and further inserted downstream thereof into an expression vector. In order to facilitate the digestion, a site for cleavage by SalI is added. However, this cleavage site by SalI is not essential.

An example of a method for synthesizing the above gene is described below. First, six kinds of polynucleotide fragments represented by SEQ ID NOs: 4 to 9 in the sequence listing were converted into DNA synthesizers (for example, Applied Bi
osystems Model 380B). next,
After annealing with a combination of polynucleotides represented by SEQ ID NOs: 4 and 5, 6 and 7, 8 and 9 in the sequence listing,
The obtained three fragments were ligated to obtain the basic gene I.
That has a translation stop codon and a SalI cleavage site downstream of
(Hereinafter referred to as "basic gene II").

Next, the obtained basic gene II is inserted, for example, between BamHI and SalI of plasmid pUC18 of E. coli,
This plasmid is introduced into, for example, E. coli JM109 strain. For example, when the host cell is Escherichia coli, the transformation of the host cell is performed according to the method of Hanahan (Hanahan, D .: J. Mol. Biol. 16).
6,557, (1983)), i.e.CaCl ₂ or MgCl ₂ or RbCl
The recombinant DNA is added to competent cells prepared in the presence of
It can be carried out by adding a body. In addition, a lambda-based phage vector can be used in addition to the plasmid.

From the transformants obtained above, those into which the gene fragment of interest was inserted were selected, and the DNA sequence of the synthesized gene was determined by the chemical modification method of Maxam-Gilbert (Maxam, AM and Gilbert, W.:"Methods in Enzymol
ogy "65,499, (1980)) and the dideoxynucleotide chain termination method using M13 phage (Messing, J. and Vieira, J .: Ge
ne 19, 269, (1982)).

Here, a tandem repeat type gene can be constructed using the basic gene II for which DNA sequencing has been performed. This flowchart is shown in FIG.

First, the basic gene between BamHI and SalI of pUC18
The plasmid into which II was inserted (FIG. 1, a) was replaced with BglII and Sa.
Cut with lI. After cutting, the basic gene is inserted between BglII and SalI.
When II is inserted, the gene coding for the precursor peptide is converted to a nucleotide sequence (hereinafter, referred to as "DNA linker") corresponding to the peptide (hereinafter, referred to as "linker peptide") connecting the precursor peptides. Can be obtained by connecting the two (FIG. 1, b).

In this case, a site linked by a BamHI cleavage site and a BglII cleavage site (FIG. 1, b: (Ba / Bg): AGATCC)
Is not cleaved by both enzymes, unlike the recognition sequences of BamHI (GGATCC) and BglII (AGATCT). The tandem repeat type gene obtained here has the above-mentioned restriction enzyme recognition sequence added so that the reading frame to amino acids does not shift when translated from the upstream.

Next, the plasmid of FIG. 1B is cut with BglII and SalI. After the cleavage, by inserting the basic gene II between BglII and SalI, a tandem repeat type gene in which genes coding for three precursor peptides are linked can be obtained. By sequentially performing such operations, a tandem repeat-type gene in which genes encoding a plurality of precursor peptides are linked can be obtained.

As an alternative to the construction method, an already prepared tandem repeat type gene can be used instead of the basic gene II to be inserted. For example, BamHI-Sa containing the tandem repeat type gene was obtained from the plasmid of FIG.
The lI fragment was extracted and this fragment was
By inserting between BglII and SalI, it is possible to obtain a tandem repeat type gene in which genes encoding four precursor peptides are linked (FIG. 1, c).

By appropriately combining the above methods, a tandem-repeat-type gene in which genes encoding a plurality of precursor peptides are linked is constructed.

The tandem repeat type gene thus constructed can be used as a vector suitable for transforming a host.
Other prokaryotic or eukaryotic host cells can be transformed by insertion into the DNA. further,
By introducing appropriate promoters and sequences related to expression into these vectors, the tandem repeat type gene can be expressed in each host cell.

Examples of prokaryotic hosts include Escherichia coli and Bacillus subtilis. Generally, pBR322 and pUC-based plasmids are often used as Escherichia coli vectors, but are not limited thereto. Also, pT is used as a vector for Bacillus subtilis.
UB228 (Ohmura, K. et al: J. Biochem. 95, 87, (1984))
Etc. are used, but the present invention is not limited to this.

Eukaryotic host cells include cells of vertebrates, insects, yeasts and the like. As vertebrates, for example, COS cells (Gluzman, Y .: Cell 23,
175, (1981)) and Chinese hamster ovary cells (CH
O) -deficient dihydrofolate reductase (Urlaub, G. and
Chasin, LA: Proc. Natl. Acad. Sci. USA 77, 4216,
(1980)) is often used, but is not limited to this.

One part of the present invention is to introduce a tandem repeat type gene into a host cell, express the gene, and obtain a polymerized protein. Preferred operations and conditions for culturing the transformant are selected according to the type of host-vector system to be expressed.

When the tandem-repeat type gene is expressed by linking it to a gene containing a signal sequence, when an animal cell or Bacillus subtilis, yeast, filamentous fungus or the like is used as a host, the polymerized protein produced is extracellular. It is secreted and accumulates in the culture filtrate. When Escherichia coli is used, it is accumulated in the periplasm fraction or the culture filtrate.

On the other hand, when linked to a gene having no signal sequence, the resulting polymerized protein often forms an inclusion body (inclusion body) in the cell as an insoluble protein component. Therefore, in this case,
In order to collect the target substance, it is necessary to centrifuge the culture solution, collect the cells of the precipitated fraction, and crush the cells. Ultrasonic treatment, lysium treatment, freeze-thaw treatment, or the like can be employed as a method for disrupting the cells.

The method for preparing a polymerized protein will be described with reference to the case of expression in E. coli as an example.

In the above-mentioned plasmid (FIG. 1), the tandem repeat type gene was inserted into the plasmid pUC18 of E. coli, and the reading lacZ gene under the control of the lac promoter of pUC18 was inserted into the plasmid. The frames are connected so that they match. This plasmid is designated as pUCal m (m: represents the number of genes encoding the precursor peptide). pUCalm
Was used to transform Escherichia coli strain YA21, and transformed strain YA21 was used.
Get / pUCal m The obtained transformant was transformed with 2 x YT medium (16 g tryptone, 10 g yeast extract,
After shaking culture at 37 ° C. for 8 hours using 5 g of sodium chloride in 1 l of each solution (containing 25 μg / ml of ampicillin), 0.5% inoculation was performed in the same medium (25 μg / ml of ampicillin). And shake culture at 37 ° C for 16 hours. At this time, the target polymerized protein is accumulated in the cells as an inclusion body.

Next, the cells are collected from the culture solution by centrifugation, washed with an appropriate buffer, and then disrupted by sonication. Since the polymerized protein is present as an insoluble component, the precipitated cell fraction is collected after centrifuging the crushed cells,
Wash with a buffer containing a surfactant such as Triton X-100. By centrifuging again, the impurities are transferred to the supernatant fraction, and the desired polymerized protein can be obtained in the precipitate fraction. At this time, the polymerized protein is obtained as a fusion protein in which a partial peptide chain of β-galactosidase is bonded to its N-terminal.

Another aspect of the present invention is to obtain a precursor peptide from the polymerized protein, which is achieved by cleaving the polymerized protein with a protease. Tandem Lipid for genes without signal sequence
When the G-type gene is linked and expressed, the polymerized protein is often produced as an insoluble protein component. In this case, when the prepared polymer protein is cleaved with protease after culturing, it is preferable to add a protein solubilizing agent to solubilize the protein. As the protein solubilizing agent, guanidine hydrochloride, urea and the like are employed.
Urea is preferred.

When a tandem repeat-type gene is expressed by ligating it to a gene having a signal sequence, it is soluble, so that it is not necessary to add a protein solubilizing agent.

Cleavage of the prepared polymerized protein by protease is performed by removing an amino acid residue represented by δ in the general formula (I).
When Arg residue is used, clostripain is employed as a protease. When the amino acid residue represented by δ in the general formula (I) is a Glu residue, V8 protease is used as the protease used.

As a specific example, the plasmid pUCa
Cleavage by a protease when expressing and preparing a polymerized protein using lm as an expression vector and Escherichia coli YA21 as a host will be described.

When the transformant YA21 / pUCal m is cultured as described above to prepare a polymerized protein, it is prepared as an insoluble component, and urea is added to solubilize it. The urea concentration is then reduced in order to carry out cleavage with clostripain. Clostripain is suitable for the cleavage reaction of the present invention because it is a protease that cleaves peptide bonds only at the carboxyl group side of Arg residues in proteins. Activated clostripain is added to the diluted urea solution containing the polymerized protein, and the cleavage reaction is carried out at about pH 8.0 at 37 ° C. for 2 to 4 hours to perform the cleavage reaction.

As described above, the precursor peptide obtained by cleaving the polymerized protein is obtained by subjecting the reaction solution to ion exchange column chromatography, gel filtration column chromatography, or the like.
The separation and purification can be carried out by using, for example, a reverse phase column chromatography.

Finally, from the precursor peptide obtained as described above, human calcitonin having the same structure as that of the natural type (the C-terminal Pro residue is amidated) can be obtained.

According to this method, the C-terminus (amino acid residue represented by δ in the general formula (I)) of the precursor peptide is changed to Ar
In the case of g residue, Arg residue is removed by the action of carboxypeptidase B under the conditions of about pH 8, 37 ° C., and 1 to 2 hours. The reaction solution thus obtained was subjected to the same column chromatography operation as described above, whereby the peptide consisting of the amino acid sequences of amino acids 1 to 33 out of the amino acid sequences shown in SEQ ID NO: 1 in the sequence listing was obtained. Obtainable.

Next, the peptide obtained as described above is
Using carboxypeptidase Y (JP-A-62-29997)
No.) and amidation of the C-terminus, human calcitonin having the same structure as the natural form can be obtained.

[0052]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Embodiment 1 Chemical synthesis of basic gene II When designing Escherichia coli as the host, the codon usage of Escherichia coli (Sharp, PM et al .: Nucleic Acid
ds Res. 16, 8207, (1988)) and secondary structure. Recognition sequences by restriction enzymes (BamHI and BglII) necessary for polymerizing the gene were added to the upstream and downstream regions of the gene encoding the precursor peptide. Further downstream, a recognition sequence by a restriction enzyme (SalI) was added in consideration of insertion into an expression plasmid. Of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, the DNA sequence is shown in nucleotide numbers 1-130.

The total synthesis of the basic gene II was carried out by using six kinds of polynucleotides having a length of 39 to 43 bases shown in SEQ ID NOs: 4 to 9 (hereinafter referred to as "F1,""F2," and "F3", respectively).
They are called "F4", "F5" and "F6". ) Was performed using Applied Biosystems Model 380B DNA synthesizer. After synthesis, each polynucleotide was purified by 10% polyacrylamide gel electrophoresis containing urea. Annealing and T
4 The ligation reaction of polynucleotides by DNA ligase
According to the method of Ikehara et al. (Proc. Natl. Acad. Sci. USA
81, 5956 (1984)).

The purified polynucleotide is 5 ′ of F2 to F5.
The ends were phosphorylated, annealed with the combination of F1 and F2, F3 and F4, and F5 and F6, and ligated with T4 DNA ligase to obtain the basic gene II.

Embodiment 2 FIG. Construction of human calcitonin polymerized protein expression plasmid in Escherichia coli FIG. 1 shows a pattern of construction of a human calcitonin polymerized protein expression plasmid.

Phosphorylated 5 'end of the basic gene II obtained in Example 1 was used to obtain BamHI and SalI of the expression vector pUC18.
To obtain pUCal 1 (FIG. 1, a). The DNA sequence of the inserted gene fragment was confirmed by the dideoxynucleotide chain termination method.

Next, between BglII and SalI of pUCal 1,
Insert a phosphorylated 5 'end of basic gene II,
pUCal 2 (FIG. 1, b) was obtained. BamHI and Bg used here
II has the same protruding end and is ligable, and the junction is not recut by both BamHI and BglII.

Next, pUCal 2 was cut with BamHI and SalI,
6% polyacrylamide gel electrophoresis was performed, and a small fragment thereof was extracted from the gel. Separately, pUCal 2 is
After digestion with BglII and SalI, 1% agarose gel electrophoresis was performed, and a large fragment thereof was extracted from the gel. By ligating the two fragments obtained here, pUCal 4 (FIG. 1, c) was obtained. Next, similarly, the BamHI-SalI small fragment of pUCal2 was ligated to the BglII-SalI large fragment of pUCal4 to obtain pUCal6. By performing this operation using the above-mentioned various combinations of plasmids, an expression plasmid in which a plurality of genes encoding the precursor peptide were arranged was obtained.

Embodiment 3 FIG. Expression in E. coli Plasmids such as pUCal1, pUCal2 and pUCal4 obtained earlier
Since the reading frame is ligated to the lacZ gene under the control of the lac promoter of pUC18, expression is possible by introducing these plasmids into the host. FIG. 2 shows a schematic diagram of the polymerized protein predicted by pUCal4, and SEQ ID NO: 10 in the sequence listing shows the amino acid sequence of the polymerized protein expressed by pUCal4.

Escherichia coli YA21 was prepared using the various expression plasmids described above.
The strain (F ^- λ ^- leu met relA) was transformed by a conventional method.
Ampicillin 25 μg /
2 x YT medium containing 16 g tryptone (Difco), 10 g
The yeast extract (Difco) and 5 g of sodium chloride were each contained in 1 l of the solution) and shake-cultured at 37 ° C. for 16 hours. YA carrying various expression plasmids
13% polyacrylamide SDS gel electrophoresis was performed on all bacterial cell proteins after culturing the 21 strains. It was confirmed by Western blotting using anti-human calcitonin antiserum that the protein expressed in a large amount was a polymerized protein containing a precursor peptide.

Embodiment 4 FIG. Preparation of Polymerized Protein Escherichia coli YA21 strain into which the above expression plasmid was introduced was shake-cultured at 37 ° C. for 8 hours in 2 × YT medium containing 25 μg / ml of ampicillin, and then 2 × YT containing 25 μg / ml of ampicillin was obtained.
0.5 ml (v / v) was inoculated into 100 ml of the medium, and cultured with shaking at 37 ° C. for 16 hours. At the end of the culture, the polymerized protein had formed an inclusion body in the cells of the YA21 strain.

After completion of the culture, the cells were collected by centrifugation at 3000 × g for 5 minutes, and 20 ml of buffer A (50 mM Tris (hydroxymeth
yl) aminomethane, pH 8.0, 1mM EDTA, 50mM sodium chloride), and add 1mg of lysozyme (Sigma).
It was kept at 5 ° C for 20 minutes. Thereafter, the cells were disrupted by sonication, and the inclusion body was obtained as a precipitate by high-speed centrifugation (11000 × g, 20 minutes). Since the precipitate fraction still contained a large amount of bacterial cell fragments, it was washed with buffer A containing 0.5% (v / v) of Triton X-100. After washing, centrifugation is performed, and the precipitated fraction is removed in a small amount of 20 mM Tris (hydr).
This was suspended in oxymethyl) aminomethane (pH 8.1), and this was used as a crude preparation of a polymerized protein.

By preparing as described above, 100 m
l Retain pUCal 4, pUCal 6, and pUCal 8 per culture
With the YA21 strain, 2.2 mg, 4.8 mg and 3.4 mg of polymerized protein could be obtained, respectively.

Embodiment 5 FIG. Preparation of Precursor Peptide from Human Calcitonin Polymerized Protein (1) In order to obtain a precursor peptide from the polymerized protein, cleavage of the polymerized protein with clostripain was performed. In 1.7 ml of a crude preparation of a polymerized protein obtained by culturing Escherichia coli using the expression vector pUCal6, 1.7 ml (total protein content: 16.6 mg, containing approximately 30% of polymerized protein), 3.3 ml of 10 M urea, 450 μl
1M Tris (hydroxymethyl) aminomethane, 150 μl β
-Mercaptoethanol was added, and the mixture was allowed to stand at 37 ° C for 1 hour. Then add 5.0 ml of 20 mM calcium nitrate and add pH
After preparation to 7.8, activated clostripain (Sigma):
12.3 Enzyme units) was added and reacted at 37 ° C. for 2 hours.

The reaction was stopped by adding 800 μl of trifluoroacetic acid, and the resulting reaction solution was centrifuged, and the supernatant was recovered.

The desired product (precursor peptide) was purified by high performance liquid chromatography. 100 μl of the above reaction supernatant was equilibrated with 0.1% trifluoroacetic acid containing 20% acetonitrile in a TSKgel ODS-120T reverse phase column (4.6
x 250mm) (manufactured by Toso Corporation) and subjected to a linear gradient method of acetonitrile (20-30% (0-5 minutes), 30-40% (5-15 minutes),
40-50% (15-20 minutes)) and eluted at a flow rate of 1.0 ml / min.
I), two major peaks were obtained at 15.54 minutes (Peak II). Each of these peak fractions was collected, and the obtained substance was subjected to amino acid sequence analysis using a protein sequencer-470A manufactured by Applied Biosystems. It was a peptide consisting of the amino acid sequence of amino acids 16 to 49 in the amino acid sequence shown in SEQ ID NO: 10 (hereinafter referred to as "hCT-Leu-Arg").

Peak I corresponds to amino acids 16 to 53 of the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing.
A peptide consisting of the amino acid sequence of hCT-Leu-Arg
(N) -Ala.Asp.Pro.Arg- (C) linker peptide was added to the C-terminal side of the sample.

When the clostripain cleavage reaction was continued for a longer time, peak I decreased and peak II increased. The peak II substance (hCT-Leu-Arg) was collected by repeating the above high performance liquid chromatography.

Next, carboxypeptidase B (Sigma) was allowed to act on the peak II substance (1.5 mg) to remove the C-terminal Arg residue. As a pretreatment for the reaction to enhance the solubility of the peak II substance, the method of R. David Cole (CHHirs: "Method in Enzymology" 11, 206 (196
According to 7)), Cys residues at the 1- and 7-positions of the disulfide bond were subjected to a reversible S-sulfonation reaction. The reaction product was subjected to high-performance liquid chromatography using a TSKgel-ODS-120T reversed phase column (4.6 x 250 mm) (manufactured by Tosoh Corporation) equilibrated with 10 mM ammonium bicarbonate. Using the concentration linear gradient method with acetonitrile (the conditions are the same as described above) 1.
Elution was performed at a flow rate of 0 ml / min, and detection of the ultraviolet light at 225 nm revealed the target product of S-sulfonation at 11.27 minutes, which was collected. Preparative liquid (containing 1.3mg precursor peptide)
After adding an equal amount of distilled water, 0.087 enzyme units of carboxypeptidase B was added and reacted at 37 ° C. for 30 minutes. The reaction solution was subjected to high performance liquid chromatography under the same conditions as above.
As a result, a peak was detected at 10.37 minutes. The obtained peak fraction material was subjected to a desulfonation reaction and then to an amino acid sequence analysis. As a result, the amino acid sequence of amino acids 16 to 48 of the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing was obtained. (Hereinafter referred to as “hCT-Leu”).

Further, in order to confirm that the C-terminal Arg residue had been removed, amino acid composition analysis was performed using a Hitachi 835-type amino acid automatic analyzer. Table 1 shows the results.

[0072]

[Table 1]

From the above results, this peak substance was found to be hCT-
The hCT-Leu was confirmed to have the C-terminal Arg residue of Leu-Arg removed. The desired hCT-Leu was collected by repeating the above high performance liquid chromatography.

Embodiment 6 FIG. C-terminal amidation reaction In order to produce human calcitonin having the same structure as the natural form from hCT-Leu obtained in Example 5, a C-terminal amidation reaction was performed. This reaction is basically carried out according to JP-A-62-299.
This was performed according to the method described in No. 97.

That is, 50 μl of S-sulfonated hC
400 μl in T-Leu solution (40% dimethyl sulfoxide solution)
l of a 5.68M ammonia solution (aqueous ammonia-hydrochloric acid, pH 9.
5), 50 μl carboxypeptidase Y solution (1.7 mg /
ml) and left at 50 ° C. for 30 minutes. Reaction is 100 μl
Formic acid and 1500 μl of 8 M urea were added and stopped. 210 μl of reaction solution was equilibrated with 10 mM ammonium bicarbonate
The product was applied to a TSKgel ODS-120T reverse phase column (7.8 x 300 mm) (manufactured by Tosoh Corporation). Using the linear concentration gradient method with acetonitrile (0-10% (0-4 minutes), 10-55% (4-60 minutes))
Elution was carried out at a flow rate of 2.5 ml / min, and detection by ultraviolet 225 nm gave a main peak at a position corresponding to the standard human calcitonin sample at 32.67 minutes in a yield of 53%. The peak fraction material was collected, and after desulfonation reaction, amino acid composition analysis and amino acid sequence analysis were performed to confirm that it was human calcitonin having the same structure as the natural type.

[0076]

According to the present invention, human calcitonin can be produced several times to several tens times at a time as compared with the conventional genetic engineering method.

[0077]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 34 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Hypothetical sequence: No Sequence characteristics: 1-32 E mat peptide 33 E Gly, Leu, Ile, Val, Phe or Ala 34 E Arg or Glu sequence Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe 1 5 10 15 Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro 20 25 30 Xaa Xaa SEQ ID NO: 2 Sequence length: 234 Sequence type: Nucleic acid Number of strands: Double stranded Topology-: Linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical sequence : No Antisense: No Sequence characteristics: 2-232 E CDS 1-6 E BamHI recognition site 229-234 E BglII recognition site Sequence G GAT CCG CGT TGC GGT AAT CTG TCT ACT TGC ATG CTG GGC ACT TAC 46 Asp Pro Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr 1 5 10 15 ACC CAG GAC TTC AAC AAA TTC CAC ACC TTC CCG CAG ACT GCA ATC GGC 94 Thr Gln Asp Phe Asn Lys P he His Thr Phe Pro Gln Thr Ala Ile Gly 20 25 30 GTT GGA GCA CCG CTG CGT GCA GAT CCG CGT TGC GGT AAT CTG TCT ACT 142 Val Gly Ala Pro Leu Arg Ala Asp Pro Arg Cys Gly Asn Leu Ser Thr 35 40 45 TGC ATG CTG GGC ACT TAC ACC CAG GAC TTC AAC AAA TTC CAC ACC TTC 190 Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe 50 55 60 CCG CAG ACT GCA ATC GGC GTT GGA GCA CCG CTG CGT GCA GAT 232 Pro Gln Thr Ala Ile Gly Val Gly Ala Pro Leu Arg Ala Asp 65 70 75 CT 234 SEQ ID NO: 3 Sequence length: 130 Sequence type: Nucleic acid Number of strands: Double strand Topology-: Linear Sequence type : Other nucleic acid Synthetic DNA Hypothetical sequence: No Antisense: No Sequence characteristics: 2-124 E CDS 1-6 E BamHI recognition site 115-120 E BglII recognition site 125-130 E SalI recognition site Sequence G GAT CCG CGT TGC GGT AAT CTG TCT ACT TGC ATG CTG GGC ACT TAC 46 Asp Pro Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr 1 5 10 15 ACC CAG GAC TTC AAC AAA TTC CAC ACC TTC CCG CAG ACT GCA ATC G GC 94 Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly 20 25 30 GTT GGA GCA CCG CTG CGT GCA GAT CTG TAA GTCGAC 130 Val Gly Ala Pro Leu Arg Ala Asp Leu 35 40 SEQ ID NO: 4 Length: 41 Sequence type: Nucleic acid Number of strands: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical sequence: No Antisense: No sequence GATCCGCGTT GCGGTAATCT GTCTACTTGC ATGCTGGGCA C 41 Sequence number : 5 Sequence length: 43 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical sequence: No Antisense: Yes Sequence GTGTAAGTGC CCAGCATGCA AGTAGACAGA TTACCGCAAC GCG 43 SEQ ID NO: 6 Sequence length: 42 Sequence type: Nucleic acid Number of strands: Single stranded Topology-: Linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical sequence: No Antisense: No sequence TTACACCCAG GACTTCAACA AATTCCACAC CTTCCCGCAG AC 42 SEQ ID NO: 7 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology-: linear Sequence type: other nucleic acid Synthetic DNA Hypothetical sequence: No Antisense: Yes sequence ATTGCAGTCT GCGGGAAGGT GTGGAATTTG TTGAAGTCCT GG 42 SEQ ID NO: 8 Sequence length: 41 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical sequence: No Antisense : No sequence TGCAATCGGC GTTGGAGCAC CGCTGCGTGC AGATCTGTAA G41 SEQ ID NO: 9 Sequence length: 39 Sequence type: Nucleic acid Number of strands: Single stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical sequence : No Antisense: Yes Sequence TCGACTTACA GATCTGCACG CAGCGGTGCT CCAACGCCG 39 SEQ ID NO: 10 Sequence length: 166 Sequence type: Amino acid Topology-: Linear Sequence type: Protein Hypothetical sequence: No Column features: 1-12 E β-Gal added peptide 16-47 E human calcitonin 54-85 E human calcitonin 92-123 E human calcitonin 130-161 E human calcitonin sequence Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Cys 1 5 10 15 Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn 20 25 30 Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro Leu 35 40 45 Arg Ala Asp Pro Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr 50 55 60 Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile 65 70 75 80 Gly Val Gly Ala Pro Leu Arg Ala Asp Pro Arg Cys Gly Asn Leu Ser 85 90 95 Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr 100 105 110 Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro Leu Arg Ala Asp Pro 115 120 125 Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp 130 135 140 Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala 145 150 155 160 Pro Leu Arg Ala Asp Leu 165

[Brief description of the drawings]

FIG. 1 shows a flowchart of construction of an expression plasmid for a human calcitonin polymerized protein.

FIG. 2 shows a schematic diagram of a polymerized protein expressed by pUCal 4.

Continued on the front page (58) Fields investigated (Int.Cl. ⁶ , DB name) C07K 14/585 C12N 15/00-15/90 BIOSIS (DIALOG) CA (STN) WPI (DIALOG) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A protein encoded by a tandem repeat type gene, which is represented by the following general formula (I): (N) -α-δ-X- [δ-β-δ-X] _n- δ-γ- (C) (I) (wherein, X is a peptide consisting of the amino acid sequences of amino acids 1 to 33 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, α, β and γ are One or two or more identical or different amino acid residues which simultaneously satisfy the following conditions ([i] to (iii)] [(i) in the DNA sequence encoding -α-δ in the formula: (Ii) a recognition sequence of a restriction enzyme is present in the DNA sequence encoding δ-γ- in the formula, and the sequence is the same as the recognition sequence shown in (i). (Iii) the DNA sequence encoding β in the formula is -α in the formula
DNA sequences coding for -δ and δ-γ- are each digested with the corresponding restriction enzymes and then ligated. ], Δ is an Arg residue or a Glu residue, n is an integer from 1 to 20, (N) is the N-terminal, and (C) is the C-terminal. ) Human calcitonin polymerized protein represented by 2. A DNA encoding the human calcitonin polymerized protein according to claim 1.