JPH03503596A - A method for preparing a protein or polypeptide, a DNA sequence encoding the polypeptide, a microorganism having the DNA sequence and the polypeptide, and use of the polypeptide as a pharmaceutical preparation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods for preparing proteins or polypeptides, DNA sequences encoding the polypeptides a microorganism having the sequence, the DNA sequence and the polypeptide, and a drug using the polypeptide. Use as a medical preparation.

Detailed description of the invention The present invention relates to a method for preparing proteins or polypeptides. This preparation method is an amino-terminal extension added to the desired polypeptide chain, preferably at least (using two amino acids).

Various valuable proteins or polypeptides (e.g. enzymes or hormones) are It can be produced biosynthetically by culturing the main bacteria. The host bacterium is a plasmid This includes vectors with NA sequences such as. This DNA sequence is the desired polypeptide. Code Chido. The resulting polypeptide is isolated from the culture medium, e.g. Purified by matoography. Polypeptides produced biosynthetically in this method Examples of tides are somatostatin, insulin and human growth hormone.

A biosynthetic method using microorganisms carrying the gene encoding human growth hormone It is known to produce more human growth hormone. human growth hormone odor The N-terminus was modified using the amino acid methionine for the purpose of translating the start code ATG. Extended. Remove methionine groups to obtain the desired protein in pure form It is necessary. This is sometimes due to enzymatic cleavage in the microorganism itself (in culture). (according to the nutritional conditions) or in the next step this cleavage is achieved enzymatically. be done. Human growth hormone occurs in a mixture of diverse polypeptides that are difficult to separate. Both cases involve the risk of various side reactions. Furthermore, the desired peptide is , present in intracellular fluids at relatively low concentrations in mixtures with other polypeptides Therefore, its isolation is difficult.

A microorganism that has a gene encoding a protein with a desired signal sequence attached to its N-terminus. (see EP0001930). The signal sequence is Because of the presence of The cells are transported into the periplasmic space where they can be isolated. This is done by lysis, which As a result, the outer membrane degenerates and intracellular fluid from the pericellular space is released and isolated.

Fluid in the periplasmic space contains only one fraction (e.g. proteins produced by microorganisms) (approximately 1%), and the desired protein is produced in a concentrated form, so the above matters are not necessary for purification. offers considerable advantages in terms of

Another advantage of transporting the desired protein into the periplasmic space is that the protein is transported within this space. substantially correctly folded and correctly folded for active protein It is to provide the original protein. This is extremely difficult to do in vitro. Production of proteins on an industrial scale is possible because there is no need to perform the difficult subsequent folding. Contains serious advantages in synthetic manufacturing.

During or after the desired protein is transported across the intracellular membrane, the signal sequence It is cleaved by enzymes contained in bacteria. However, due to the culture method, Depending on the species, lopsided non-specific cleavage of the signal sequence may occur, or subsequent cleavage may occur. an enzymatic reaction occurs, which binds the N-terminus of the desired protein to the peptidase in the periplasmic space. The cleavage is not completely specific as it is degraded to an uncontrolled extent by .

For the reasons mentioned above, this well-known method generally yields difficult-to-isolate groups of polypeptide molecules. This will result in a specific result.

Finally, the desired protein with a small (both two amino acid) amino-terminal extension It is known that the desired ripe protein can be produced by microorganisms that encode the protein. (see DK patent application 3448/84). The desired protein with an extension is located at the cell periphery. It is collected intraluminally, isolated from the pericellular space, and purified. This extension is correct N In order to obtain a desired protein with terminals in a pure state, for example, the enzyme DAP l and/or specifically cleaved using DAP4. In addition, the purification effect One or more charged amino acids to be used in such a presequence ) can be inserted into the This results in a protein with an extended amino terminus. Chromatographic analysis takes advantage of the difference in charge between the protein and the fully ripe protein produced by enzymatic cleavage. It can be refined into

When using a bacterium that has a DNA sequence encoding a polypeptide with the following general formula: In some cases, the advantages of both of the last mentioned methods can be obtained without the disadvantages associated with the above. We now know what can be done.

5-E-P (wherein S is a signal sequence, E is at least two amino acids, at least one of which is an amino extension with a charged charge), and P is the desired mature protein) In such cultures, the desired polypeptide is synthesized with associated extensions. be done. Depending on the signal sequence, the protein with its amino terminus extended closes the cytoplasm. It will be transported through the filling membrane.

The amino terminus of the following general formula makes it possible to isolate extended polypeptides. The signal sequence is cleaved during this step in order to

-P (wherein E and P are as above) This amino-terminally extended polypeptide The putido occurs in a correctly folded form. Used by microorganisms with a periplasmic space If the desired amino-terminally extended polypeptide is be gathered inside. Certain conditions (circuIIlce) that are not fully understood Under s), this protein similarly crosses the extracellular membrane into the culture medium.

The isolated amino-terminally extended polypeptide is then treated with the enzyme DAP, for example. It can be used as a substrate for P to produce the desired polypeptide P. .

During this step, the signal sequence may not be completely cleaved correctly, or it may be exposed after cleavage. Even if cleavage of individual N-terminal amino acids is performed in the final purification, the enzyme Between proteins with an extended amino terminus that can survive elementary cleavage and mature proteins. By taking advantage of the difference in charge between the It is possible to use amino-terminally extended polypeptides to produce be.

The method of the invention substantially provides for any protein having an enzymatically removable amino-terminal extension. It can be commonly used to prepare any desired protein. Manufactured by the method of this invention Examples of proteins that can be used include IGF 1 with an extended amino terminus; 22K hGH with amino terminal extension, 20K hGH with amino terminal extension, Lengthened IL L and amino-terminally elongated insulin.

The amino-terminal extension, E, preferably consists of two amino acids as described, especially preferably contains 4 to 16 amino acids.

According to an advantageous embodiment of the invention, the amino-terminal extension comprises at least one Contains charged amino acids. The presence of one or more charged amino acids increases the effectiveness of the desired protein. enables efficient purification and chromatographic isolation.

This invention relates to a DNA sequence, a plasmid containing this DNA sequence, and It also relates to microorganisms into which such plasmids have been inserted.

Any microorganism capable of inserting plasmids can be used in the method of this invention. Ru. E. coli is well suited for this purpose, but other microorganisms can also be used. Ru. An example of this is B, 5ubtils. This microorganism is E. does not have an extracellular membrane as in coll, and therefore does not have a pericellular space. I don't have it. with an inserted plasmid containing a DNA sequence of the type of this invention. When subtilis is cultured, the resulting polypeptide is transferred to the culture through the cell membrane. It is transported into a solution and cleavage of the signal sequence occurs simultaneously. This polypeptide is Correctly folded and chromatographed by methods known per se (chromatography on ion exchange resins) can be isolated from the culture medium by

Polypeptides with amino-terminal extensions produced by the method of this invention are It is suitable as a starting material in preparing the protein P. Prefers amino-terminal extension With proper selection, it is possible to obtain a specific enzymatic digestion of the extension. for example, One of the enzymes DAP 1 or DAP 4 can be used for this purpose.

The enzyme preferably opens stepwise by simultaneously removing two amino acids. tear apart The resulting ripe protein is then processed by well-known methods such as chromatography. The polypeptide is isolated from unreacted or partially reacted extended polypeptide.

Furthermore, this invention provides interleukin 1 (IL-1) with an extended amino terminus. ) and formulations containing this biologically active polypeptide.

Interleukin-1 (IL-1) is produced in various animal cells such as monocytes. It is a polypeptide that is Complete amino acid sequences of IL-1 and IL-1 precursors The columns are known.

IL-1 has diverse biological effects. That is, IL-1 is a Langerha It has a cytotoxic effect on the pancreatic islets (i.e., β-cells of the pancreas) and has an inhibitory effect on insulin. This causes drug-dependent diabetes mellitus (IDDM). This effect is due to the specific receptor of β-cells. Occurs in connection with the attachment of Ru 1 to the body.

A variety of biological influences result in overproduction of IL-1.

For example, preventing overproduction of IL-1 to treat or prevent IDDM It is highly desirable to either interfere with IL-1 toxicity, or to interfere with IL-1 toxicity.

Embodiments of the present invention show that amino-terminally elongated ratio-1 is present in β-cells in the pancreatic region. Based on the discovery that it interferes with the cytotoxic effects of Ru1 on Ru.

IL-1 with an extended amino terminus binds to the IL-1 receptor on β-cells, and this It appears to block these receptors. This results in the cytolytic effect of Roux 1. is prevented.

That is, the present invention also relates to IL-1 with an extended amino terminus. Furthermore, this The invention relates to formulations containing amino-terminally elongated Lu 1. This amino terminal The expanded IL-1 can be used, for example, for the treatment or prophylactic treatment of IDDM. Can be used.

Furthermore, this invention provides amino-terminally extended IL-1 or amino-terminally extended IL-1. The present invention relates to a DNA structure for encoding an IL-1 derivative.

The extensions of IL-1 and derivatives of IL-1 can have different lengths. . It can also include, for example, most of the preliminary sequences of naturally occurring IL-1. Wear. However, preferably relatively short amino-terminal extensions (e.g. 2-6 amino acids) are used.

A typical example of a polypeptide of the type of this invention is To1eL-Glu-Ala- Glu-IL-1Ala-C: 1u-IL-1 and ty”Met-Glu-Al a-Glu-Phe-Asp-IL-1, and all polypeptides are substantially It has a reduced cytotoxic effect. In certain cases, this effect occurs naturally significantly lower than IL-1.

If desired, this polypeptide can be modified in a manner known per se, For example, by substituting one or more amino acids with other amino acids, or Modifications can be made by deleting one or more amino acids at the terminus.

The polypeptides of this invention are suitable for example (i, a,), diabetes and autoimmune diseases. can be used therapeutically to combat or prevent these. Ma In addition, due to the action of endotoxin antibodies (endoLoxianLibodies), This polypeptide can also be used to treat patients suffering from multifaceted shock. It is proposed that For this purpose, pharmaceutical preparations, especially injectable preparations, agent is used. This formulation comprises a polypeptide in a pharmaceutically acceptable carrier solution. including.

The polypeptide of this invention can be obtained from microorganisms or microorganisms containing the gene encoding the polypeptide. or produced biosynthetically by culturing cell lines. Then this polypep Tido is recovered and purified to make the desired formulation.

By inserting into a host cell and culturing the host cell, the present invention The production of suitable plasmids for carrying out the procedure involves recombinant techniques known per se. more executable.

Such production involves the construction and production of a variety of plasmids suitable for the practice of this invention. is illustrated in FIGS. 1-4.

The plasmid named pHD165 is a hybrid of plasmid p^T153. Based on (Mandel, M., Higa, A., 1970. J.M. ol, Biol,.

53.159-62). Restriction site E, between coRI/Bam old, promo ter, Shine-Dalgarno sequence and outer membrane protein A (Chen, R, et. al., 1980. Proc, Natl, Acad, Sci, 77.4592- A DNA segment containing a signal sequence from a DNA fragment (see 96) is inserted, followed by restriction enzyme treatment. A synthetic compound containing the recognition sequences of elementary NaR1, Bgll, Pvull and BamHI NA linker is inserted.

Plasmid pHD167 is likewise a derivative of plasmid pAT153. child The plasmid encodes the promoter, Shine-Dalgarno sequence, and IGFI. This includes a synthetic gene for activating a gene, and a transcription terminator. The above sequence is pAT15 E in 3 is inserted between the coRI and Bam old restriction sites. Plasmid pHD17 B is a transcription terminator isolated as an Accl/BamHI DNA fragment. and the gene for IGFI from plasmid pHD176 and the amino-terminal extension. In addition to encoding a short synthetic NA linker, a plasmid site and a promoter site , and the signal sequence from plasmid pHD165.

That is, plasmid pHD17B encodes the outer membrane protein A signal sequence. site, followed by the amino-terminal extension A I a-G I u-A l a-Glu and and IGPI encoding sequences.

Plasmid pHD141 shown in Figure 2 is a derivative of plasmid pHD187. It is. The sequence encoding IGFI in this plasmid pHDIB7 is The amino terminus has been extended to 20-nGH and replaced by the sequence encoding nGH.

Plasmid pHD223 contains a synthetic NA linker encoding an amino-terminal extension. In addition, the sequence contained between C1a I/EcoR1 of plasmid pHD141, C1al/Narl DN A from pHD165 encoding the signal sequence Contains fragments. Therefore, pHD223 encodes the outer membrane protein A signal sequence. site, and the amino terminal extension A l a-G l u- A l a-G I u; Following this, <20> has a sequence encoding 10GH.

Plasmid pHD10B-9 shown in Figure 3 is a derivative of vector pUc18. be. This vector pUc18 contains 88% of the E. coli fimbriae protein. (Gaastra, W. et al., 1982. Microbial, Re The region encoding the signal sequence from It is inserted between the restriction site Bam old/Pst1.

Plasmid pHD117-45P13 is a derivative of plasmid pHD167. Ru. In this plasmid, the IGPI-encoding site is located at the amino terminus or at the extended 22 is substituted with a site encoding -hGIl.

Therefore, plasmid pHD148-22-hGH is present in the fimbriae protein at 8 the signal sequence from g followed by the amino-terminal extension A l a-G l u -A l a-G I u-A l a-G l u, followed by <22- It has a site encoding hGH.

Plasmid shown in Figure 4 1) HD183 is derived from plasmid pHD167. It is the body. In this plasmid, the IGPI-encoding site is located at the amino terminal It has been replaced with a region encoding expanded IL-1.

Plasmid p11D185 is a derivative of plasmid pAT153.

This plasmid contains the restriction enzymes Na10, Bglll, Pvu[l and Ba[ In addition to the recognition site for IIHl, the region encoding the outer membrane protein A signal sequence A synthetic NA fragment containing was inserted between restriction site E, coRI/Baa+ ing.

Therefore, plasmid pHD163/185 extracts the signal sequence from outer membrane protein A. coding site followed by an amino-terminal extension Met-Glu-Ala-Glu -Phc-As+) and a site encoding interleukin 1. Ru.

The method of this invention will be more fully illustrated by the following several examples. Dew.

Example 1 The plasmid pHD165 containing the promoter and the above signal sequence was added to the enzyme N The plasmid fragments were then purified using a+I/Bamold. for example , a sequence encoding human IGFI, followed by a transcription terminator. plasmid pHDL67 was cut using the enzyme Accl/Ban+Old, and then A fragment with approximately 600 base pairs was purified. Then with these two pieces , an amino terminal with restriction enzyme overhangs for the enzyme Nar I/Accl Connect DNA linker encoding end extension to form plasmid pHD176 did. The binding mixture (Ligantion m1x-ture) was made from bacteria E, Co 11 substantially transformed into MC1061 and plated on ampicillin-containing agar plates. It was smeared and cultured at 37°C overnight.

Select multiple bacterial colonies and culture them for further characterization by restriction enzyme analysis did. Then, these clones pHD176-D and -F were selected. clone pH D17B-F was cultured on a large scale and the bacteria were then collected by centrifugation. next The bacteria were then partially ruptured by osmotic shock. This allows the cell periphery to Proteins present in the marginal space were released from the bacteria. Removed partially ruptured bacteria Later, the amino-terminally extended IGPI was purified. This material was then converted into DAP 1 as a substrate, thereby specifically removing the N-terminal extension.

The expression level in the culture of the above bacterium HD176-F is 0D600 of approximately 1. Approximately 5 mg of amino-terminally extended IGFI per liter of bacterial culture solution. there were.

After purification by ion-exchange chromatography, pure ICIC eco-isolated Ta.

Example 2 Plasmid pHDI41 containing the gene encoding human growth hormone in 20, Cut using the enzyme C1al/EcoRI and then purify this plasmid fragment. did. Plasmid pHD165 was cut using the enzyme C1a1/Narl, and then A fragment having approximately 120 base pairs was purified using a method. Plasmid pHD223 To form these fragments, these fragments are digested with enzymes for the restriction enzyme Narl/EcoR1. The plastics are substantially bonded together in the presence of a synthetic NA clinker with bar hangs. Sumid pH0223 was formed. This binding mixture is E, Co11 MCll0 61 and plated onto ampicillin-containing growth plates.

Culture and isolation of growth hormone from the amino terminus 20 obtained thereby. was carried out as described in Example 1.

This expression level is the same as that described in Example 1 and the signal sequence described above is The contained plasmid pHD10B-9 was digested using the restriction enzymes Baa + old/Hindl. I cut it. A fragment with 190 base pairs was then purified. Enzyme this fragment Cut using BstNl to create a blunt end. treated with rease, extracted with fenoflu, crystallized with ethanol, then Cleavage was performed using enzyme C1!1. A further 60 base pair fragment was purified.

Plasmid pifD117-45P13 was cut using the enzyme Cl5l/EcoRI. The plasmid fragment was then purified. Plasmid pHD14g-22-h To form GH, the two purified fragments were combined with Narj/Hindl rinsing. substantially bonded together in the presence of a car. This binding mixture was combined with E, Co11 MC 1061 and plated onto ampicillin-containing growth plates. −Day and night culture After that, multiple colonies were further analyzed by restriction enzyme analysis and DNA sequencing. selected for characterization.

Cultivation and isolation of the resulting amino-terminally extended 22K hGH were carried out. A similar method as described in Example 1 was carried out.

Example 4 Plasmid I) having a sequence encoding human IL-1 with the amino terminus extended HD163 was cleaved using the enzyme C1al, treated with phosphotase, and purified. Manufactured.

Plasmid pH0185, which has a sequence encoding a signal sequence, was added to the enzyme C1xl. /Narl and then purified a DNA fragment of about 90 base pairs. Next These two fragments were then combined to form plasmid pHD183/185. joined together.

inserted into. The obtained amino-terminally extended human IL-1 was purified and DAP was added. It was converted to human IL-1 by the reaction used.

The following example describes the preparation of amino-terminated interleukin 1 and its pharmaceutical formulation. Explain its use.

Built. Metsenger RN of high-level and low-level expressed genes in bacteria Comparative studies of A sequences revealed strong relationships between codon usage and expression efficiency, respectively. It was found that there was a strong correlation. That is, high level expression of E, Co11 genes It was decided to use the most frequently used codons.

Useful cloning sites were inserted at both ends of the gene and within it. This legacy The gene has an amino-terminally extended Met-G l u-A I a-Glu (MEAE ) was extended using a DNA sequence encoding This gene has 80 to 10 stepwise between the restriction sites of oligonucleotides with an average length of 0 nucleosides. Manufactured by cooling. Gene sequence for MEAE-IL-1 The columns are shown in FIG.

E, Co11, glycosylation or other modification of IL-1 has been reported. It was selected as the host microorganism because it is not present. This expression is driven by a synthetic promoter ( sp13) and an optimal Shine-Dalgarno sequence. This use For the expression of bhGH in E., Col. It was selected because it is known to be effective (more than 20% of total bacterial protein). (Biotechnology, Vol. 5. Feb. 1987. p. 160). this The clone is called pHD230.

Whole cell extracts from pHD230/MC1061 were analyzed with SDS gel gel. -By coloring with blue, a strong band of the expected size can be obtained. Things have changed. IL-L-specific El.

By analyzing the bacterial extract with Is^, the sample was determined to have a bacterial density (OD60 0) is an immunoreactive substance corresponding to about 10 μL of IL-1 per 11 bacterial cultures. It was shown that it contains quality.

Extraction of IL-1 was performed in Biotechnology, Vol. 4. Dec, 198B, p.

1078. N-terminally extended IL-1-β inducer Conductor purification is performed using FP-S Sepharose, PF-Q Sepharose and gel filtration coats. Each was executed on Ronnie. The derivative in question was analyzed by amino acid analysis and N-terminal sequence analysis. , the film atomic bombardment method entmethod) (Parper M et al. (198L) J, Chem, Soc ,,Chem,Comm, Vol, 1981. p, 325-327), SDS and molecular weight determination by conventional electrophoresis.

Example 8 Clone encoding Met-Glu-Ala-Glu-Phe-Asp-1 preparation of The region encoding phenylalanine-aspartic acid was generated by in vitro mutagenesis. The DNA section containing the above was inserted into clone pHD230. This is IL-1 The sequence is extended with Met-Glu-Ala-Glu-Phe-Asp-. Inserted by method. The genes for IL-1 with an extended amino terminus are E, C o11 and purified as described above.

This clone was created by in vitro mutagenesis on plasmid pHD230. It was prepared by IL-1 with an extended amino terminus is expressed as described above, Refined. The N-terminal methionine group is cleaved in vivo by E and Co11 enzymes. It was done.

Example 10 1 Preparation of a clone encoding amino acids 72-269 of L-1 In vitro mutation By mutagenesis, the sequence of amino acid numbers 4-5 (DAN sequence AAGCACCCTGT ), a sequence was introduced to code for a similar amino acid, but a unique N An arl restriction site (DNA sequence AGGCGCCGGT) was induced.

The obtained clone pHD228 was then cut using the enzyme Narl/C1al. Furthermore, a fragment encoding the following amino acid sequence was inserted. The gene is the above It was expressed and purified in E. Co11.

The inserted amino acid sequence has the following structure. :MeL-Lys-Leu- Arg-Lys-Met-Leu-Val-Pro-Cys-Pro-Gln- Thr-Phr-Gln-C:1u-Asn-Asp-Leu-5er-Dinghr -Phe-Phe-Pro-Phe-11e-Phe-Glu-Gl　u- 1u-P ro-I l e-Phe-Phe-Asp-Th r-T rp- Asp-Asn-Glu-Al　a-T　ychichi| υal-His-8 sp.

Instead of end-extended Met-Gl-u-A Ia-Glu to amino, amino It has a region encoding the amino acid Met-Ala-Tyr-Vat-His-Asp. The DNA fragment was inserted onto Coulomb pHD230 by in vitro mutagenesis. did. This amino-terminally extended IL-1 was prepared by E, Co11 as described above. expressed and purified within.

Specific biological activity (units/nglL-1-β) nic) Simultaneous biological responsiveness in mouse thymocyte proliferation assay (LAF) Measurements also revealed that the IL-1-β protein in response to IL-1-β Eliza (Eli) sa).

1. LAF assay is performed as described above (Scand, J., 1 mmun o1.28:611, 1987, 5cand, J, 1mmuno1. :In the literature interleukin 1, tumor necrosis factor alpha and prostaglandin E. Endotoxin-stimulated mononuclear cell secretion of , shows stable interindividual differences. vinegar).

Thymocytes were derived from CH3/he mouse species (Charles River, 5- to 7-week-old male mice from Al Republic of Germany) removed from the After washing the cells, they were diluted with 90% RpMl-18 4 and 10% stock human serum inactivated by heat. into a shallow microtiter plate (rafcro). Titer plates) (Nunc, Roskilde).

This culture solution is added to the sample according to a dilution series, which consists of 20 volumes. Starting from volume %, it is diluted in 8 steps with l:2. This test series is repeated 3 times. returned. 5 μg/ml of PIIA (Dif’co) each wells. After 48 hours of incubation at 37°C, 1 μCi of chloride was added per well. Lithium labeled thymidine was added. Furthermore, the cells were cultured for 18 hours using a semi-automatic cell culture machine. After that, the culture solution was passed through a glass fiber filter (skeleton (5 carton)). , Norway). The filter is then placed inside the beta counter. It is counted.

This result is calculated as follows. Calculate the average of 23 times. Background For each dilution series containing three times or more counts of regressiori).

The highest in the dilution series only if the count is 10% higher than the count in the next dilution step. A new count has been introduced. Linear regression can be performed either semi-logarithmically or log-logarithmically. be exposed. This linear regression Parallel curves that allow directly the quantification of biological responsiveness to standards consisting of Gives a group of lines. Furthermore, this result can be expressed in units/ml. WHO sign Standard contains 100 U/ng.

Performed by Ikit (Bio Rad, Protein assay kit) It will be done.

Results The results are shown in the table below. The specific activity of MEAE-IL-1-β is It was found to be 50 to 100 times weaker than the modified intact IL-1-β. It is noteworthy that This result is obtained from three independent experiments.

Example 13 Diabete volJ6. p, B41-847; 1987: Interloiki Islet of Langerhans cytotoxic effect, culture conditions, and islets of Langerhans donor of N-1 Impact due to the characteristics of Islet Cytotoxicity of’ Inte r-1eukin-11nfluence of C1tureConditi ons and l5let Donor Characcerisics, ).

Pancreas was removed from 5- to 7-day-old Wistar (ν1star) rats. and islets are isolated by collagenase disruption.

The islets of Langerhans were incubated at 37°C in RPMI-1640 + lo% NC3, humidified. cultivated in the atmosphere. After one week of incubation, the islets of Langerhans are R Washed with PMI-1640 + 0.5% H3, and further treated with IL-1 and IL- Incubate for 6 days without 1. This incubation from Langerhans Surin secretion is determined by radioimmunoassay (RIA). Insert IL-1 Insulin secretion from incubated islets of Langerhans is significantly different from control IL. −1 expressed as the percentage (%) of insulin secretion from the islets of Langerhans without Ru.

MEAE- IL-1 is one-tenth as active as AE-IL-1, and λE-IL-1 is rlL It was shown that the activity was one-tenth that of IL-1 (see Figure 6). Figure 3 shows that IL- 1 standard curve is shown. The insulin component contains an extremely low concentration of rlL-1 (0,00 1 ng/ml) (approximately 130%). On the other hand, insulin secretion is r It is inhibited by about 50% at a high concentration of 1 L-1 (0, Ing/ml).

Engraving (no changes to the content) Ba'rl(I U) Koshitodo v9 insulin h$r, xqb%' control D-p! Surin now Tsum ρ s’qi %2, name of the invention Use as a pharmaceutical preparation.

3. Patent applicant Address: Denmark, DK-28805, date of submission of amendment June 1, 1989 6. List of attached documents (1) Translation of written amendment 1 copy of claims (wherein P is the desired polypeptide and E is 2 to 16, preferably 2 to 6 an amino-terminal extension having several amino acids, at least one of which is a charged amino acid. A method for preparing a protein or polypeptide (wherein S is a signal sequence) , E and P are as above). eukaryotic bacteria of the general formula 5-E-P, or with simultaneous cleavage of the signal sequence. The polypeptide passes through the membrane that confines the cytoplasm of the microorganism and enters the periplasmic space or the culture medium. A preparation method characterized by culturing in a culture substrate under conditions in which it is transported in a nutrient solution. Law.

(2) A claim characterized in that the bacterium is E, Co11 or B, subtilis. The method described in Section 1.

(3) P is polypeptide ICF+, 22K hGH, It, 1 or human in Claim 1 or Claim 2, characterized in that it is one of surin. How to put it on.

(4) E is a general formula, Met-Glu-Ala-Glu Ala-Glu-R: Peptide containing one of Met-Glu-Ala-Glu-Phe-Asp- According to any one of claims 1 to 3, characterized in that it is a card arrangement. Method.

(5) Produced by the method described in any one of claims 1 to 4. polypeptide.

(6) The polypeptide according to claim 5 and an additional pharmaceutically acceptable carrier. A pharmaceutical preparation comprising:

(7) The pharmaceutical according to claim 6, which is prepared as an injection preparation. formulation.

3. Person who makes corrections Relationship to the case Patent applicant Name: Novo-Nordisk Knee/varnish October 9, 1990 (shipment date) Translation of drawings (Figl, Fig2-4, Pig6-7) 7, Contents of amendments international search report 1m*nm+eMI Aeekelm, N-, PCT10K8810 [IIO1

Claims (20)

[Claims] (1) General formula: E-P (where P is the desired polypeptide and E is the amino terminal extension) A method for preparing a protein or polypeptide having the general formula: S-E-P (wherein S is a signal sequence, E and P are as above) A polypeptide of the general formula S-E-P is completely isolated from bacteria having the encoding DNA sequence. Cytoplasmic confinement with simultaneous cleavage of the complete or partial signal sequence A preparation method characterized by culturing in a substrate under conditions of transport through a membrane. (2) A claim characterized in that the amino terminal extension comprises at least two amino acids. The method described in Section 1. (3) A claim characterized in that the amino terminal extension contains 4 to 16 amino acids. The method described in Section 1. (4) characterized in that the amino terminal extension comprises at least one charged amino acid; 4. The method according to claim 2 or claim 3. (5) General formula: S-E-P (wherein S, E and P are as described in claim 1) A DNA sequence for insertion into a plasmid characterized by having codons for Column. (6) A plasmid characterized by having the DNA sequence according to claim 5. Do. (7) A microorganism characterized by having the plasmid according to claim 6. (8)E. coli or B. coli. Claim 7, characterized in that the plant is a subtilis. microorganisms. (9) In the general formula according to claim 5, P is the polypeptide IGF1, 22K h A DNA sequence characterized by being one of GH, IL1 or human insulin Column. (10) Interleukin with an amino-terminal extension of 1 to 45 amino acids A polypeptide consisting of IL-1 (IL-1) or an IL-1 derivative. (11) The extension consists of 1 to 30 amino acids, preferably 1 to 10 amino acids. 11. The polypeptide according to claim 10. (12) Claim 10, characterized in that the extension consists of 2 to 6 amino acids. polypeptides. (13) The polypeptide according to claim 10, characterized in that the extension consists of one amino acid. peptide. (14) The following general formula [There is an array], [There is an array] or [There is an array]. 13. The polypeptide according to claim 12, which is one of the following. (15) The polypeptide according to any one of claims 10 to 13 For the expression of an amino-terminally extended polypeptide encoding DNA sequence. (16) D according to claim 15, having a structure as shown in FIG. NA sequence. (17) The gene organ (gene apparatus) is defined in claim 14 or A microorganism characterized by comprising the DNA sequence described in any one of claim 15. thing or cell. (18) The polypeptide according to any one of claims 10 to 14 and an additional pharmaceutically acceptable carrier. . (19) A therapeutic preparation, which is prepared as an injection preparation. (20) From administration of the pharmaceutical preparation according to claim 18 or claim 19. A new treatment method.