JPS59501095A - Production and expression of genes for calcitonin and its analogs - 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] Gene production and expression background for calcitonin and its analogues The present invention generally relates to human canolecitonin and and specific DNs useful for recombinant procedures that enable the production of polypeptide analogs thereof. Regarding the production of A array.

For providing prior art information regarding recombinant DNA techniques, Yitz Hak Stabinsky's Lucitonin is a 32 amino acid polypeptide In Mont. It is secreted by coarse blood. Other polypeptides exhibiting honoremon activity (e.g. As in the case of ACTH and endonolefin), canolecitonin is more It is a product that is excised in vivo from a large molecular weight precursor molecule.

So far, calcitonin has been produced uniformly in species i, and there are seven forms of calcitonin. It has been confirmed that one of these is a fecal structure. 7 canoles whose structures have been revealed It also contains human canolecitonin, which has the amino acid sequence below. That is, 2 025 IIis-Thr-Phe- Pro-Gl n-Thr- A la- + t e-G ly- Va l-G l y- A la- Pro-CONH2 Has [→Nyoko + 'A. Edited by Pecile, Calcitonin 1980, Chemistry, Biology Physical, Pharmacological and Clinical Aspects J I Exerpta Medica , Amsterdam' (1981)].

All biologically active forms of canolecitonin ever isolated (this Yes, {Dysnolephaid that binds cystine residues 1 and 7. There is a proline at the carboxyl terminus of the nopeptide. It has an amide group. Based on tests of polypeptides with modified structures, these structures Both the structural features and the important biological activity of Lehenore G seem to be essential.

In humans. The main role of calcitonin is to release G during canolecium stress. , appears to have a specific effect during pregnancy, but the drug efficacy per mg is not very large. The duration of kiku and 1 乍 use is also longer. Therefore, it is more synthetic than human calcitonin. Salmon Canorecitonino is currently suffering from Beziet's disease and hypercalcemia. Used and 0.

Unfortunately, some patients treated with salmon calcitonin may Some people have severe systemic hypersensitivity reactions to mons, and even local hypersensitivity reactions at the site of administration. Focal inflammatory reactions have been reported in over 10% of patients. pageant disease Some patients initially receive calcitonin after 18 months of TFA. Circulating antibodies have been reported. Occasionally, very high antibody titers However, they usually relapse and have low calcitonin levels in salmon. Does not respond to mu blood effects.

Salmon calcitonin has high efficacy and duration of action, but it does not have the above-mentioned unfavorable immune effects. Attempts have been made to develop human canolecitonin analogs not shown. polypef Systematic synthesis and testing of detide analogs suggests that efficient methods of their production exist. have been greatly hindered by not doing so. Small amounts of analogues are a well-known benefit Field Procedures (Merr, J. Am. - Chem. Soc.. 85. pp. 2149-2154 (1963)) However, this approach is difficult in terms of the quantities produced and the time required to prepare the analogues. This procedure is not satisfactory.

Hito Calcitonin cDNp&! Structure and parts of recombinant plasmid containing A The determination of the characteristic properties has recently been reported (Craig et al., Nature, 295 , pp. 345-347 (1982) - Structure for human calcitonin A gene that differs from it in the type and/or position of one or more amino acids Gene production for more than one polypeptide; Setup for cloning and expression Replacement DNA technology has never been applied to this problem.

concise summary What is provided by the present invention is that human bacteria in selected host microorganisms are It is a manufactured gene that has the ability to control the synthesis of lucitonin. manufactured genes In the desired form. The salt alJ may be added to the intended host microorganism, e.g. E. c. Based on the preferential expression characteristics of codons within the oli. The cost of using the same amino acid Contains one or more codons selected from among the alternate rodons. The produced gene Other desirable forms include (1) base codons in the polypeptide being synthesized; Specification of additional amino acids to make them biologically active human calcitonin one that facilitates separation (e.g., the initial FIet residue, or Lys-Arg[ EJIJ), and/or (2) a base code that specifies human calcitonin. restriction endonuclease cleavage of the DNA B prior to and/or after the A sequence of bases with a portion of salt (for example, PstTmh comes and the result is includes those that facilitate the formation of expression vectors.

What is further provided by the present invention is that (11) It's human.

Manufactured genes capable of directing the synthesis of calcitonin polypeptide analogs and the aforementioned analogs differ from humans in the type and/or position of one or more amino acids. ・Different from calcitonin polypeptide (e.g. (Val 8) human ・Calcitonin and (Asn 15) human calcitonin), and (2) this With a fusion gene containing the produced gene according to the invention.

If the above-mentioned produced gene is human calcitonin polypeptide or lucitoids (e.g., the ability to control the synthesis of β-lactamase and β-galactosidase) It is fused to a second, more powerful gene.

When practicing the invention to produce a polypeptide product, the produced gene The DNA sequence containing the offspring can be inserted into a viral or circular) lasmid DNA vector. and form a hybrid vector, and the Vibrift Vector bacteria (e.g. E, coli) or host microorganisms such as yeast cells. used for The transformed microorganisms then receive appropriate nutrition. The polypeptide products of the present invention are expressed under conditions of growth.

The novel DNA recombinant of the present invention, preferably Yitzhak 5 tabins United States Patent Application No. 375.4 jointly owned and filed eleven times by Ky. 93 "Manufacture and Expression of R Structural Genes" (250 for Proxy Seikanmon) Synthesized from nucleotides according to the method described. To summarize, the general method is as follows: Consists of steps.

and/or a single strand with a tail of 3 to 7 selected bases at the other end of the strand Anneal to one or two different duplex chains with unterminated sequences. This is it single-stranded terminal region of a double-type DNA chain with a single continuous double-stranded head or tail terminal region. at least three base pairs formed by complementary association of regions.

In the preferred general process of manufacture, at least three different duplex DNs the Aste DNA sequence has a duplex region of at least 42 selected base pairs; This is due to the complementary association of the single-stranded terminal region of the double-type chain cleaved in step (11). at least two non-adjacent segments of three or more base pairs formed by Including.

The dual DNA preparation step (11 is optional) of the DNA sequence production process described above. Alternatively, it consists of the following steps.

(a)! The first and second straight lines have five or more bases in a selected linear sequence. Create deoxyoligonucleotide fragments. Linear sequence of bases in the second fragment consists of a complete complement of the base sequence of the first fragment, but at least one of the second fragment One end has 3 to 7 selected bases in addition to those that perfectly complement the first fragment. or three complementary to the terminal sequence of the first fragment. Either the second fragment has a linear sequence of seven bases, but the second fragment Does not have one additional base sequence or no base sequences at both ends shall be taken as a thing.

(b) The first fragment and the second fragment are combined under conditions that promote dual-type association between the fragments. one or more triblend codons selected from recombinant codons.

Detailed explanation As used in this specification, the term “manufactured” refers to a DNA sequence or The term refers to a product that is completely chemically synthesized by the assembly of nucleotide bases. biological products or other chemically synthesized products refers to the product obtained from iJ. As such, this term is cDNA or genomic cloning methods using physical starting materials According to the theory, ``synthetic products are excluded.

The following abbreviations are used herein to refer to amino acids. Alanine- Aha, argini-7-Arg, asparagine-Asn, aspartic acid-Asp , Stein-(ys, Glutamine-Gin, Glutamic acid-<　]U, G Linone-Gly, Histidine-Hls, Inleucine-11e, Rhinone-Le u, lysine-Lys, methionine-Met, phenylalanine-Phe, proly N-Pro, Serine-5er.

Threonine-Thr-Trp, Tyrosine-Tyr, and Vari N-Val. The following abbreviations are used for nucleotide bases. 8-Adenine, G- Guanine, T-thymine 1U-uracil, and C-cytosine.

To facilitate understanding of the present invention, Table 1 below lists the 64 triblets of DNA. ・Correlation table between 7 nucleotide base codons and 20 amino acids, and Indicates the transcription termination (“stop J”) function specified in .

Table 1 1st position 2nd position 3rd position CAG Phe Ser Tyr Cys T T　Phe　Ser　Tyr　Cys’ CLeu　Ser　5top 5to p ALeu Ser 5top Trp GLeu Pro His Arg T (: Leu Pro 1lis Arg CLeu Pro Gin Arg A Leu Pro Gin Arg G 11e Thr Asn Ser T Met Thr Lys Arg G Val Ala Asp Gly T GValAhaAspGlyC Val Ala Glu Gly A Val Aha Glu Gay G The following examples show nine deoxyoligonucleotides used in the production of the DNA sequences of the invention. 1 illustrates a desirable general procedure for preparing.

First example Is the deoxyoligonucleotide made by II? 1utteucci et al., J. Am, Chem, Soc, 103', pp3185-3192 ( (1981) and Beaucage et al. Tetrahedron Lett ers, 22,’ IIp. 4859-1862 (1981) and its It is carried out according to the general methods published in the documents referenced therein. synthesis Derived from high performance liquid chromatography grade silica gel for proper protection. The first step is to include the nucleotides. Deoxyoligonucleotide is a carpooxylate covalently bonded to the silica gel via the 3°-hydroxyl group. attached to the silic acid functional group. used to add one nucleotide to this support. The chemical procedure used is as follows. (1) In nitromethane/methanol Detrichlation using ZnBr (4 minutes). (2) Nucleus bonded to support 5'-p-ayucyl-phenylmethyl deoxynucleo with cleosides Condensation of sido-3”-methoxy-N,N-dimethyl-aminophosphine (5 minutes) . (3) Remove unreacted support-bound nucleoside hydroxyl groups with acetic anhydride. (5 minutes). (4) Oxidize phosphite to phosphate using 12 (2 minutes) . The synthesis is performed by a single technician in a simple molten glass funnel. one The synthesis cycle takes 20 to 3 hours, and up to 30 molecules can be produced in less than 15 hours. Deoxyoligonucleotides containing oligonucleotides can be obtained in high yield. .

Whenever possible, redundancy in the genetic code should be used to Even in leotide, the base sequences of +lR and cylindrical nucleotides are not formed far apart from each other. The complementarity of the bases allows the strands to achieve the desired result by eliminating one opportunity for folding. increases the yield of linear chains.

Furthermore, the intended host for the expression of the produced DNA sequence is '. It is a microorganism That's why. Alternative codon selection 14. coli-based on codon preference. It was done. (For example. Grantha Ittei, Nucleic ACids R esearct++ 8 + pp. r49-r62 (1980); G rantha4, Nucleic Acids Research, 8, pp. 1893-1912 (1980); and Grantham et al. Nucleic Acids Research, 9-. pp. r43 -r74 (1981). ] Purification of deoxyoligonucleotides and separation is accomplished by the following procedure. Terminal gp-anisi by acid After the final condensation step, which includes the removal of the phenylmethyl group, deoxyoligon The silica gel containing cleotide was thoroughly washed with methanol and air-dried. be done. To remove the methyl group from the phosphotriester, each polymer (100 mg, containing 2-5 micromoles of oligonucleotide) At room temperature with 2 ml of Nor:Et3N:dioxane (172:2) solution. 753}. Following this step, concentrated ammonium hydroxide The deoxyoligonucleotides were treated at 20°C for 2 hours using Hydrolyzes the ester bonded to. Deoxyoligonucleosynthesis by centrifugation After collecting the supernatant containing the tide, the base protecting group was removed for 50 minutes in a sealed test tube. It is removed by heating to 241 Jl at °C. Ammonium hydroxide solution is that It is then dried by fan. It is redissolved in Hx02m, filtered, and The solution was then washed three times with n-butanol (3×2mβ). product final For standard purification, use Tris-borate buffer (pH 8) containing 7M urea at 20% concentration. This is done by electrophoresis on a lyacrylamide gel. 80% formamide Approximately 100 ml of paddy material is contained in 30 μl. D. Unit (260 nm) is 2.5 It is filled into a cm-wide well, and electrophoresis is performed at 16 V/cm. DN^ To detect the bands, the gel was placed on a fluorescent TLC plate (Sigma Chemical Company). It was placed on top of it and was irradiated with i-wave Shikukyo. Visual inspection of deoxyoligonucleotides You can do it, but it looks like a strong absorbing hand for each lane. desired bar DN^ was excised from the gel, DN^ was 0.5M ammonium acetate, 10 mM MgCI2, 0.1% SOS. and with 0.1 mM EDTA It is eluted. After washing twice with n-Pukunor. deoxyoligonucleo Chido is. Cefadenox G5 using 10mM TEAB (pH 7.0) Desalted on a 0/40 column (45 x 2.5 am). Rough DN^100D ．． Generally, the amount that can be recovered from the unit is 1.0 to 2.0.0. D. Unit (26 0 mm).

The following example is an illustration of the structure of DNA control, and the above-mentioned DNA control is , (Lys-2', Arg-1) 9 genes containing the code for fin lucitonin and this sequence was added to the β-lactamase gene of pllR322. Insert at the Pst'1 site, approximately 540th base pair downstream of the lactamase initiation portal, It has a terminal base sequence that makes it easier.

First example The deoxyoligonucleotide described below was synthesized according to one example. silica ・Starting with 5′-dimethoxytrityldeoxynucleotides bound to the gel Ta.

i. 5'-T('.CGGTAACCTGTCTACCTGCAT-3'2. 5'-G (GOGGGCACCTATACTCAGGACTT-3'3.5"- CAACAAAATTCCATACCTTTCCCGC-3'4.5゜-AGACC GCTATCGGTGTTGGTGqTCCG-3'7. 5’-GGTCT GCCGGG^^GGTATGGAATTT-3'8. 5'-GCACCAA ACGC-3' to CACcGATAGC-3'DE15'-^ DE 3 5'-TGATGATGCA-3'DB 4 5'-TCATCA CGGA-3’DE75”-CGCAGCGTTTTTGC^-3+Fully protected A 0.5 nmol aliquot of the missing eNA was added to 20 µM of quination buffer. Melted by Lolita. Phosphorylation of the 5'-OH end is +'32P-ATP and T-4 polynucleotide kinase using cold carrier TP catalyzed by. To avoid the formation of undesirable coupling products later on, DE1 and DE4 were not treated with kinase. degree of quination Ion exchange filter paper chromatography on Wasotoman ED~81 Strinobu monitored by. After the quination is complete, a step is taken to denature the kinase. The reaction mixture was then boiled. A pair of DNA mats undergoes boiling and subsequent slow cooling. Annealed by DEI, DE7. DEI. DE5. DE2, DE6. DE3, DE7. DE4, DE8. and DE3, DE4.

The annealed pairs were mixed in the following manner. That is, DEI, DE7 and and DEI, DE5. DE2, DE6 and DE3, DE7. DE4, DE8 and DE3, DE4. The mixture was heated to 37'C, Chilled to 4°C for 2 hours. Add T4-DNA Riga to a cold mixture of three (New England High Labs, 3 Wise {ffi, .nTr) and ATI) lJ, with a final concentration of 20 mM DTT and 0.8 mM It became ATP. Binding was carried out for one interval at 4°C. change the ligase The reaction mixture was boiled for clarification.

Table n shows the products of the deoxyoligonucleotide annealing reaction described above. The double-stranded DNA sequence obtained is shown below. Individual fragments are indicated in parentheses. Been formed The amino acids specified by the codons are shown above +mlJ. illustrated In the sequence, Pstl' cohesive end J (3'-A C G T-5' and 5'-T G C A-3')! It was formed in the terminal area of Shinobu 1, but this arrangement Note that does not include the hexasalt wst + the remaining bases of the recognition site.

Hidden expression One reaction mixture was boiled to denature the ligase. Reaction mixture after cooling was passed through a 10m1 column of Sefa Soks G-150-40. Desired result The reservoirs containing the synthesis product were pooled and dried for 1 minute, then 20 microliters of 6. The final ligation product was resuspended in binding buffer and treated with ligase as before. The product contained a band corresponding to 112 bases on the polyacrylamide gel. Ba DNA was excised, eluted with DNA, precipitated with ethanol, and buffered. A13 is resuspended in the solution and bound as A13 mp8 or mp9 duplex DNA. It was.

Bacterial vesicles are infected and subjected to one rotation of the heavy chain DN &amp; salts shown in Table ■. mlJ was confirmed.

After confirming base IIJ, the prepared sequence was inserted into an expression vector (pBR322 ) can be expressed using the commercially available R1Δ kit Gl'jl. Cytonin 1125RI Yaki Nodo”, catalog number 2500. Immu or new -Clear Corporation, Stainwater 5 Minnesota) This can be determined by performing radioimmunoassay of the cell product.

The example below is (Lys-2, Arg-LGly33.Lys34. Lys 35. Arg 36) The gene that codes for human calcitonin Although it is intended to prepare DNA samples containing DNA, as described above, A terminal base that facilitates insertion of the sequence into β-lactamase of pBR322. Contains arrays.

First example Deoxyoligonucleotides were prepared in the same manner as in Example 2, except that fragment D E4 and DF5 were excluded and instead variant fragments DE5 and DF6 were prepared. This fragment was used in an annealing reaction. Alternative fragment shiokunimentsu1] is.

DE55'-GGTAAGAAGCGCTGATGCA-3'DE 6 5' -TCAGCGCTTCTTACCCGG8-310 fragment DEI and D days are keys Not treated with Naze. [Pairs of lNA strands are brought to a boil and then cooled slowly.] annealed by DEI, D mouth. DEI, DF5. D F2. DF6゜D bean paste.

DF7゜DF, 4. DF8° and DF5. DF6° annealed pair were mixed in the following manner. That is, Dul, 1) ET and (IEl, ( lE5. DF2. [lE6 and DF3. Dl7゜DF4. D[,8o and Dl, 5. The DF6° mixture was warmed to 37°C for m minutes and heated to 4°C for 2 Chilled for an hour. Add T4-DNA ligase (two-in-one) to a mixture of three cold Grand Hyoraps, 3 Weiss single (ffi, DTT and ATP added) The final concentrations were 20'mM DTT and 0.8mM ATP.

Bonding was carried out over 12 FJ at 4°C. 9 to denature ligase The reaction mixture was boiled. After cooling, the reaction mixture was mixed with Cephadex G-150. It was passed through a 10mff1 column of -40. The reservoir containing the desired conjugation product is dried, then resuspended in 20 microliters of binding buffer. , treated with ligase as before. The final bonded product is polyacrylamide It contained a band corresponding to 121 bases on the gel. As in the second example, the band cut out.

The DNA is eluted, precipitated, resuspended in buffer, and ligated to a confirmation vector. Ta.

The base discrepancies in the produced DNA sequence were confirmed, and the 7th base was inserted into pBR322. It was. E, colt transformed by Noichifubafudo Vector The expression of the desired product can be confirmed by radioimycosine analysis. I can do it.

The DNA that was confirmed was the same as that prepared in the second example, but The region containing the code for the terminal amino acid is as follows (amino acid position 31 starting from the alanine codon).

The following example is (Ala-2, Net-L, Val 83 human calcitoni shows the preparation of a manufactured DNA sequence containing the gene encoding the , the aforementioned sequence constitutes a base pair recognition site for PstI restriction endonuclease cleavage. "Contains a blunt J-terminus. To facilitate rM production of hybrid vectors, The terminal portion described above was treated with Pstl before being inserted into the expression vector at the Pst1 site. You can.

1st reciprocal example Deoxyoligonucleotides were prepared in the same manner as in Example 2, except that annealed In the ring reaction, fragments 1, 2 and DEI to DF4 are excluded and the first generation is Instead, fragments C, CA3 and CA4 to CA7 were used. Prepared a The contracted operators were as follows.

2, 5'-GCTGGGC^απATACTCAGGACTT-3'3, 5' -CAIIcAAATTCCATAACCTTTCCCGC-3'4,5'-AGA CCGCTATCGGTGTTGGTGCTCCG-3゜6,5'-GTTG AAGTCCTCACTATAGGTGCC-3'7.5'-GGTCTGCC GGGAAGGTATGGA^↑TT-3'8.5'-GCACCAACACC GATAG(1;-3'CA 1 5'-TGCGGTAACCTGTCTAC CTGCGT-3’CA35”-CAGCACGCAGGTAGACAGGTT AC-3’CA45”-CGGCTGCAGCAATGCA 5 5’-CGC ACATTGCTGCAGCCGCA 6 5'-TGATAACTGCAGC The pair of ATCACGGA DNA strands to CCGC 875'-CGGCTGCAGTT is Annealed by boiling and then slowly cooling. CA4゜C A5゜C^L CA3゜CA2. CA6゜CA3. CA7゜CA4. CA 8. and CA6. The CA7° annealed pairs were mixed in the following manner. . That is, CA4. CA5 and CA1. CA3゜CA2. CA6 and CA3. CA7゜CA4. CA8 and CA6. CA7° mixture is 37°C It was warmed by crossing over to Sugihikida. The three mixtures are then combined into one mixture. It became a compound, and after 1t)5+Ya was sucked at 37°C, it was 2 o'clock at 4°C. Healed. Cold Nkonta Matsuni T4-DNN Soligase New England Ba Iolafize 13 Weiss Mono (ffi), DTT and ATP are added and the final The concentrations were 20mM DTT and 0.8mM ATP. Bonding is at 4°C It was carried out for 12 hours. To denature the ligase, the reaction mixture is It was boiled. After cooling, the reaction mixture was diluted with 10% of Sephadex G-150-40. Passed through the m1 column. The final conjugation product is a polyacrylamide gel A band corresponding to the upper 125 bases was included. As in the second example, the hand is cut out. The DNA was eluted, precipitated, and analyzed by NA. Confirmation vector still has crony has not been updated.

IINA e+J, which is estimated to have been assembled, is shown in Table 2 below. beginning The expected function of the Ala specification codon in the female is a reconfigurable six base pair pair for Ps The hope was to bring about the G-C base pair that would help create the recognition site. Any amino acid could be used as long as it resulted in a G-C pair (e.g. For example, please note that GTT using valine).

first day table 5top Stop ? The following example is a gene encoding [Met-1■νa1B] human calcitonin. Although the above-mentioned 100 examples show the development of DNA sequences containing the gene, the Xba I restriction end The first base resulting in a 6-base pair A”aVA stand for cleavage by a nuclease Sequences and 6 Shioya IEcoRI restriction endonucleases yielding parts of the intestinal site base, and provides a 6-base recognition site for cleavage by BglIt. Contains terminal jll1 and 6eamHI restriction sites.

first day example The deoxyoligonucleotides described below were prepared exactly as in Example 2.

2. 5’-GCTGGGCACCTATACTCAGGACTT-3”3, 51-C88cuAnccarAccTrcccc-3”4.5’-AG ACCGCTATCGGTGTTGGTGCTCCG-3'6.5'-GTTG 88 GTCCTCACTATAGGTGCC-3'7.5'-Gl, TCTGC CGGGAAGGTATGGAATTT-3’8.5”-11,CACCAAC ACCGATAGC-3”CA 1 5’-TGCGGTAACII, TGTC TACCTGCGGT-3'C835'-ε8[, CACGCAGGTAGACA GGTTAC-3'CA 15 5'-AATTCTCTAGAATG-3' CA165'-CGCACATTCT8GAG-3'CA 17 5'-TGA TAGATCTG-3'C8185'-GATCCAGATCTATCACGG -3'ON chain pairs are annealed by boiling and then cooling slowly. was nged. CA15, CA16. C^1, C^36CA2, CA66C^3 ,9^7. C^4, CA8. and CA17, C818.

The annealed pairs were mixed and warmed to 37°C for one time, then heated to 4°C. It was chilled for 2 hours. Add T4-DN8 ligase (2j, 1-in) to the cold mixture. Grand Bioruff's, 3 wythes, DTT and ATP added + 7M at the end of the drum was 20mM DTT and 0.8mM ATP.

Binding was carried out at 4'c for 12 hours. to denature the ligase 1. The reaction mixture was boiled. After cooling, the reaction mixture was converted to Sephadex G-1. Passed through column 101 of 50-40. The fraction containing the desired combined product is pooled. dried, resuspended in 20 microliters of binding buffer, and Treated with kinase as described above. Final product number, urea-polyacryl It contained a band corresponding to 121 bases on the mid-gel. As in the second example, the band The DNA is excised, the DNA is eluted, precipitated, resuspended, and ligated into a confirmation vector. combined. The cloned fragment has not yet been confirmed1. Estimated pole 5 1 is shown in the following table ■ Table N 1020 Leu Gly Thr Tyr Thr Gin Asp Phe Asn Lys　Phe　t{is　Thr3032 Phe Pro Gin Thr Ala IleGly Val Gly A la Pro Stop Stop The following example is (Ala -2t Glu -1, Asn 15) To the DN containing the gene encoding human calcitonin The preparation of the sequence is shown. Terminal base sequence and DN resulting in the 6-base Tanrin Matadate portion for Zee EcoRI At the other end of A is a piece of 6-salt Asahi α tuna for Pstl restriction endonuclease. Contains the terminal base sequence that results in

Fifth case Following the steps generally described in the previous examples, Although cloned and confirmed, the aforementioned Bl is The coding site contains the asnomaragin specification codon (AMC) and is shown below. It differs from IIJ in Table Ⅰ in its method.

and It is clear that a dide product is produced. Examples of polypeptide products include the second This includes the example and the third example. These ↓ cysteine at amino acid position number 1 It has Lys-Arg table 'J in front of , and the component part of this distribution + key gene and It is first synthesized in microorganisms. In this case, use an appropriate protease. If the fusion protein is processed using Polybebutitka is destroyed. As an example of a suitable protease, see Mandibular Protease. Enzyme commercially available as ``Ase'' (Pierce Chemikanoray., Ronokufo (Hard, Illinois). Trypsin can also be used, but amino acids B. To prevent procarese from acting on the lysine present at position number 18. Appropriate measures shall be taken. Other examples of polypeptide compounds include the third example. There is a broline with the amino acid sequence number υ followed by Gly-Lys. -Lys-Arg has icV resistance. The product number of this quiff, Kanoreshitoni. When treated with carpoxamide group-forming proteases endogenous to synthetic organisms, the ) Couminanol Pro-C-NH in the form of biologically active luntonin This results in a product with two groups. [The above-mentioned C-terminal] Calcitonin is the main source It is the first case that becomes a clear peptide, it is formed by Mekunol ammonia treatment. It is possible to This treatment forms carpogisamide groups with other acid residues (e.g. For example, changing aspartic acid at amino acid position 15 to asparagine) It will be done. ] Shown in the 6th example (Hi-l- with glutamine before Cys I'J) ・Polypephants of canoleboxamide Cystine can be used as the starting amino acid for one summer.

Further polypeptide products of the invention include Examples 4 and 5. There are some that have an initial methionine that precedes the ``natural J'' (Cysl). Before Treatment of the above product with cyanogen bromide results in the formation of silane as the first amino acid in the polypeptide. Restore Sutin.

The aforementioned human species in which one or more amino acids occur before or after the natural amino acids. In addition to polybebutide analogues of lucitonin. The present invention provides an amino acid within r of the natural sequence. It also provides different human calcitonin analogues. The aforementioned compounds include. Fourth Examples and 5th example (Val g) class A 9 compounds and 6th example (Asn 1.5) compound is included.

It is possible to tag the products of the invention and/or their antibodies. For example, it can be combined with 5 enzymes to make it radioactive (for example, +125). make use of). Or a fluorescent marker [{e.g. qualitative and/or quantitative analysis of the aforementioned products and/or the aforementioned antibodies. Useful reagents for assay and/or diagnostic testing for iltl1 determination bring. The aforementioned antibodies may be used in one or more animal species (e.g., mouse, rabinote, goat, etc.). can be obtained from human inoculation or from monoclonal antibody sources. Reagent materials mentioned above Either alone or with a suitable culture medium, e.g. on glass or brass. Can be used with chinoku grains or beads that have been removed. .

The novel DNA el of the present invention, specifically shown in the examples described above, is usually expressed The recognition site for DNA cleavage by enzyme 1 nuclease, which makes insertion into S difficult Contains all or part of the base stock that results.

If the specific examples described so far are named 1.F, those who are proficient in this technology will be able to implement the present invention. It is likely that many improvements or changes can be thought of. Therefore, the present invention tFd-J shall be deemed limited only to the extent reflected in the claims. shall be provided.

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Claims (1)

[Claims] 11 Synthesis of human calcitonin polypeptide in selected host microorganisms Based on the characteristic of preferential expression of function codons that govern Contains one or more codons selected from among the following. 3. The produced gene according to claim 1, which contains bases,” in September 5th. of the codon ((based on the legal expression characteristics, alternative codons specifying the same amino,] acid) Contains one or more codons selected from among these. 4. The produced gene according to claim 1, which has a base sequence as follows. 5. A salt for human calcitonin in the produced gene of claim 1. 渠州”1 is the beginning and/or bz i includes the 9th codon at the end, and the above codons are Additional initial and/or terminal amino acids within each synthesized polypeptide Easy separation of biologically active human calcitonin What to do. 6. A salt for human calcitonin in the produced gene of claim 1. There is a base line immediately before and/or immediately after the base line /, and this IIJ force line I] Limited endonucleases are molecules that form part of a base sequence that results in cleavage. of. 7. Human calcitonin polypeptide analogs in selected host microorganisms A manufactured gene capable of directing the synthesis of one or more of the aforementioned analogs It differs from human calcitonin polypeptide in the type and position of the acid. What to do. 8. The produced gene of claim 7, which represents the base fraction, but does not contain the expected host microorganisms. Based on the characteristics of preferential expression of a codon within an organism, alternative codes for specifying the same amino acid are proposed. Contains one or more codons selected from among the dons. 9. The produced gene according to claim 8, whose base sequence is the same as that in E. coli. Among the alternative codons that specify the same amino acid, based on the preferential expression properties of the codon. One or more codons selected from: 10. The synthesized gene according to claim 7, which has the following nucleotide sequence: 2. 5'-T G CG G T A A CCT G T CT A CCT G CG T G CT G-3’-A CG CCA T T G G A C A G A T G G A CG CA CG A CG G CA CC T A T A CT C-A G G A CT T CA A CA A A T-c c a T a c A T A T G A c TcCT c A A c T T G T T T A-TCCATACCTTTCCCGC AGACCGCTATCGG-AGGTATGGAAGGGCGTCTGGCG ATAGCC- and 5'-T G CG G T A A CCT G T CT A CCT G CA T G CT G-3’-A CG CCA T T G G A C A G A T G G A CG T A CG A C-GGCACCTA TACTCAGAACTTCAAACAAAT-CCGTGGATATGAGTC TTGAAGTTGTTTA-TCCATACCTTCC(:GCAGACCG CTATCG’G-AGGTATGGAAGGGCGTCTGGCGATAGC Those with one C-. 11. The produced gene of claim 7 specifies human calcitonin. The base sequence includes an initial and/or end codon, and each of the aforementioned codons Additional initial and/or terminal amino acids within the polypeptide being synthesized , which facilitates the isolation of biologically active human calcitonin analogs. What to do. 12. The produced gene of claim 7 specifies human calcitonin. There is a base bush just before and/or after the base, and this Toshio Rika (DN A salt that provides a recognition site for endonuclease cleavage of the A sequence? Part of IJ What constitutes 13. A fusion product containing the produced gene according to any one of claims 1 to 7. A synthetic gene that produces human calcitonin polypeptide or human calcitonin polypeptide. a second polypeptide in a manner that allows the synthesis of a fusion polypeptide containing the polypeptide A second relic that has the ability to control the synthesis of Chido (fused with the prince). 14. A fusion gene of claim 13ql, in which the second gene is β to galactyl. A gene that controls the synthesis of tosidase enzyme. 15. The fusion gene according to claim 13, wherein the second gene is a β-lactamer. A gene that controls the synthesis of enzymes. 16. A biologically functional A microorganism tiger containing the produced gene according to claim 1. nsformation hector. 17. Biologically functional DNA microorganism containing the produced gene according to claim 7 Transformation vector. 18. Biologically functional DNA microorganism containing the produced gene according to page 1 of the claims Transformation vector. 19. Claim No. 161Jl, Section 17 or Section 18Q4 It is a circular DN^ plasmid. 20. By the vector of either Claim 1 σ-dense Clause 17 or Clause 18 A transformed microorganism. 21. A process for producing human calcitonin polypeptide, claimed in claim 1. microorganisms transformed by raw pasture eNA containing the produced genes of It involves growing under suitable nutritional conditions, thereby achieving the above-mentioned microbial strength statement. A gene that expresses the gene and produces human calcitonin polypeptide. 22. ``The microbial force grown by the process of claim 21''. With microorganisms some stuff. Edict. The scope of the claim is a process for producing a human cyclotonin polypeptide analogue. Transformed by biologically functional DNA containing the genetic material produced in Section 7. It involves growing the cultured microorganisms under suitable nutritional conditions, thereby The aforementioned microorganism expresses the aforementioned gene and produces human calcitonin polypeptide. What to do. 24. In the process of claim 21, the microorganism grown is E. coli micro Something that is a living thing. 25. A polypeptide for expression of the produced gene according to claim 1 in a microorganism. Tide product 26. Expression of the produced gene of claim 7 in a microorganism Polypeptide product of claim 261, (Val 8) The one that is lucitonin. 28. The product of claim 26, wherein (Asn 15) human calcitoni something that is 29. Reagent material containing the produced gene of claim 1 that is radioactive am fee. darkness. The reagent material according to claim 2, wherein the radioactive label is 125. 31. Having an antibody against the polypeptide of claim 5 Reagent material applied to the surface of plastic beads.