JPS61500250A - Microbial expression of type I transforming growth factors, their polypeptide analogs, and hybrid EGF/TGF polypeptides - 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] “Type! Transforming Growth Factor, its Polypeptide Analogs, and Hybrid EGF/TG ``Microbial Expression of F Polypeptides'' The present invention was developed by Banks et al. in 1983. Co-owned and Co-Pending U.S. Patent Application No. 547.48, filed November 1, 2013 This is a partial continuation application of No. 4.

, back---east TECHNICAL FIELD This invention relates generally to the manipulation of genetic material, and more particularly to the manipulation of genetic material. nsforming Growth Factor), its polypeptide analogs , and a hybrid polypeptide comprising a sequence portion of the amino acid residues of a transforming growth factor. of specific NA sequences useful in recombinant engineering to establish microbial expression. Regarding production.

Usually normal mammalian cells (including cells removed from tissues or cell lines) grows only in monolayers and not in soft agar. This is because it does not adhere to cells. This is because it requires solid support. However, the transformed phenotype, Namely, loss of density dependence due to suppression of cell proliferation in monolayer culture, excessive proliferation, changes in cell morphology, and acquisition of anchorage-independent proliferation (acqui site of anchorage-independent grow th) is a normal mammalian cell by one or more mammalian families. It is possible to add polypeptide growth factors, transforming growth factors (TGFs) to It is Noh.

Polypeptides having properties of TGFs can be used in IItrHI cells of different origins, e.g. For example, murine leukemia virus Therefore, transformed rat embryonic fibroblasts crat embryo fibroblasts blast) (Twardzik+D, R,, et al, +5cien ce 216+ 894-897 (1982)), human metastatic melanoma cell line A 205B (human metastatic melanoma cell) 1ine A205B) cells CFlarquardt, H0+ et al ,,J. Mouse 3T3 cells infected with Moloney mouse sarcoma virus (Moloney murine sacroma virus-4nfected +mouse 3τ3 cells) (DeLarco, J., et at.

J. Biol. Chem., 255.3685-3690 (1980)) Therefore, it is secreted and partially purified. The intracellular shape of TGFs is also It has also been determined in tumor cells in culture and in vivo [Rober et al. ts, A, et al, PNAS (USA), 77, 3494-3 498 (1980)).

TGFs are classified into several types based on their biological and physical characteristics. It's been coming. The classification of TGF that can be evaluated is Type I TGF. TGF) (alternately, τGF: a, eTGF, or EGF-like TGF) It is only mentioned. Furthermore, in order to promote the proliferation of IT tumor cells, immediate i.e. transformed cells in fibroblasts and epithelial cells when grown in tissue culture. In order to have the ability to reversibly induce the phenotype, Type I TGF must be Binds to cellular EGF receptor Epidermal growth factor (EGF) (Marquardt, H., et al., PNA) S tis A 8 Kano 4684-4688 (1983)).

Type I TGF is functionally equivalent to epidermal growth factor (“EGF”, “urogas EG) and can be divided into several common properties. It is approximately the same size as F, has about 50111 amino acids, and has 5DS-polymer. Molecular weight of 7.400 daltons as determined by acrylamide gel. E Like GF, Type I TGF forms three disulfide prefixes. EGF has six cysteines at similar positions (Ha plate L J, Bi ol, Chem, + 5upral at 5224], but EG Unlike F, Type I TGF does not affect cells with a transformed phenotype. However, EGF is only found in non-transformed types. Ty peI TGF expression, unlike EGF expression, is associated with active oncogenes (active It seems to be related to the expression of oncogene.

On the filing date (November 1, 1983) of the patent application of the same applicant as above, T isolated from cell lines derived from solid tumors in mice, rats and humans. The amino acid sequence of ype I↑GF was only partially found in the literature. . The part of the polypeptide used to identify the sequence must be identical up to amino acid number 40. By all identical residues in certain mouse and rat Type I TGFs , were determined to be almost identical (ilarquardt, et al., Until recently, human Type I The sequence of TGF was incomplete (mouse and dental within the first 34 amino acids, At least two types of deformations derived from Type I TGF are known; The other four residues are located beyond the 0 position number 34, which was only experimentally confirmed. The line was unknown. ), the complete human Type I TGF sequence is De rynk, et al, + Cedan, 3fl: 287-297 (1984 (August 2013) and is shown below.

Val Val Ser His Phe Asn Asp Cys Pro Asp Ser His Thr Gln Phe Cys Phe His G ly Thr Cys Arg Phe Leu Vat GInGlu As p Lys Pro Ala Cys Val Cys His Ser Gl y Tyr Va + Gly Ala Arg Cys Glu His Ala Partial activation of Type I TGF in Asp Leu Leu Ala rats The amino acid sequence is from Marquardt.

et al, PNAS USA, 80: 4684-4688 (1983 ), in which the sequence up to amino acid residue 43 is shown. and no residues have been identified at positions 26.30.36.40 and 42. Ca1ifornia In5 posture of Technolo Gy Nori, Hood and his collaborators, etc. located 26.30 in September 1983. ．． have reported the confirmation of amino acid residues at 36, 40, and 42, and The construction of the remaining sequences from acids 44 to 50 is reported. Rat type I The complete sequence of TGF was subsequently published in New York on September 8-13, 1983. Co1d Spring H in Co1d Spring Harbor of k arbor Laboratories Cancer Conference (Eleventh Conference) rance on Ce1l proliferation and Canc George J, ↑0da, in the “Growth Factors” subcommittee of the conference during the Submitted by ro. Marquardt, et aL,, 5cie nce,’ll,.

3:1079-1082 (1984), the complete sequence is shown below.

Val Vat Ser His Phe Asn Lys Cys Pro Asp Ser) Iis ThrGln Tyr’Cys Phe His Gly Thr Cys Arg Phe Leu Val GInGlu Glu Lys Pro Ala Cys Val Cys His Ser Gly　Tyr　Val If available in biologically active form in large amounts, the above Type I TGF (as well as polypeptide analogues) with the sequence *TVpe I TGF can be used to increase antibodies. i.e. malignant cells that secrete RIAsSType I TGF. To detect or characterize the presence of a tumor, or ablishment) or to potentially promote immunization of the animal against progression. It is used for diagnostic tests. Rat T [The amino acid sequence of TGF is sufficiently identical to the human Type I TGF sequence. (i.e., 92% agreement), i.e., rat Type I TGF. It is expected that the antibody will cross-react with human Type I TGF. . Therefore, human tissue, i.e. blood or urine, is susceptible to rat Type XTGF antibodies. The used can be screened for the presence of malignancy. Similarly, Type e　I　No symptoms due to the synthesis of large amounts of TGF! ! Tumor (subclinical) A common screen for the detection of tumors has been developed, and cancer Therefore, it becomes possible to monitor the therapeutic effects of patients undergoing treatment.

Furthermore, when used as an additive in cell culture media, Type ITGF Growth of refractive cell types Potentially useful in promoting and for a variety of medical or research purposes , which has the potential to allow cell culture to grow in large quantities in vitro. can have a specific For example, Type I TGF and polypeptides Certain possible uses of analogs include for skin grafts, etc. to stimulate the growth of large cultures of epithelial cells. (Tsao, M. ,,et al,,J,Ce11. P., Danhong 219-229 (1982) reference〕.

Furthermore, in order to maximize the therapeutic potential, i.e. to improve the healing of severe wounds. In order to find a way to utilize the common properties of EGF and Type I TGF, For example, Chemical Week, 5ept, 28+ 1983+ Pages 40-48.

The applicability of recombinant DNA technology to the production of EGF is based on the substantial efforts of the applicant. This is the gist of the research. Coding microbial expression of EGF and EGF analogs The production of the gene for Acture and Expression of Genes C.

ding for Urogastrone and Polypeptide Co-pending patent application entitled “Analogs Thereof” No. 486,091. The disclosure of the aforementioned application is specifically incorporated herein by reference. It was taken as a light. Also, European Patent Publication W issued on February 17, 1982, HN.

, 0046039.

Until now, Type I TGF has been classified by fractionation, Gelbermesicon chromatography, 4-(gel per+*ation chromatography) and Reverse phase high pressure liquid chromatograph 4-(reverse phase high p Cost including result l1quid chromatography) various origins in microgram quantities by a time-consuming and time-consuming chemical operation. It was isolated from a llt tumor. Production of structural gene of Type ITGF, cloning The first public report of the use of recombinant DNA technology for coding and expression was After the filing date of Applicant's U.S. Patent No. 547,484, Derynk, et al. al., 5 upra. Other methods for creating polypeptide analogs No such technology has been reported to date. te)” or “fused” or “hybrid” ``a polypeptide, i.e. a portion, which has the biochemical and immunological properties of EGF, and In part, polypeptides possessing the biochemical and immunological properties of Type I TGF There have been no public reports of any attempts to generate putido.

Therefore, pure, biologically active Type I TGF, polypeptides 1 and above. and species polysaccharide containing the amino acid residues of EGF and Type I TGF. Well-operated methods and materials for large-scale production of peptides are in the art. It continues to be requested.

Next day - one stroke The present invention provides Type I transformation within a host microorganism. This is an engineered gene that can manipulate the synthesis of growth factors. The fabricated remains of the present invention In a preferred embodiment of the gene, the base sequence is of the human or rat type. I A polypeptide that replicates both TGF ('hTGF' and "rTGF") Codon preferences within the encoded and designed host microorganism, e.g. E, col+, Select from alternative codons specifying the same amino acid based on specific expression characteristics contains one or more codons. Other preferred aspects of the created gene The first step is to create a special protein within the encoded polypeptide that is expressed directly within the microorganism. There are base codons that specify specific amino acid residues (eg, the starting Met residue).

In yet another preferred embodiment of the generated gene, a desired polypeptide is specified. The sequence of base codons that define one or more molecules that allow for the formation of proteins or the generation of structural genes for polypeptide analogs. contains a sequence of more bases, i.e., transcription termination or promoter/regulatory Sequence of bases providing promoter/regulator region and/or selection of one or both ends or an intermediate position of the structural gene. restriction enzyme cleavage site cleavage (5ue)).

Preferably, the sequences at one or both ends of the structural gene are A restriction enzyme recognition site that occurs only once within a vector donuclease enzyme recognition 5ite) Code. Intermediate sequences within the gene are preferably not present within the vector structure. Like potatoes, it encodes unique restriction enzyme recognition sites for its own genes. .

Also provided by the present invention are identical amino acid residues of one or more amino acid residues. Type I differs from Type I TGF in terms of gender and/or location. Manipulable microbial expression of transforming growth factor polypeptide analogs It is a gene. Certain polypeptide analogs according to the invention more closely resemble human T ype I Approximate to TGF sequence, for example, (Asp7) rTGF; (P he’Yu) rTGF; (Ala) rTGF; and (Asp”) rTGF including.

Further aspects of the present invention include the prepared Type I TGF gene and the present invention. by itself a second biologically active polypeptide [e.g. strone% epidermal growth factor (epidermal grOWih fact or), i.e., a second gene or genes capable of manipulating the synthesis of "EGF")) A gene containing a gene fragment fused to the fragment is provided. The method is to I Part or all of the transforming growth factor polypeptide or polypeptide analog a fused polypeptide comprising part or all of a second polypeptide, such as EGP, By allowing microbial expression of hybrid polypeptides. Such a work An example of a hybrid polypeptide product of the generated gene is S'rT"/-JJ"E. GFap☆’, “hEG膓//rTGsil”h T G F, -,, / h E G F...

1. Hybrid polypeps shown in “h E G F, , / h T G F and d Contains chido.

In the practice of the present invention to produce polypeptide products, the produced DNA Hybrid vectors are created by inserting the sequence into a virus or circular plasmid DNA vector. and further transform the hybrid vector into bacterial (e.g., E. coli) or yeast cells. used to genetically transform host microorganisms, such as genetically trait The transformed microorganisms are then grown in suitable nutritional conditions and treated with the polypeptides of the present invention. Expressing de-products.

Also encompassed by the present invention are the Type I TGs disclosed herein. F. Antibodies raised against polypeptides and their polypeptide analogs and fragments thereof It is the development of substances.

Other aspects or advantages of the present invention will become apparent upon consideration of the following detailed description. It will be.

Nymph ■buddanhada As used herein, "created" (m) applied to DNA sequences, that is, genes. The term "manufactured" refers to an assembly of nucleotide bases. chemically synthesized in its entirety by or of products thus chemically synthesized. Indicates products from biological replication. As such, the term is of biological origin. The cDNA method or the genome cloning method (geno mic cloning methodology), synthesized ( "synthesized)" products are excluded.

The abbreviations below indicate amino acids used herein. That is, Alani ne, AlaHAArginine+Arg; Asparagine, A sn; Asparatic acid, Asp; Cysteine, C ys; Glutamine, Gln; Glutamic acid, G luHGlycine, GlyHHistidine, HisHl5ole usine.

11e; Leucine, Leu; Lysine, LysHFleth ionine, Met; Phenylanine, Phe; Prol ine, Pro; 5erine, Ser; Threonine, Thr; T ryptophan + Trp; Tyrosine, TyrHValine , Val. The abbreviations below indicate nucleotide bases. That is, A is adeni adenine, G is guanine, T is thymine (t hymine)% U is uracil, C is cytosine sine).

To facilitate the understanding of the present invention, Table 1 below lists 64 alternative trials of DNA. triplet nu Table between cleotide base codon) and 20 types of amino acids The transcriptional stop identified by this correlation (“5top”) Show function.

5-upper 1st W, 2nd 3rd layer pipe CAG Phe Ser Tyr Cys T Phe Ser Tyr Cys C T Leu Ser 5topStop ALeu Ser Sto Tr G Leu Pro His Arg T Leu Pro His Arg C CLeu Pro Gln Arg ALeu Pro Gln Ar G 11e Thr Asn Ser T 11e Thr Asn Ser C A Ile Thr Lys Arg AMet Thr Ls Ar G νal Ala Asp Gly T Val Ala Asp Gly C (, Val　Ala　Glu　Gly　AVal　Ala　Glu　GI　G Preferably, the production of the structural gene according to the present invention is carried out by “Manufacture a nd Expression of 5structural Genes' Filed by Yitzhak 5 Tabinsky on May 6, 1982 under the title by the operations disclosed in co-owned co-pending U.S. patent application Ser. No. 375,493. (PCT International Publication No. 0831, published November 24, 19B3) 04029]. That application is incorporated herein to describe the steps of the operation. It was.

The following examples include Type I hTGF, Type I rTGF, and the like. polypeptide analogues of, and Type r TGF/EGF hybrid polypeptides An example of the implementation of the invention in the production of a DNA sequence encoding the microbial expression of Also shown is the construction of an expression vector for direct expression of the desired polypeptide. .

The following examples illustrate oligonucleotide fragments for the production of structural genes according to the present invention. The general operations used to synthesize pieces are shown.

SuJfJL-1 Oligonucleotide fragments were synthesized using a four-step procedure and several washes. . Polymer in the glass filter (sintered glass funne+) Bound dimethoxytrityl (polymer bound di+netho xytrityl) protected nucleosides are 3% trichloroacetic acid for 1-1/2 minutes. Using dichloromethane containing cid), The 5'-protecting group (5'-protecting group) methoxytrityl) was removed. Washed polymer with solid acetonitrile It was washed, left to stand in the presence of argon, and subjected to the following liquefaction treatment. 10mg tetra Add 0.5 ml of the acetonitrile solution containing the sol to the reaction vessel containing the polymer. added. Then, 30-g of the protected nucleoside phosphoramidite (ρro tected nucleoside phosphoramidite) 0.5 ml of acetonitrile was added. The reaction was stirred during this reaction, and the reaction was continued for 2 minutes. I made it happen. The reactants are then removed by suction and the polymer is washed with acetonitrile. Washed with. This is followed by an oxidation step in which 0.1 mol of 1 ml of a 1:2:2 solution of 2-6-lutidine/H20/THF containing I:L 1 was reacted with the polymer-bound oligonucleotide chain for 10 seconds. Subsequently, T) IP wash camping (THF rinse cap of the nucleotides in the reaction) ping) is dimethylaminopyridine (dimethylaminopyridine). dine) (6.5 g in 100 ml TIIF) and acetic anhydride, 2.6-l The test was carried out for 2 minutes using a solution containing tidine (ni 1) in a ratio of 4:1. Following this , methanol cleaning and THF cleaning were performed. Then, add trichloroacetic acid again. Processing was performed by CH, CI, and the cycle was started. cycle is desired oligonucleotides were obtained.

The final oligonucleotide strand was prepared with thiophenol, triethylamine, Treated with dioxane (L:1:2) for 45 minutes. Then dioxane, After washing with methanol and diethyl ether, the oligonucleotides are stored in a room. Cleaved from the polymer using hot concentrated ammonia. solution from polymer After pouring out, the concentrated ammonia solution was stored in a sealed tube for 16 hours. It was heated to 0°C.

Each oligonucleotide solution was then extracted four times with l-butanol.

The /8 solution was subjected to 20% polyacrylamide 7 molar urea gel electrophoresis, and the appropriate The product bands were isolated. The example below codes Type I TGF. Regarding the production of structural genes for polypeptide analogues thereof, Shows how to prepare a child.

1 By the operation of Example 1, 14 specific deoxyoligonucleotides (deo xyoligonucleotide) (numbers 1 to 14) were synthesized . Oligonucleotides were purified by polyacrylamide gel electrophoresis and Then phosphorylate at the 5° end using ATP and T,z polynucleotide kinase. It was done. That is, 1 nanomole of DNA, more than twice as much ATP, 1 unit (un It was phosphorylated in a standard reaction using 20 μM of buffer containing T dakinase. Ta. The buffer was 50 Lnj of oxyethylpiperazineethanesulfonic acid ChV drOxyethylpiperazine ethane 5ulfontc acid), 10a+M? 1gCl, 10mM dithiothreitol (dithiothreitol), prepared in pit 7.6. reaction Afterwards, the kinase was destroyed by boiling for 5 minutes. These phosphorylations in buffers The resulting oligonucleotides are then directly ligated. It was used for.

Oligonucleotides in 20 μl of standard buffer combine to form short duplex structures. did. Each duplex combines equimolar amounts of two complementary sequences and boils the mixture , and then slowly cooled to room temperature for 1/2 hour or more. in this way, Seven duplex structures were formed.

These seven double structures were bonded in succession, and each set of double structures was heated at 37°C for 5 minutes. , annealed until the final structural gene becomes a single tube suitable for ligage runs. I nged. The oligonucleotide mixture was then made up to 150 μM with ATP and 8 Treated with 4 units of Ty DNA ligase for 16 hours at 4°C. Lige enough The modified structural genes were then analyzed by polyacrylamide gel electrophoresis. Purified.

Table ■ below shows polypeptides having the main structure of rat Type I TGF. The final assembled structural gene encoding direct microbial expression of It shows.

! 1et Val Val Ser His Phe Asn Lys Cys Pr.

1-1-1-2- 5'-G ATCCAG ATG GTA GTT TCT CAT TTC AACAAA TGCCCG3’-GTCTACCAT CAA AGA GTA AAG TTG TTT ACG GGC" Average L Asp Ser His Thr Gln Tyr Cys Phe His Gly Thr Cys Arg Phe-1111 lower--3--------------------- 7-----GACTCT CACACT CAG TACTG (: TTCC AT GGT ACT TGCCGT TTCCTG AGA GTG TGA GTCATG ACG AA to GTA CCA TGA ACG GCA AAG□910 ” Vomit L Leu Val Gin Glu Glu Lys Pro Ala Cys Val　Cys　Hts　Ser　Gly-4=-Tm-5 CTG GTA CAG GAA GAG AAA CCG GC^τGCGT A　TGCCAT　TCT　GGT[;ACCAT　CTCCTT　CTCTT T GGCCGT ACG CAT ACG GTA AGA CCAb U- Tyr Val Gly Val Arg Cys Glu) Its Ala Asp Leu Leu Ala6 7□ TACGTT GGCGTA CGT TGCGAA CAT GCA, GA T CTG CTG GCT TAAATG CAA CCG CAT GCA ACG CTT GTA CGT CTA GACGACCGA ATTTA C ATCAGCT Table ■ below shows the polypeptides having the main structure of human Type I TGF. The final assembled structural gene encoding the direct expression by microorganisms It shows.

Met Val Val Ser His Phe Asn Asp Cys Pr.

1-1-1-2- 5'-G ATCCAG ATG GTA GTT TCT CAT TTC AACGACTGCCCG3'-GTCTACCAT CAA AGA G TA AAG TTG CTG ACG GGCNeo[ Asp Ser His Thr Gin Phe Cys Phe His Gly Thr Cys Arg PheGACTCT CACACT CAG TTCTGCTTTCCAT GGT ACT TGCCGT TTCCTG AGA GTG TGA GTCAAG ACG AAG GTA CCA T GA ACG GCA AAGLeu Val Gln Glu Asp Ly s Pro Ala Cys Val Cys His Ser Gly-4- 1cho-5------11--------rCTG GTA CAG GAA GA CAAA CCG GCA TGCGTA TGCCAT TCT GGTGA CCAT GTCCTT CTG TTT cGc CGT ACG CAT 'Acc GTA AGA CCA-1112□ Jika Uji Tyr　Val　Gly　Ala　Arg　Cys　Glu　I(is　Ala Asp Leu Leu AlaTACGTT GGCGCA CGT TC CGAA CAT GCA GAT CTG CTG GCT TAAATG CAA CCG CGT GCA ACG CTT GTA CGT CTA GACGACCGA ATTAG ATCAGCT Included in each table is the D,'lA sequence inserted into the appropriate mucomicrobial expression vector. A sequence with the number of amino acid residues specified when each polypeptide sequence Thus, naturally occurring rat and and human Type I TGF. Ru.

In the table, the codons that specify each amino acid are listed below the identified residue. It is being I) The grouped region of the NA sequence consists of the 14 initially formed It shows the separated oligonucleotides and also shows the intermediate duplex structure ( For example, 1, 2, 3, 4, etc.). If it is consistent with the manipulation of the structure of the structural gene As always, the codon usage is as follows: Grantham, et al., ) iucle ic Ac1ds Re5search+ 8. pages 149-162 ( 1980); Grantham, et al.

Nucleic Ac1ds Re5earch, 8. pages 189 3-1912 (1980); and Grantham, et al., Nuc leic Ac1ds Re5search + iLpages 143474 ( Based on the data shown in (1981), the planned expression hosts (e.g. L. Selected based on "preferences". Bennetze n, et al, J, Biol, Chem, 257. pp, 302 6-3031 (1982); and Grosjean, et al., Gen. e, 18. See also PP, 199-209 (1982).

The codons used to generate the Type I TGF structural gene in Tables ■ and Table ■ are , among the codons that allow maximum expression in E. coli. many strange In addition, Type I TGFs (contained within the resulting structural gene encoding the U product) performed in order to enable expression and/or operation of Is possible.

For example, by using alternative codons, two unique It is now possible to introduce a third and penultimate internal restriction site (i.e. , & Tei and Takumi). This accommodates further species-specific changes in the amino acid sequence. Each gene can be modified or spliced if desired. to be done). The LLLLIn recognition site preceding the stop codon This allows additional arrays to be added if desired. Codon changes are not allowed. It can also be used to change the second structure present in the created structural gene. It is Noh.

The end of each gene (Met) specifies the amino-terminal residue of the polypeptide to be expressed. , Val, etc.), the selected control v! sticky end of enzyme recognition site (schematically referred to herein as the Bamt II terminal end) ). At the end of each gene specifying the carboxy-terminal residue of the polypeptide (A la), translation stop codons (e.g. TAA, TAG), and similarly selected The bases that form the sticky end of the restriction enzyme recognition site (herein shown schematically) The plate will cause sticky edges. One or all double endonuclease recognition The recognition site is enzymatically treated to form a selected sticky end within the vector. It will be understood that this is effected at the possible end.

strands encoded to provide alternative restriction enzyme recognition sites. d) Promoter with Type I TGF sequence selected at the 51 end of / operator so that it can be inserted after the ``leader'' der)” DNA sequence is provided. For example, the leader DNA described below The A sequence is a synthetic T5 early promoter replication DNA sequence (synthetic T5 5 early promoter replica DNA) (especially the present invention) “Enhancement of Micr”, which is incorporated by reference in the specification. obial Expression of Polypeptides” Co-owned by BankS, etc., filed on August 10, 1983, under the same title, T5A-1ac promoter/disclosed in US Patent Application No. 521,959 operator sequence), or λPL promoter [e.g., Shimatake , et al, , "Nijikan h" n: 128-132 (1981)], Or commonly used for direct expression! Type I TG adjacent to promoter Allows insertion of the F gene.

Met Lys Lys Tyr Trp Ile Gln Met5 °-G AA TTCTCT AGA ATG AAG AAA TAT TGG AT Discharge to CCAG ATG-3' [XbaT-Riku! Moon- )l The Xba1 site is a short “pr” that can be incised with Met by cyanogen bromide.

“In order to create a polypeptide with a polypeptide sequence, the polypeptide code EcoRI recognizes The leader DNA sequence with the site is a fusion peptide with β-galactosidase. It also allows for the development of EcoRI recognition sites in the middle of the genetic coding sequence. It also makes it possible. As mentioned above, the leader DNA sequence is the same as the structural gene. or by inserting the structural gene into a plasmid into which the sequence has already been introduced. It is possible to add

Microbial production of a polypeptide sequence that differs from the natural Type I TGF sequence The novel Type I TGF gene encoding the present To change the base sequence of the oligonucleotide and to obtain a new gene. can be prepared by repeating the gating steps. For example, rat type By changing oligonucleotide numbers 2 and 9 of e r TGF, the remaining A codon specifying group 7 lysine is placed at position 7 of human Type I TGF. Aspartic acid, which is a residue associated with a protein, can be changed to a specific codon. oligonucleo The change in codon number 2 changes 5'-AAA-31 to 5°-GAT-3' or 5'-GAC-3'.

Change in oligonucleotide number 9 of rat Type I TGF is 31-↑T Changing T-5' to 3''-CIA-5' or 3'-CT[;-5' It is.

Similarly, oligonucleotide numbers 3 and 10 of rat Type I TGF were changed. and changing the tyrosine at residue 15 to phenylalanine By doing so, the sequence becomes more similar to the human sequence. oligonucleotide The codon change for code number 3 is 5’-TAC-3’ to 5’-TTC-3’. It is to be. Complementary codon changes in oligonucleotide number IO is to change 3'-ATG-5' to 3'-AAG-5'.

Several codon changes are made to change the sequence of the desired amino acid to another amino acid. It is possible to do so.

The following examples demonstrate the expression of certain Type I TGF polypeptide products of the invention. Microbial host to establish! Form of expression vector used for transformation of llI cells Concerning operations for

Human Land Week-Yu A, Plasmid ADH15 gene Table of Example 2 of Type l rTGF gene created with 166 base pairs ■ The BamHI- and chain-adhesive strands are shown in the same sequence derived from the cloning vector. It is inserted into E. coli pBR322 as shown. The cloning vector The target is L total - and ■ located at 5° to the BamHI restriction enzyme recognition site. It contains the leader DNA sequence of Example 2 having a restriction enzyme recognition site. the The resulting plasmid pADH12 contains the following O already present in the original vector. Contains Type I TGF gene sequence 3' for the leader sequence to N .

5　°-GAA　TTCTCT　AGA　ATG　AAG　AAA　TAT　T GG ATCC-3'EcoRT Xbal BamH The gene clone confirms the calculated molecular weight of the Type I TGF structural gene. were characterized by polyacrylamide gel electrophoresis.

pADH12 was cut with the restriction enzyme Xba and a 166 base pair T ype I TGF coding sequence and the base encoding the leader sequence following it A small fragment containing the pH1l1322-derived expression plasmid p Inserted into lnt-γ-txB4. The plasmid is tryptophan. ■) Structural genes encoding promoter sequences and other polypeptides For Shine/Dalgarno sequences followed by children Contains the Xba T site 3°. “The Manufacture and Expression of Large Filed on April 15, 1983 entitled ``5structural Genes'' See co-pending U.S. patent application Ser. Teru (pcT International Publication No. WO104053 published on November 24, 1983) ), thus the resulting plasmid i.e. pADH15 carries the m promoter It has a Type I rTfl;F gene placed under the control of -. correct The insertion of the leader sequence and type I rTGF gene in the Confirmed by restriction enzyme analysis and molecular weight measurement using lyacrylamide gel electrophoresis. I was bitten.

Vector pADH15 is a polypeptide containing the following 8 amino acid "pro" sequence: Used as a host hector within the Ecoli host to produce putido products .

Nl2-Met-'-Lys-'-LysE T y r' T r p E 1 1e'-Gln'-Flet-'-(TGF　)-COOHB, Boosmid A [5 of 1H96 Plasmid pA! 11115 with a valid promoter/operator (■■) In conjunction with the prepared Type I rTGF structural gene, the desired protein Further manipulations were performed in an attempt to generate expression vectors that yielded high levels of L2 expression. It was done. These operations were described in a 1983 book entitled “DNA Plasmids”. Co-pending U.S. Patent Application No. 5, co-owned by Morris, filed August IO. 21, 964, including the use of plasmids. Disclosure of the aforementioned application is specifically incorporated herein. Briefly, plasmid pcFt1414 (A, T, C, C, 40076), i.e. the plasmid used to form the In transversion, host cell cultures grown at 30°C have fewer plasmid copies. , plasmid copy number increases 50-fold in host cells with an increase in culture temperature above 34°C. (i.e., "runs away").Growth at 37°C normally It's deadly.

The specific operations involved in the formation of vectors using A, T, C, C, 40076 are listed below. As written. Plasmid pADH15 contains 7ype I rTGF structural gene. approximately 4 including the child, leader sequence codons, and associated m promoter/operator. To form a base pair fragment of 80 Ill, EcoRI (t■ promoter 5") and was enzymatically cleaved in a dish. Plasmid pcFM414 was The large fragments were separated by enzymatic cleavage with 2- and 2-niji. Derived from pADH15 The Rrufudan fragment containing the hpeI TGF gene was then transformed into the plasmid pA Lid with the large EcoRI/■1 fragment of PCFM414 to form DH96. Gated. This formation maintains a complete Shija-region &^ part, 5BJI and Although ligation of the complementary adhesive ends of the binder does not repair both recognition sites, the PC The flexural area adjacent to the damaged area of FM414 is repaired. T in the transcription stop sequence ype I rTGF structural gene 5', namely Toop, in pcPM414 It is also worth noting that this is the result of placing 3' in the rainbow part of .

By the above operation, before ejecting and inserting into the rainbow-cut pcFM414. , the first and second intermediate plasmids (pAD) 112 and pA of the genes in Table ■ DI (+5, in which the gene first cooperates with the leader sequence and then the pressure promoter pADH96 results in the formation of pADH96. photo Shino's plasmid is K! “Linker (14nker)” arrangement including iL adhesive end Adding a column to the gene in Table 1 with the terminal sticky end and the starvation occurring in the correct direction. The two sticky end sequences were generated by enzymatic cleavage of plasmid pADH59. can be formed by inserting it into a fragment. Plasmid pADH5 as described above 9, “The Manufacture,” the disclosure of which is incorporated herein by reference. ture and Expression of Genes for Ur ogasLrone and Polypeptide Analogs Th by the above-mentioned applicant, filed on April 25, 1983, entitled Co-pending U.S. Patent Application No. 486,091 (Published November 24, 1983) It is disclosed in pcT International Publication-083104030).

In E. coli JM10311 cells deposited as ATCC39335 The existing plasmid pAD1159 encodes urogastrone The generated gene, the leader sequence described above, and pint-γ-txB4 Contains the derived trp promoter/regulator, all inserted into pCFM414. entered. If the urogas trone gene follows the same enzyme digestion If the large fragment remains an exact copy of pADH96, it can be cut with the enzyme. It can be cut out. Therefore, "Mosuo" [-Sat A, Plasmid ADH123-5 166 flill bases shown in Table II of Example 2 with BamHI and Germany The Type I hTGF gene created in pairs was derived from plasmid pADH120. It is amplified by inserting it into a similarly cut M13mp9 vector. So After that, the Type T hTGF gene was transformed into pADl+1 with BamHI/5ail. Inserted into pA[1)125 which was cut from 20 and digested with Ban) II/Satan. It was done. pAD) 125, also described in the aforementioned U.S. Patent Application No. 486,091. It is listed. Briefly, pADH25 is one promoter/regulator. The prepared Binghong site is followed by the tar sequence, and the internal Xba I recognition site shown below The DNA "tinker" sequence formed and the unga produced A pBR322-derived plasmid (plNT-γ-Tx Contains part b4).

K lice L "Blood I Ba plate L GAA TTCTCT AGA ATG AAG AAA TAT TGAGA TCT TACTTCTT ATA ACCTAGpAIIH25 is un with BamHI/5alt to remove the sequence encoding gastrone. After enzymatic cleavage, BamHI/5all Type I hTGF sequence is inserted into it. is inserted to form the expression vector pADH123. Vector pADl(12 3 is a polypeptide containing the same 8 amino acid "pro" sequence as in Example 3A above. used as an expression vector in an E. coli host to form a putid product. It will be done.

B, Plasmid ADH131-5 A similar procedure to increased expression of Type I rTGF in Example 3B above was used. This was followed for the prepared Type I hTGF sequence. Plasmid pADH12 3 is Type I hTGF structural gene, leader sequence codon, and related In order to form an approximately 480 base pair fragment containing the promoter/operator, For this purpose, enzyme cut with EcoRI (5' to trp promoter) and 5all It was cut off. Plasmid pcFM414 was enzymatically cleaved with Liao J- and Xhol. Type I hTGF gene derived from pADH123 from which a large fragment was isolated The EcoRI/Xhol fragment containing the offspring was then transformed into plasmid pA[1H131 ligated with the larger EcoRl/Xhol fragment to form the As a result, Type I hTGF structural gene 5' is added to the transcription termination sequence, that is, T oop, place 3' in the male part of the pcFM414. This formation completes All retain the recognition site, but the ligation of the complementary sticky end of Fudan and Xhol Both recognition sites are not repaired by the application. In the Kyotei part of pcFM414 Adjacent Kanyu [one part is retained.

The following examples demonstrate how desired Type I TGF polypeptides of the present invention can be administered to microorganisms. Regarding the expression of

z upper plate-1 n Uni Fallen Birth Village Plasmid pADH96 (Example 3B) was transformed into L. and grown in L-medium. Clone with correct Type I rTGF fragment were separated and characterized by polyacrylamide electrophoresis. Type The precisely determined product of I rTGF is made visible by staining the gel. It was.

Plasmid 9AO) 1131 (Example 4B) was derived from h Kasada strain J! +103 shape The cells were transformed and grown in L-medium. Contains the correct Type I hTGF fragment Clones were isolated and characterized by polyacrylamide electrophoresis. T Precisely calculated products of ype I hTGF are visible by gel staining It was done like that.

The following examples demonstrate the biological activity of the TVpe I TGF polypeptides of the invention. Regarding detection.

Shazenkan-ko Prior to performing the following three analyses, pADl (in E. codan (Example 4)) 96 and 8 amino@R+x expressed by ρADH131.

Type Ir TGF and Type hTGF polypeptides containing AX , Enhancement of Bi, specifically incorporated herein by reference. Logical Activity of Microbially Exp dated 10 August 1983 entitled 'Polypeptides' Co-pending U.S. Patent Application No. 521,908 filed Banks Co. It was recovered from host cells by the disclosed solubilization and sequential degradation and oxidation treatments.

A, EGF・、Recebu-ノ＼ EGF radioreceptor analysis y) is either Type I rTGF or Type [hTGF of the present invention] polypeptide and A431 human epidermal carcinoma 1t! (A431 human epi Using dermoid carcinoma) cells, Mrquardt, e. tal,, PNAS (USA), - Nail L” bale P1atpage 4685 Performed in accordance with the disclosed procedures. Type I rTGF and T of the present invention ypeI hTGF attaches EGF receptor to A431m cells. By competing with I-EGF, each has no biological activity in this assay. I was caught.

substantially Hollenberg, M., et al., J., Biol, C. hem, +2503846-3853 (1975). Therefore, 3H-thymidine absorption analysis shows that the Type I rTGF polypeptide of the present invention and human foreskin fibroblasts It was done using .ast).

The Type I rTGF of the present invention was found to be mitogenic in this analysis. nic) and was therefore found to be biologically active.

C., Coronin The Type r rTGF and Type I hTGF polypeptides of the present invention are Marquardt, H, et al, J, Biol, Chem, , 257.5 upra, at page 5221. and rat kidney fibroblasts. Biological activity was analyzed using soft agar colony formation assay. Type I rT The biological activity of GF and Type I hTGF polypeptides was determined in this assay. Confirmed by colony formation on agar.

D, body length Current research efforts are underway to generate antibody materials directed against the polypeptides described and disclosed herein. is being paid. In particular, Type IhTGF and Type IrTGF Efforts are currently underway to generate monoclonal antibodies against. Of course, Jithio dithiothreit.

1) Recombinant Type Ih reduced by (breaking three binary bonds in the molecule) Mice immunized with TGF have polyvalent antibodies in their serum. It has been previously known that this method produces a tibody. In preliminary tests, this antibody Type I obtained from true biological sources when reduced with dithiothreitol It was proven that rTGF can be recognized. Furthermore, this antibody Recombinant dithiothreitol reduction Type I rTGF (recombinant dithiothreitol-reduced Type I rTGP) , and its immunogen recombinant dithiothreitol-reduced Type I hTGF ( immunogen recombinant dithiothreitol -reduced Type [hTGF) was recognized. However, currently antibodies have not shown recognition of Type I rTGF in their natural biological tissues.

Multivalent and monoclonal antibody materials are commonly used in immunological studies as known to those skilled in the art. Polypeptide analogs and polypeptides of rTGF and hTGF can be prepared using conventional techniques. Techniques for de velopment of hybridom cell 1ines des crtbed in Paul (ed,) + Fundamental Imm unologV+ Ch, 28+ pp, 751-7'65. Raven Press, New York (1981) and examples cited therein. See also]. For the quantification and detection of Type I TGF from biological sources In terms of its potential as a reagent for characteristic analysis, Type I TGF polypeptide The formation of monoclonal antibodies against DNA, analogs, and fragments is of interest.

The following example shows the amino acid sequence portion of rough) Type I TGF, and human u A hybrid polypeptide that combines the amino acid sequence part of rogas trone (hEGF). To ensure the expression of hybrid polypeptide products of the invention containing peptides, It relates to the construction of expression vectors used in the transformation of biological host cells.

Disaster relief-1 A, Fu Σ Nuni L JIAD) + 55 and ADHloo's ≦Type I rTG the first 32 amino acids of the amino terminus of F and the carboxy terminus of human EGF. A hybrid gene encoding the last 22 amino acids was formed by the following procedure. It was. As disclosed in the aforementioned U.S. Patent Application No. 486.091, the The gene encoding rogas trone (EGF) is followed by E and the promoter. Plasma with a DNA sequence encoding an amino acid sequence and an 8-amino acid “pro” sequence. Sumid pADH25, a plasmid derived from pBR322, is was enzymatically cleaved. The coding region for the first 32 amino acids of human EGF is Plasmid pADH25 was then excised and then injected with bacterial alkaline phosphatide. treated with Tase. Type I Plasmid p containing TGF + structural gene ADH12 (Example 3A) was similarly enzymatically cleaved with hL and dish, resulting in 101 The two 1M-fragments were separated by polyacrylamide gel electrophoresis. It was. The Type I TGF fragment isolated from pADH12 was then h starvation enzyme cleavage fragment derived from plasmid pADH25 containing the promoter. By mixing and transforming into E, co and JM103, Type I TGF-E DN to yield plasmid pADII55 encoding the GF hybrid protein. Cultured with A ligase.

J! pADH55 isolated from the '1103 culture was then transformed into the complete m promoter. - and Type I The first 32 amino acids of TGF and the last 22 of EGF EcoRI- and and 5a11. This fragment was then converted to pAD of Example 3B above. 196, the promoter gene fragment is transformed into a temperature-sensitive high expression plasmid. EcoRI/Xhol enzyme-cleaved pAD) 1100 Ligated within CF gate 414.

The Type I hTGF sequence was similarly designed into Type I rTGF. Since they have the same button/restriction site, a person skilled in the above technology can create a hybrid type. e　I　hTGFz書/　h　E　G　Ij, to similarly form and express Can be done.

B, The amino-terminal beginning of boothmid ADC530 and ADC533 and human EGF. 31 amino acids of and the last 18 of the carboxy terminus of Type I rTGF A hybrid gene encoding the following amino acids was formed by the operations described below. . Plasmid pADII25 (Example 7A) contains the last 22 amino acids of EGF. Enzymatic cleavage in the dish and n to excise the acid coding region, followed by pressure promolysis. pADH25 containing the protein was treated with bacterial alkaline phosphatase.

Type r Plasmid pADHI2 containing the TGF structural gene is also After enzymatic cleavage at the button and chain ■, the force 1uidL fragment was electrolyzed on a polyacrylamide gel. Separated by electrophoresis. The isolated fragment derived from pADH12 was then enzymatically cleaved. promoter-containing fragment derived from plasmid pADH25, and plasmid pADH25. Cultured with DNA ligase to yield ADc530. In addition, the above pA[1 The isolated fragment derived from H12 was transformed into E coli JM103. Encodes GF-Type I TGF hybrid protein.

pA[1C530 isolated from JM103 was then transformed into the complete trp promoter. -, and the first 31 amino acids of EGF and the last 1 of TVpe I TGF To cut out the gene encoding the eight amino acids, we used a combination of enzymes with oRI and chain enzymes. It was severed. This fragment was then cut into EcoRr/XhoI#N as described above. Temperature-sensitive high expression plasmid ligated into the cut pCF gate 414 A promoter gene fragment is provided in pADC533.

As mentioned above, a similar hybrid hEGF, -77/TVpeI hTGF, was also used. It can be planned, and it can also be expressed using the operations cited above.

The following examples illustrate desired Type I TGF/EGF hybrid polypeptides of the present invention. Regarding the microbial expression of

1st class 1 T e I TGF/EGF i E, Plasmid 11A transformed into codan strain JO3 and grown in L-medium. DH100 is a hybrid polypeptide (“Type f　rTGF/P5　/hTGFjJ-#'), that is, Type I Contains the first 32 amino acids of TGF and the last 22 amino acids of EGF Expressing a hybrid polypeptide.

E. Temperature-sensitive high expression protein transformed into codan JM103 and grown in L-medium. In lasmid pADC533, the first 31 amino acids of EGF and I hEGF, a hybrid polypeptide containing the last 18 amino acids of TGF, - , / Type I r TGF,; ) is approximately 80 mg100- Quantitated in 1 iter.

The foregoing illustrative examples demonstrate direct expression and fusion product generation in E. co and host cells. Currently passed Type I TGF and similar polypeptides and TVpe r T The main purpose is the formation of engineered structural genes for GF. made These genes are particularly amenable to direct expression in yeast cells through the use of yeast-preferring codons. 5 creation of Exog 19 entitled Enous Polypeptides from Yeast’ Co-owned and co-pending U.S. patent filed by Bitter on April 22, 1983 487,753 leads to the secretion of the polypeptide product into the growth medium. can be used to ensure yeast expression. In such cases, Methi Translation initiation ATG code for Type I TGF specific codon that specifies onine It is not necessary to bring about Don 5''.

The foregoing illustrative examples specifically relate to Run 1-Type I TGF and human EGF fragments. Concerning the synthesis of hybrid polypeptides containing fragments of the human or other species Type It will be appreciated that 9M species containing ITGF sequences are within the scope of the present invention.

Furthermore, the examples show that about a portion of the residue sequence of Type I TGF or EGF is - 1 and part 2 of the residue sequence of an alternative growth factor, but other Hybrid polypeptides in the form of It should be understood that it can be synthesized to Hoop and Woods , PNAS (USA), 78°3824 (1981). For example, naturally occurring Type I TGF contains three main hydrophilic regions. It shows. These are located at or near residues 8.28 and 44, respectively. It is location. Previous research on synthetic fragments of EGF has shown that the EGF receptor The central region of the polypeptide is crucial for binding the polypeptide to cells. Confirmed experimentally. Therefore, the amino and carboxy termini of Type I TGF amino and carboxy terminal portions that overlap, and a central portion that overlaps EGF. A hybrid polypeptide containing Nevertheless, it had the same desirable characteristics as those of naturally occurring EGF.

The products of the invention and/or antibodies thereto may be produced in a liquid sample as described above. analysis and/or for qualitative and/or quantitative measurement of the substance and/or the aforementioned antibodies. or properly “tagged” to bring useful reagent materials to a characterization test device. ged)”, e.g. enzyme-conjugated radiolabeling (e.g. I) or fluorescent labeling. You can attach a bell. The aforementioned antibodies may be used in one or more animal species (e.g. (e.g., mice, goats, humans, etc.), or simply from cloned antigens. Before The reagent substances mentioned above can be independently prepared in a suitable substrate, such as glass or plastic particles or Can be used with beads.

Various modifications and changes may occur to those skilled in the art in carrying out the invention. This may be possible by considering the above-mentioned exemplary embodiments. Therefore, The invention is to be considered limited only to the scope of the appended claims.

・1m-+le-“1747 to Mekabenji CT8S 84

Claims (45)

[Claims] 1. Synthesis of Type I transforming growth factor polypeptide in a selected host microorganism A created gene that can be manipulated. 2. The produced gene according to claim 1, which has one or more base sequences. selected and designed from alternative codons that contain codons and specify the same amino acid. Based on the preferential expression characteristics of codons within the host microorganism. 3. The produced gene according to claim 1, which has one or more base sequences. codons selected from alternative codons specifying the same amino acid; co Based on the preferential expression characteristics of codons within li. 4. The produced gene of claim 1 specifies the Type I transforming growth factor. The determining base codon may be used as an additional initiation and/or Contains a start and/or stop codon that specifies the stopping amino acid. 5. In the generated gene of claim 4, before specifying the additional starting amino acid. The start codon mentioned above is the codon that specifies the start methionine residue. 6. In the generated gene of claim 4, before specifying the additional starting amino acid. The start codon described above is a codon that specifies the amino acid leader sequence below, i.e. [There is an array]. 7. In the constructed gene of claim 6, the leader start codon described above is Contains the sequence of bases shown below, that is, [there is a sequence]. 8. The produced gene of claim 1 specifies the Type I transforming growth factor. The specified base codon precedes and/or follows and/or is used for restriction enzyme cleavage. It contains a sequence of bases that includes a base sequence portion that provides a recognition site for. 9. The rat tape according to claim 1, 4, or 8 The gene that specifies the I transforming growth factor has the following nucleotide sequence: [There is an array] including. 10. differing in the identity and/or position of one or more amino acids Selected host microorganisms are capable of synthesizing Tape transforming growth factor polypeptide analogs. A created gene that can be manipulated within an object. 11. The produced gene according to claim 10, which has one or more base sequences. Alternate codons containing the above codons and specifying the same amino acid are selected and planned. based on the preferential expression characteristics of the codons within the host microorganism. 12. The produced gene according to claim 11, which has one or more base sequences. E. Based on the preferential expression characteristics of codons in E. coli. 13. The produced gene according to claim 10, [Asp7]rTGF; he15], rTGF; [Asp28] rTGF; [AIa41] rTGF; Met-1]rTGF; and [Met-8, Lys-7, Lys-6, Tyr- 5, Trp-4, IIe-3, GIn-2, Met-1] Selected rat Type I transforming growth factor polypeptide analogs are identified. 14. The produced genes of claim 10, [Met-1] hTGF and [Met-8, Lys-7, Lys-6, Tyr-5, Trp-4, IIe-3 , GIn-2, Met-1] hTGF. Identifying peI transforming growth factor polypeptide analogs. 15. The prepared gene according to claim 10, Type I transforming growth factor Base codons specifying polypeptide analogs are present in each synthesized polypeptide. start and/or stop codons specifying additional start and/or stop amino acids; include. 16. In the constructed gene of claim 10, specifying an additional starting amino acid. The start codon described above is a codon that specifies the amino acid leader sequence: [There is an array]. 17. In the constructed gene of claim 16, the above-mentioned amino acid pro sequence is The aforementioned leader initiation codon to be specified has the following base sequence, namely: Contains [There is an array]. 18. The prepared gene according to claim 10, Type I transforming growth factor A base codon specifying a polypeptide analog precedes and/or follows and/or or contains a base sequence portion that provides a recognition site for restriction enzyme cleavage of the DNA sequence. Contains a sequence of bases. 19. Contains the produced gene according to claim 1 or 10. A fusion gene, the fusion gene is the produced gene according to claim 1 or 10. A fusion hybrid polypeptide containing a polypeptide product encoded by a gene to manipulate the synthesis of the second polypeptide in such a way that the synthesis of the second polypeptide can be manipulated. fused to a second gene that can produce 20. The fusion gene is created in the selected host microorganism, and the fusion gene is Selected portions of the amino acid sequence of ypeI transforming growth factor and epidermal growth factor to manipulate the synthesis of polypeptides containing selected portions of the amino acid sequence of It is possible. 21. The fusion gene is the most prominent in the selected host microorganism. The first 32, or amino terminal, amino acid residues of the Type I transforming growth factor. group, and the last 22, i.e., the carboxy terminus, i.e., the amino terminus of human epidermal growth factor. It is possible to engineer the synthesis of polypeptides containing acid residues. 22. The fusion gene is the most prominent in the selected host microorganism. The first 31, or amino terminal, amino acid residues of human epidermal growth factor; The latter 18, i.e., the carboxy-terminal, i.e., the amino acids of the Type I transforming growth factor. It is possible to engineer the synthesis of polypeptides containing acid residues. 23. Contains the produced gene according to claim 1 or 10. Biologically functional DNA microbial transformation vector. 24. A biologically functional DNA microorganism containing the fusion gene of claim 19 material transformation vector. 25. The fusion gene according to any one of claims 20, 21, or 22 A biologically functional DNA microbial transformation vector containing. 26. The vector of claim 23, wherein the vector is a circular DNA plasmid. be. 27. The vector of claim 24, wherein the vector comprises a circular DNA4 plasmid. It is de. 28. The vector of claim 25, wherein the vector is a circular DNA plasmid. be. 29. A microorganism transformed with the vector of claim 23. 30. A microorganism transformed with the vector of claim 24. 31. A microorganism transformed with the vector of claim 25. 32. A method for the formation of a Type I transforming growth factor polypeptide, comprising: A biologically functional product containing the produced gene of either Scope 1 or 10. Microorganisms transformed with competent DNA are grown in suitable nutritional conditions, thereby The above-mentioned microorganism expresses the above-mentioned gene to produce Type I transforming growth factor polypeptide. Produces tide. 33. By the method of claim 32, the microorganism grown is E. coli microorganism be. 34. A microorganism containing the produced gene according to claim 1 or 10. Polypeptide products of expression within. 35. The product of claim 34, wherein the product is [Met-1]rTGF; [Met-1]hTGF; [Asp7]rTGF; [Phe15]rTGF; AIa41]rTGF; [Asp28]rTGF; [Met-8, Lys-7, Lys-6. Tyr-5, Trp-4, He-3, Gln-2, Met-1] rTGF; and [Met-8, Lys-7, Lys-6, Tyr-5, Trp- 4, Ile-3, Gln-2, Met-1] selected from the group consisting of hTGF be done. 36. Polypeptide for expression of the produced gene in a microorganism according to claim 19 De product. 37. The produced product according to any one of claims 20, 21, or 22 Polypeptide products of expression of genes in microorganisms. 38. A radiolabeled preparation according to claim 1 or 10. A reagent material containing a gene that has been identified. 39. Radiolabeled according to claim 34, 36, or 37 Reagent material containing polypeptide products. 40. The reagent material of claim 38, wherein said radiolabel is I125. 41. The reagent material of claim 39, wherein said radiolabel is I125. 42. Claims 34 and 36 coated on the surface of plastic beads, or a reagent material comprising a tagged antibody against the polypeptide of paragraph 37. 43. Type I transforming growth factor polypeptide or polypeptide analog; or an antibody substance capable of binding to a fragment thereof. 44. The antibody substance according to claim 43, wherein the antibody substance is a multivalent antibody. 45. The antibody substance according to claim 43, wherein the antibody substance is a monoclonal antibody.