JPH02502604A - Microbial production method for peptide oligomers - Google Patents

Abstract

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

Description

[Detailed description of the invention] [Name of invention] is a microbial production method for petit oligomers. 〔Technical field〕 The present invention relates to the microbial production of oligomers obtained by application of these methods. Poly relates to putido products and microorganisms for use in such production.

Other aspects of the invention include methods for genetic engineering of such microorganisms and such methods. Uhiko Concerns lids on plasmids for use in genetic engineering.

[Background technology]

Methods for genetically engineering microorganisms are well known. These methods are suitable for this application. G and D explain the aspects of the species.

Stormo, T, D, 5eheider, M, Gold, Nu Clay Tsukushizu Research 10.2971-2996 (19g2); A, Shatzman, Y, S, Ho, M.

Experimental manipulation of Rosenberg gene expression, ppl-14, M.

Edited by Inouye (Academic Press, 1983); A.

Rattray, S+Altuvia, G, Mahagna, A, B , Oppenheim and M., Gottesman, Journal of Modern biochemical advances in bacteriogenetic technology provide new opportunities for interspecies gene transfer. brought about the introduction of new technology. In many of these technologies, the hosts are microorganisms and plasmids. Based on the use of vectors. These vectors can be controlled under the control of bacteria. allows the establishment and expression of heterologous genes in microorganisms. r, G, 5ute Genetic Engineering by F. Liffe and F. M. Aubel, pp83. -111, edited by A. M. Chakrabarty (CRC Press, 1978 ) and R, Wu, L, -H.

Genetic Engineering Technology by Guo and R, C, Scarpellm, pp3- 21, edited by P., C. Huang, T. Kuo and R. Wu (A. (Kademic Press, 1982). Naturally associated regulatory DN The gene with the A sequence or one that functions under the control of regulatory DNA sequences specific to the parent plasmid. A number of plasmids are currently available that allow the cloning of both genes. is. , many of these plasmids contain large numbers of genes, gene fragments or have been used to isolate, verify, and express gene promoter sequences. grams In bacteria such as Escherichia coli, which is a sexually active bacterium, cloned from plasmid vectors and Most of the genes that have been expressed are enzymatic or have physiological functions (e.g. (e.g., hormones, blood factors, cell growth factors, etc.).

All structural proteins such as components of the extracellular matrix in multicellular higher organisms. There are relatively few genes, or gene fragments, that encode have been cloned; these proteins include the collagen group, elastin, , fibronectin, laminin and other fibrous tongue/moji substances. Physics Other structural proteins whose physical or chemical properties are of interest include those found in higher organisms. Thick, medium.

Thin filament proteins or glycotanomics, annelids or arthropods silk, cell flagella, resilin, eukaryotic eggshell proteins, insect epidermal proteins and Contains structural proteins involved in eukaryotic differentiation such as tissue formation and tissue formation.

These cloned genes are rarely expressed in heterologous bacterial hosts; Isolation, purification and/or biochemical analysis of protein/quaternary protein products associated with its expression. It has not been.

Those who have been and have an interest in optimizing heterologous gene expression from plasmid vectors. First, researchers using recombinant DNA technology using the bacterial host E. coli are Various strategies have been used to increase protein production. These strategies include plasmid Runaway replication of vectors, heat generation of heterologous gene expression control promoter DNA sequences or chemically induced, foreign to E. coli or its natural or artificial mutants Highly active promoters such as lac, trp or IPP promoters - Use of arrays, etc. Examples of such efforts include B. Uhhin , S.

Molin, P., Gustafison and Nordstrom + Di. 6.91-106 (1979); K., Backman and M. Ptashne, Cell 13.65-71 <1978); Nords trom, S, Mo1in and J, Light, plus 5tinas ens, E+Remaut and W, p'1eyB, Gene 36.211 -223 (1985).

-35 consensus sequences and 5' flanking regions (one promoter and one region sequence and a second natural or synthetic promoter/orator DNA sequence. Is the promoter/o containing the Shine-Dalgarno sequence part of the promoter sequence? The hybrid promoter that is commonly used in E. coli It has proven to be particularly useful for level expression. hybrid trp-1ae pro For motors H, A, DeBoer, L, J, Com5tock and Bi M, Vaaser Proceedings Op the National Academy of the Society 80.2l-25 (1983); E, Amann, J, Brosi us and M.

Ptaihne, Gene 25.167-178 (1983); L A, B U.S. Patent No. 4.551 published by eBoer on November 5, 1985 ．． No. 433; and R, Arantzen and S, R, Petteway. European Patent Application No. 0136090 by Jr. (filed on August 24, 1985) ) please refer to the literature. cI repressor protein from pL promoter Temperature-sensitive expression of dominant activity and initiation by bacteriophage N protein Bacillus in association with additional elements such as the nutL locus for anti-transcription termination activity. A plasmid vector that utilizes the control elements of the theriophage lambda pL promoter. It has also been shown that E. coli expresses heterologous genes at high levels in E. coli. Comparable to other strong promoters such as the lac UV 5 promoter in bacteria It was also revealed that Maroku was even more powerful than that. Especially E, Remaut, P, 5tanasens and W, Fiers,’; -715 , 8l-93 (1981): M, Rose published on March 25, 1986. U.S. Patent No. 4,578,355 by Nberg: J.A. Raut emberger.

D, Court and T, S, Papas, Gene, 75-84 (1 983); and H, Aviv, M, Gorecki, A.

Levanon, A, Oppenheim,' T-Vogel, E-Z European Patent Application No. 0131843 by Eelon and M. Zeevi ( (filed March 7, 1984); and C. A. Can1eott and M. Ph. odes, ) Lens in Biotechnology 4.142-146 (1986) Please refer to Most of these publications place the start codon ATG within the reading frame. Cloning of heterologous genes and lambda pL oL promoter/oL promoter the N protein-nutL interaction and the lambda cII gene protein sequence. describes the production of fusion proteins under the control of protein binding sites.

The product fusion protein is derived from bacteriophage lambda cII protein/microcyte protein. Contains some portion of the min-terminal sputide sequence.

Names of T, S, Papas and J, A, Laut@nberger U.S. Patent Application No. 6-5 dealing with plasmid pJL 6 filed by the U.S. Department of Health and Human Services in No. 11,108 f: Filed on July 6, 1983. Portions of this application apply to the U.S. Obtained from Department of Commerce 5 National Technical Information Service. The scope of the patent claims is Unobtainable and kept secret. Summarizes the available parts of this application. Ru. The construction of pJL6 has been described, and cloning for its heterologous gene has been described. Molecular cloning is commonly used as a lid on oncogenes and expression. The appropriateness drawn from the experiment is misleading in the example. Use of non-recombinable bacterial hosts The available application portion encodes a synthetic gene or structural protein. or C1a I site or C1a I-BamH Regarding cloning into restriction enzyme recognition sites in pJL 6 other than 1 site pair It doesn't say anything. Therefore all non-cloned into pJL6 Homologous genes necessarily produce fusion and tetraplasmic products, which in turn result in heterologous genes. The child product contains an amino radical residue on the amine terminus derived from the lambda CII gene. 　　　'

A, 5eth, P, Lapis, G, F, Van de Wood e and T, S, Papas #2'; -:y 42.49-57 (19 86), Nilamda bacteriophage PLOL in order from 5' to 3' Promoter/o flanks the promoter sequence, N gene site and start codon ATG Expression vector pJ to obtain some plasmid vectors containing restriction enzyme recognition sites for describes an improvement of L6, which requires at most one foreign amino acid residue. A heterologous protein is inserted into reading frame along with the start codon to encode a protein product with Allows insertion of genes. A, plasmid constructed by 5eth et al. The code is designed to be cut with an appropriate restriction enzyme and treated with S1 nuclease. specific Hp that has been cloned and has been described as useful for cloning heterologous genes. a second NdeI restriction site downstream of the BamHI'i or KpnI restriction site Not only the restriction enzyme recognition site but also the NdeI restriction enzyme recognition site including the start codon ATG'i. I have. This paper is intended for purposes of synthetic gene cloning or biochemical research. Nothing is described about the production of structural proteins for external purposes. These plastics The benefits of using the E. coli non-recombinant bacterial host for SMIDS have not been described in the literature. and is not discussed by these authors.

H, Aviv et al. (in the above-cited text) Fi5' to 3': bacteriophore A DNA sequence containing the promoter and promoter-pLoL from dilambda, N gene utilization site for anti-transcription termination N protein binding produced by host cells This allows the mRNA of the desired gene to bind to liposomes within the host cell. A DNA sequence containing a liposome binding site for the ATG initiation codon or vector DNA sequence that is converted into an ATG initiation codon by insertion of the desired heterologous gene into , and the desired heterologous gene into reading frame within the vector along with the ATG start codon. A vector containing a restriction enzyme recognition site for insertion is claimed as a composition. Ru. This type of vector contains an undesired nucleotide at the amino terminus that cannot be easily removed. Fusion protein f with a mino-removal residue: inevitably has the potential disadvantage of producing embryos. This patent application, which does not include synthetic genes or genes with repetitive amino acid sequences, cloning of structural proteins or proteins with interesting physical properties. large intestine for gene expression from requested plasmid vectors. There is no mention of the efficacy or suitable use of recombinant, incompetent bacterial hosts. stomach.

In the technology of molecular genetics, gene fusion and (J, K, Setlo (edited by W and A. Hollaender: Prena 4 Press, 1984) and J, H, Ke 11y and G, J.

Darlington, Manual Review Optoelectronics 19,273 -296 (1985) for a review. Also J, L, Bitt World Patent Application WOS 3103547 by Le and R, A, Lerner (U.S. national priority date April 14, 1982), 5etippa Clinical and Research Fund. WO 35102611 by R, A, Houghten (December 1984) A, C arson, G. Rhodes and R.A. Houghten, W. P 86101210 (filed in August 1985), and Biogen N, V , CoWeissman and) 1. European patent application EPA by Weber 0141484 (UK priority date 10 June 1983), Beecham glue P, L, C-I's H, Fevres, R, A, G, Sm1th and A , J. Garrnan, EPAO 152736 (UK priority date 1984 November 1, 2017), and Ce1ltech Ltd. ．． EPA0161937 (UK priority date 198 See also May 16, 2004). All of these patent applications are Fusion fc for drugs, enzyme conjugates and diagnostics and kits Describes protein production. However, all of these applications For the production of proteins whose physical or structural properties are favorable, see and the need for production of recombinant products in recombinant-incompetent bacterial hosts. Not discussed. Some of these applications contain oligomers of natural proteins. Patents claim boptide or protein products with internally repeating amino acid sequences. However, as discussed in the applicable application without exception, these products are A compound that is active as a drug or as an antigen.

Other aspects of molecular cloning techniques that are suitable for the invention described herein Note that several groups have successfully cloned synthetic genes. I don't have much. Zuputids or protein insects with internally repeated amino acid sequences These cloning efforts have not been focused on. Polymerized form of oligo R peptide, In particular, a synthetic gene encoding di-R peptide and mono-aspartyl-L-phenylalanine. The cloning of offspring was carried out by M, T., Dos 1 et al. Search 8.4575-4592 (1980). soreculark Large am strain HB 101 widely used in rowing technology (required for gene owner use) The necessity was recognized. However, these researchers have demonstrated the ability to polymerize short oligoR peptides. It merely describes a method for producing a mold, which can then be chemically or enzymatically produced a short Rigo is degraded to putido, with the potential for direct production and use of the polymer Zuputi F°. It is not directed towards the pursuit of advantage. The method described in this reference also A partial oligo polymerization can be further annealed to N-person sequences. A] Two completely complementary oligodeoxynucleotides to create an ibrit ° Synthetic genes that can be constructed by leotigi annealing include f! Ill limit; raru . This reference describes the oligomerization of synthetic gene products into larger synthetic genes. It does not describe how to do so.

Molecular cloning and protein expression techniques Other publications have addressed the issue of DNA segment oligomerization. internal retaliation Placement 01 - Coat in a manner that does not disturb large petited or tanned materials. specifically and efficiently insert equivalent DNA segments into long DNA sequences that contain Some of these documents present strategies for combining. J, L, Ha rtl@y and T, J, Gregory';-713.347-353 ( 1981); T., A., Willson et al., Genetic Analysis Techniques 2.77 -82 (1985); and T. Kempe et al. Generi, 239-245 (1985). In comparison with the present invention, none of these documents shows the polymerization state. for the production of synthetic genes encoding repetitive amino acid sequences that are of intrinsic value in However, the structure for plasmid expression vectors with synthetic genes is not described. There is also no mention of the preferred use of non-replaceable bacterial hosts. These papers discuss The examples discussed are those that are or have been shown to be pharmaceutically active. Only aggregates or oligomers of proteins or R-peptide products with no activity In British patent application GB 2162190 (issued July 8, 1985), S. Petty-5aphon and J, A.

Light claims a method for producing polypeptide products made from silk components. However, the silk protein containing these has the sequence (Gly-Ala-Gly-Al a-Gly-8er). However, no examples have been shown. It is not possible to grant permissions because the user has not done so. i.e. appropriate plasmid expression vector microorganisms or plant or animal cell hosts. was not identified, and the above sequence (Gly-Alt-Gly-Ala-Gly-3 Methods for producing or isolating natural or synthetic genes encompassing the set of not present.

[Disclosure of the invention]

Summary of the invention One aspect of the invention is that the plasmid contains repeated amino acid sequences for insertion into the lid. This relates to a method for preparing a double-stranded DNA fragment 1 encoding a sequence as a whole. Rashi, consisting of the following steps: (1) At least two complementary phosphorylated DNA oligodeoxynucleotides This deoxynucleotide is added to the mixture to anneal the mixture. When heated, the base pairs form and partially overlap), and then from 5' Forms stable base pairs between complementary sequences oriented antiparallel with respect to 3' polarity Cool the mixture slowly to: (b) The above-mentioned annealed DNA oligodeoxynucleotide mixed *1 ligase treated with enzymes to create adjacent molecules of the same 5' to 3' polarity into longer DNA segments. (e) The covalent bond is linked to the oligodeoxynucleotides of A double oligodeoxynucleotide linker D DNA is enzymatically linked to provide a double-stranded DNA fragment with a linker attached to the end. the linker is a repeating oligodeoxynucleotide of the double-stranded DNA fragment. Linker not observed within the sequence - Contains at least one unique restriction enzyme recognition site However, it has one degree in the plasmid sequence of Kanashi, and the linker has one degree in the plasmid sequence. The D adapted to maintain one or more repetitive amino acid sequences of the NA segment. ing.

Other aspects of the invention relate to double-stranded DNA sequences prepared by the method of the invention. a preferred embodiment of the method of the present invention is the DNA fragment or the The linker-end multimers on the oligomeric form of the DNA fragment are removed. It is characterized by cutting the car with a restriction enzyme.

In another preferred embodiment of the method of the invention, covalently linked base-paired complementary DNA I) Completely or partially complete the breaks and gaps in the NA sequence by base-pairing synthesis. Further treatment with DNA 4 limerase prior to ligation of the linker to remove It is characterized by. Yet another preferred embodiment of the method of the invention comprises step (b) and <c) t- It can be done at the same time. Further preferred embodiments of the method of the invention is the rate and speed sufficient to base-pair the formation of hybrid oligodeoxynucleotides. Cool the oligonucleotide mixture of step (1) of the present invention to a degree of It is characterized by maximizing the amount of overlap between godeoxynucleotides.

In yet another preferred embodiment of the method of the invention, further: (d) one or more duplexes attached with linkers as in step (C); A mixture containing stranded DNA fragments, the mixture optionally containing compatible ends for ligation, (containing another double-stranded fragment that encodes and disguises a repetitive amino acid sequence), is sufficiently low cooling to a temperature to join the double-stranded DNA sequences into longer double-stranded DNA fragments; and (e) treating the cooled mixture of double-stranded DNA fragments with a ligase prime; The double-stranded DNA fragments are covalently linked to each other, and one to six amino acids are linked together by covalent bonds. characterized by forming linked double-stranded DNA fragments encoding adjacent repeats of a sequence shall be.

Another aspect of the invention provides that the plasmid (lid and one or more of the above) Concerning a method for creating a recombinant plasmid consisting of the above double-stranded DNA fragment. The method consists of the following steps: (a) Preparing the plasmid vector. Cut at the restricted light purple site determined by: and (b) adding one or more double-stranded DNA sequences at said site to said plasmid; the genetic code reading frame of said DNA fragment with respect to the translation initiation DNA sequence in the vector; and repeat amino acid sequences throughout and between any joined DNA. is enzymatically linked so as to maintain the DNA sequence in the plasmid. It is composed of known amino acids that can be expressed in microorganisms and cloned in microorganisms. to form a polyg peptide.

The present invention also provides recombinant plasmids and recombinant microorganisms produced by the method of the present invention. related. The invention further provides that any of the polysptate products of the recombinant method of the invention related.

Brief description of the diagram Figure 1 is a physical map of the expression vector PAVI.

DESCRIPTION OF THE PREFERRED EMBODIMENTS One aspect of the invention is that a linker-DNA terminus is inserted into a suitable plasmid vector. A technique for producing a double-stranded DNA fragment having ends and encoding a desired repeating amino acid sequence. Regarding the process. The first step of the method involves encoding or at least two synthetic oligodeoxynucleotides that can function as anticoding strands Otide is prepared. Oligodeoxynucleotides have adjacent deoxyribose A covalent bond between the C5' and C3' atoms of the sugar moiety and through the phosphodiester bond. A polymeric DNA sequence that is a linear chain of deoxynucleotides linked together.

There are many synthetic methods for preparing such oligodeoxynucleotide sequences. example Each can be chemically synthesized, for example, by several available solution or solid phase techniques. most First S, L, Bemucage and M, second stop by Caruthers The phosphorus described in Uhedron Letters 22.1859-1862 (1981) As a review of suitable solid-phase synthesis techniques based on foramidite chemistry, M, H, Ca Ruthers et al., Genetic Engineering, Volume 4 (J-Setlow and A, Hol Plenum Press, 1982).

Properties of synthetic oligodeoxynucleotides prepared for use in the practice of the invention Quality is important. The oligodeoxynucleotides prepared are base-paired and partially at least two oligodeoxynucleotides capable of forming double-stranded DNA. It must be configured. Synthetic oligodeoxynucleotides selected or prepared accordingly At least one of the cleotides is granin, which is well known in the field of nucleic acid biochemistry. Other selections and preparations using cytosine and adenine and thymine base pairing rules s3b, two complementary synthetic oligonucleotides Oligodeoxynucleo considering the antiparallel polarity of the godeoxynucleotide chain Must be a cyclically permuted sequence of characters. Therefore, for example, synthetic ori Godeoxynucleotide sequence: 5'-a-b-c-d-eAmmth egh-3' (in the formula &s b% e% as @%f, g and h are each one of the four purine or pyrimidine nucleotides ) When the other synthetic oligodeoxynucleotides are 3l-dl-el・, , fl-gI-h'-, I-bI-ct, -51 (formula d and d', shun and e', etc. represent bases that form appropriate pairs) It would be expressed as Circulation for at least one synthetic deoxynucleotide The selection of permutations of the two synthetic oligodeoxynucleotides is performed in a later step of the method. When the strands are annealed to each other, the number of paired and unpaired bases on each strand is equal. It is restricted to an arrangement that will remain strong if it changes. Further unpaired after annealing It is also required that the number of combined bases is not zero. Thus, in the above example, The number of bases represented by the sequence 5'-de, , fgh-3' is the sequence 5'-abc Not equal to the number of bases represented by -3'. Pole of unpaired base after scallop annealing In order to control the orientation (i.e. after the annealing step in current methods) Unpaired bases are present at the 5' or 3' end of each synthetic deoxynucleotide )-! At least two complementary oligos that will leave no unpaired bases The amount of deoxynucleotide silver prevents hybridization, thereby subsequently or to prevent efficient oligomerization during the annealing step of the present invention. , the DNA sequence of the oligodeoxynucleotide is selected according to these rules. .

The selection of nucleotide sequences in synthetic oligodeoxynucleotides is the basic repeating unit. is governed by the order of amination in the product polyR peptide, whereas directly the product polyR peptide repeating oligomers are desired. the desired basic repeating peptide unit or this code. one or more synthetic oligonucleotides to encode a cyclic permutation of the code sequence. Oxynucleotides can be selected. The coding sequence is the genetic code and described in this invention. Based on preferred codon usage in the host microorganism in which the identified synthetic gene is to be expressed. selected. Code selection for optimal code usage in selected host microorganisms In situations where the choice is unknown or ambiguous, one or more coding sequences may be selected. . The length of the oligodeoxynucleotide chosen or prepared can vary widely. It's different. Minimum oligodeoxynucleotide length used in the method of the invention is linked by covalent bonds equal to three times the number of amino acids in the basic repeating peptide unit. It's the number of nucleotides. The maximum length is not strict, but is an integer multiple of this number of bases. The use of synthetic deoxynucleotides is also acceptable, and It is preferred if the number of amino acids is less than about 4.

Synthetic oligodeoxynucleotides are generally terminated with a 5' hydroxyl chemical group. If this part had all the phosphorous salt chemical groups, then the 5′ Phosphorylation of the chain ends may be required. The phosphorization of the 5′ hydroxyl chain end is Preferably, adenosine triphosphate (ATP) is phosphorylated to the 5' hydroxyl site. It can be conveniently carried out by any enzyme capable of transferring groups. The preferred enzyme is T4 polynucleotide kinase (E, C, 2,7° 1.78), but as appropriate Other phosphorylating enzymes, such as phosphatases, can also be used for phosphorylation under favorable substrate conditions. Can be used.

In the first step of the method, the phosphorylated oligodeoxynucleotide is annealed. to form complementary oligomeric forms. These complementary synthetic oligodeoxynucleotides Reotide contains two or more oligodeoxynucleotides, at least two (the two are complementary) are annealed by heating in a suitable buffered salt solution. blend The final temperature at which the synthetic oligodeoxynucleotide is heated can vary widely, but preferably Above the temperature at which leotide stably forms hydrogen bonds and base pairs, but below 100°C. ・Ru. The heated mixture of synthetic oligonucleotides is left to form base pairs between complementary strands. Cool slowly to the desired temperature. For example, using the nomenclature introduced earlier, the two A possible raw JliilED DNA hybrid duplex sequence can be expressed as follows: 3 /-dl eI 9. fl　g/　hI　, 1bI　Ct,, 51 synthesis ori Other structures are possible if the internal sequence between godeoxynucleotides is degenerate; The structure of is likely to have no, or at worst, few base-pairing mismatches. formed The number of structures identified is the smallest unique structure contained within an oligodeoxynucleotide. As a result of the sequence and number of synthetic oligodeoxynucleotides in the reaction mixture, Dew. For example, if the sequence 5'-a-b-e-de, , f-g-h-3' is Due to the internal degeneracy, only two synthetic oligodeoxynucleotides are used. Then, only the two base-paired structures shown above are the two synthetic oligodeoxynucleotides. It may be a molecule generated by thermal annealing between cleotides.

Final temperature suitable so that hybrid structures form only with a defined single-strand polarity is selected (i.e. 5 or 3' base overhang), but the most stable base The cooling process is slow enough to allow complete formation of only the paired hybrid structure Kagishi can also use other temperatures below that value. Complementary and overlapping synthetic oligodeos Individual nucleotides that are base-paired in a partially offset manner to two oxynucleotides. The oligomer type of the redundantly synthesized NA chain with oligodeoxynucleotides is the sample This method will subsequently form if the temperature is lowered further. This method By lowering the sample temperature at this stage, overlapping base pairing synthesizes oligodeoxynucleotides. By further annealing the partially shifted ends of the base pairs, other overlapping base-pairing synthetic oligonucleotides can be used. A combination of oxynucleotides forms stable base pairs. Annealing result The length of the hybrid duplex DNA segment will vary widely. Complete Since it cannot be hybridized by alignment, it cannot be duplicated before obtaining the essential length. Two synthetic oligodeoxynucleotides are selected to prevent premature termination of chain elongation. selected, so that in a preferred embodiment of the invention the length of such fragment is Generally essential. Oligomeric DNA strands produced during the annealing process Is the basic repeat at the end of the oligomeric DNA strand that encodes a portion of the peptide unit? The selected elementary repeats will encode contiguous repeats of peptide units. a Judicious selection of kneeling temperature and base sequence of synthetic oligodeoxynucleotides Alternatively, the oligomeric DNA strands are either both 5' or both 3' overbanks. The two ends are self-complementary. For example, the previously introduced life Using the method, the oligomer duplex produced by the annealing step of the method The DNA would be shown as follows.

(3) 5'-a-b-c-de,, f-g-h a-b-e-de, ,, f-g-h-3'3/dl eI,,, fl〆h' a' b' c’ d’ e’e @ IIf’ 7 h’ a’ b’ c’ 5’ Aniichi The number of complementary oligodeoxynucleotides is determined by will vary between each set of oxynucleotides and the system responsible for annealing. , one to many repetitive base pairing and overlapping synthetic oligodeoxynucleotides. The size will be distributed within the range.

Method Fi using two or more complementary oligodeoxynucleotides as described above I have described it as such. Some embodiments of the method of the invention include oligomeric DNA fragments. More than two oligodeoxynucleotides are annealed in the preparation of a piece will be required. The implementation of such embodiments is subject to all specifications. or the optimal codon usage for a given host for protein expression is unknown. Ambiguous cases or related to the amount of transfer RNA (tRNA) molecules in bacterial cells May be suitable when tight translational control limits the rate or extent of protein synthesis . If between any given pair of annealed complementary oligonucleotides The unpaired bases of the complementary synthetic oligodeoxynucleotides in the reaction mixture are is equivalent in sequence and polarity to the unpaired base in all other pairs. Complete base pairing according to nin-thymine and guanine-cytosine base pairing requirements A combination of one or more pairs of complementary oligodeoxynucleotides forms an oligomeric DNA fragment hair. Can be converted into Neil. For example, using the nomenclature introduced earlier, the above Examples of two pairs of synthetic oligodeoxynucleotides that can be oligomerized are: (4) 5'-a-b-e-de++*f-g-h-3'3idl eI, 0. fl gI hI, I bI Cl-51 and (5) 5'-a-be-e-j-, , f-g-h-3'3/-j/ kloo, fl gI hl-, I-bI, , Cl-5/, in which the unpaired The sequence and polarity are equivalent to the combined bases abe and aI bl Cl. If one All such synthetic oligodeoxynucleotides with the exception of The unpaired synthetic deoxynucleotide A nucleotide sequence that differs from a godeoxynucleotide by at most a few positions an odd number of synthetic oligodeoxynucleotides equal to or greater than 3 if It is also possible to use otide and is included in the invention. In this embodiment of the invention Base-pair mismatches can also be resolved through hydrogen bonds by annealing at appropriate temperatures. must still allow stable formation of That is, the base pair mismatch in the resulting binary molecule is so small that the chosen annealing temperature does not destabilize the binary formation. It must be.

In the second step of this embodiment of the invention, the oligomerized DNA base pairing synthesis molecules oriented parallel to each other with respect to their 5' to 3' polarity. Rigodeoxynucleotides are covalently linked. The ligase enzyme used is A hole between two DNAds terminates in a 3' hydroxyl and a 5' phosphorus group, respectively. Any of several enzymes capable of forming sulfodiester bonds. suitable enzyme is T4 DNA ligase (, E, C, 6, 5, 1, 1) and E. coli DNA These enzymes include ligase (NAD", E, C, 6, 5, 1, 2'). The compound can be prepared using suitable cofactors and substrates or methods known in the art. Appropriate substrate concentrations for enzyme activity will be used. any ligating enzyme and In particular, as a cofactor for T4 DNA ligase, in the presence of T4 DNA Alj gauze , the enzyme T4 RN, which is known to stimulate the formation of linearly linked DNA products. A ligase (A, Sugino et al., Journal Oppaiolo (see Dical Chemistry-252゜3987-3994 (1977)), and is polyethylene glycol, sR lumidine ficoll or bovine serum albumin. (B, H , Pheifler and S., B. Zimmerman, Nucleiqua (See Shitsu Research L, 7853-7871 (1983)).

The double-stranded DNA molecule that results after enzymatic treatment with a DNA ligase enzyme is a have one or more cuts or gaps on either or both sides Let's go. These breaks or gaps are defined by the chemistry of the oligodeoxynucleotide. Incomplete removal of the 5' or 3' strand ends of synthetic oligodeoxynucleotides during synthesis protection, incorrect base pairing during the annealing step of the claimed method, and during chemical synthesis. Premature chain termination or failure of elongation causes one or several bases to shorten the strand. contamination of synthetic oligodeoxynucleotides with Incomplete attachment of the phosphoryl group to the end of the roxyl chain, in the ligation step of the claimed method. Synthetic oligodeoxynucleotides with preceding or subsequent contaminating nucleases It can occur by several mechanisms, such as non-specific degradation. Not all of it Even many of these problems involve linking double-stranded synthesized DNA fragments to DNA,! ? Remeller can be substantially reduced or eliminated by treatment with nick tiger Through the integration method, DNA polymerase converts existing DNA strands from 5' to 3' (R, G, Kelly et al., Journal Opbiology) Calchemistry-245, 39-45 (1970)). known for this technology A variety of DNA polymerases or fragments thereof can be used in this step of the claimed method. A useful and preferred enzyme is DNA polymerase I (E, C).

2, 7.7.7) Escherichia coli that retains t or holoenzyme polymerase activity Any protein/microlytic fragment of DNA polymerase I.

Synthetic duplexes prepared in certain embodiments of the invention following DNA polymerase treatment Stranded DNA fragments are fractionated and collected to a minimum size for use in subsequent steps. Isolate only those fragments that are present. This purification method can be used to identify natural genes and gene fragments. As described below, in some embodiments of the methods of the invention, specific genes or It is also necessary for the DNA copy of Metsenger RNA for gene fragments or gene fragments. It's probably necessary. Purification methods include size exclusion chromatography and ion exchange chromatography. Choose from a variety of biochemical techniques including affinity chromatography and affinity chromatography. You can choose. The current preferred method is size exclusion chromatography on a suitable separation matrix. chromatography; many such matrices are commercially available.

Any natural or synthetic double-stranded DNA selected or prepared for the method of the invention The fragment is long enough to encode a polypeptide with the desired macromolecular properties. generally have the same Strength, elasticity, thermoplasticity, bonding or arrangement to other molecules The choice of a particular polymer, such as Optimization of the peptide will determine the appropriate relationship between structure and function activity. deer While increasing the chain length the physical properties are optimized to some extent in the polymer. It is a general property of polymers that the quality is usually increased. Therefore, the present invention Double-stranded DNA used in the present invention within the scope of molecular cloning It is generally desirable to maximize the size of the fragments or fragments. There is also a minimum Cloning of DNA fragments of different sizes is possible using plasmid vectors containing the DNA fragments. for subsequent identification of bacterial hosts, including microorganisms, and as described below. For optimization of ligation reactions of natural or synthetic NA fragments or fragments into DNA It's also convenient. In a preferred embodiment of the invention, the length of the fragment is at least about 7 5 base pairs, and in particularly preferred embodiments of the invention the fragment is at least about 1 The length was 00 base pairs.

The third step of the basic method of the invention involves the synthesis of double strands or naturally occurring DNA. The ends of the fragments are modified prior to cloning. The ends of the DNA fragments are Then, a DNA linker is attached with or without enzymatic treatment of the DNA fragment. The ends of the DNA fragments are modified by attaching them to blunt ends or flattened ends.

The ends of the synthetic double-stranded DNA fragments were prepared using the molecular clones of T., Maniatis et al. (Coldsfrinkbarber, Pl).

113-114, and other similar references. Enzymatically flattened. DN A I as defined for the purpose of explaining the present invention A J-linker contains at least one restriction enzyme recognition sequence, or contains a specific restriction enzyme recognition sequence. Any unpaired salt equal to the unpaired base observed at the end of the duplex DNA following the action A double-stranded oligodeoxynucleotide containing a terminal sequence of groups. Description of this specification The matching term in the text is the term DNA! Equal to J car.

Attach the DNA linker to the double-stranded DNA fragment using a suitable method or gauze light purple (preferred method). (NAD-dependent E. coli DNA ligase or T4 DNA IJ gauze) will be implemented. The resulting linker-attached double-stranded DNA fragment is then converted into a double-stranded DNA fragment. The number of linker DNA molecules attached to any one end of a DNA fragment is defined as the number of linker DNA molecules attached to any one end of the DNA fragment Recognition sequences within the oligomeric form of the linker DNA to limit the number of linker molecules to one linker molecule. Requires thorough digestion with restriction enzymes.

As described below, any particular double-stranded DNA fragment and/or plasmid The type of linker selected for use with the lid is determined by the method of the present invention. It's a fixed thing. not present within repeating oligodeoxynucleotides, preferably D There is only one small number in the plasmid vector into which the NA fragment will be inserted. The selected linker contains one restriction enzyme recognition site, either terminally or internally. There must be. For example, if the preparation of platform DNA fragments of the type described in the present invention The repetitive oligodeoxynucleotide sequences used for (6) 5’-GGT GTT GGT GTT CCG-3’3’- If GGCCCA　CAA　CCA　CAA-5', this double-stranded deoxy When the nucleotide oligomerizes itself, the amino acid sequence glycine - Glycine - Parine - Proline (3 letter amino acid code Gly-Val -Gly-Val-Pro) Linker-DNA molecule: (7) s’-aaa ccc cca-313’-GGCCCCGG G-5' can be attached to the oligomeric form of the interdecanucleotide, followed by linker-DN It cleaves within the A molecule, but cleavage occurs within the oligomeric form of R-ntadecanucleotide. Cut with restriction enzyme Apa I (recognition sequence GGGCCC).

These DNA fragments are then inserted into the specific ApaI system of an appropriate plasmid vector. It can be inserted into a restriction enzyme recognition site.

The DNA') anchor used in carrying out the method of the invention has other specific properties. must be maintained. For example, which nucleotides contained within a DNA linker The sequence is inserted into a plasmid vector into which the DNA fragment or fragments are inserted. Appropriate information regarding any regulatory genetic elements, such as translation initiation DNA sequences, found DNA fragments joined (as defined by the genetic code) in the continuation frame or Fragments must be placed. Any DNA fragment that binds to a DNA linker The repetitive amino acid sequences encoded by the fragments are consecutively encoded by the DNA linker. It is also preferred that the amino acid sequence be within and within the amino acid sequence. For example, DNA The DNA fragment to which the linker is attached is sequenced. (8) 5’-GTT GGT GTT CCG GGT-3’3’- If it is an oligomer type of CCA CAA CCA CAA GGC-5' ( The oligomer type is the opposite of the amino acid sequence Val-Gly-Mal-Pro-Gly. A suitable linker related to the present invention is (9) 5'- GTT GGG GTG CCG GGT-3'3'-CCA CAA CCC CACGGC-5'de chiro.

In this representative example, the linker DNA is inserted into the DNA fragment. preferably within the plasmid vector into which this DNA fragment is to be inserted. Specific recognition sequence for restriction enzyme Ban I (GTGCC) not observed in Contains. This linker further includes the amino acid sequence Val-Gly-Val- Encodes repeats of Pro-Gly into and through the linker DNA The reading frame of the DNA fragment is maintained so that the reading frame of the coding sequence is further contiguous. It also has the ability to guarantee that it will survive. That is, the linker DNA is also The amino acid sequence Val-Gly-Val-Pro-Gly f encodes .

Open in the proper orientation to maintain genetic coding capacity for the desired repetitive amino acid sequence. To ensure that only the oligomerized DNA segment is bound, Double Linker - The single-stranded ends of DNA are self-complementary rather than identical sequences. Even more preferred (ie the terminal sequence does not have a two-fold axis of rotation). for example, If the desired repeating amino acid sequence is the oligomerization nucleotide shown as (3) above, If encoded by a code sequence, the selected DNA linker is αQ　5'-a-b-e-j-1cm1-m-n,,,op-q-3'3' −j’ k’ 1’ m’ n/, 6 + 6’ p’ q’ a’ b’ e '-5' is 5'-a-be-e is not equal to 5'-a'-b'-a' required.

This results in binding to the oligomerized DNA with the wrong polarity and an open position on the anticodon strand. A DNA linker sequence encoding an unneeded amino acid sequence from the open reading frame I will avoid it. An example of such an anticodon strand is the sequence 5'-c shown in θQ. '-b'-a'-q'-p'-o'+ + on'-m'-1'- to '-j'- It is 3'.

Two DNA fragments encoding different amino acid sequences may be used in some aspects of the invention. are attached to the same linker-DNA at the same time. In this example, the linker-DNA is at least one of the sequences encoded by one of the two combined DNA fragments is forced to maintain the amino acid sequence of fragment-linker-fragment D. NA is covalently linked to adjacent repetitive amino acid sequences encoded by other fragments. Polyspeptedo with repeated amino acid) sequences encoded by each fragment Code chain?

or shared by an amino acid residue or residues present in both repeat amino acid sequences The two sequences encode poly-R peptide chains that are linked by bonds and have overlapping sequences. Li Linked by a linker DNA and encoded by each DNA fragment Examples of DNA fragments within the scope of the invention that provide overlapping repetitive amino acid sequences include: double stranded deoxynucleotide α 5’-CCG CCG GGT CCG CCG G GT-3'3'-CCA GGCGGCCCA GGCGGC-5'Pro Pro Gly Pro Gly and (2) 5’-GTT GGT GTT CCG GGT-3’3’-CC A CAA CCA CAA GGC-5’Val C1y Val Pro 6D is a DNA fragment formed as an oligomer of Gly, which in turn car (2) 5’-GTT GGG GTG CCG GGT-3’3’C CA　CAA　CCCCACGGC-5’Val　Gly　Val　Pro It can be bonded by G, ly.

In this thorny example, the linker DNA is a double-stranded deoxynucleotide. Recognition sequence for restriction enzyme Ban I that does not exist in the oligomer type (GGT GCC ). The combined DNA fragments encode polypeptides, some of which are Mino awakening sequence (Pro-Pro-Gly). -(Val-Gly-Val-Pro -Gly)rn i or (Val-Gly-Val-Pro-Gly)rn-( Pro-Pro-Gly)n, where the common proline -Two repeated amino acid sequences are duplicated in glycine di and peptide, and two original D A recognition sequence for Ban 1 is introduced between the NA fragments for use in the method of the present invention. Several groups of linker DNAs are suitable for this purpose. DNA linker is attached The DNA fragments used may have blunt or cohesive ends, but suitable types of DNA fragments may be used. The ends are cohesive ends. The linker DNA for blunt-ended DNA fragments is preferably small. Both have one blunt end and a 9.1-degree linker-DNA that guides the DNA fragments to the same end. Once A is attached to the DNA fragment, the linker-specific internal site within the DNA is then It is cut with an appropriate restriction enzyme that recognizes. In this example, at least two non- The use of linker DNA with overlapping limited light purple recognition sites is particularly preferred, and with appropriate controls If the restriction enzyme cuts the linker DNA, it will not result in a sticky end, and following the cutting, any two Sticky ends produced by two suitable restriction enzymes are complementary and non-complementary to base pairing. are equivalent. It also contains a restriction enzyme recognition site that is non-flea-based. Recognized and cleavable by lintrogenic or multi-recognition site restriction enzymes. The use of linker DNA in the method of the present invention also applies to 7IC! Suitable for Ff. the Examples of such enzymes are Ace I, Afl m, Aha II, Ava I, Ban I, Ban I [.

Bgl l, Hae II, Hg1AI, Wine L Nsp Bn.

Xho If, Bbv 1. Bsm 1. Fok I, Gsu I, Hgal.

Hph T, Mbo If, Mnl 1. Sfa N I, Sfi I and Tth 111 II. Such is the binding of the NA linker and non-equivalence of the DNA fragment ends following cleavage, such that one of the NA fragments or two or more such base-pairing matches for both ends. DNA fragments or other DNA fragments with sticky ends can only bind to each other in one direction. Ru. The non-equivalence of cohesive ends on the:whirlNA fragment also indicates that two or more Repeat amino acids encoded by covalently linked aggregates of the above DNA fragments. This ensures the desired diversity of types of wake-up sequences. That is, D in such a binding association Each of the NA fragments and linker DNA contains a polynucleotide of the desired contiguous repeating amino acid sequence. will likely express sequences from the R peptide or the appropriate NA coding strand, DNA fragments or linker D that may bind with the wrong polarity in larger aggregates NA is not expressed. Linker DNA with multiple restriction enzyme recognition sites can also be used. Wear. The DNA IJ linker contains at least one non-overlapping restricted light purple recognition site. However, in order to minimize the size of the linker DNA that is produced on a platform, two or six Particularly preferred is the use of linker DNAs with such recognition sites as However, in some embodiments of the invention, a linker with two or more such recognition sites may be used. car DNA will be used. D with at least two restriction enzyme recognition sites N A 17 The linker is cleaved with an appropriate restriction enzyme after binding to the DNA fragment, resulting in non-isolated A DNA fragment with cohesive ends is obtained.

In another type of embodiment within the scope of the invention, the DNA linker is a plasmid. ligated to an expression vector and prepared and/or selected by the method of the present invention. Provides a novel insertion site for heavy@DNh fragments. Such a linker is Linearization plus technology that is familiar to those who have implemented the technical cloning Natural or synthetic DNA that can bind to mid-DNA and has a complementary DNA IJ linker sequence Used as a binding site for fragments.

and oligomers for synthetic oligodeoxynucleotides that are oligomerized. For linker D, N'A, which is attached to synthetic oligodeoxynucleotides It is also possible to combine the ligation reactions into a single step, which is included in embodiments of the present invention. It is rare. This method is based on the ratio of double oligodeoxynucleotide and linker DNA. Several methods, including controlling the size distribution of synthetic genes, can be used to change the give an advantage. At least one linker-DN is attached to this DNA fragment during enzymatic conjugation. If A is incorporated, it will become circular during the oligodeoxynucleotide ligation step. The DNA strands obtained can be subsequently linearized to provide clonable DNA fragments. It has the advantage of

When linking a DNA IJ linker to a double-stranded DNA sequence, a type of multimeric linker is often formed. As used herein, the term "multiple embedding" refers to oligomeric DNA sequences. More than one linker is attached to the end. In this case, double elevation, The linker-DNA molecule attached to the end of the DNA fragment is Recognition sequences are preferably present within the oligomeric form of the linker DNA to limit the linker to the linker. Double-stranded DNA sequences are subjected to exhaustive digestion with appropriate restriction enzymes.

Here, the double-stranded DNA sequence with a linker attached to the end can be directly replicated by a suitable method. A suitable restriction enzyme in a cloned vehicle (preferably a plasmid vector) Can be cloned to a single recognition site or to a set of sites. However, as pointed out earlier The length of double-stranded DNA fragments and the vorisp expressed from such fragments are as follows: There is a direct relationship between the molecular weight of fragments and the amount of protein expressed from such fragments. between the molecular weight of the polybutide and the degree of quality of the desired physical properties of the polybutide product. There is also a direct relationship. Therefore, in a preferred embodiment of the invention, plus The length of the DNA fragment can be further extended prior to insertion into the midvector. Often desired. Through the linker end, a polypeptide of larger molecular weight can be attached. Enough to cause binding of the DNA fragment into a long double-stranded DNA sequence that encodes Stir the DNA linker-bound DNA fragments, which have been cooled to a low temperature, and then add the This increase in length can be conveniently obtained by treating the compound with gauze enzymes. It will be done. Although the desired temperature varies widely, in preferred embodiments the temperature of the mixture is The temperature is low enough to be above freezing. In this preferred embodiment, , the cooling process is also slow enough to allow complete formation of the most stable array structure. It is. After cooling, the mixture is soaked with gauze enzyme (optionally DNA polymerase) at this low temperature. (with limerase) to covalently link all double-stranded DNA fragments with the desired sequence. Make it a long array.

For example, T. Maniatis et al. Ng Harbor 11982), pp-243-246 (herein attached as a reference) of two or more using conventional methods of joining such as those described in Join the fragments together, each having at least about 75 base pairs: greater than or equal to N, A Fragments and eggplants.

A preferred gauze for this step of the invention is T4 DNA ligase. these The larger, combined DNA fragments encode identical or different repeated amino acid sequences. contains one or more repetitive oligodeoxynucleotide sequences that can , and for at least one repeated amino acid sequence throughout and between the DNA fragments. The genetic code is linked in such a way that it maintains a continuous reading frame. larger size The symmetry and arrangement of the combined DNA fragments in the NA fragment can vary, and the It leads to polypeptides encoded by larger NA fragments, but they are random. It is a temade chika or alternating block peptide copolymer. Large NA fragment preferably has a cohesive end.

It also most preferably has non-equivalent cohesive ends. The above with complementary and equivalent sequences An example of a suitable cohesive end found at each end of a large NA fragment is: α 4 5' C, , , , , , , , , , , , - GGGCC-3'3 '-CCGGG, , , , , , , , , , , , -C-5' and (2) 5'-AATTC,,,,,-,-,,----・,,,,G -3'3'-G, , , , , , , , , , , 0. CTTAA-5' , they each base pair with the DNA end remaining after cleavage with the restriction enzyme ApaRI. Can be combined. Examples of suitable cohesive ends that are non-equivalent and non-unique (lintroms) include: The following sequence is generated by cutting the linker DNA with a restriction enzyme having multiple recognition sequences. Examples include: restriction enzyme α@5'-GTGCC,,,,,,,,,,,,,,,,G-3' Ban I3'-G, , , , , , , , , , , , , , CCACG- 5'(1η 5'-C, , , , , , , , , , , , , GTGCC-3' Bsp 12863'-ACGGG, , , , , , , , , , , , , C- 5' larger NA fragment, natural gene or gene fragment, or double-stranded DNA Used for the preparation of complementary DNA copies of all or part of a natural gene in the form of fragments. Another source of double-stranded DNA fragments is well-known from the technique of molecular cloning. It can be isolated using the technique described. Most or all of these DNA fragments are It is limited to those encoding deconstructed amino acid sequences, but is required in the genetic code. When considering triplet grouping of nucleotides, each DNA chain Natural genes or genes useful in the practice of the invention, excluding the upper nucleotide Examples of fragments include proteins collagen, elastin, keratin, trobonin C, etc. Intermediate filament tan, 4 lithium (E, Lazarides, Nature 283.249-256 (1980)) or any form of silk fibroin. something that encodes part or all or an isolate and is found within a protein sequence Contains most or all of the amino acid sequence exhibiting a degree of repeatability. Repetitive The degree of leukemia can be determined using various theoretical techniques of plastic biology. This can be determined from the quality sequence homology. For example, S, B, Needleman and and C, D, Wun+sh, Journal (1970), and Eise. See Nberg et al. Examples of useful complementary DNA copies of the present invention is reverse transcribed from Messenger RNA and the DNA strand is copied by an appropriate reverse transcription process. During the process of copying the DNA strand, the tsenger RNA is intermediate filaments or It is transcribed from genes encoding proteins such as silk fibroin. child An exemplary list of these includes suitable double-stranded DNA fragments that can be prepared by any of the methods of the invention. Some of the holes do not contain all the proteins you get. this Their natural DNA fragments match the cohesive ends on the synthetic DNA fragments. Fragments can be well prepared for isolation using restriction enzymes that leave sticky ends on them. cormorant. Alternatively, the natural DNA fragment is preferably linked with a DNA linker and multiple linkers. adapted or modified and subsequently ligated to the native DNA fragment.

When specifically cut with one or more restriction enzymes, one or more synthesis If one or more sticky ends appear that match the sticky ends of the NA fragment, or inherently have it.

In the basic method of the present invention, shorter DNA fragments and longer DNA fragments are The encoded repeat amino acid sequences depend on the shorter DNA fragments selected for binding. It can vary widely. Some preferred amino acids encoded by shorter DNA sequences The mino-gan sequence (in the 3-letter amino starch code), poly (Gly), 4-li (A) 1 m), poly (cxy-Alm), 4 li (Ala-Lys), ;s rli (Gly-Ala-Gly-Ala-Gly-8er), poly(Gl y-Aim-Pro), Poly (Gly-Pro-Alm), J Ri (Gly-Pro-Pro), Poly (Gly-Va l-Gly-Vat-Pro), poly(G17-Lys-Leu-Glu -Ala-Leu-Glu), Poly(Alt-Lys-Pro-Tbr- Tyr-Lys), poly(Ala-Lym-Pro-3er-Tyr-P ro-ProThr-Tyr-Lye) and its analogs, in the formula Each amino acid residue has an L-amino acid conformation. These sequences of hydroxyl Formulated forms are also suitable and are included in certain additional embodiments of the invention. The present invention In some embodiments, partially proline-containing or zololine-rich amino acids A short DNA fragment encoding the sequence is preferably selected, and then the oligo merization and DNA fragment-linker bonding in larger NA fragments. It occurs adjacent to the Australian proline codon.

The desired repetitive amino acid sequence or fc is a DNA encoding the linked repetitive amino acid sequence. The fragments and larger NA fragments are inserted into suitable plasmid vectors using conventional methods. can be entered. Such techniques are well known in this field and are discussed in detail here. will not be listed. Larger NA fragments contain complete bases at their cohesive ends. inserted into a specific site or set of sites in a plasmid vector that allows pairing. Ru. In such insertions, any gaps between the plasmid vector and the inserted DNA fragment You may or may not get a restriction enzyme recognition sequence on the junction. present invention In a preferred embodiment of the present invention, in other applications of the invention, if desired, the Such restriction enzyme recognition sequences may be constructed or Reconfigured. The site of DNA fragment insertion is preferably a strong site in the plasmid vector. 3' to the promoter/operator sequence, which is the position of the inserted DNA fragment. (must be inserted correctly in the reading frame and in the correct orientation) would regulate the production of sufficient amounts of polypeptides. Suitable plasmid vector Examples of controllers include pAsl (described in U.S. Pat. No. 4.57 & 355). ), pKc30 (R, N, Rao, Gene 31.247-250 (1 984)), and pKN403 (described in U.S. Pat. No. 4.495.287). ).

A suitable plasmid vector contains Fip JL6 (J, A, Lauten - Berger et al., Gene 23.75-84 (1983)), pA Vl (its construction is described below), ptae12H (E, Amann et al., G. 725.167-178 (1983)) and pKK233-2 (E. , Amann and J., Brosius, Gene, in press).

a suitable bacterial host or other suitable microorganism in which the fragments can be expressed using established techniques. Using conventional methods known in the field of seven-dimensional cloning, plasmids are If the vector and the inserted DNA fragment or larger NA fragments or fragments are 'l', Mani atis et al%'VQz 5-20-ning: Laboratory manual (Goldsp Ring Harbor 11983), Pp 249-255; Andori, Eana Han.

Journal of Molecular Biology-166゜557-580 (1983 ) (incorporated herein by reference) describes the technique. helpful Bacterial species may vary widely, but include Escherichia coli, Bacillus subtilis, and similar species. It's probably a well-known strain.

A preferred bacterium is a strain of Escherichia coli that contains an inserted DNA fragment with six internal repeats. It prevents recombination from occurring between the various segments of the fragment. It is something. A particularly suitable strain of E. coli has a genotype of ree A-, and especially M Techniques similar to those used in the construction of HO3 (ree A-1 TetrM) IO1 Strain N4 produced by transduction from PI strain N6240 830 derivative), DC1138 (pro″″, 1eu-1λΔ5rlRre c A301:: Tn 10, λdef CI”), DC1139A (λ defΔBan H1ΔH1c1857f: Same as DC'1138), J M109 and DHB 9 (F' lae I'Z"Y", ree A, sr 1:: Tn 10, phoR1ΔphoR1Δmm1F, Δarale u1 ΔIac, gal Elgal K; derived from MC1000) I'll go.

After transformation, clonal isolates of transformed cells can be isolated using conventional methods, e.g. Screening by hybridization technology using oligodeoxy probes can be screened and selected using Screened bacterial colonies plasmid vectors are selected and isolated once determined to contain useful plasmid vectors. Test whether the inserted DNA is expressed as a polypeptide with a repetitive amino acid sequence. do. If the cloned bacterium is derived from the DNA fragments used in the method of the present invention, If it is possible to express chido.

Additional bacteria are grown under fermentative conditions, and these bacteria have the specific plasmid utilized. The desired polyzotide vector is expressed under conditions appropriate to the bacterial host gene expression system. You can induce sea urchins. The desired polypeptides are grown in bacterial growth medium or are isolated from bacteria. Examples of useful bacterial growth and bacterial product collection methods are very For more information, see European Patent Application No. 0131843, which is incorporated herein by reference. It is listed.

If the cloned bacterium has a polybore of the desired length or the desired repeat amino acid sequence, If no peptides are produced, one cut only the associated linker-DNA. plasmid vectors containing such inserts can be isolate one or more DNA fragment inserts from cells containing such inserts; PolyR peptides of appropriate length or sequences of appropriate lengths can be obtained by oligomerization of input DNA. Larger NA fragments can be obtained that encode one. Newly created shidai The technology for oligomerization and transformation of the Kinari NA fragment is based on the techniques detailed above. It is a kana wavelength. This method uses more than one encoding sequence of different repeat amino acids. It can be applied to create vibrator and lid DNA fragments containing DNA fragments. DNA fragment Oligomerization and recloning can be performed several times to obtain genes with desired properties. This can continue until a genetic construct is formed. For example, a repeating amino sequence ( Gly-Pro-Pro) and (Gly-Val-Gly-Val-Pro) Individual DNA fragments encoding or alternating block copolymerization consisting of repeating units of these amino acid sequences of various lengths. A combined polypeptide is obtained. Sequence (C; 1y-Pro-Pro)n is eukaryotic It is similar to the protein collagen, and therefore forms a helical macromolecular association. It exhibits physical properties of high tensile strength and low elasticity. Sequence (Gly-Val-Gl y-Val-Pro　)m is a known amino acid of eukaryotic elastin. It is a common sequence extracted from the sequence, and among its various physical properties, it has high elasticity. ing. The hybrid copolymer polysputide of these two repeating amino acid sequences is Suitable hybrid copolymerized polysaccharides are prepared by the method of the present invention encoding peptides. The tensile strength and/or is expected to exhibit elasticity.

[Industrial applicability]

The method of the invention has many uses. For example, this method has many useful polymorphisms. It can be used to produce or create bacteria that produce peptide products. Examples of such products As collagen, elastin, keratin, thick, medium or or thin filament proteins or glycoproteins, silk fibroin, Vomyosin, trobonin C, resilin, eukaryotic eggshell protein, insect epidermal tan Naturally occurring proteins such as proteins and eukaryotic building proteins Similar things can be mentioned.

The following examples are presented to specifically illustrate the invention: Example 1 Preparation of synthetic genes for collagen analogs without DNA linkers The complementary and overlapping oligodeoxynucleotides listed below are Beaucage and The homophasic phosphoramidite described by Caruthers (cited above) prepared on an Applied Biosystems model 380 DNA synthesizer using standard chemistry. : A, 5'-CG GGT CCG CCG GGT CCG C-3'B, 3'-GGCCCA GGCGGCCCA GGC-5' each oligodeo Oxynucleotides were electrophoresed on a 20% polyacrylamide gel containing 8M urea. and isolated from the shorter strands (products of failed extension) by elution. final student The products are end-labeled oligodeoxynucleotides separated by analytical gel electrophoresis. 95% by densitometry of autoradiodrum prepared from the tide product. The purity was determined to be greater than . 5' ends of oligodeoxynucleotides A and B The reaction was carried out in separate reaction vessels, each containing 8.6 nmol of phosphoric acid. 3 oligodeoxynucleotides and 20 units of T4 polynucleotide kinase 5-45μt of buffer (66mM Tris-HCL, pI (7, 6, 1mM Spermidine, 10mM Mgc12゜15mM dithiothreitol, 20 0μf/zd bovine serum albumin (BSA) and 1mM (r-32P :l　A　TP　P (specific activity of 0.2Ci/mmol)). These anti The reaction mixtures were heated at 37°C for 2 hours, then they were combined and incubated overnight at 14°C. start. During this time, oligodeoxynucleotides A and B are annealed. , a 17 base heteroduplex with perhaps 1 base pair overhanging the 3' end, or A 10 base pair heteroduplex is formed with an 8 base pair overhang at the 5' end. It probably is. Add T4 DNA ligase (40 units) and incubate at 14°C. Continue using cupagemone for 3 days, and use multiple doses of tripeptide (Gly-Pro-Pro). 2) 7-body DNA hair-annealing oligodeoxynucleotide Increase the amount of punch. These synthetic genes were added to TE buffer (10mM) Lis-H. C1,p)i 7.5. Dialyze against 1mMEDTA) and remove unincorporated ozone. Remove the oligodeoxynucleotide and buffer components. The end of the synthetic gene is After the following: 600 μM each of dCTP, dGTP, dATP and T TP: 50 rnM) Lis-HCL, pH 7,8: 9 mM MgC22: 10mM 2-mercaptoethanol; and 50pt/mlBSA Klenow cleavage of Escherichia coli DNA polymerase ■ in a reaction solution (50 μt in total) containing Use 3 units of the piece and make a smooth end. The reaction mixture was incubated for 30 minutes at 14°C. 150 μt of 510 mM TE buffer with subsequent addition of Na5EDTA. Also add. The synthetic gene was purified using a DE-52 column and then ethanol precipitated. do. These synthetic genes were prepared in an essentially similar manner and pre-transfected with Sepharo Exclusion of synthetic genes from other batches passing through the Gas 6B (Pharmacia) column. Combine with subfractionation.

The combined synthetic gene is size fractionated using a Sepharose 4B (Pharmacia) column. . The size distribution of the synthetic gene was determined by electrophoresis on a 5-th polyacrylamide gel. established.

Highly multiplexed synthetic genes modify the relative molecular weight distribution of enriched fractions (i.e. (containing 8M urea) and on a non-denaturing 5% polyacrylamide gel. this In their gel, the molecular weight distribution of the single-chain synthetic gene was different from that of the heterogeneous binary synthetic gene. It was shown that the size of the break in the double-stranded heteroduplex DNA was smaller than expected. and/or a gap exists. 1.2 μ2 synthetic gene cut Eyes and interstices each 167 μM dcTP.

In the presence of dGTP, dATP and TTP (others as described for the blunt-end reaction above) 1 unit of E. coli DNA polymerase (with buffer components) for 20 minutes at 10°C The synthetic gene (05 μ2 heterologous duplex DNA) was prepared without further manipulation (C 1aI-digested and blunt-ended pJL6 plasmid DNA (2,0/j); 5 units of T4DN in buffer for the phosphorylation and binding reactions described above A! J gauze (10 μtm volume). The reaction mixture was brought to 14°C. (incubated overnight) diluted to 200 µt in TE buffer and directly incubated with E. coli strain MH. Used for transformation of O1.

A highly repetitive synthetic gene is excised from the expression vector by host-initiated homologous recombination. Transfer into strain N99cI+ using a solution that ensures that it is not expelled. This special fa carry genes for chloramphenicol resistance (cml) and high temperature (42 (°C), forming distinct plaques (clr).

At low temperatures (32°C), the plaque is cloudy. N6240 Harbor, 1972) The recA mutation is introduced into N99cI+ as described in . new 5d of N99cI+ cultured overnight was mixed with an equal volume of MC buffer (0.1M MgSO< , 5 mM CaC42). Cells were aerated for 15 minutes at 37°C. do. 100 pt of suspended cells were added to 100 µt of Plffi bacterial material 10-1 or 1 Add to 0-2 dilution. After incubation for 20 minutes at 37°C, each test Add 200 μt of 1M sodium citrate to the tube. Contents 125μf/- Plate on LB plates using 3mR top agar containing tetracycline. Incubate overnight at 39°C. Confirm that it is not an accidental PIM source Treat each tetracycline-resistant colony with chloramphenicol at 300 Screen for susceptibility. Presence of recA mutation increases sensitivity to UV light be tested and confirmed. Each potential bacterial transductant crosses the LB plate. Perform set cultures and expose different parts of the streak to UV light for 0.10 or 20 seconds, respectively. . The agar plates are then incubated overnight at 30°C.

Has abnormal UV sensitivity relative to its parent (indicated by growth only in the 0 second exposed portion of the streak) One strain was saved and designated MHol.

Identification of a plasmid carrying a synthetic collagen analogue gene without a DNAIJ anchor MHOI transformed cryocompetent cells by the method of Hanahan (D , Hanahan.

Journal Opmolecular Biology, 166:557-580 (1983 ) (where FSB buffer is 10mM potassium acetate, pH 6, 4, 100mMKC4, 15mMnC4z: 10mMCaC 1z and 3mM hexamine cobalt chloride) and transform. about 12 5nr DNA was used for each transformation; cells were subsequently infected with Select based on tracycline resistance. The transformants were replica cultured and grown on nitrocellulose. Transfer to a filter and use radiolabeled oligodeoxynucleotide A as a probe. Plates carrying synthetic gene inserts by colony hybridization used as Identify those containing lasmids. 20% formamide, 5XSSC, 0 , 1% SDS, 1 mM Na2EDTA, IXX non-rut bath solution, and 250μf/mgi in a solution consisting of sexed and sheared salmon sperm DNA. Perform hybridization for 2 hours at 7°C. nitrocellulose filter Wash with 5X SSC, 0.1% SDS 3 times at 55°C for 20.10 and 1 min. and then rinsed for 2 minutes in 2X SSC at room temperature. Plasmids that hunt inserts are Bacterial clones that obtain positive hybridization signals are subsequently isolated.

These plasmids were digested with the enzymes Hindm and Ndel”e to obtain the restriction fragments. The cleavage products are analyzed by electrophoresis on an agarose gel to determine the size of the insert.

Some super carp that do not have a DNA IJ anchor and are waiting for a synthetic collagen-like gene. Direct sequencing of the 5' junction of synthetic gene inserts in R, plasmid DNA I performed it as described by J. Zagursky. 5′ used here and 3' gene orientation respectively refer to the lambda PL promoter located in pJLS. means close and distant joints with respect to each other. The following oligodeoxy Nucleotides were prepared by solid-phase automated synthesis and modified by Zagursky et al. DNA sequencing reaction based on the Sanger dideoxynucleotide sequencing method Prepare: C, 5'-CTTACATATGGTTCGTGCAA-3' The prepared synthesis reaction using oligodeoxynucleotides is C1 of pJL6. a Gene inserted at the I site in the reading direction towards Hindm of pJL6 allows for the sequencing of Correct reading frame and correct coordinates of 5' and 3' junctions Based on the code information, one of these plasmids was designated pAC1 and was further investigated. Ru.

To determine the junction sequence of the 3' end of the synthetic collagen-like gene in pACl, pA cl was restricted with Hindm and the linearized plasmid was transformed into a T4 polynucleotide kit. The labeled plasmid was radiolabeled with [γ-32P)ATP using Nasase. The DNA was digested with the enzyme NdeI and added to 5% polyacrylamide gel containing 8M urea. After purifying the synthetic gene fragment on a chemical cleavage method (Maxam and G11ber) t, 1980. Menz in Enzymology, 65:499-560) Example 5 pACl-code collagen 7 analogue gene metsenger RNA Northern profiling cut analysis Total RNA was prepared from the following three strains: DC10λdefΔBamHrΔH 1cI857), DC1139A (pJL6), and DC1139A (pA cl). Cultures were grown overnight at 30°C in 20 mL LB broth to 0 Daoo = 3. urge Now split the culture and half to activate the λPL promoter. Raise to 41°C for 1 hour. Following incubation, the culture is cooled to 0°C.

0℃.

Centrifuge the cells for 5 minutes at 8000Xf. ST of the beret so o pt E buffer (100mM NaCl, 10mM Tris-HCl, pH 7.0, Resuspend in 1mM Na2EDTA) and transfer to a 1.5m Etzpendorf centrifuge tube. . Add 500 μt of hot (65°C) phenol that has been equilibrated with distilled water and add to the tube. After vortexing, the tubes were incubated at 65° C. for 10 minutes. Eh After centrifugation for 5 minutes in a Pendorf microcentrifuge tube, the aqueous phase was removed and 50 μt of thermophenol was added. Add mol. Add another 500 μt of STE buffer to the first 7-enol phase. After vortexing both tubes, incubate both tubes at 65°C for 5 minutes. .

After a further 5 minutes of centrifugation, both aqueous phases are pooled. Updated phenol extraction Repeat 3 times. Final extraction was performed with 7 enol:chloroform (1:1) at room temperature. be exposed.

The sample is extracted with ether and the RNA is precipitated with ethanol. 30 RNA 0 pL RNA storage buffer (21゜pt 100% ethanol, 90p12 Phosphate buffer consisting of 0mM sodium phosphate* pH 6.5+ 1mM Redissolve in Na2EDTA, 99.5% ethanol). RNA preparation sample The quality of the sample was 1.2% in 10mM sodium phosphate buffer, pH 7.0. Monitor by electrophoresis on an agarose gel. 0D260 and Record 0D280.

pl) 202-203. The RNA sample is Electrophorese on a 10% agarose-formaldehyde gel at 30V overnight. next morning , gels were stained with acridine orange to visualize RNA, and Barinaga et al. Transfer of RNA to solid support by 5chleicher and 5chnel Perform Northern hybridization analysis according to the method described in l) . Agarose-formaldehyde gels are plotted onto DBM paper overnight. No The hybridization solution was as described by Baringa et al. It is prepared by grinding. The DBM paper containing the transferred RNA is 17-prehybrid. Incubate overnight at 42° C. in diization solution. northern plot The probe was radiolabeled with T4 polynucleotide kinase in the presence of [γ-32P]ATP. The oligodeoxynucleotide of Example 1 was identified.

25% formamide, 5XSSPE, 0.05% SDS, 1mM Na2EDT A, IX Denhardt's solution and 750 p? /- from salmon sperm DNA The hybridization solution is constructed (T., Maniatis et al., cited above). for the definition of IX5SPE). probe and high Mix the hybridization solution and the DBM paper containing the transferred RNA at 37 °C. and incubate overnight). DBMM 1t(7)4XSSPE, 0.1% 20 min at 55°C with SDS, 10 min at 55°C with 800-4XSSPE, 20 min at 55°C. 0 m 4XSSPE at 55°C for 1 min.

and 500d of 2XSSPE for 2 minutes at room temperature. The plot continues The film is dried overnight and the X-ray film is exposed overnight. The autolog obtained by this exposure Lamb was highly sensitive to DC1139A (pAcl) RNA in cultures induced at 41°C. It showed strong probe hybridization. DC1139A (pAcl) All other strains and hybridizers under other culture conditions, including growth at 30° zation was minimal. These data clearly show that, as expected, and the DC1139A (pAcl) strain only have the collaboration from the λpL promoter. -gen-like oligonucleotide B-4) Strength of heterogeneous messenger RNA synthesis This indicates that induction has occurred.

fixed To examine the protein encoded by plasmid pAC1, commercially available pairs were used. A new transcription-translation system (Amarjam) was used. The reaction mixture was placed in a 25μ2 plate. Contains lasmid DNA and was prepared according to the method supplied by the manufacturer. pAC The parental plasmid pJL6 was also studied in parallel reactions for comparison with 1. These plastics In vitro transcription-translation stimulated by Sumid DNA, pAcID DNA An aliquot of one sample containing Treat for 30 minutes at °C.

Each reaction mixture was mixed with an equal volume of loading buffer (0.08 M) Lith-HC4, pH 6. , 8,0,1M dithiothreitol, 2SDS, 10% glycerol, 0,1 dilute with cape/-bromophenol blue) and heat to 100°C for 5 minutes.

Half of each sample was incubated overnight at 50 V on an IZ5%5DS-polyacrylamide gel. pneumophores. The molecular weight marker protein running in parallel lanes is bovine serum albumin. Ovalbumin, carbonic andolase and the method described Fluorography is performed on En3Hance using Gel to X-ray film After overnight exposure, two prominent bands could be visualized on the autoradiogram.

The first band is pJL6 DN A, similar to the lane containing pACI DN A. Also present in lanes containing This is probably encoded on both plasmids Represents beta-lactamase enzyme. The second band is unique to pAcI DNA. It is a 22,000 dalton protein based on its electrophoretic mobility. this The protein is the product of the synthetic collagen-like gene pAC1. this Evidence supporting this conclusion is that pACI treated with collagenase prior to electrophoresis The fact is that this band is not visualized in DNA-containing samples.

Example 7 Synthetic collagen-like gene without DNA linker contained in plasmid pAC1 encoded by a synthetic gene inserted into the peptide expression plasmid pACl from Expression of collagen-like peptides on the human body was determined using a whole-cell labeling protocol. It was shown using a file. DC1139A (pJL6) and DC113 at 30°C Prepare overnight cultures of 9A (pAcl). The next morning, add 50 μl of the overnight culture. 20 ml of I, B gloss (102 tryptone, 52 of yeast extract and 52 of NaCL in 1 liter of water). Ru.

The culture is grown at 30°C until 0Dsoo = 0.4. 1d trial Remove and wash twice in M63 salt solution.

Pellets with 0.2 tyglucose, lpy/rnt vitamin Bl.

and 1d M6 with addition of all amino acids except proline at 100μ2/- 3. Resuspend in medium. After preincubation of the culture for 20 min at 41°C Add 2 μCi of [14c]proline to each culture tube and incubate for an additional 3 minutes. Continue incubation. Approximately 1 cape of unlabeled proline was added to all cultures; After an additional 3 minutes of incubation, pellet the cells of the gold culture. Terminate the incubation by Cell pellets - Kid's M63 salt The remaining unincorporated (14c) proline is removed by washing with water. Ultimate Cell pellets were added to 50 μt of SDS loading buffer (80 mM) in Lis-HC4, pH 6. , 8, 100mM dithiothreitol, 2% SDS, 10% glycerol, 10 Resuspend in 0μ2/bromophenol blue) and immediately soak in a boiling water bath for 5 minutes. Heat. Part of 20 μttz: 125% 5DS-polyacrylamide gel Electrophoresis on top. Transfer the resulting gel to En3Hance in New England. Nuclea) and exposed to X-ray film overnight. Autoradiolog obtained Lamb showed a protein band in the lane containing pAC1 protein, which is pJL. Not observed in the 6 protein lanes: This band is based on electrophoretic mobility. This shows that it is a protein with an apparent molecular weight of 22,000 Daltons. are doing. This protein was therefore identified in the paired transcription-translation system of Example 6. It is the same size as the pAcl-specific protein.

The effect of temperature during pre-incubation of the cultures was also determined. Experimental protocol The authors investigated the preincubation temperature in the range of 30' to 47°C. Same as before except for. The obtained autoradiogram shows the largest collagen class. Preincubation temperatures leading to similar peptide expression are between 41' and 44°C. It shows that there is.

: Measured molecular weight of Colladev analog encoded by pAcl (2 2,000 Daltr) and pAC1 from the physical map and DNA sequencing data. There appears to be a discrepancy between the calculated molecular weight of 12,000 Daltons. So Therefore, collagen analogs migrate abnormally on 5DS-polyacrylamide gels. We started an experiment to determine whether or not to do so. 10%, 12.5% or 15% point Three 5DS-polyacrylamide gels were prepared and irradiated with polyacrylamide concentration. Heat-induced DC1 labeled with proline (:14c) as well as sex-labeled marker protein Replicate samples from 139A (pAcl) cultures were added to all three 5DS-PolyAcl cultures. Electrophoresis was performed on a lylamide gel. The results showed that the collagen 7-like peptides This shows that the car protein migrates unusually slowly compared to the car protein. Therefore, Korra The true molecular weight of the -gen-like peptide is less than 22,000 Daltons.

Additionally, many genetically engineered proteins form insoluble aggregates called inclusion bodies. collagen-like peptides enter the soluble or insoluble protein fraction of cells. It will also be of interest to determine whether the phenotypes segregate.

Perform a gentle lysis method to remove any concomitant proteins present in the high salt pellet. Analyze in the same way as in the clear liquid.

Two 1- A sample of Understand. One sample of each culture is processed as before and subjected to unfractionated extraction.

The other parts were pelletized following instantaneous label tracking experiments and 100% of TES buffer (4Q Wash with (mM) Lis-HC1, pH 8, 0゜1mM EDTA, 25% ShoS) do. Resuspend the pellet in 250μt TES buffer and add 1 Misaki Norizozyme. Add. Samples were frozen in dry ice and thawed in a 37°C water bath to facilitate lysis. Repeat twice to do this. Next, add lysis buffer to the following concentration.20. 5 Chinoridet P 40. 10mM MgC4, 50mM Nact.

By adding Escherichia coli DNase l enzyme, the viscosity of the cell lysate is 20μ9/rat. Decrease. Samples were left on ice for 30 minutes and then incubated at 4°C in an Etzbendorf microcentrifuge tube. Centrifuge for 1o. Trick the resulting supernatant to a final concentration of 10%. Add roloacetic acid and place on ice for 15 minutes. These acidified samples were centrifuged (again at 4 After washing the white pellet twice with 100% ethanol, 50 μt Resuspend in SDS loading buffer and heat in a boiling water bath for 5 minutes. High salt beret is 5 Suspend in 0 pl SDS device buffer and wash with TES buffer before heating.

Before heating in a boiling water bath, add a sufficient amount of IM, pH 8. , 0 to change the color of those samples of yellow hue back to blue. Same as unfractionated control sample The fractionated samples were also electrophoresed on a 12.5% polyacrylamide gel. ance and - exposed to X-ray film overnight. The autoradiograph of this gel 4 in the lane containing the De1139A9 (pACl) protein. different view The main band with an apparent molecular weight of 22,000 daltons is mainly from the supernatant liquid. was observed in the fraction and was the main labeled band in that lane.

Example 8 Preparation of expression vector pAVl When the gene inserted into the ClaI site of the expression vector pJL6 is expressed, λc Protein, a fusion polypeptide containing the first 13 amino acid residues of ll protein. Produces protein. Protein code for cll protein from pJL6 A novel product with the exception of sequence and the introduction of specific Apal luminal restriction endonuclease positions. A current vector was designed and constructed; this new expression vector was designated pAV1. Ru.

Plasmid pAV 1 still contains the λpL promoter and just above the translation start codon. Ndel and Hind of pJL6 are used, but the current DNA sequence variants are used. The plasmid vector DNA between the m sites and the chemically synthesized DNA sequence has been changed, so λcl! rather than the first 13 amino acids of a protein. The tripeptide Met-G+y-pro is created. New Apal Lumil Restriction Nuku The lyase position is located within this sequence and therefore encodes the amino acid residue Pro. The DNA that is present is cut. Unpaired base sequence: Ends with the song GGCC-3' Any synthetic or natural gene or gene fragment can be inserted into the Apa1 site of pAVl. can be cloned, and the above tripeptide is converted into λP in pAV by gene expression. It will occupy the amino terminus of the peptide produced under the control of the L promoter.

The oligodeoxynucleotides listed below are applied as the first step in pAvl construction. Synthesized using Biosystems Model 380A automatic DNA synthesis machine: D. 5’-TAAGGAAATACTTACATATGGGGCCCTAAGC TTTAATGCGGTAGTT-3' E, 5'-TAAAGCTTAGGGCCCCATATGTA-3' oligo Deoxynucleotide E is completely complementary to a portion of oligodeoxynucleotide D and generates DNA fragments with overhang ends on both the 5' and 3' ends. Ru.

In the case of annealing, oligodeoxynucleotides D and E are heterologous and unlinked DNA. Release the restriction enzymes and recognition enzymes for both Ndel and Hind [10]. The recognition area is located. The most direct way to construct pAVl from D and E is , after digesting the synthetic heteroduplex with Ndel and Mindful, the heteroduplex product into pJL6, and then NdeI-Hlndl[l The small NA fragment produced by double digestion has been excised. pAV1fi During the construction process, Ndel mostly removes the synthetic heteroduplex formed by D and E. It has been decided not to perform any or all limited degradation and require the additional steps described here. Essential. Oligodeoxynucleotides D and E (270 pmol each) Anneal (75°C) in 35μt of 10mM TE buffer (see Example 1). by slowly cooling the solution from to room temperature). A part of this synthetic heterogeneous binary [γ-32P] Radiated by T4 polynucleotide kinase in the presence of ATP Gender marking. After completion of the radioactive labeling, the synthetic heteroduplex WODE-52 cellulose (wax) was added. chromatography (9) on a 100% chromatography method, and precipitated with ethanol. . A labeled synthetic heteroduplex is added as a tracer to an unlabeled substance, and the combined fraction is Further purification on a NENSORB-20 column (DuPont) and concentration by evaporation . Another 270 pmol each of oligodeoxynucleotides D and E were added to the concentrated solution. annealing by adding it to the solution and slowly cooling the solution from 98°C to 4°C. repeat the reaction. Gel electrophoresis on 16% polyacrylamide of a portion of the reaction mixture to monitor proper annealing.

Synthetic heteroduplexes were prepared at 50mM Nact, 50mM) Lis-HCt, p ) (s, O, l OmM MgCl2 and loopt7mtBSA buffer Restriction digestion was performed with 75 units of Hind[I restriction enzyme at 37° C. for 5 hours. after that Add 9 units of Ndel enzyme and add buffer components to 200 mM NaCl, 6 0mM) Lis-HC4, pH 8, 0, 17mMget, and 200 pt/*Bs*KJ), and incubation at 37°C is continued overnight. reaction The mixture was stored at -20°C, followed by 75 units of HindI[l enzyme and 3 units of of Ndel enzyme is added and the mixture is incubated again, this time at room temperature overnight. . The reaction mixture was then diluted twice with phenol:chloroform (1:l) and then with ether. After extraction once, chromatograph through Sephadex G-25 (7 Almasia) Purify by water. The excluded fractions were pooled, and the synthesized heteroduplex was 150 m M NaC4, 10mM) Lis-HC1.

pH 7, 8, 7mM MgCl2. 6mM 2-mercaptoethanol and Restriction digestion with 15 units of Ndel enzyme was carried out in Perform at room temperature for 24 hours. The reaction mixture was diluted once with phenol:chloroform (1:1). The extracted and synthesized heteroduplex was further treated with Sephadex G-25 (Pharmacia). Purified by chromatography. The excluded fractions were pooled again and 50 mM NaC4, 50mM Tris-HCj, pH8.0, 10mM MgC 4z and 100 units of Hindm enzyme in 100pt/ml BSS A Process at room temperature for 24 hours. This incubation is followed by a phasing of the reaction mixture. Nol:chloroform (1:1) extraction was performed, and the synthesized heteroduplex was extracted with Sephadex. Purified by chromatography through G-25 (Pharmacia).

The excluded fractions were pooled and a portion of the pooled fractions was injected into denatured and non-denatured 16-tipolymer Both acrylamide glucose (8M urea, respectively, are analyzed for presence or absence).

A portion of the pooled material was cleaved and produced by both Hindnl and Ndel enzymes. NAC3PREPAC mini column (Bethesda Research Lab) according to manufacturer's instructions It binds to 10 μm of pJL6 DNA purified in a 5-fold molar excess. It shows that Chimeric plasmid joined at common Hindm site - Synthetic heterogeneous binary DNA was separated from unbound material by NAC3PREPAC minicolumn (vessel). Purified by chromatography on Suda Research Laboratory). f# The prepared 1,000-mera DNA was then lysed with 3.7 units of T4 DNA ligase and 33 mM) Acetic acid, pa 7.9, 66mM potassium acetate, 10mM magnesium acetate Mu + O-5mM dithiothreitol and 100μf/me BSA for 5 minutes at 37° C. in a 10 μt reaction mixture containing: Na3 EDTA Terminate the reaction by adding up to 10mM.

The solution was extracted once with phenol:chloroform (1:1), llil mini-chill; Any remaining traces of ether are removed under vacuum. Heat the chimeric DNA to 65℃ and slowly Cyclization is effected by cooling the reaction mixture to 4°C.

The resulting circular chimera contains a single strand gap on each side of the annealing region; is filled with the Klenow fragment of E. coli DNA polymerase I. This means 50m M) Lis-HC4, pH 7, 8, 60mM MgC4, 1mM dATP.

1mM dCTP, 1mM dGTP, 1mM TTP, 10mM 2-mercaptoe Tanol, 50μ97d (DBSA and 1 unit of E. coli DNA polymerase) was achieved with 5μ2 of chimeric DNA in a 50μt solution containing the Klenow fragment of Ru. The reaction mixture was incubated at room temperature for 15 minutes before adding 10 μt of 10X binding. Buffer (10X=0.66M) Lis-HC1,I) H7,6,10mMATP, 10mM Spermidy, 0.1M MgCl2. 150mM dithio threitol and 2 capes/m/BSA), 3 units of T4 DNA ligase and water (to bring the total reaction volume to 100 μt). This reaction mixture is directly Incubate for 3-4 hours at 16'C before being used to transform S. enterica strain MHOI. (100 npDNA per transformation).

Colonies carrying the desired plasmid construct are identified by colony hybridization. is identified. Oligodeoxynucleotide F to T4 polynucleotide kinase and [γ-32P)ATP to radioactively label and probe colony plots. Use as a backup. To lytic colonies fixed on nitrocellulose filter Hybridization was performed in 37% formamide, 5X Dennolt's solution.

250μf/mA yeast + RNA, 1.0M NaC4,0,1M) Su-HCt, pH 8.0, in 6mM NaxEDTA and 0.1% SDS, This occurs overnight at 37°C. Radiolabeled probe heno-bridized colonies The plasmid prepared from Apa I restriction site is removed by treatment with Apa I enzyme. Check for presence and analyze on agarose gel.

One plasmid containing the Apa1 site was described by Chen and Seeburg, 1 985. DNA4: 165-170. Plus according to the method described by underwent mid-DNA sequencing. Counterclockwise direction for normal pBR322 physical map ]) BR322Hind I [1-site 16-mer primer New England BioLabs) was used for these DNA sequencing reactions. array The decision result is Guo and Wu, 1982 r Nureitsukumasitsuzurisa Confirmed and extended by the method described in 10:2065-2084. did. Briefly, a plasmid containing the Apa1 site was digested with EcoRV restriction enzyme. and subjected to restriction digestion with E. coli exonuclease m. dATP, dCT P.

All four standard dideoxynucleotides (ddA rp, ddcT'p, ddcTp and ddTTP) in the presence of E. coli Performed using the Klenow fragment of DNA polymerase I. Plasmid here A val restriction enzyme and the resulting DNA was run on a polyacrylamide sequencing gel. Analyze by electrophoresis. DNA around the Apa I recognition site of this plasmid Sequence determination revealed that between Ndel and Hind111 on the coding strand in pJL6. The DNA had been replaced with the following sequence (coding strand): 5'-TATGGGGCCCTA-3' Furthermore, all gene translation initiation code A mutation from A to G occurred during plasmid construction at the 5' position to the The region produces a base sequence that can be read as follows: The bases marked with an asterisk are the bases that have mutated, while the underlined regions represent this new base. Shine-Dalgarno sequence and translation initiation code in each constructed plasmid. It is. This plasmid is designated pAV1; the physical map of pAV1 is provided. This is shown in the figure below.

Example 9 The following complementary and overlapping oligodeoxynucleotides were prepared as described in Example 1. 2F prepared as follows: 5'-TTCCGGGTGTTC; GTG-3' G, 3'-CCACAACCCACAAGGC-5' final product is >97% The purity was determined as described in Example 1. unmarked RAT As described for a similar reaction in Example 1, except that P was added (1 mM) Add 0.57°C mol of oligodeoxynucleotide to 20 μt of buffer equivalent to 5′ ends of oligodeoxynucleotides F and G in separate reactions containing otide Add phosphoric acid to. Combines phosphorylated oligodeoxynucleotides and kinases Heat at 70°C for 15 minutes to inactivate the enzyme. Prepared oligodeoxynus Parts of each of nucleotides F and G are separately linked to T4 polynucleotide kinase and and [γ-32P)ATP. Each radiolabeled oligodeoxy 2 pmol of nucleotides were added to unlabeled oligodeoxynucleotides F and G. into the reaction mixture and slowly reduce the temperature of the mixture from 70°C to 1°C overnight. let them do it.

Annealing oligodeoxynucleotides F and G under these conditions This helps form the most stable heteroduplex. In this case, five bases each 10 bases that are linked together at the 5' end of each oligodeoxynucleotide chain Pairs are preferred heteroduplexes. The 5' overhang ends are base-paired to each other. was further created, and the sequence Val-Pro-Gly-Val-Gly was created by enzymatic binding. Generated a long synthetic NA gene encoding an elastin analog consisting of repeats of do. After annealing of oligodeoxynucleotides F and G, new ATP (63 μM) and T4DNAIJ gauze enzyme (5 units) were added to the reaction mixture. Ru.

The temperature of the reaction mixture was gradually increased to 14°C over 2 hours until the mixture was infused. Continue incubation at 14°C overnight. The enzymatic Ochiai product is Sepharose 4B ( Size fractionation is performed by chromatography on a Obtain the NA gene. The relative size of the synthetic gene DNA in each fraction is described in Example 1. (determined by each step. The DNA break and/or gap is 0.5 μ2 using the method described in Example 1 (however, dATP, dCT except that the final concentration of each of P, dGTP and TTP was 0.33 mM. ) removed.

The DNA product was prepared directly as described in Example 1 (C1al-digested and blunt-ended). ligated into pJL6 plasmid DNA. Binding reaction solution is TE buffer up to 200μ as described in Example 3 (directly used for transformation of E. coli strain MHOI). be done.

Bacterial transformants containing plasmids carrying synthetic gene inserts have been identified and Physical location using restriction enzymes as described in Example 3 for gene-like genes Characterized by diagramming. In these experiments, oligodeoxynucleotide F was used as a radioactive label] and hybridization probe. This pro The hybridization temperature was 27°C using a buffer wash, and the buffer wash was 55°C. It was carried out at 30°C instead.

The following complementary and overlapping oligodeoxynucleotides were prepared as described in Example 9. Specifically prepared and purified: H,5'-GGGCCCCG-3' 1, Addition of 3'-GGCCCCGGG-5' phosphate and oligodeoxynucleotide For annealing of leotide H and As described in Example 9, except cooling to room temperature. Apa l! Appropriate annealing of oligodeoxynucleotides H and I to form the j-linker. The rings were tracked by electrophoresis of the samples on a 20% polyacrylamide gel. .

The following oligodeoxynucleotides were chemically synthesized as described in Example IK. and purified: J, 5'-GGTCCGCCGGGTCCGCCG-3'o A purified preparation of Rigodeoxynucleotide J was determined as in Example 1. contains no detectable impurities. Oligodeoxynucleotide J is an example It is complementary to B in 1 and overlaps with it. Ligation of oligodeoxynucleotides B and J Examples include acid addition and annealing and ligation to form large synthetic genes. 9 for oligodeoxynucleotides F and G. This is accomplished in the same way. Using these annealing conditions, oligodeoxy The preferred heteroduplex between the oligodeoxynucleotides B and J is the oligodeoxynucleotide of each A nucleotide chain with three bases overhanging at the 3' end and containing 15 base pairs. It's like going. The 3' overhang ends further base pair with each other, resulting in a ligation. A long synthetic sequence encoding a repeat of the amino acid sequence Gly-Pro-Pro Forms a collagen-like gene. Synthetic NA gene formed in this way was subsequently cyclized by chromatography on Sepharose 4B (Pharmacia). The relative amounts of synthetic gene DNA in each fraction were determined as described in Example 1. Evaluate size. The final length of the synthetic gene DNA that can be synthesized during the previous step One factor that may be limiting is the 5'-phosphorus at the growing end of each of these molecules. It is a linkage of oligodeoxynucleotides lacking acid groups. large synthetic gene dna as previously described in some examples to ensure that it contains a phosphate group at its 5' end. The kinase reaction is repeated as expected. ATP concentration in the kinase reaction mixture 4 units of 74 DNA IJ gauze enzyme was added after adjusting to 2mM and this material (approximately Apal linker (23 pmol) is bound to a part of 0.23 pmol, ) be done. After incubating the reaction mixture overnight at 14°C, any breaks or and/or gaps with 37μt 5X polymerase buffer (5X = 250mM). Hct, pH 7, 8, 45mM MgC4, 50mM 2-mercaptoe Tanol, and 250μ? /lnl BSA), dATP.

dcTP, dGTP and TTP final K 1 mM, and 7 units of E. coli D Add NA polymerase I to a final volume of 185 μt. be removed. The reaction mixture was incubated for 1 hour at 14°C and then incubated with phenol. Extract once with chloroform (1:1).

Precipitate the DNA and add 5mM) Lis-HC4, pH 7, 4, 6mM NaCl, 6 mM MgCl2, 6 mM 2-mercaptoethanol, and 100μ Add Apal enzyme (up to 100 units) for up to 2 days in a reaction mixture containing f/d BSA. ) to digest. 1st degree phenol: chlorophor! After extraction with (1:1), DN A product is Apa digested through a Sepharose 4B (Pharmacia) column. Remove the Lumi linker.

The resulting synthetic collagen gene cassette carrying the Apa I linker terminus is The fractions rejected from the Rose 6B column are pooled and then ethanol precipitated.

Transformation of DC1138 The collagen-like gene cassette carrying the Apal luminary linker was digested with Apal. is ligated into pAVI DNA. Typical binding reactions are approximately 0.3 to 1 ．． 2 prnol collagen-like gene cassette and 0.03 to 0.12 arrays 1 Apal-digested plasmid +° with a reaction volume of 10-17 μt.

The reaction buffer and other conditions are the same as in the previous example. The reaction solution is diluted with Then, dilute the solution μ with TE buffer to 1 nP of total DNA per solution, E. coli strain DC1138 is transformed as described in Example 3. each shape Use 3 to 10 ny of mRNA on transformation plates. Collagen-like genetics Identification of transformant colonies containing plasmids carrying child cassettes is described in Example 3. Act as if it were. Furthermore, the size of the synthetic gene inserted into pAV In order to roughly estimate the It was used. Restriction enzyme Nde1. and an interesting synthesis using HindIIl Once the gene inserts have been sorted into more accurate size order, the collagen-like genes are The size of the gene cassette was determined by combining the recombinant plasmid DNA with enzymes Banl1 and/or Or digest with Apal and check. Plasma carrying a collagen-like gene cassette A number of transformants containing Sumid were identified. Characterization of some of these revealed that Gene insertions identifiable as single gene cassettes in analyzed bacterial clones have been shown to range in length from about 170 to about 350 base pairs.

Identification of bacterial clones containing Ligation and characterization of colonies containing plasmids carrying multi-collagen-like gene cassettes Conversion and identification are as described in Example 11. plasmid expression vector Using a 10 to 1 molar excess of collagen gene cassette to DNAk, many The likelihood that the cassette will be incorporated into a single plasmid is increased in this example. is adding to it.

One plasmid, when digested with the restriction enzymes Ndel and Hindl[I, has approximately It was identified as containing a 440 base pair synthetic gene insert. This plasmid ( Apal digestion of pAC95) produces two collagen-like gene cassettes. One is about 200 base pairs long and the other about 230 base pairs long. Ndel - Sequencing the H’rndm synthetic gene cassette fragment Vector Bluescript M l3+ (Stratadiene, San Diego, CA) and synthesized An RNA transcript of the gene cassette fragment is prepared.

These RNA transcripts were prepared using Trimyoplastosis Reverse Transcriptase and the manufacturer's instructions. sequenced with appropriate oligodeoxynucleotide primers. p p surrounding two Apa sites external to the inserted DNA contained within Ac95. Sequence analysis of the NdeI-Hlndm region of AC95 reveals two gene cassettes. is the Apa1 site of pAVl in the appropriate orientation with respect to the translation initiation signal sequence of the vector. This shows that two gene cassettes have been inserted in tandem into the cell.

The tandemly arranged gene cassettes in pAC95 therefore contain the peptide Met- (Gly-Pro-Pro)*gives a-GJy-Pro.

Engraving (no changes to the content) FIG. 1 Submission of translation of written amendment (*Verdict M Article 184-8) July 7th, 1989 Yoshi Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1. Display of patent application PCT/US87103369 2. Name of the invention Microbial production method of peptide oligomers 3. Patent applicant Address: New Chassis, USA 07960. Maurice Counte Columbia Road and Park Avenue, Morris Township - (@Jinai 7 people, Allied Corporation 4, agent) Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo Phone (270) 6641-6646 5. Date of submission of written amendment December 1988, Patent The scope of the claims (1) (a) Two or more complementary DNA oligodeoxynucleotides, The DNA oligodeoxynucleotide is phosphorylated at the s' end to form a salt. Since they partially overlap when forming a base pair, any two oligodeoxy groups in the mixture The number of complete base pairs formed by annealing between nucleotides is the annealing is not equal to the number of unpaired bases in the identified strand; and is not equal to the number of unpaired bases in the strand; and encodes the desired amino acid sequence, (1) Linker DNA, The linker DNA is not present within the DNA segment and Contains one or more restriction enzyme recognition sites that exist only once in the DNA of the Sumid vector. 1 when said segment is enzymatically tandemly joined to said plasmid vector; Maintaining the repeating amino acid sequence of the above-mentioned DNA segment and reading the genetic code adapted to maintain the frame; Complementary linker DNA and and/or DNA segments oriented antiparallel with respect to their 5' to 3' polarity. annealing the mixture to form stable base pairs between the agents; and (b) Treat the annealed DNA oligodeoxynucleotide with ligase Treatment with enzymes converts adjacent oligodeoxynucleotides into identical 5' to 3' Duplex linker-DNA is attached to the ends by polar covalent linking. longer double-stranded DNA segments that meet or incorporate ; Double-stranded DNA encoding a polypeptide consisting of a repetitive amino acid sequence consisting of each step Method for producing A fragment.

(2) To remove the multimeric form of the linker end on the DN A fragment, It is further characterized by partially or completely cleaving the mark/car with a suitable restriction enzyme. The method according to claim 1.

(3) Completely or partially fill the breaks or gaps in the double-stranded DNA segment. The double-stranded DNA segment is treated with a DNA polymerase enzyme to remove the double-stranded DNA segment. 2. The method of claim 1 further comprising:

(4) In step (a), hybridization results in DNA oligodeoxynucleotides. base pairing to provide the maximum amount of overlap between the oligodeoxynucleotides. The method according to claim 1, wherein the mixture is cooled at a rate sufficient to Law.

(5) A claim in which the mixture consists of two complementary oligodeoxynucleotides. The method described in box 1.

(6) When the linker DNA binds to the double-stranded DNA, it presents non-equivalent ends. A method according to claim 1, wherein the method is selected to provide.

(7) The DNA oligodeoxynucleotide and the linker DNA are synthesized The method according to claim 1, wherein the method is a product.

(8) the mixture consists of two or more complementary DNA fragments, and therefore The oligomerized DNA fragments described above exist as random block peptide copolymers. A heteropolymer encoding a polypeptide with two or more amino acid sequences The method according to claim 1, characterized in that it is a reamer.

(9) The DNA fragments of two or more species are further mixed and the ligators claim 1, which is bound into the oligomerized double-stranded DNA fragment by enzyme enzyme In the method described, Each species of said DNA fragment mixture contains at least one other species of different species of DNA fragments. at least one single-stranded end that is compatible and base-paired with the single-stranded end of the An oligomerized double-stranded DNA fragment such as A resultant that encodes a heteropolypeptide consisting of a random block repeating amino acid sequence. binding fragment, or a polypeptide with a single repeated amino acid sequence (binding DNA1vr polypeptide (with a higher molecular weight than the polypeptide encoded by the polypeptide) A method consisting of joined fragments that code.

(A larger double-stranded DNA fragment of 1 is linked to two or more Coating a heteropolypeptide consisting of a random or block repeating amino acid sequence 10. The method according to claim 9, which comprises:

ω) The species of the DNA fragment is a mixture of synthetic and natural DNA fragments. The method according to claim 10, wherein the natural DNA fragment is a natural DNA gene or is a complementary DNA copy of a gene segment or natural messenger RNA sequence , and the oligomerized DNA fragment consists of adjacent random consists of the aforementioned synthetic fragments of alternating repeating blocks and encodes a heteropolypeptide. Two or more natural DNA fragments to be coded are joined together, and adjacent random or is a method that consists of alternating blocks of repeating amino acid sequences.

(2) Claims 1 to 9 further characterized in that the DNA fragment is isolated. The method described in section.

(Claim 1) wherein the isolated DNA fragment has a length of about 75 base pairs or more. The method described in Section 2.

0→Claim 1, wherein the isolated DNA fragment has a length of about 100 base pairs or more. The method described in item 13.

(g) The linker DNA provides equivalent ends when treated with an appropriate restriction enzyme. capable of cleaving at least one enzyme recognition site in the linker DNA provided. 15. The method of claim 14, wherein the method is selected such that the

The linker DNA of 0 is the sequence 5'-GGGCCCCG-3' and 5'-GGGCCCCG-3 ’ The method according to claim 1, comprising the oligodeoxynucleotide of

(b) If the linker is treated with an appropriate restriction enzyme, it will provide non-equivalent ends. selected to be able to cleave at least one enzyme recognition site in the car DNA The method according to claim 1.

(The linker-DNA sequence is 5'-GGGCCGCCAGGGCCGCCG-3' and 5'-CGG Is it an oligodeoxynucleotide with CCCTGGCGGCCCCG-3'? 18. The method according to claim 17, comprising:

Q9) The DNA is a repeating fullergen-like tripeptide sequence, a repeating elastin, etc. Pentapeptide-like sequences or linked adjacent collagen-like tripeptides and random or alternating polypeptides consisting of elastin-like pentapeptide sequences. The method according to claim 1, characterized in that it is a tide copolymer.

(2. A DNA fragment produced by the method described in claim 1.

(21) A DNA fragment produced by the method according to claim 11.

(22) The fragment according to claim 20, having a length of at least about 75 base pairs.

(23) The fragment of claim 22 having a length of at least about 100 base pairs.

(24) A replicable plasmid cloning vehicle, D of claim 20. If said vehicle containing the NA fragment is cloned into a suitable host microorganism. The carrier is a carrier capable of expressing the DNA fragment.

(25) Replicatible plasmid cloning vehicle according to item 24 and forming a recombinant plasmid consisting of the DNA fragment according to claim 20. , the method comprising the following steps (a) and (b): (a) before Plasmid cloning operations are performed using previously determined restriction endonuclease recognition sites. and (b) the DNA fragment is used for the plasmid cloning. 1 at said site so as to be under the control of a regulatable gene promoter sequence in the vehicle. enzymatically inserting one or more of the DNA fragments of claim 20, and Thereby, the inserted DNA fragment can be expressed, and the recombinant plasmid can be one or more repeated amino acids when cloned into a suitable host microorganism A method of forming a polypeptide consisting of units of sequence.

(26) The DNA fragment with respect to the translation initiation DNA sequence in the plasmid vector to maintain the genetic code reading frame of the fragment and throughout the inserted DNA fragment. and one or more repeated amino acid sequences between them. more than one of said DNA fragments are inserted in tandem into said plasmid cloning vehicle. 26. The method according to claim 25.

(27) The two or more DNA fragments have identical repeating amino acid sequences throughout 27. The method of claim 26, wherein said insert fragment encodes can be expressed to form a polypeptide consisting of repeat units of said sequence.

(28) a repeat in which the two or more DNA fragments are different in whole or in part; 27. The method of claim 26, wherein the method encodes an amino acid sequence. The inserted fragment consists of block-like or random repeating units of the amino acid sequence. Methods in which polypeptides can be expressed to form block or random heterologous polypeptides. .

(29) Naturally occurring DNA fragment or naturally occurring metsenger RNA complementary DNA copies of all or part of said one or more DNA fragments. 26. The method of claim 25, further comprising enzymatically inserting:

(30) A recombinant plasmid prepared by the method according to claim 25.

(31) Forming microorganisms capable of producing polypeptides consisting of repetitive amino acid sequences A method, wherein the method comprises producing a recombinant protein using a recombinant plasmid according to claim 30. A method consisting of the step of transforming a functional microorganism.

(32) The method according to claim 31, wherein the microorganism is Escherichia coli.

(33) A microorganism transformed according to the method according to claim 31.

(34) A method of producing a polypeptide consisting of a repeating known amino acid sequence: (a) The microorganism according to claim 33 is grown in a culture medium to produce the achieving expression of said fragment or fragments comprising a coding sequence for a polypeptide; thereby forming said polypeptide; (b) isolating a fraction containing the polypeptide from the microorganism; and (c) A method characterized by a step of purifying the fraction to provide the polypeptide. Law.

Procedural amendment (formality) 1.Display of the incident PCT/US87103369 2. Name of the invention Microbial production method for peptide oligomers 3. Person who makes corrections Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo 5. Date of amendment order: February 27, 1990 (old dispatch) 6. Subject of amendment Translation of the written amendment written by type printing 7, Contents of the amendment As shown in the attached sheet (note that there is no change in the content of the above document) 1.Display of the incident PCT/US87103369 2. Name of the invention Microbial production method for peptide oligomers 3. Person who makes corrections 5. International search report of the amendment order [Attachment 1, February 270, 1990 (shipment date)] 1m--C^-1 (True Lies N/PCT/IJS 87103369 International Investigation report υS 8703369 Page 2 SA 20153

Claims (36)

[Claims] (1) (a) Heating a mixture of two or more complementary DNA oligodeoxynucleotides By cooling the 5′ to 3′ polarity, two or more complementary DNAs by forming stable base pairs between complementary sequences The oligodeoxynucleotides are annealed and the DNA oligodeoxynucleotides are annealed. Reotide is phosphorylated at the 5' end; it partially overlaps when base-paired. annealin between any two oligodeoxynucleotides in the mixture. The number of perfect base pairs formed by unequal and zero; and encode one or more desired amino acid sequences Ori, (b) The annealed DNA oligoteoxynucleotide is treated with a ligase enzyme. treatment to align adjacent oligoteoxynucleotides with identical 5' to 3' polarity. longer double-stranded DNA segments by covalently linking with ; (c) enzymatically attaching a double-stranded linker DNA to said double-stranded DNA segment; , create a double-stranded DNA fragment with a linker at the end, The linker DNA is not present within the DNA segment and is present in certain plasmids. It has one or more restriction enzyme recognition sites that exist only once in Pector's DNA. , the linker allows the segments to be enzymatically linked in tandem to the plasmid vector. maintains the repetitive amino acid sequence of one or more of said DNA segments when combined; and Repeated amino acids, each step adapted to maintain the genetic code reading frame A method of producing a double-stranded DNA fragment encoding a polypeptide consisting of an acid sequence. (2) To remove the multimeric form of the linker end on the DNA fragment, further characterized in that the linker is partially or completely cleaved with a suitable restriction enzyme. The method according to claim 1. (3) Completely or partially fill the breaks or gaps in the double-stranded DNA segment. The double-stranded DNA segment is treated with a DNA polymerase enzyme to remove the double-stranded DNA segment. 2. The method of claim 1 further comprising: (4) In step (a), hybridize to obtain DNA oligodeoxynucleotides. base pairing to provide the maximum amount of overlap between the oligodeoxynucleotides. The method according to claim 1, wherein the mixture is cooled at a rate sufficient to Law. (5) A claim in which the mixture consists of two complementary oligoteoxynucleotides. The method described in box 1. (6) After adding the linker DNA, perform the ligation reactions in steps (b) and (c). The method according to claim 1, which is carried out simultaneously. (7) When the linker DNA binds to the double-stranded DNA, it presents non-equivalent ends. A method according to claim 1, wherein the method is selected to provide. (8) The DNA oligodeoxynucleotide and the linker DNA are synthesized The method according to claim 1, wherein the method is a product. (9) the mixture is composed of two or more complementary DNA fragments, and therefore the mixture is composed of two or more complementary DNA fragments; Two oligomerized DNA fragments exist as a random block peptide copolymer. or a heteropolymer encoding a polypeptide with an amino acid sequence of The method according to claim 1, characterized in that: (10) DNA fragments of two or more species are further mixed and ligated claim 1, which is bound into the oligomerized double-stranded DNA fragment by enzyme enzyme In the method described above, each species of said DNA fragment mixture comprises at least one different species of DNA fragments. also has at least one single-stranded end that is compatible and base-paired with one other single-stranded end; and such oligomerized double-stranded DNA fragments contain two or more linked A heteropolypeptide consisting of adjacent random block repeat amino acid sequences is a binding fragment that encodes a binding fragment, or a polypeptide with a single repetitive amino acid sequence (binding D said polypeptide having a higher molecular weight than the polypeptide encoded by the NA fragment; A method consisting of combined fragments that code for (11) The larger double-stranded DNA fragment has two or more linked phases. Heteropolypeptides consisting of contiguous random or block repeat amino acid sequences 11. The method of claim 10. (12) The species of the DNA fragment is a mixture of synthetic DNA fragments and natural DNA fragments. The method according to claim 11, wherein the natural DNA fragment is a natural DNA gene. or the complementary DNA sequence of a gene segment or natural messenger RNA sequence. and the oligomerized DNA fragments consist of adjacent random or consisting of the above-mentioned synthetic fragments in which blocks are repeated alternately, and which generates a heteropolypeptide. Two or more encoding natural DNA fragments are joined together to form adjacent random or a method in which the blocks consist of repeating amino acid sequences. (13) Claims 1 to 1, further comprising isolating the DNA fragment. The method according to item 10. (14) The isolated DNA fragment has a length of about 75 base pairs or more. The method according to paragraph 13. (15) The isolated DNA fragment has a length of about 100 base pairs or more. The method described in box 14. (16) When the linker DNA is treated with an appropriate restriction enzyme, equivalent ends are generated. cleavage of at least one enzyme recognition site in the provided linker DNA; 16. The method according to claim 15, wherein the method is selected such that: (17) The linker DNA has a sequence 5'-GGGCCCCG-3' and 5'-GGGCCCCG-3' The method according to claim 1, comprising the oligodeoxynucleotide of (18) When the linker is treated with an appropriate restriction enzyme, it provides non-equivalent ends. selected to be able to cleave at least one enzyme recognition site in the linker DNA. The method according to claim 1, wherein the method is selected. (19) The linker DNA has a sequence 5'-GGGCCGCCAGGGCCGCCG-3' and 5'-CGGCCC A chain consisting of oligodeoxynucleotides with TGGCGGCCCCGG-3' The method according to item 18. (20) The DNA is a repeating collagen-like tripeptide sequence, a repeating elastin Similar pentapeptide sequences, or linked adjacent collage-like tripeptides Random or alternating polypeptides consisting of polypeptides and elastin-like pentapeptide sequences. A method according to claim 1 which encodes a peptide copolymer. (21) A DNA fragment produced by the method according to claim 1. (22) A DNA fragment produced by the method according to claim 12. (23) The fragment of claim 21 having a length of at least about 75 base pairs. (24) The fragment of claim 23 having a length of at least about 100 base pairs. (25) A replicable plasmid cloning vehicle, D of claim 20. If said vehicle containing the NA fragment is cloned into a suitable host microorganism. The carrier is a carrier capable of expressing the DNA fragment. (26) The replicable plasmid cloning vehicle and and forming a recombinant plasmid consisting of the DNA fragment according to claim 21. a method, said method comprising steps (a) and (b): (a) previously determined; Cut the plasmid cloning vehicle at defined restriction endonuclease recognition sites. and (b) said DNA fragment is prepared in said plasmid cloning vehicle. one or more at said site so as to be under the control of an articulable gene promoter sequence. enzymatically inserting the DNA fragment of Claim 20 above, and thereby The inserted DNA fragment can be expressed, and the recombinant plasmid can be expressed in a suitable host microorganism. unit of one or more repetitive amino acid sequences when cloned into a A method of forming a polypeptide consisting of. (27) The DNA fragment with respect to the translation initiation DNA sequence in the plasmid vector to maintain the genetic code reading frame of the fragment and throughout the inserted DNA fragment. and one or more repeated amino acid sequences between them. more than one of said DNA fragments are inserted in tandem into said plasmid cloning vehicle. 27. The method according to claim 26. (28) The two or more DNA fragments have identical repeating amino acid sequences throughout. 28. The method of claim 27, wherein said insert fragment encodes can be expressed to form a polypeptide consisting of repeat units of said sequence. (29) a repeat in which the two or more DNA fragments are different in whole or in part; 28. The method of claim 27, wherein the method encodes an amino acid sequence. The inserted fragment consists of block-like or random repeating units of the amino acid sequence. methods that can be expressed to form block or random heteropolypeptides . (30) Naturally occurring DNA fragment or naturally occurring messenger RNA complementary DNA copies of all or part of said one or more DNA fragments. 27. The method of claim 26, further comprising the step of enzymatically inserting: (31) A recombinant plasmid prepared by the method of claim 26. (32) Forming microorganisms that can produce polypeptides consisting of repetitive amino acid sequences A method, wherein the method comprises recombinant and non-recombinant plasmids according to claim 30. A method consisting of the step of transforming a functional microorganism. (33) The method according to claim 32, wherein the microorganism is Escherichia coli. (34) A symptomatic organism transformed according to the method set forth in claim 31. (35) A method for producing a polypeptide consisting of a repeating known amino acid sequence: (a) The microorganism according to claim 33 is grown in a culture medium to Achieving the expression of said fragment or fragments containing the coding sequence for the peptide, said polypeptide is formed by; (b) isolating a fraction containing the polypeptide from the microorganism; and (c) A method characterized by the step of purifying the fraction to provide the polypeptide. (36) A polypeptide produced by the method according to claim 35.