WO1986002947A1 - Gene synthetisant un lysozyme humain - Google Patents

Gene synthetisant un lysozyme humain Download PDF

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Publication number
WO1986002947A1
WO1986002947A1 PCT/JP1984/000546 JP8400546W WO8602947A1 WO 1986002947 A1 WO1986002947 A1 WO 1986002947A1 JP 8400546 W JP8400546 W JP 8400546W WO 8602947 A1 WO8602947 A1 WO 8602947A1
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tct
aga
ttg
aac
gtt
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PCT/JP1984/000546
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Japanese (ja)
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Morio Ikehara
Makoto Kida
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Takeda Chemical Industries, Ltd.
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Priority to PCT/JP1984/000546 priority Critical patent/WO1986002947A1/fr
Priority to EP85114427A priority patent/EP0181634A3/fr
Priority to JP60252899A priority patent/JPS61158793A/ja
Publication of WO1986002947A1 publication Critical patent/WO1986002947A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion

Definitions

  • the present invention relates to a recombinant DNA technology for producing human lysozyme. More specifically, the present invention relates to the chemical synthesis and expression of human 'lysozyme gene, recombinant plasmids and transformed cells related to those techniques, and products resulting therefrom.
  • Lysozyme is a relatively small enzyme protein with a molecular weight of about 14,000, which is widely distributed in living organisms and has a lytic effect on many bacteria. It is thought that it is a concept. It is believed that the main function of this action is the hydrolysis of polysaccharides on the cell wall by the activity of 1-dalcosidase. Nitrile egg white contains a particularly large amount of lysozyme, and high-purity lysozyme can be isolated relatively easily, so that it can be added to cheese, sausage, seafood, or used for milk preservation. It is used for the purpose of emulsification of mothers [Katsuya Hayashi, Taiji Imoto, Lysozyme, Nankodo (1974)].
  • pharmacological actions such as hemostasis, anti-inflammatory, tissue regeneration, and antitumor properties are known (for example, "Recent New Drugs, Vol. 34", p. 107, Pharmaceutical Daily, Tokyo, 19). 8 3) It is commercially available as an anti-inflammatory enzyme agent.
  • Human lysozyme is composed of 130 amino acids (Table 1), and 53 amino acids differ from those of hen egg white lysozyme.
  • the optimal sequence was determined according to the following criteria. i) Use the most permissive codons in yeast that are deemed appropriate for the expression of the synthetic gene. ii) Placing a specific restriction enzyme recognition site (XbaI in this case) in the gene to serve as an index for cloning or to facilitate gene modification after the construction of an expression vector . iii) Avoid sequences that are self-complementary on either strand or duplex, or complementary to other sequences (other than the correct sequence). Assuming that the above conditions are satisfied, The nucleotide sequence was determined.
  • ACG AGT CGA AAC GAA GTC CTG TTG TAA CGA
  • the present invention provides a synthetic gene for expressing human lysozyme represented by the above DNA sequence.
  • the DNA of the present invention is the same as the DNA of the present invention except that the above-mentioned synthetic arrestor itself is linked to a certain restriction enzyme recognition site, start codon, and stop codon at its 5 ′ and 3 ′ ends. It can be used for convenience of operations such as recombination of arrested children. As an example of this,
  • XhoI cleavage site has a start codon ATG, and at the 3 'end, TATAGAGC
  • XhoI cleavage site and those having TAA and TAG, which are stigcodons.
  • recombinant plasmids incorporating these DNAs are also included in the category of the present invention.
  • the gene can be produced, for example, by synthesizing a plurality (about 2 to 80) of oligodoxynucleotides and linking them, for example, as shown in Table 2. For example, by dividing into 52 oligonucleotide nucleotides' blocks, for example, [H.I to, ⁇ . ⁇ ,, I ke, S. I kuta, K. I takuraj Nucleic Acids Res., 10, 1755 (1992)). Oligonucleotide blocks are hybridized using known methods and linked enzymatically (e.g.,
  • the method of division into these fragments is not limited to the above, as long as care is taken to avoid the above-mentioned self-association, and various divisions are possible. is there.
  • the next floor is to sub-clonal the first step by incorporating it into an appropriate vector in order to increase the number of synthetic arrestors obtained in this way. Any vector may be used as long as the arresting element can be incorporated into the vector.
  • Transformation methods are known per se, and when Escherichia coli is used as a host, for example, the method of Choen et al. [Cohen, S.N.eta1.Proc.Nat1.Acad.S USA, 69, 2110 (1972)], when yeast is used as a host, for example, Hin Nat. A eta 1. Proc. Nat 1. Acad. Sci. USA, 75, 1927 (19778)], when using Bacillus subtilis as a host, for example, Protoplast method
  • Transformation can be carried out, respectively, according to CDubna, D. & Abelson, R. D, J. Mo1. Biol. 56, 2009 (1971)).
  • E. coli 294, E. coliw 3110, E. coli C600, B. subti 1 is 1 A1, B. subtilis 1 A33 39, B. subtilis 1A340 can be used.
  • the plasmid DNA into which the gene has been inserted is subjected to, for example, an alkaline extraction method [Birnboim, HC, & Dolly, J .: Nucleic Acids R. es., 7_, 1513 (19779)].
  • the inserted plasmid is cut out by treating the obtained plasmid DNA with an appropriate restriction enzyme, and the gene can be isolated by, for example, agarose gel electrophoresis or polyacrylamide electrophoresis. .
  • These series of operations are known, and are described in detail in the literature, for example, [Moe ecu ar CI Oning (1992), Old Spring Harbor Laboratory].
  • the simulated arrestor is indirectly ligated with the promoter downstream of the promoter of an appropriate expression vector to construct an expression plasmid.
  • an appropriate expression vector to construct an expression plasmid.
  • Any expression vector may be used as long as it can be actually used for expression of the gene.
  • the aforementioned p PHO 17, PGLD 906, P trp 770 (JP-A-58-210 796), PRC 23 (JP-A-58-18991) 9 7), P BTM 1 3 4, P TEX 2 0 but 1 and the like, PPHO 1 7 and PGLD 9 0 6 Gayo Ri preferred arbitrariness.
  • Animal cells and other eukaryotic cells can also be used as expression hosts.
  • a vector a plasmid having an XhoI cleavage site downstream of the promoter is more preferable.
  • the host cell is transformed with the obtained expression plasmid to obtain the desired recombinant.
  • the transformant thus obtained is cultured by a method known per se.
  • the culture medium may be, for example, an M9 medium containing glucose and casamino acids (Millu, J., Experiments in Molecu 1 ar Genetics, 4 3 1-4 3 3 ( C old S spring H abor L aboratory), Ne
  • the reaction is carried out at C for 10 to 96 hours, preferably 24 to 72 hours, and may be aerated or agitated as necessary.
  • the medium include L-broth and BHI (Brain Heart Infusion).
  • the culture temperature is usually about 15 ° C to 42 ° C, more preferably about 24: to 37 ° C, and the pH of the culture medium is about 5.0 to 9 with the initial pH. 0, and more preferably about 6.5-7.5.
  • the cultivation time is usually about 2 to 72 hours, and more preferably 3 to 48 hours. After completion of the culture, the cells and the supernatant are separated by a method known per se.
  • transformants of Escherichia coli and Bacillus subtilis are used alone or in combination with the freeze-thaw method, the lysin treatment method, the ultrasonic treatment method, etc.
  • yeast transformants the cells are destroyed by adding Zymolyase (manufactured by Kirin Brewery Co., Ltd.) or by mechanical soiling with glass beads or the like.
  • lysozyme produced by adding a surfactant such as Triton X-100, deoxycolate or a protein denaturant such as urea or guanidine hydrochloride to this. Can be extracted.
  • Lysozyme produced in or outside the cells in this manner can be purified by conventional protein purification methods such as salting out, isoelectric focusing, crystallization, ion-exchange chromatography, and high-performance liquid chromatography. -(HPLC, FPLC, etc.) to obtain the desired lysozyme.
  • FIG. 1 and FIG. 2 show construction diagrams of the plasmids PPH0117 and PGLD906 shown in Reference Examples 1 and 2, respectively.
  • FIG. 3 is a restriction map of the promoter cloning vector PBT M126 shown in Reference Examples 3 and 4, and a restriction map of the PBTM128 incorporating the promoter.
  • FIG. 4 shows the nucleotide sequence of the promoter DNA obtained from pBT Ml28. '
  • FIG. 5 shows a construction diagram of the plasmid pBTM13 shown in Reference Example 6.
  • FIG. 6 is a restriction map of 'pBT Ml 27 incorporating a neutral protease arrestor.
  • Fig. 7 and Fig. 8 are diagrams showing a subling of a child with a neutral proteathesis.
  • Fig. 9 is a restriction map of DNA containing a neutral protease arrestor.
  • Bg is Bgin
  • T is Taql
  • Sa is Sac ac
  • Ha is HaeIH
  • S is Sau3AI
  • A is Accl
  • Be is Be1I
  • H Indicates Hind HI.
  • FIG. 10 shows a part of the nucleotide sequence of the neutral protease gene.
  • FIG. 11 and FIG. 12 show the construction diagrams of the plasmids PT ⁇ X 201 and ⁇ ⁇ 374 shown in Reference Example 10 respectively.
  • Fig. 13 and Fig. 14 show a part of the sequence coding for the mature protein of Penicillinase in ⁇ ⁇ ⁇ ⁇ 374.
  • FIG. 4 is a diagram showing a method for synthesizing a human lysozyme gene by assembling and linking genes.
  • the sample was electrophoresed in 0 mM Tris-HCl, 100 mM boric acid, 2 mM EDTA (pH 8.0)] at 140 V for 2 hours. After the electrophoresis, the gel fragment containing the 0.63 kb DNA fragment was sealed in a dialysis tube, submerged in a buffer for electrophoresis, and the DNA fragment was eluted from the gel electrically! : DcDonell, M.W. et al., J.Mo ".B
  • 1 g of the plasmid PSH 19 was added to 2 units of the restriction enzyme BamHI and 2 units of the restriction enzyme Sa1I in a reaction solution of 201 [10 mM MTris-HCl (pH 8 . 0), 7 m MM g C 1 2, 1 0 0 m MN a C 1, 2 m M 2- mercapto Bok ethanol] 3 7 ° C in two hours after the action, the reaction mixture 0 Electrophoresis was carried out on an 8% agarose * slab gel under the conditions described above. After electrophoresis, 8.
  • the 0 kb DNA fragment was separated from the gel by the above-described method, deproteinized with phenol, and precipitated with cold ethanol (see FIG. 1).
  • 3 g of plasmid p PH012 DNA was mixed with 2 units of restriction enzyme Sa1I in a reaction mixture of 201 (10 mM MTris-HC1 (P H 7. 5), 7 m MM g C 1 2, 1 7 5 m MN a C 1, 0. 2 MEDTA, 7 m M 2- mercapto Toeta Nord] 3 in 7. C. After acting for 2 hours, the proteins were deproteinized with phenol, and the DNA was precipitated with cold ethanol. To 3 g of this DNA, 12 units of BAL31 nuclease (Bethhesda Research Laboratories) was added to a reaction mixture of 501 [20 mM MTris-HC1 (pH 8.
  • XhoI linker d (CCTCGAGG) (New Zealand Bio Labs) was added 3 units of T4 poly, nucleotide 'kinase [Takara Shuzo Co., Ltd.].
  • a 50 1 reaction mixture C 50 mM MT ris-HCl (pH 7.6), 10 mm MM g C 12, 10 mM 2 — Menolecaptoethanol, 100 MATP] The reaction was carried out at 37 ° C for 1 hour to phosphorylate the 5 'end.
  • Pgap 491 Holland, J. P. et al., J. Biol. Chem., 2558, 5291 (1983)
  • GLD GLD from the N-terminal side 5′—AGCAACTCTAACCAT—3 ′
  • Crea R. et al.
  • Ci was added to 1 g of the oligonucleotide.
  • Bruno - [JZ P] ATP (a mersham Co.) and 1 0 units T 4 poly Nuku Reochi de kinase and the 3 0 1 of the reaction solution [5 0 m MT ris -.
  • HC 1 (P H 7 6 ), 10 mM MM g C 12, 10 mM M 2 —mercaptoethanol] for 37, 30 minutes, and labeled at the end with, 200 mM MEDTA ( pH 8.0) was added, and the protein was deproteinized with 1 volume of phenol.Then, TEN buffer (1 Om MTris-HC1 (pH 8.0), 200 mM Na C 1, 1 m MEDTA] Sepharose 4B (? 1131: 111 & (1: 1 & Co.)) * Column (0.25 x 25 cm) ) To collect the labeled oligonucleotides that are eluted near the voidvolume was used as a probe for screening.
  • P TR26 2 (Roberts, T, M. et al., Gene 12, 123 (1980)) 0.1 g digested with Hin (i III) and DNA 0 of domain number 7 And 2 g were mixed with each other by the action of T4 DNA ligase under the conditions described in Reference Example 1. Using the reaction mixture, E. coli DH1CManiatis, T. et al. loning, ColdSpring * —Harbor Laborator 2 5 4 —2 5 5 (1 9 8 2)] was transformed to obtain about 1,200 ⁇ of tetracycline shochu transformant.
  • Plasmid PGLD 9 DNA 1 0 0 g to 5 0 Yuni' Bok restriction enzyme H ind III 2 0 0 1 of the reaction solution [1 O m MT ris - HC 1 (. P H 7 5), 7 m MM g C l 2 , 60 m MN a C 1]
  • the DNA polymerase I large 'fragment was allowed to act on 1 g of the 0.36 kb DNA fragment under the conditions described in Reference Example 1 to change the cohesive end of Taq I to a blunt end.
  • 1 g of this fragment and 50 ng of the phosphorylated XhoI linker described in Reference Example 1 were mixed, and T4 DNA ligase was allowed to act under the conditions described in Reference Example 1.
  • Escherichia coli-Yeast Shuttle 'vector-pSHl95 (5 g) and 6-unit restriction enzyme Sa1I in a reaction solution of 201 (1 Om MTris-HC1 (pH 7.5), . 7 m MM g C 1 2 , 1 7 5 m M a CI, 0 2 m MEDTA, 7 m M 2 - mercapto Toeta no
  • Plasmid p SHl9 Reaction solution of 50 units of 10 units of restriction enzyme BamHI and XhoI in 10 g of 1 DNA [10-m 'MTris-HCl (. p H 7 5), 7 m MM g C l 2, 1 0 0 m MN a C 1, 7 m M 2 - Menorekapu Toeta Nord] 3 in 7. C, and allowed to act for 2 hours, and then subjected to electrophoresis using 1.0% agarose slab gel under the conditions described in Reference Example II. After the electrophoresis, a 8.0 kb DNA fragment was separated from the gel by the method described in Reference Example 1.
  • the 8. O kb DNA fragment 5 0 0 ng was mixed with 0. 3 6 kb DNA fragment 2 0 0 ng and 0. 7 5 kb DNA fragment 2 0 0 n g according to 2 of Example 2, Reference The binding was effected by the action of T4 DNA ligase under the conditions described in Example 1. Escherichia coli DH1 is transformed using the reaction solution, and a recombinant having the plasmid pGLD906 to which three types of DNA fragments are bound is separated from the ampicillin-like transformants. did. WIPO ⁇ Reference example 3
  • Plasmid PBTM1226 was prepared as follows according to the method of Wi11ams et al. Obtained from Fermentation Research Institute
  • DNA was prepared from Bacilluspumilus NCIB 860 (IF.O-12089), and the DNA (6.5 g) was combined with 40 units of restriction enzyme EcoRI and 37, 1 hour. After the reaction and cutting, the mixture was heated at 68 for 15 minutes, and subjected to ethanol precipitation. On the other hand, plasmid PUB110 (2.0 g) was reacted with 20 units of restriction enzyme EcoRI at 37 ° C for 1 hour, cut, and then heated at 68 for 15 minutes. Ethanol precipitation was performed. Both precipitates were dissolved in water and mixed, and the reaction mixture (1001) containing SO nmole ATP, 100 units of T4 DNA ligase (Takara Shuzo) and ligase buffer was added.
  • Plasmid was prepared from a transformant of Chloram phenicol shochu, which was named PBTM124.
  • PBTM124 plasmid PBTM 124 (2.5 g) was reacted with 14 units of restriction enzyme Pstl for 37 hours for 37 hours, cut, and heated with _68 T1 for 15 minutes, followed by ethanol Knol precipitation was performed.
  • PBTM 125 2.5 ml of Brasmid PBTM 125 was mixed with 18 units of restriction enzyme BamHI and 15 units of restriction enzyme Bg for 1 hour at 37 ° C and 1 ° C. The mixture was cut by reaction, heated at 68 for 15 minutes, and then ethanol precipitated. After dissolving the sediment in water, 1 I in a reaction solution (1001) containing 66 nmo 1 e of ATP, 13 units of T4 DNA ligase (Takara Shuzo) and ligase buffer was added. : For 28 hours, and used to transform Bacillus' Sacillus MI114. Plasmid was prepared from a kanamycin shochu transformant, and the plasmid was named PBTM126 (see FIG. 3). Reference example 4
  • the promoter cloning vector pBTM126 (2.1 ⁇ g) obtained in Reference Example 3 was treated with the restriction enzyme Pstl (8 units) 37. C, cut for 1 hour, cut with restriction enzyme EcoRI (5 units) for 37, 1 hour, heat at 688 for 15 minutes to stop the reaction, Was performed.
  • the chromosome (6.2 g ) of Baci 1 1 ussubti 1 is JB — 1 166 (IFO — 141 4 4) was analyzed by P stl (24 units) and Eco RI, respectively. It was cut for one hour at 37 ° C, heat-treated at 68 ° C for 15 minutes, and then subjected to ethanol precipitation.
  • the plasmid pBT Ml28 (221g) described in Reference Example 4 was 37 with Ps ⁇ 1 (208 units) and EcoRI (220 units), respectively. After cleavage with C for 1 hour, the mixture was subjected to 10% polyacrylamide gel electrophoresis. The gel was immersed in ethidium bromide solution for staining, and the promoter DNA fragment detected by an ultraviolet lamp was recovered. The DNA fragment was electrically eluted from the gel, and then extracted with ethanol and ether, and precipitated with ethanol. Slow precipitation
  • Plasmid PBTMI28 (7.7, "g) was reacted with the restriction enzyme Pstl (51 udites) for 37 hours, cut for 1 hour, and then cut into 0.75 units of E. coli alkaline phosphatase.
  • the reaction product was collected by phenol extraction and ether extraction, and then ethanol precipitation, and the precipitate was dissolved in a small amount of water.
  • Chromosome DNA was prepared in accordance with the following formula: MethodsinEnzymo1ogy, 68, 342 (19779). 8.8 g of the chromosomal DNA was incised by reacting with 36 units of restriction enzyme Bgin for 37, 50 minutes, heat-treated at 65 ° C for 15 minutes, and added with ethanol to add DNA. Was precipitated. On the other hand, cloning vector pBTM119 (Japanese Patent Laid-Open No. 59-55887) (2.9 g) was added to 30 units of restriction enzyme Bg1 1 and 37 ° C. C.
  • BTM127 This plasmid is located at the Bgin site of the plasmid pBTMl19, and contains a Bgin fragment containing the neutral protease gene of B. amy1 o1 iquefaciens (IFO-141). K b) is imported
  • the plasmid pBTMl27 (80, "g) obtained in Reference Example 7 was reacted with the restriction enzyme Bgin (200 units) 37 for 1 hour, followed by cleavage for 1 hour. A 7% agarose gel was electrophoresed, and the gel of the band with the smaller molecular weight was cut out. The DNA in this gel was electrophoretically eluted. , 1982), about 10 g of a Bgin fragment (3.9 kb) was obtained. The obtained Bgin fragment (3 g) was digested with restriction enzyme Hindm (6 units).
  • Plasmid PBTM 515 is a plasmid in which the A and C fragments are inserted in the Hindm site of plasmid PBTM 126 in opposite directions, and plasmid pBTM 508 is plasmid The B fragment was inserted into the Hindill site of PBTM126 (see Fig.
  • Plasmid PBTM127 (1 g) was treated with EcoRI (3 units) and Bgin (2 units), and plasmid pUB110 (l, "g) was treated with EcoRI. (3 units) and B am H
  • a halo-forming strain was selected from the kanamycin shochu transformants, and the plasmid was selected as pBTM5.
  • the EcoRI-Bg1 ⁇ fragment is composed of the Bg1 P of PBTMI27, a part of the B fragment of the fragment, and the C fragment.
  • PBTM 5 15 (100, "g) was digested with restriction enzymes Bgin (200 units) and HindE (200 units) at 37 ° C for 1 hour, and cut. After 1% agarose gel electrophoresis, about 1.9 Kb of this gel DNA was recovered by electrophoretic elution (manual for arresting children, edited by Yasutaka Takagi, Kodansha). 0 g of the C fragment was obtained.
  • FIG. 9 shows a restriction map of the C fragment.
  • B, subti 1 is 1 A2 7 4 (Bacillus subti 1 is 1 A274 / p BTM5 15), which retains the plasmid pBTM5 15 Four Deposited as 377 7 and deposited under the Budapest Treaty with the Research Institute of Microbial Industry and Technology (FRI) of the Ministry of International Trade and Industry of Japan as FERMBP-622.
  • FIG. 10 shows the nucleotide sequence from the Bg1 site of the C fragment and the translated amino acid sequence.
  • TTGCAG TTGCAG
  • E ⁇ gamma a promoter
  • E 6 37 AGTTTT
  • GAG AT TGCT the 10 region
  • TAGTTTTATA the 10 region
  • the fact that the Bgin—Sau3A1 fragment (530 bp) (see FIG. 9) has promoter activity is based on the fact that the promoter-cloning vector PFTB 281 (Yoshimura et al. The above-mentioned promoter is expected to be functioning because it was confirmed by using the Proceedings of the Annual Meeting of the Showa 58 Meeting, page 28). SD area and open frame exist, from 1st methionine to 27th or 28th alanine The amino acid sequence after the second alanine is the neutral protease sequence reported in the literature !; P. L. Levy et al., Proc. atl. A cad.
  • Plasmid p BTM containing the promoter of the neutral protease gene on the C fragment of BTM5115, a region encoding the signal peptide, and a region encoding the upstream of the peptide.
  • Plasmid PTR10 (see Fig. 11) formed by inserting the Sau3A1 fragment (about 530 bp) into the BamHI site of PFTB281 was digested with EcoRI. An EcoRI fragment of about 53 CTb p was isolated. This fragment is digested with the restriction enzyme Haem, and an EcoRI-Haeltl fragment (about 340 bp) containing a region encoding the promoter and signal peptide up to the C-terminal region is isolated. did. In this fragment? : Hoi linker (CTCTCGAGG) was ligated using T4DNA ligase.
  • the plasmid pMB9 [F. Bolivareta 1., Gene, 75 (1977)] has a plasmid PEN 1 (Osaka University The SacI-PstI fragment (8.5 Kb) was isolated from (obtained from Prof. Yasuji Oshima) (see Fig. 10). This fragment contains the latter part of the signal peptide of penicillinase and the region encoding the mature protein, and the PstI site corresponds to the vicinity of the middle of the region encoding the signal peptide. I do.
  • PUB110 was digested with restriction enzymes Sphl and EcoRI, and a large SP hi-EcoRI fragment (3.5 Kb) was isolated by agarose gel electrophoresis and electrophoretic elution (No. See Figure 1 1).
  • the EcoRI-XhoI fragment (340 bp, 0.5 g) containing the promoter for the neutral protease arrestor and the signal peptide-encoding region obtained above, PENX
  • the Xhol — Sphl fragment (1.9 Kb, 0.5 g) derived from 374 and the Sphl — EcoRI fragment (0.5 g) derived from pUBllO were ligated to T4 DNA ligase. Subsequent ligation was carried out to effect the transformation of B.
  • subti 1 is 1 A274, and among the resulting kanamycin shochu recombinants,
  • PTEX201 5.7 Kb
  • B. subtilisl A 274 B aci 11 ussubtilis 1 A 274 / PTEX 210
  • IF 0—1 4 3 It has been deposited as FERMBP-619 with the Ministry of International Trade and Industry under the Ministry of International Trade and Industry's Institute of Industrial Science and Technology (FRI) under the Butapest Treaty.
  • Each of the oligonucleotide blocks was prepared by solid-phase synthesis, but the nucleosides used as raw materials were immobilized.
  • thymidine resin Aminomethylpoly * styrene resin [1% divinylbenzene, amino group: 0.21 meq Zg, 100-250 mesh) known methods (for example, Miyoshi et al., N ucleic A cids Res., _8_, 5507 (1980))] and prepared by combining 5'-dimethoxytritylthymidine- 3 '-—- succinate
  • the method of dinucleotides is known by the known method [Fig. 15, C. ⁇ . Reese, L.
  • the phosphorylated oligomer solution at the 5'-position was divided into groups [I] to [V] as shown in Fig. 17 and mixed.
  • the total liquid volume was adjusted to be 2361, 66 mM MT ris hydrochloric acid (PH 7.6), 6.6 mM magnesium chloride, 500, "MATP. C for 3 minutes, quench with ice, and soak again in 75 ° C hot water.
  • the sample was left to stand at 15 ° C for 10 minutes, then 0.2 M mercaptoethanol (1311) and T4 DNA ligase (EC 6.5.1.1.1) were added. ; 2.5 U / 1, 1 ⁇ 1) and incubated for 20 hours at 15 ° C.
  • group (IV) (approximately 74 bp) showed a large number of bands due to side reactions.
  • each group (1.5-2, " g ) was ligated with T4 DNA ligase and the complete human lysozyme arrested (approx. This product was obtained on a 5% polyacrylamide gel, about 404 b.
  • both 5 'ends of the DNA were enzymatically phosphorylated by a conventional method.
  • the restriction enzyme Xhol (50 units) was added to the plasmid PGLD906-6 (10 ⁇ g) described in Example 3 in a reaction mixture of 501 (reaction solution for XhoI, supra). After working for 1 hour, the gel was subjected to 1.5% agarose 'slab gel electrophoresis (150 V, 1 hour). After the electrophoresis, the gel piece containing the 409 b DNA fragment was sealed in a dialysis tube. And immersed in the electrophoresis buffer. Eluted (Dc Done 11, M.W., et al., Supra) Extract the solution in the dialysis tube with phenol and ether, then add NaCl to 0.2 M. Subsequently, 2 volumes of cold ethanol were added and the mixture was precipitated at 120.
  • the restriction enzyme Xhol (5 units) was added to the plasmid PPHO17 (1 g) constructed in Reference Example 1 at 37 ° C in a 101 reaction mixture (Xhol reaction mixture). After allowing to act for 1 hour, the protein is deproteinized with phenol, and 50 ng of the DNA fragment containing the human lysozyme gene is mixed with the DNA (10 ng) precipitated with cold ethanol and mixed. DNA was bound according to the procedure described above. Escherichia coli DH-1 was transformed according to the method described in Example 3, and the PH05 promoter and the human lysozyme gene in the very direction were located at the XhoI site of pPHO17 from among the transformants. Into which the introduced plasmid p PH 0 1 ys 101 was obtained.
  • a plasmid PGLD906-5 was obtained in which a GLD promoter and a human lysozyme gene were inserted in the XhoI site of PGLD906.
  • the lysate was centrifuged at room temperature at 5,000 rpm for 10 minutes to obtain a supernatant (2 ml). Then M eyer's way! : The supernatant was acidified according to J. Biol. Chem. 113, 303 (19336)], cooled ethanol was added, heat treatment was performed, and the supernatant was collected again. Further, ethanol was added thereto, and the mixture was centrifuged to collect a precipitate. To the precipitate was added 0.4 ml of 50 ⁇ -phosphate phosphate buffer ( ⁇ 7.4) to dissolve the precipitate, and 0.1 ml of the solution was used to determine the activity. Served in quantity.
  • Human lysozyme has pharmacological actions such as anti-inflammation, hemostasis, tissue regeneration, and antitumor properties, and is used as an anti-inflammatory enzyme in eye drops and as a food preservative. Moreover, when used for medical purposes, it does not cause side effects due to an immune response like lysozyme derived from nit egg white, but conventionally, only a small amount has been obtained. '' By providing the human lysozyme synthesis gene of the present invention, it has become possible to supply a large amount of human lysozyme useful as a pharmaceutical product as described above. It is.

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Abstract

ADN possédant un gène synthétisant un lysozyme humain, procédé de préparation, cellules transformées avec l'ADN, procédé de préparation du transformant, et procédé de préparation du lysozyme humain par culture du transformant. Il est possible de produire en grandes quantités un lysozyme humain possédant des effets anti-inflammatoires, hémostatiques, régénérateurs des tissus et antinéoplastiques, utilisé comme agent enzymatique anti-inflammatoire pour bains oculaires ou analogues, sans effets secondaires de réactions immunitaires, ou comme antiseptique.
PCT/JP1984/000546 1984-11-14 1984-11-14 Gene synthetisant un lysozyme humain WO1986002947A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/JP1984/000546 WO1986002947A1 (fr) 1984-11-14 1984-11-14 Gene synthetisant un lysozyme humain
EP85114427A EP0181634A3 (fr) 1984-11-14 1985-11-13 Gène synthétique pour lysozyme humaine
JP60252899A JPS61158793A (ja) 1984-11-14 1985-11-13 ヒト・リゾチ−ムの合成遺伝子

Applications Claiming Priority (1)

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PCT/JP1984/000546 WO1986002947A1 (fr) 1984-11-14 1984-11-14 Gene synthetisant un lysozyme humain

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WO1986002947A1 true WO1986002947A1 (fr) 1986-05-22

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Kogyo Gijutsuin Kenkyu Kikan, Dai 20 Kai Kobunshi Kenkyu Seika Happyokai Shiryo "Kobunshi Zairyo to Life Science no Setten" Zaidan Hojin Nippon Sangyo Gijutsu Shinko Kyokai, 31. January. 1985 (31.01.85) Muraki Michio and three others, "Hito.Rizo Team Idenshi no Kagaku Gosei to Daichokin niokeru Hakken" p.52-59 *

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