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WO2003100065A1 - Processus de production industrielle de proteines de soie et de proteine de type soie genetiquement modifiee a fonction impartie - Google Patents

Processus de production industrielle de proteines de soie et de proteine de type soie genetiquement modifiee a fonction impartie Download PDF

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WO2003100065A1
WO2003100065A1 PCT/JP2002/005010 JP0205010W WO03100065A1 WO 2003100065 A1 WO2003100065 A1 WO 2003100065A1 JP 0205010 W JP0205010 W JP 0205010W WO 03100065 A1 WO03100065 A1 WO 03100065A1
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Prior art keywords
silk
sample
protein
dna
medium
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Japanese (ja)
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Tetsuo Asakura
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Tokyo University of Agriculture and Technology NUC
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Tokyo University of Agriculture and Technology NUC
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Priority to JP2004508303A priority Critical patent/JPWO2003100065A1/ja
Priority to PCT/JP2002/005010 priority patent/WO2003100065A1/fr
Publication of WO2003100065A1 publication Critical patent/WO2003100065A1/fr
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Priority to US11/197,315 priority patent/US20060019348A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms

Definitions

  • the present invention relates to a method for mass-producing a silk protein and a genetically modified silk-like protein provided with a function, and more particularly to a method for mass-producing a genetically modified silk-like protein provided with cell adhesion or elasticity or strength.
  • Silk is a high-strength, high-elastic fiber that is composed of amino acids contained in living organisms and is biocompatible, so it is used not only in clothing but also in various fields such as food and cosmetics. I have.
  • the primary structure of silk is characterized by a block copolymer in which several types of amino acid sequences (motifs) are repeated a dozen times.
  • motifs amino acid sequences
  • vectors obtained by fusing a strong T7 promoter to an expression vector have been widely used.However, because of the strong expression, the target protein is a strong stress against Escherichia coli. In such a case, there is a drawback that the protein has an effect on the growth of cells and the expression efficiency is not improved.
  • the present inventors have designed four types of functional silk-like proteins by arbitrarily selecting functional site motifs of silkworm silk-in, wild silkworm silk-in, elastin and fibronectin. After selecting an expression vector, selecting a host E. coli, and optimizing expression conditions for a functional silk-like protein having a repeat sequence, mass production of the designed functional silk-like protein using E. coli was performed. And found that this method can be applied to general silk proteins, and arrived at the present invention.
  • an object of the present invention is to provide a method for mass-producing silk proteins and silk-like proteins to which functionality has been imparted. Disclosure of the invention
  • An object of the present invention is a combination of two or more proteins selected from among silkworm silk fiproin, wild silkworm fibrous mouth, elastin, and fibronectin, wherein at least one of the above silkworm silk fibroin or wild silkworm silk fibrin is required.
  • a silk-like polymer consisting of a combination is designed, the minimum unit of the silk or the designed polymer is synthesized, and the synthesized polymer of the minimum unit is extracted from an expression vector containing a T7 promoter.
  • Integrate into at least one selected expression vector and then transfer the expression vector to BL21 (DE3) pLysS or BLR (DE 3) p Lys S, which is achieved by a method for producing a silk-like protein, wherein the Escherichia coli is grown on a medium selected from a complex medium. Was done.
  • the temperature for growing Escherichia coli be lowered by 2 to 7 ° C. below the optimal temperature for growing Escherichia coli.
  • an expression vector containing a T71ac promoter as an expression vector containing a T7 promoter is preferably used. It is preferable to use them.
  • Figure 1 shows the results of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) when SLP 2, 4, and 6 were expressed in E. coli strain BL 21 (DE 3) p Lys S. .
  • FIG. 2 shows the results of separation of SLPA4 by SDS-PAGE and detection by Western plot using a His-Tag antibody.
  • FIG. 3 shows the results of Western blot using His-Tag antibody after SELP8 was separated by SDS-PAGE.
  • FIG. 4 shows the results of separation of SLP F5 by SDS-PAGE followed by detection with a ⁇ ⁇ stamp mouth using a His-Tag antibody.
  • the term “silk 5” is a protein secreted from the posterior silk gland of the silkworm (Borabyx mori), and the term “silk 5” is a protein secreted from the posterior silk gland of the silkworm. .
  • Elastin is a type of elastin that exists in the tissues of various organisms.
  • elastin there is a high frequency of a five-residue amino acid sequence Val-Pro-Gly-Val-Gly (Sequence Table 1).
  • sequence Table 1 For example, for elastin in chicks, Bressan, G. k., Argos, P. and Stanley, KK Repeating structure of chick tropoelastin revealed by complementary DNA cloning, Biochemistry 26, 1497—1503 (1987), See Raju, K. and Anwar, RA Primary structures of bovine el astin a, b and c deduced from the sequences of cDNA clones, and J. Biol. Chern. 262, 5755-5762 (1987)). Therefore, the functional site motif of elastin in the present invention means the amino acid sequence of the above five residues.
  • fibronectin is a cell-adhesive protein that exists in the extracellular matrix of various organisms.
  • This cell-adhesive protein is composed of four residues of Arg_Gly—Asp—Ser (Sequence Table 2).
  • the amino acid sequence is expressed in association (Reference: Pierschbacher MD, Rouslahti E, Nature 309 30-33 (1984)). Therefore, the four amino acid sequence Arg-Gly-Asp-Ser contained in fibronectin is a functional site motif of fibronectin.
  • Thr-Gly-Arg_Gly_Asp-Ser-Pro-Ala (Toroki-Gly-Asp-Ser) is used in order to allow Arg-Gly-Asp-Ser to retain a secondary structure necessary for expressing cell adhesion.
  • System IJ Table 3 was used as the functional site motif. Therefore, the sequence of these eight residues is not necessarily required.However, considering the biocompatibility when used as artificial skin, etc., the extra sequence described above is the Arg contained in human fibronectin. — Gly — Asp — Serative amino acid sequence around the Ser sequence.
  • the silk proteins proposed by Lewis et al. Designing protein treasures based on the idea that the physical properties and functionality of silk will change by changing the types of motifs and how they are combined.
  • motifs contained in natural silk fibers, elastin, and functions specific to fipronectin they each exhibit heat responsiveness (the property of being insoluble in water by increasing the temperature) and cell adhesion).
  • Various motif sequences are combined.
  • SLP silk-like protein
  • SELP sirolimus and elastin-like protein
  • SLPF silk-like protein with fibronectin
  • SLP Abbreviation of Silk-like Protein: Amino acid sequence (Gly Ala Gly Ser Gly Ala) 3 (Sequence Table 4) contained in silkworm silk and amino acid sequence Gly in rich region of glycine contained in silkworm wild silk Gly Ala Gly Ser Gly Tyr Gly Gly Gly Tyr Gly His Gly Tyr Gly Ser Asp Gly Gly (Sequence Table 5) Combination
  • SLPA abbreviation for Silk-like Protein with poly-alanine: Amino acid sequence contained in silkworm silk Gly Val Gly Ala Gly Tyr (System U Table 6), Gly Ala Gly Ala Gly Tyr (SEQ ID NO: 7), Gly Val Gly Ala Gly Tyr and Gly Ala Gly Val Gly Tyr (SEQ ID NO: 8) and amino acid sequence (A) 18 (SEQ ID NO: 9) similar to the polyalanine region contained in wild silkworm silk combination
  • SELP Abbreviation for Silk and Elastin-like Protein: Amino acid sequence contained in silkworm silk (Gly Ala Gly Ser Gly Ala) 3 (Sequence Table 4) and amino acid sequence contained in elastin ( Gly Val Pro Gly Val) 2 (Sequence Table 10) Combinations
  • SLPF Silk-like Protein with Fibronectine: Amino acid sequence contained in silkworm silk (Gly Ala Gly Ser Gly Ala) 3 (sequence table 4) and amino acid contained in fipronectin Combination of IJThr Gly Arg Gly Asp Ser Pro Ala
  • Fibers must have crystalline and amorphous domains, and when designing new silk-like proteins, it is necessary to combine motifs to form these domains simultaneously.
  • motifs that form a crystalline region or an amorphous region in Eri silkworm silk, which is a type of domestic silk or wild silk, are respectively combined.
  • SELP and SLPF the functional stability motifs of elastin and fibronectin are combined with silk proteins for use as biomaterials, not just as fibers.
  • further new functions can be provided.
  • pET30a containing a T7 promoter as an expression vector, BL21 (DE3) pLysS or BLR (DE3) pL of an inducible expression type as a host E. coli for expression.
  • Select ys S. By combining these, until the addition of IPTG (isopropylthio-1 ⁇ -1D-galactoside) as an expression inducer, the target protein downstream of the ⁇ 7 promoter is expressed because ⁇ 7 RNA polymerase is not expressed. Therefore, stress on E. coli due to large overexpression is reduced.
  • IPTG isopropylthio-1 ⁇ -1D-galactoside
  • plasmid pLysS further expresses ⁇ 7 lysozyme and inactivates ⁇ 7 RNA polymerase, two-step suppression can be expected.
  • the expression of the target protein can be gently progressed by intentionally removing the optimal culture conditions for the growth of Escherichia coli. Can increase the yield. Therefore, in the present invention, it is preferable to set the culture temperature to 2 to 7 ° C. lower than the optimum temperature for the growth of E. coli.
  • the medium used in the present invention is particularly preferably a TB medium.
  • the concentration of I PTG added is preferably from 0.2 to 1.0 mM.
  • the pH is preferably 6.7 to 7.0.
  • the synthesized film-like oligonucleotide is defined as Tris (Tris) EDTA (10 mM Tris-HCl (pH 8.0), ImM EDTA (pH 8.0): hereinafter referred to as TE.
  • Tris Tris
  • EDTA 10 mM Tris-HCl (pH 8.0), ImM EDTA (pH 8.0): hereinafter referred to as TE.
  • the SLP monomer DNA and pUC118 vector sample were mixed at a ratio of 10: 1 (weight ratio), and the mixture was mixed with Takara Ligation Kit ver 2 slution I 16. C for 1 hour. After completion of the reaction, transformation was carried out using Combinent Cell DH5. The presence or absence of the inserted gene is confirmed by color selection using X—gal (5-promo 1—black 1—indolyl 1—j3—D—galactobyranoside), and DNA sheeting is performed on those containing the inserted gene. The sequence was confirmed, and a plasmid pUC-SLP (1) containing SLP monomer DNA was obtained. Construction of PUC-Link>
  • the cloning vector PUC118 used in this study does not contain a region digested by the restriction enzymes NheI and SpeI. Therefore, for the purpose of adding a recognition region for restriction enzymes Nhe I and Spe I to pUC118, an adapter was designed (Sequence Table 19), and pUC118-Link (a plasmid containing the designed adapter) was constructed. In addition, codons encoding methionine residues were arranged on both sides of the adapter in addition to the recognition regions for the restriction enzymes Nhe I and Spe I.
  • methionine residues are added to both sides of the inserted gene of the protein obtained by expression, and the methionine residue is specifically cleaved using cyanogen bromide, so that the sequence derived from plasmid is not included.
  • a sample was obtained.
  • the synthesized oligonucleotide was dissolved in TE (10 raM Tris-HCl (pH 8.0), ImM EDTA (pH 8.0)) at a concentration of 1 ⁇ g / ⁇ 1. After equimolar mixing of the respective complementary strands and heat treatment at 99 ° C for 30 seconds, the mixture was cooled to 37 ° C over 1 hour and left standing for 30 minutes to construct double-stranded DNA. After mixing equal amounts of each double-stranded DNA, the closing vector pUC118 (manufactured by Takara Shuzo Co., Ltd.) was digested with restriction enzyme Xba I at 37 ° C for 1 hour 30 minutes, and CIAP , And the treatment was performed at 37 for 30 minutes.
  • TE raM Tris-HCl (pH 8.0), ImM EDTA (pH 8.0)
  • the reaction solution was extracted and purified using a mixed solution of phenol: cloth-form: isoamyl alcohol (25: 24: 1) (weight ratio). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterile water to obtain a vector sample.
  • the adapter DNA and pUC118 vector sample were mixed at a ratio of 10: 1 (weight ratio), and ligated at 1 ° C. for 1 hour using Takara Ligation Kit ver. After the completion of the reaction, transformation was performed using the competent cell DH5a.
  • X The presence of the introduced gene by color selection using gal After confirming the absence, the DNA containing the introduced gene was subjected to DNA sequencing, and the sequence was confirmed to obtain a plasmid pUC-Link containing the adapter.
  • Both ends of the SLP monomer contain the Spe I and Nhe I restriction enzyme recognition regions.
  • the protruding ends of the fragments digested with the restriction enzymes Spe I and Nhe I are both synonymous and can bind to each other.
  • the newly combined sequence differs from both the Spe I and Nhe I restriction enzyme recognition regions and is not digested by Spe I and Nhe I. Utilizing this property, we constructed a plasmid pUC-Link SLP (n) containing DNA that encodes SLP n times by polymerizing SLP monomer in one direction.
  • Combinent cell DH5 ⁇ was transformed with pUC-SLP (1), and cultured at 37 ° C for 18 hours in a 2 ⁇ medium. Plasmid was extracted from the culture solution by the alkali-SDS method and dissolved in TE. The sample was simultaneously digested with Nhe I and Spe I (both from Takara Shuzo Co., Ltd.) for 1 hour and 30 minutes at 37 ° C., and SLP (1) was isolated from plasmid. The reaction solution was concentrated to 5 ⁇ l using MicroCon (Millipore), and subjected to electrophoresis using 1.5% agarose gel to cut out an insert DNA band. For extraction of Geno force, DNA of UltranoDA ( ⁇ Noretrafree DA) (manufactured by Millipore), the extract was concentrated to 51 using a regeneration microcon to obtain an imported gene sample.
  • UltranoDA ⁇ Noretrafree DA
  • pUC-Link was digested with NheI
  • CIAP was added and treated with alkaline phosphatase.
  • the reaction solution was extracted and purified using a mixed solution (25: 24: 1 in weight ratio) of phenol: cloth form: isoamyl alcohol. Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterile water to obtain a vector sample.
  • the DNA concentration in the inserted gene sample and the vector sample was confirmed by electrophoresis using a 1.5% agarose gel, and the inserted gene sample and the vector sample were mixed so that the force became 10: 1. Takara Ligation kit ver2 solution I was added, and the cells were ligated with 16 for 1 hour.
  • a competent cell DH5 ⁇ was transformed.
  • the cells were inoculated on an LB plate containing ampicillin and screened.
  • the colonies generated were picked up, inoculated on a 2 ⁇ medium, and cultured at 37 ° C for 18 hours.
  • Plasmid was extracted from the culture medium by the alkali-SDS miniprep method and dissolved in TE to obtain a sample. After simultaneously digesting the sample with Nhe I and Spe I, the presence and size of the inserted gene were confirmed by electrophoresis. Thereafter, DNA sequencing was performed, and the sequence was confirmed to obtain plasmid pUC-Link SLP (1).
  • pUC-Link SLP (1) was digested with NheI, CIAP was added thereto and treated with an alkaline phosphatase solution.
  • the reaction solution was extracted and purified using a mixed solution of phenol: chloroform: isoamyl alcohol (weight ratio: 25: 24: 1). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterile water to obtain a vector sample.
  • the DNA concentrations of the inserted gene sample and the vector sample were confirmed by electrophoresis using a 1.5% agarose gel, and the inserted gene sample and the vector sample were mixed at a weight ratio of 10: 1.
  • An equivalent amount of Takara Ligation kit ver2 solution I was mixed with the mixed solution, and allowed to bind at 16 ° C for 1 hour. After completion of the reaction, it was transformed Konbiten Toseru DH5 ⁇ .
  • the cells were inoculated on LB plates containing ampicillin and screened. The colonies generated were picked up, inoculated into a 2 ⁇ medium, and cultured at 37 for 18 hours. Plasmid was extracted from the culture medium by the alkali-SDS muprep method, dissolved in ⁇ , and used as a sample.
  • the pUC-Link SLP (2, 4, 6) obtained as described above was digested with restriction enzymes BamHI and Hind III (both from Takara Shuzo). After concentrating the reaction solution to 5 ⁇ 1 using a microcon, the insert DNA band was cut out by electrophoresis using a 1.5% agarose gel. UltrafreeDA was used to extract the DNA from the gel force, and the extract was again concentrated to 51 using a microcon to obtain an inserted gene sample. Use the expression vector pET30a (Novagen) with BamHI and HindIII? After shading, CIAP was added and treated with alkaline phosphatase.
  • reaction solution was extracted and purified using a mixed solution of phenol: chloroform: isoamyl alcohol (25: 24: 1 by weight). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.
  • the ligation reaction mixture was transformed with the competent cell DH5cII, inoculated on an LB plate containing kanamycin, and screened. The generated colonies were picked up, inoculated on 2 XYT medium, and cultured. Extract the vector from the medium by alkaline SDS method, dissolve in TE And After digesting the sample simultaneously using Nhe I and Spe I, the presence and size of the insert DNA were confirmed by electrophoresis, and the rooster S row was confirmed by DNA sequencing, and the expression vector pET-SLP (2, 4, 6) were constructed.
  • Plasmid pET—SLP (2, 4, 6) obtained as described above, and host E. coli BL21 (DE3) pLysS (Novagen) having each were added to 1 ml of 2 X TY (25 ⁇ l). g / ml kanamycin, 25 ⁇ g / ml chloramphenicol) The cells were cultured at 37 ° C for 16 hours in a liquid medium. Next, 100 ⁇ l of the culture solution was added to an L-shaped tube containing 5 ml of the 2xTY (0.25 ⁇ g / ml kanamycin, 25 Ug / inl chloramphenicol), and the mixture was heated at 37 ° C. in 1 hour (0D 6 .. 0.
  • IPTG-dependent bands were observed at 19 kDa for SLP2, 29 kDa for SLP4, and 40 kDa for SLP6. This proved that the addition of IPTG induces the SLP gene and obtains a strain that overexpresses SLP.
  • host E. coli BL21 (DE3) pLysS with plasmid pET-SLP (2, 4, 6) was added to 2.5 ml of 2 x TY (25 ⁇ g / ml kanamycin, 25 ⁇ g / ml (Chloram-Fuecol)
  • 2 x TY 25 ⁇ g / ml kanamycin, 25 ⁇ g / ml (Chloram-Fuecol)
  • the cells were cultured in a liquid medium at 37 ° C for 16 hours.
  • the culture solution was added to 250 ml of 200 ml of 2xTY (25 ⁇ g / ml kanamycin, 25 ⁇ g / ml chloramphene-col) in 500 ml
  • IPTG final concentration lmM
  • the cells were further cultured for 2 hours and harvested (5000 rpm, 10 minutes, 4 ° C) to obtain cells.
  • the obtained cells were stored at 130 ° C.
  • Table 1 shows the yield of each protein with a molecular weight of 19 kDa, 29 kDa, and 40 kDa.
  • the amino acid sequence of the N-terminal several residues was determined by the N-terminal amino acid sequence.
  • Each protein was separated from the contaminating proteins by electrophoresis using a polyacrylamide gel, and transferred to a PVDF (polyvinylidene fluoride) membrane using Saltprot 2-S (manufactured by Sartorius). After the transfer, the cells were stained with a staining solution for 5 minutes, and the band of the target protein was cut out using scissors washed with methanol and washed.
  • the N-terminal amino acid sequence was determined using an ABI 473 gas-phase Edman sequencer. This result was consistent with the expected amino acid sequence, confirming that the expressed protein was a protein derived from plasmid.
  • methionine residues are arranged on both sides of the inserted gene by adapters. Since SLPs 2, 4, and 6 themselves do not contain methionine residues, proteins that do not contain plasmin-derived amino acid sequences such as tags can be obtained by chemically cleaving methionine residues. The methionine residue of the protein was cleaved, and the amino acid sequence of the N-terminal several residues was determined by the N-terminal amino acid sequence.
  • SLP 6 was taken in a 10 mg eppendorf tube and dissolved in 90% formic acid. After confirming complete dissolution, miliQ water (ultrapure water) was diluted to a final concentration of 70% formic acid. After adding 1 O mg of cyanogen bromide to the sample solution and dissolving it, it was completely shielded from light with aluminum foil and allowed to stand at room temperature for 12 to 48 hours. After adding 10 times the amount of miliQ water to the reaction solution and terminating the reaction, dialysis was performed using distilled water as an external solution, followed by freeze-drying to obtain a white powder. The obtained white powder was dissolved in 2 ⁇ sample buf fer, and the molecular weight was compared by SDS-PAGE.
  • the synthesized film-form oligonucleotides were dissolved in TE (1 OmM Tris-HCI (pH8.0), ImM EDTA (pH8.0)) to a concentration of 1 ⁇ g / ⁇ 1. . Equimolarly mix each of the complementary strands, heat treat them at 30 ° C for 30 seconds, cool them to 37 ° C over 1 hour, and let them stand for 30 minutes to be represented by Sequence Listing 24 and 25 Two double-stranded DNAs encoding the amino acid sequence were constructed.
  • the cloning vector pUC118 was digested with the restriction enzyme BamHI at 37 ° C for 1 hour and 30 minutes, added with CIAP, and treated at 37 ° C for 30 minutes.
  • the reaction solution was extracted and purified using a mixed solution (25: 24: 4 in weight ratio) of phenol / form / isoamyl alcohol. Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterile water to obtain a vector sample.
  • Each double-stranded DNA was mixed with the pUC118 vector sample at 10: 1, and the mixture was mixed with TaKaRa Ligation Kit ver 2 solution I 16. 1 hour at C Ligation was performed to prepare a double-stranded DNA encoding the amino acid sequence represented by Sequence Listings 24 and 25. After the completion of the reaction, transformation was performed using the competent cell DH5a. The presence or absence of the inserted gene is confirmed by color selection using X-gal, DNA sequencing is performed on those containing the inserted gene, and the sequence is confirmed, whereby the DNA sequence encoding polyalanine (Sequence Table 24) can be identified.
  • a plasmid pUC-GX containing a ⁇ A sequence encoding an alternating copolymer of plasmid pUC-ALA and glycine GX (X Ala, Tyr, Val) (SEQ ID NO: 25) was obtained.
  • pUC-ALA was transformed with the competent cell DH5 ⁇ and cultured in 2xYT medium at 37 ° C for 18 hours. From the culture broth, the alkali was extracted by the SDS method and dissolved in TE. The sample was digested simultaneously with Nhe I and Spe I at 37 ° C. for 1 hour and 30 minutes, and ALA was isolated from plasmid. After concentrating the reaction solution to 5 ⁇ l using a microcon, the insert DNA band was cut out by electrophoresis using a 1.5% agarose gel. UltrafreeDA was used to extract the DNA of the gel force and the like, and the solution extracted using a regenerative microcon was concentrated to 5 ⁇ l to obtain an imported gene sample.
  • CIAP was added and treated with alkaline phosphatase.
  • the reaction solution was extracted and purified using a mixed solution of phenol: chloroform: isoamyl alcohol (weight ratio: 25: 24: 1). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.
  • Combinent cell DH5 ⁇ was transformed using pUC-SLPA (1), and cultured in a 2C medium at 37 ° C. for 18 hours. Plasmid was extracted from the culture solution by the alkali-SDS method and dissolved in TE. The sample was digested simultaneously with Nhe I and Spe I at 37 for 1 hour and 30 minutes to isolate SLPA (1) from plasmid. After concentrating the reaction solution to 5 ⁇ l using a microcon, the insert DNA band was cut out by electrophoresis using a 1.5% agarose gel. The gel force was extracted using Ultrafree DA, and the solution extracted using a microcon was again concentrated to 5 ⁇ l to obtain an inserted gene sample.
  • pUC-SLPA (1) was digested with NheI, CIAP was added thereto and treated with alkaline phosphatase.
  • the reaction solution was extracted and purified using a phenol: clog-form: isoamyl alcohol mixed solution (weight ratio: 25: 24: 1). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a solid sample.
  • the inserted gene test was performed by electrophoresis using 1.5% agarose gel. Check the DNA concentration in the sample and the vector sample, mix the introduced gene sample and the vector sample so that the ratio becomes 10: 1, add an equal amount of Takara Ligation kit ver2 solution I to the mixture, and add 16 ° C. C for 1 hour.
  • a competent cell DH5 ⁇ was transformed.
  • the cells were inoculated on an LB plate supplemented with ampicillin and screened.
  • the colonies generated were picked up, inoculated on a 2 ⁇ medium, and cultured at 37 ° C. for 18 hours.
  • Plasmid was extracted from the culture medium by the alkali-SDS miniprep method and dissolved in TE to prepare a sample. After simultaneously digesting the sample with Nhe I and Spe I, the presence and size of the introduced gene were confirmed by electrophoresis. Subsequently, DNA sequencing was performed, and the sequence was confirmed to obtain plasmid pUC-SLPA (2) (dimer).
  • pUC-SLPA (4) (tetramer) was constructed by inserting SLPA (2) into pUC-SLPA (2).
  • pUC-SLPA (4) was digested with BamHI. After concentrating the reaction solution to 5 ⁇ l using a microcon, the band of the insert DNA was cut out by electrophoresis using a 1.5% agarose gel. Ultrafree DA was used to extract DNA from the gel, and the solution extracted using a microcon was concentrated to 5 to obtain an inserted gene sample.
  • CIAP was added and treated with alkaline phosphatase.
  • the reaction solution was extracted and purified using a mixed solution (25: 24: 1 by weight ratio) of phenol: cloform form: isoamyl alcohol. Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.
  • the DNA concentration in the inserted gene sample and the vector sample was confirmed by electrophoresis using a 1.5% agarose gel.
  • the mixture was mixed so that the mixture became 10: 1, an equal amount of Takara Ligation kit ver2 solution I was added to the mixture, and the mixture was allowed to bind at 16 ° C for 1 hour.
  • the ligation reaction was transformed using DH5a, inoculated on an LB plate containing kanamycin, and screened. The generated colonies were picked up, inoculated on 2 XYT medium, and cultured.
  • the vector was extracted from the culture medium using the alkaline-SDS method and dissolved in TE to obtain a sample. After digesting the sample simultaneously with Nhe I and Spe I, the presence and size of the insert DNA were confirmed by electrophoresis. Next, the expression vector pET-SLPA (4) was constructed by confirming the sequence by DNA sequencing.
  • Plasmid pET_SLPA (4) Host E. coli BL21 (DE3) pLysS, each containing 1.5 ml of 2 x TY (25 g / ml kanamycin, 25 ⁇ g I ml chloramphenicol) liquid medium at 37 ° C For 16 hours. Next, add the culture medium ⁇ ⁇ ⁇ ⁇ ⁇ to a test tube containing 5 ml of 2 x TY (25 g / ml kanamycin, 25 g / ml chloramphenicol) at 37 ° C for 1 hour. (0D600 0.5 to 0.7 (Shimadzu UV-160)).
  • IPTG final concentration ImM
  • 100 ⁇ l of the medium was collected in an Eppendorf tube every hour and cultured for up to 4 hours.
  • the collected medium was separated by centrifugation (14,500 rpm, 5 minutes, 4 ° C), the supernatant was discarded, the pellet was dissolved in 2X sample buffer, and heat-treated at 100 ° C for 5 minutes to prepare a sample for SDS-PAGE. .
  • SLPA4 was detected by Western blot using His-Tag antibody (Fig. 2).
  • Plasmid pET—Host E. coli BL21 (DE3) pLysS with SLPA (4) was added to 1.5 ml of 2xYT (25 ⁇ g / ml kanamycin, 25 ⁇ g / ml chloramphenicol) liquid medium.
  • the cells were cultured at 37 ° C for 16 hours.
  • the culture solution was added to a test tube containing 12 ml of 2xYT (25 ⁇ g / ml kanamycin, 25 ⁇ g / ml chloramphenicol) and cultured at 37 ° C for 16 hours.
  • Buffer B To the resulting precipitate (lOOmM NaH 2 P0 4, 10mM Tris- Cl, 8M urea, pH 8.0) was added and subjected to sonication. The cell suspension obtained here was centrifuged (10,000 rpm, 40 minutes, 4 ° C), and the supernatant was recovered.
  • the obtained supernatant was used as an additional sample, and purification was performed by affinity monochromatography (flow rate: 15 to 2 Oml / h) using a column packed with Ni-agagarose beads previously equilibrated with the same buffer.
  • Each eluate was fractionated, and the fraction containing the target protein was confirmed by SDS-PAGE and collected.
  • the obtained fraction was dialyzed while appropriately exchanging the external solution with distilled water for 2448 hours, and then lyophilized to obtain a white powder.
  • the yield was 34.2 mg ZL.
  • the synthesized oligonucleotide in the form of a film was dissolved in TE (1 OmM Tris-HCI (pH 8.0) ImM EDTA (pH 8.0)) to 1 ⁇ g / ⁇ l. Equimolarly mix the respective complementary strands, heat-treat at 30 ° C for 30 seconds, cool to 37 ° C over 1 hour, allow to stand for 30 minutes, and represent the sequence listings 31 and 32. Two double-stranded DNAs encoding different amino acid sequences were constructed. After mixing equal amounts of each double-stranded DNA, use TaKaRa Ligation Kit ver 2 solution I to bind at 16 ° C for 1 hour, and prepare double-stranded DNA represented by coding SELP monomer.
  • the cloning vector pUC118 was digested with the restriction enzyme BamHI at 37 ° C for 1 hour and 30 minutes, and CIAP was purified and treated at 37 ° C for 30 minutes.
  • the reaction solution was extracted and purified using a phenol: cloth form: isoamyl alcohol mixed solution (weight ratio: 25: 24: 1). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.
  • SELP monomer DNA and pUC118 vector sample were mixed at a ratio of 10: 1 and ligated at 16 ° C for 1 hour using Ta KaRa Ligation Kit ver 2 solution I. After the completion of the reaction, transformation was performed using the competent cell DH5a. Confirm the presence of transgene by color selection using X gal Then, the DNA containing the inserted gene is subjected to DNA sequencing, and the sequence is confirmed to confirm that the plasmid containing the SELP monomer DNA pUC— SELP
  • the transformant cell DH5a was transformed and cultured in a 2xYT medium at 37 ° C for 18 hours. Plasmid was extracted from the culture solution by the alkaline SDS method and then dissolved in TE. Samples 37. C, digested simultaneously with Nhe I and Spe I for 1 hour and 30 minutes, and isolated SLP (1) from Plasmidoca. After concentrating the reaction solution to 5 il using a microcon, the insert DNA was cut out by electrophoresis using a 1.5% agarose gel. Ultrafree DA was used to extract the DNA of Ge / Reca, and the extracted solution was again concentrated using a microphone-mouth cone to 5 ⁇ l to obtain an inserted gene sample.
  • pUC-Link was digested with NheI
  • CIAP was added and treated with alkaline phosphatase.
  • the reaction solution was extracted and purified using a mixed solution of phenol: chloroform: isoamyl alcohol (25: 24: 1 by weight). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a sample of one vector.
  • the transformant cell DH5a was transformed.
  • the cells were inoculated on LB plates containing ampicillin and screened. The colonies generated were picked up, inoculated into 2xYT medium, and cultured at 37 ° C for 18 hours. Extract the plasmid from the culture medium using the alkaline SDS miniprep method. And dissolved in TE to obtain a sample. After simultaneously digesting the sample with Nhe I and Spe I, the presence and size of the introduced gene were confirmed by electrophoresis. Subsequently, DNA sequencing was performed, and the sequence was confirmed to obtain plasmid pUC-Link SELP (1).
  • DH5 ⁇ was transformed and cultured in a 2 ⁇ medium at 37 ° C for 18 hours. Plasmid was extracted from the culture solution by the alkaline SDS method and dissolved in TE. The sample was digested simultaneously with Nhe I and Spe I at 37 for 1 hour and 30 minutes and SELP (1) was isolated from plasmid. After concentrating the reaction solution to 51 using a microcon, the insert DNA nodes were cut out by electrophoresis using a 1.5% agarose gel. Ultrafree DA was used to extract the DNA of the gel force and the like, and the extract was again concentrated to 5 ⁇ l using a microcon to obtain a transfected gene sample.
  • pUC-Link SELP (1) was digested with Nhe I, CIAP was treated with alkaline phosphatase.
  • the reaction solution was extracted and purified using a mixed solution of phenol: chloroform: isoamyl alcohol (weight ratio: 25: 24: 1). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in bacterial water to obtain a vector sample.
  • the competent cell DH5 was transformed.
  • the cells were inoculated on LB plates containing ampicillin and screened.
  • the generated eggs were picked up, inoculated into 2xYT medium, and cultured at 37 ° C for 18 hours. Extract the plasmid from the culture medium using the alkaline SDS miniprep method.
  • pUC-SELP8 was digested with BamHI and HindIII. After concentrating the reaction solution to 5 ⁇ l using a microcontroller, the insert DNA band was cut out by electrophoresis using a 1.5% agarose gel. UltrafreeDA was used to extract DNA from the gel, and the extract was again concentrated to 5 ⁇ using a microphone-mouth to obtain an inserted gene sample.
  • the ligation reaction was used to transform competent cell DH5a.
  • the cells were inoculated on an LB plate containing kanamycin and screened. The resulting colonies were picked, inoculated into 2XYT medium, and cultured.
  • the cells were cultured at 37 ° C for 16 hours.
  • add 100 ⁇ l of the culture solution to a test tube containing 5 ml of 2 X YT (25 g / ml kanamycin, 25 ⁇ g / ml chloramphenicol), and add 0D at 37 ° C. 6 . . 0.5-0.7.
  • IPTG final concentration ImM
  • SELP8 100 ⁇ ⁇ of the medium was collected every hour into an Eppendorf tube and cultured for up to 4 hours. The collected medium was centrifuged (14,500 rpm, 5 minutes, 4), and the supernatant was discarded. C for 5 minutes to give a sample for SDS-PAGE.
  • SELP 8 was detected by Western blot using His-Tag antibody (Fig. 3).
  • the cells were cultured at 7 ° C for 16 hours.
  • the culture solution was cultured at 37 ° C. for 16 hours in 12 ml of 2 ⁇ YT (25 g / ml kanamycin, 25 ⁇ g / ml chloramphenicol) liquid medium.
  • IPTG 0.2 mM final concentration
  • the temperature was lowered to 30 ° C, and the cells were cultured for another 4 hours, and the cells were collected (8500 rpm, 30 minutes, 4 ° C). Then, the cells were obtained.
  • the obtained cells were stored at 120 ° C.
  • the obtained fraction was dialyzed while appropriately exchanging the external solution with distilled water for 24 to 48 hours, and then lyophilized to obtain a white powder.
  • the yield for the 35 kDa protein was 38.8 mg.
  • the synthesized oligonucleotide in the form of a film is dissolved in TE (1 OmM Tris-HCI (pH 8.0), ImM EDTA (pH 8.0)) to a concentration of 1 ⁇ g / ⁇ 1.
  • TE 1 OmM Tris-HCI (pH 8.0), ImM EDTA (pH 8.0)
  • Equimolarly mix each of the complementary strands heat-treat at 30 ° C for 30 seconds, cool to 37 over 1 hour, allow to stand for 30 minutes, and represent the sequence listings 38 and 39. Two double-stranded DNAs encoding different amino acid sequences were constructed.
  • the cloning vector pUC118 was digested with the restriction enzyme BaraHI at 37 ° C. for 1 hour and 30 minutes, and then CIAP was added, followed by treatment with 37 at 30 minutes.
  • the reaction solution was extracted and purified using a mixed solution (25: 24: 1 in weight ratio) of phenol: chloroform: isoamyl alcohol. Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.
  • the SLPF monomer DNA and pUCl 18 vector sample were mixed at a ratio of 10: 1, and the mixture was mixed with TaKaRa Ligation Kit ver 2 solution I 16. C was used for 1 B gap. After the completion of the reaction, transformation was performed using the competent cell DH5a. The presence of the inserted gene is confirmed by color selection using X-gal, the DNA containing the inserted gene is subjected to DNA sequencing, and the sequence is confirmed to confirm that the plasmid containing the SLPF monomer DNA pUC-SLPF (1) I got
  • Both ends of the SLPF monomer contain the Spe I and Nhe I restriction enzyme recognition regions.
  • the protruding ends of the fragments digested by Spe I and Nhe I are both complementary and can bind to each other.
  • the newly joined sequence differs from both the Spe I and Nhe I restriction enzyme recognition regions. It is not digested by Spe I and Nhe I. Utilizing this property, pUC Link SLPF (n) was constructed by polymerizing the SLPF monomer in one direction.
  • pUC-SLPF (1) was transformed using the DH5 cell, and cultured in 2xYT medium at 37 ° C for 18 hours. Plasmid was extracted from the culture solution by the Alkali-rich SDS method and dissolved in TE. The sample was simultaneously digested with Nhe I and Spe I at 37 for 1 hour 30 minutes to isolate Plasmidoka, et al. SLPF (1). After concentrating the reaction solution to 51 using a microcon, the insert DNA zone was cut out by electrophoresis using a 1.5% agarose gel. For extraction of the DNA of Geno force, et al., UltrafreeDA was used, and the liquid extracted using a regenerative microcon was concentrated to 5 ⁇ l to obtain an imported gene sample.
  • reaction solution was extracted and purified using a mixed solution (25: 24: 1 in weight ratio) of phenol: cloth form: isoamyl alcohol. Purified reaction solution Ethanol was added, and the resulting precipitate was dissolved in sterilized water to make one vector sample.
  • the competent cell DH5 ⁇ was transformed.
  • the cells were inoculated on an LB plate containing ampicillin and screened.
  • the generated colony One was picked up, inoculated into 2xYT medium, and cultured at 37 ° C for 18 hours.
  • Plasmid was extracted from the culture medium by the alkali-SDS miniprep method and dissolved in TE to obtain a sample. After digesting the sample simultaneously with Nhe I and Spe I, the presence and size of the inserted gene were confirmed by electrophoresis. Next, DNA sequencing was performed and the sequence was confirmed to obtain plasmid pUC—Link SLPF (5).
  • pUC-SLPF (5) was digested with BamHI and HindIII. After concentrating the reaction solution to 5 t1 using a microcomputer, the insert DNA band was cut out by electrophoresis using a 1.5% agarose gel. To extract the DNA of the gel force, et al., The extract was concentrated to 51 using UltrafreeDA and a reproduction microcon to obtain an imported gene sample.
  • reaction solution is a mixed solution of phenol: chloroform: isoamyl alcohol (25: 24: 1 by weight)
  • the cells were inoculated on an LB plate supplemented with kanamycin and screened. The generated colonies were picked up, inoculated on 2 X YT medium, and cultured.
  • the vector was extracted from the culture medium by the alkaline-SDS method, dissolved in TE, and used as a sample. After digesting the sample simultaneously with Nhe I and Spe I, electrophoresis The presence and size of the insert DNA were confirmed by DNA, and the sequence was confirmed by DNA sequencing to construct the expression vector pET-SLPF5.
  • the cells were cultured in the medium at 37 ° C for 16 hours.
  • add 100 ⁇ l of the culture solution to a test tube containing 5 ml of 2 X TY (25 ⁇ g / ml kanamycin, 25 g / ml chloramphenicol), and add 0D at 37 ° C. 60 . 0.5-0.7.
  • IPTG final concentration ImM
  • 100 ⁇ l of the medium was collected every other hour in an Eppendorf tube and cultured for up to 4 hours.
  • the collected medium was centrifuged (14,500 rpm, 5 minutes, 4 ° C), the supernatant was discarded, the pellet was dissolved in a 2X sample buffer, and then heat-treated at 100 ° C for 5 minutes for SDS'— PAGE sample.
  • the cells were cultured for 16 hours.
  • the culture solution was cultured at 37 ° C. for 16 hours in 5 ml of a 2 ⁇ YT (25 ⁇ g / ml kanamycin, 25 ⁇ g / ml mouth rampunicol) liquid medium.
  • the obtained fraction was dialyzed while appropriately exchanging the external solution with distilled water for 24 to 48 hours, and then lyophilized to obtain a white powder.
  • the yield was 38.8 mg ZL for the 23 kDa protein.

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Abstract

La présente invention concerne un processus de production d'une protéine de soie ou d'une protéine de type soie par utilisation de escherichia coli. Un polymère de soie ou un polymère de type soie comprenant au moins une protéine sélectionnée parmi une fibroine de vers à soie domestiqué, une fibroine de vers à soie sauvage, une élastine et une fibronectine comprenant essentiellement la fibroine de vers à soie domestiquée ou la fibroine de vers à soie sauvage susmentionnée est conçu et l'unité minimum de la soie ou le polymère ainsi conçu est synthétisé. Le polymère de l'unité minimum ainsi synthétisé est intégré dans au moins un vecteur d'expression sélectionné parmi des vecteurs d'expression contenant le promoteur T7. Puis, ce vecteur d'expression est intégré dans E. coli BL21 (DE3) pLysS ou BLR (DE3) pLysS. Enfin, on fait croître cette souche E. coli dans un milieu sélectionné parmi des milieux composites.
PCT/JP2002/005010 2002-05-23 2002-05-23 Processus de production industrielle de proteines de soie et de proteine de type soie genetiquement modifiee a fonction impartie Ceased WO2003100065A1 (fr)

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JP2004508303A JPWO2003100065A1 (ja) 2002-05-23 2002-05-23 絹タンパク質、及び、機能性を付与した遺伝子組換え絹様タンパク質の大量生産方法
PCT/JP2002/005010 WO2003100065A1 (fr) 2002-05-23 2002-05-23 Processus de production industrielle de proteines de soie et de proteine de type soie genetiquement modifiee a fonction impartie
US11/197,315 US20060019348A1 (en) 2002-05-23 2005-08-05 Method of mass producing silk protein and gene recombinant silk-like protein with added functionality

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6358101A (ja) * 1986-08-29 1988-03-12 Hitachi Ltd 位置検出装置
JP2009509550A (ja) * 2005-10-05 2009-03-12 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼイション 絹タンパク質
WO2019065968A1 (fr) * 2017-09-29 2019-04-04 Ｓｐｉｂｅｒ株式会社 Cassette d'expression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2162190A (en) * 1984-07-06 1986-01-29 Pa Consulting Services Improvements in or relating to production of silk
WO1988003533A1 (fr) * 1986-11-04 1988-05-19 Syntro Corporation Structure d'adn synthetique et son emploi dans la synthese de polypeptides de grande dimension
WO1991005866A1 (fr) * 1989-10-19 1991-05-02 Schering Corporation Nouveaux systemes d'expression utilisant des promoteurs de bacteriophage t7 et des sequences de gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2162190A (en) * 1984-07-06 1986-01-29 Pa Consulting Services Improvements in or relating to production of silk
WO1988003533A1 (fr) * 1986-11-04 1988-05-19 Syntro Corporation Structure d'adn synthetique et son emploi dans la synthese de polypeptides de grande dimension
WO1991005866A1 (fr) * 1989-10-19 1991-05-02 Schering Corporation Nouveaux systemes d'expression utilisant des promoteurs de bacteriophage t7 et des sequences de gene

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Molecular tools for life science research", NOVAGEN 2000 CATALOG, 2000, pages 68, XP002955878 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6358101A (ja) * 1986-08-29 1988-03-12 Hitachi Ltd 位置検出装置
JP2009509550A (ja) * 2005-10-05 2009-03-12 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼイション 絹タンパク質
JP2015002746A (ja) * 2005-10-05 2015-01-08 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション 絹タンパク質
WO2019065968A1 (fr) * 2017-09-29 2019-04-04 Ｓｐｉｂｅｒ株式会社 Cassette d'expression
US11851684B2 (en) 2017-09-29 2023-12-26 Spiber Inc. Expression cassette

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