WO2003100065A1 - Process for the mass production of silk protein and genetically modified silk-like protein having function imparted thereto - Google Patents

Abstract

A process for producing a silk or silk-like protein by using Escherichia coli. A silk or silk-like polymer comprising at least one protein selected from among domesticated silkworm fibroin, wild silkworm fibroin, elastin and fibronectin and essentially comprising the above-described domesticated silkworm fibroin or wild silkworm fibroin is designed and the minimum unit of the silk or the thus designed polymer is synthesized. The polymer of the minimum unit thus synthesized is integrated into at least one expression vector selected from expression vectors containing T7 promoter. Then, the expression vector is integrated into E. coli BL21(DE3)pLysS or BLR(DE3)pLysS. Subsequently, the E. coli is grown in a medium selected from among composite media.

Description

 Description Method for mass production of silk proteins and functionally modified recombinant silk-like proteins

 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.

Background art

 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.

 Detailed structural analysis of silk has been performed to find the origin of these excellent physical properties. 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. In recent years, as the secondary structure of each motif has been revealed, results on the correlation between the structure and physical properties of silk fibers are accumulating.

In recent years, studies have been made to synthesize artificial silk-like proteins from synthetic DNAs encoding silk proteins. Since silk proteins have a repeating structure of the same amino acid sequence, the characteristic feature is that the number of occurrences of a specific amino acid increases. This can result in errors in terminating protein synthesis due to the depletion of several aminoacyl-tRNAs. Also, repetition of DNA sequence may cause sequence recombination in E. coli. Therefore, it was still difficult to obtain a large amount of silk-like protein having a repetitive sequence using E. coli.

 Therefore, in order to increase expression efficiency, 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.

 Therefore, 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.

 Accordingly, 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.

 In the present invention, it is preferable that the temperature for growing Escherichia coli be lowered by 2 to 7 ° C. below the optimal temperature for growing Escherichia coli.In particular, an expression vector containing a T71ac promoter as an expression vector containing a T7 promoter is preferably used. It is preferable to use them. BRIEF DESCRIPTION OF THE FIGURES

 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 ゥ ヱ stamp mouth using a His-Tag antibody. BEST MODE FOR CARRYING OUT THE INVENTION

 In the present invention, 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. .

 These are described in the Terminology of Sericultural Sciences, edited by The Japanese Society of Sericultural Sciences (19779).

Elastin is a type of elastin that exists in the tissues of various organisms. In the primary structure of elastin, there is a high frequency of a five-residue amino acid sequence Val-Pro-Gly-Val-Gly (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.

 In addition, 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. Thus, in the present invention, 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.

In the present invention, 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. In the present invention, it is considered that 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.

 Specifically, SLP (silk-like protein) has been developed in order to design proteins with new physical properties and functions not found in natural silk fibers by recombining the motifs contained in natural silk fibers. , SLPA (silk-like protein with polyalanine), SELP (silk and elastin-like protein), and SLPF (silk-like protein with fibronectin) were designed as follows.

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 (Abbreviation for 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. For example, in the case of SLP and SLPA, 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. In the case of SELP and SLPF, the functional stability motifs of elastin and fibronectin are combined with silk proteins for use as biomaterials, not just as fibers. In addition to the functions such as the properties, further new functions can be provided. In the present invention, 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-1β-1D-galactoside) as an expression inducer, the target protein downstream of the Τ7 promoter is expressed because Τ7 RNA polymerase is not expressed. Therefore, stress on E. coli due to large overexpression is reduced. In addition, since plasmid pLysS further expresses Τ7 lysozyme and inactivates Τ7 RNA polymerase, two-step suppression can be expected. In the present invention, it is preferable to select an expression vector from expression vectors containing a T71ac promoter, and particularly to use pET30a. Preferably.

 After the induction of expression, stress on E. coli is reduced by optimizing culture conditions such as culture temperature, IPTG addition concentration, and pH using a medium selected from the complex medium. In the present invention, 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. Further, the pH is preferably 6.7 to 7.0. Example

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. In the following, unless otherwise specified, “%” represents “% by weight”, and the ratio means a weight ratio. The meaning of each symbol in the text is as follows. Example 1.

Construction of SLP gene>

 Four oligonucleotides shown in Sequence Listings 12 to 15 were synthesized by Asahi Techno Glass Co., Ltd.

 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.

) And dissolved at a concentration of 1 μg / 1. Each complement The chains are equimolarly mixed, heat-treated at 99 ° C for 30 seconds, cooled to 37 ° C over 1 hour, and allowed to stand for 30 minutes to be represented by Sequence Listing 16 and 17 Two double-stranded DNAs encoding amino acids were constructed. After mixing equal amounts of each double-stranded DNA, use TaKaRa Ligation Kit ver 2 slution I (Takara ligation kit purge ion 2 solution I (trade name, manufactured by Takara Shuzo Co., Ltd.)) at 16 ° C. After binding for 1 hour, double-stranded DNA encoding SLP monomer was prepared (SLP amino acid sequence IJ; Thr Ser [Gly Gly Ala Gly Ser Gly Tyr Gly Gly Gly Tyr Gly His Gly Tyr Gly Ser Asp Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser] _n (n = 2, 4, 6), see Sequence Listing 18 (when n = 2)).

 Using the cloning vector pUC118 (Takara Shuzo Co., Ltd.) with the restriction enzyme BamHI 37. Add 1 hour 30 minutes with C, add CIAP (Calf Intestine Alkaline Phosphatase (Alkaline phosphatase from small intestine of larva) (Takara Shuzo Co., Ltd.)), and treat with 37 for 30 minutes. (Hereinafter referred to as “alkaline phosphatase treatment”). The reaction solution was extracted and purified using a mixed solution of phenol: clog form: isoamyl alcohol in a ratio of 25: 24: 1 (weight ratio). Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.

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. As a result, 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 µg / µ1. 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. 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.

<Construction of SLP (n)>

 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. In addition, 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 DH5α was transformed with pUC-SLP (1), and cultured at 37 ° C for 18 hours in a 2χΥΤ 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 μl 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 (ゥ Noretrafree DA) (manufactured by Millipore), the extract was concentrated to 51 using a regeneration microcon to obtain an imported gene sample.

After 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.

 After completion of the reaction, a competent cell DH5α was transformed. The cells were inoculated on an LB plate containing ampicillin and screened. The colonies generated were picked up, inoculated on a 2χΥΤ 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).

 After 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 _α. The cells were inoculated on LB plates containing ampicillin and screened. The colonies generated were picked up, inoculated into a 2χΥΤ medium, and cultured at 37 for 18 hours. Plasmid was extracted from the culture medium by the alkali-SDS muprep method, dissolved in ΤΕ, and used as a sample. Simultaneously use Nhe I and Spe I After digestion, the presence and size of the introduced gene was confirmed by electrophoresis. Thereafter, DNA sequencing was performed, and the sequence was confirmed to obtain a plasmid pUC-Link SLP (2) (dimer).

 Insert SLP (2) into pUC-Link SLP (2) to construct pUC-Link SLP (4) (tetramer), then insert SLP (2) into pUC-Link SLP (4) pUC—Link SLP (6) (hexamer) was constructed.

Construction of expression vector pET—SLP (n)>

 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. 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 vector sample.

 Confirm the DNA concentration in the inserted gene sample and the vector sample by electrophoresis using a 1.5% agarose gel, mix the inserted gene sample and the vector sample so that the ratio becomes 10: 1, and mix with the mixture. The amount of Takara Ligation kit ver2 solution I was calorie-coupled with 16 for 1 hour.

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.

Expression of pET—SLP (2, 4, 6)>

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 μl). g / ml kanamycin, 25 μg / ml chloramphenicol) The cells were cultured at 37 ° C for 16 hours in a liquid medium. Next, 100 μl of the culture solution was added to an L-shaped tube containing 5 ml of the 2xTY (0.25 μg / ml kanamycin, 25 Ug / inl chloramphenicol), and the mixture was heated at 37 ° C. in 1 hour _{(0D 6 .. = 0. 5 ~} 0.7 (Shiniadzu UV- 1 6 0)) were cultured. In this case, IPTG (final concentration ImM) was added to induce the expression of SLP, and 1 Ο Ο μΙ of the medium was collected every other hour into an eppendorf tube and cultured for up to 3 hours. The collected medium was centrifuged (14,500 rpm, 5 minutes, 4 ° C), the supernatant was discarded, the pellet was dissolved in 2X sample buffer (sample lysis buffer), and then heat-treated at 100 ° C for 5 minutes. And SDS-PAGE samples. As shown in FIG. 1, unique 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.

Next, host E. coli BL21 (DE3) pLysS with plasmid pET-SLP (2, 4, 6) was added to 2.5 ml of 2 x TY (25 μg / ml kanamycin, 25 μg / ml (Chloram-Fuecol) The cells were cultured in a liquid medium at 37 ° C for 16 hours. Next, the culture solution was added to 250 ml of 200 ml of 2xTY (25 μg / ml kanamycin, 25 μg / ml chloramphene-col) in 500 ml In addition, the cells were cultured at 37 ° C for 1 hour (0D ₆ = 0.5 to 0.7 (Shimadzu UV-160)). At this time, IPTG (final concentration lmM) was added to induce protein expression, and 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.

 Gently thaw the above cells stored at 130 ° C on ice and place in Lysis buffer (5 OmM Tris-HCl, 300 mM NaCl, 1 OmM imidazole). The suspension was suspended and sonicated on ice (Out put 3.5, Duty 60% (TOMY UD201)) four times for 1 minute each with a cooling time of 1 minute. The obtained bacterial cell disruption was centrifuged (10,000 rpm, 10 minutes, 4), and the supernatant was recovered.

 Using the obtained supernatant as an additional sample, purify by affinity chromatography (flow rate: 15 to 2 Oml / hour) using a column packed with Ni-NTA agarose beads previously equilibrated with the same buffer. went . Each eluate was fractionated, and the fraction containing the target protein was confirmed and recovered by SDS-PAGE.

 The obtained fraction was dialyzed against distilled water for 24 to 48 hours while appropriately exchanging the external solution, and then lyophilized to obtain a white powder. Table 1 shows the yield of each protein with a molecular weight of 19 kDa, 29 kDa, and 40 kDa.

 [Table 1] Yield (mg) Yield (%)

SLP6 21 52.5

 SLP4 24 60.0

 SLP2 15 50.0

<Identification of SLP> For each protein, 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. Using this sample, 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.

 <Cleavage of tag sequence by cyanogen bromide>

 In designed SLPs 2, 4, and 6, 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. As a result, a decrease in the molecular weight was confirmed as compared with the sample before the treatment with cyanogen bromide. SLP6 purified in this manner was separated from impure proteins by electrophoresis using polyacrylamide gel, and transferred to a PVDF membrane using Zaltoblock 2-S (Sartorius). After the transfer, the cells were stained with a staining solution for 5 minutes, decolorized with methanol, and the band of the target protein was cut out using washed scissors. Using this sample, the N-terminal amino acid sequence was determined using an ABI 473 gas-phase Edman sequencer. The determined N-terminal amino acid sequence was consistent with the expected amino acid sequence, and it was confirmed that the target protein was SLP6.

Example 2.

Construction of SLPA gene>

 Four oligonucleotides shown in Sequence Listings 20 to 23 synthesized by Asahi Techno Glass Co., Ltd. were designed.

 The synthesized film-form oligonucleotides were dissolved in TE (1 OmM Tris-HCI (pH8.0), ImM EDTA (pH8.0)) to a concentration of 1 μg / μ1. . 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 丽 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 DH5α 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 μl 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 μl to obtain an imported gene sample.

 After digesting PUC-GX with Spe I, 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.

Confirm the DNA concentration in the inserted gene sample and the vector sample by electrophoresis using a 1.5% agarose gel, mix the inserted gene sample and the vector sample so that the ratio becomes 10: 1, and mix with the mixture. An amount of Takara Ligation kit ver2 solution I was caloried and allowed to bind at 16 ° C for 1 hour.

After completion of the reaction, it was transformed Konbiten Toseru DH5 _α. Ampicillin The seeds were inoculated on an LB plate to which was added and screened. The colonies generated were picked up, inoculated into 2xYT medium, and cultured at 37 ° C for 18 hours. Plasmid was extracted from the medium by the alkali-SDS mi-prep method, dissolved in TE, and used as 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. Then, DNA sequencing is performed to confirm the sequence.

-SLPA (1) was obtained (SLPA amino acid sequence; [Ala Ser (Ala) ₁₈ Thr Ser Gly Val Gly Ala Gly Tyr Gly Ala Gly Ala Gly Tyr Gly Val Gly Ala Gly Tyr Gly Ala Gly Val Gly Tyr Gly Ala Gly Ala Gly Tyr] _n , see Sequence Listing 26 (when n = 4)).

<Construction of SLPA (n)>

 Combinent cell DH5α 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 μl 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 μl to obtain an inserted gene sample.

 After 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.

 After completion of the reaction, a competent cell DH5α was transformed. The cells were inoculated on an LB plate supplemented with ampicillin and screened. The colonies generated were picked up, inoculated on a 2χΥΤ 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).

Construction of expression vector pET—SLPA (4)>

 pUC-SLPA (4) was digested with BamHI. After concentrating the reaction solution to 5 μl 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.

 After digestion of the expression vector pET30a with BamHI, 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.

 <Expression of pET-SLPA (4)>

 Plasmid pET_SLPA (4) Host E. coli BL21 (DE3) pLysS, each containing 1.5 ml of 2 x TY (25 g / ml kanamycin, 25 μg I ml chloramphenicol) liquid medium at 37 ° C For 16 hours. Next, add the culture medium Ο Ο Ο μ に 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)). In this case, IPTG (final concentration ImM) was added to induce the expression of SLPA4, and 100 μl 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. .

 After SDS-PAGE, SLPA4 was detected by Western blot using His-Tag antibody (Fig. 2).

As is clear from the figure, a band was observed at 29 kDa in SLPA4. These results indicate that the SLP gene is induced by IPTG addition, It was proved that it could be obtained.

 Plasmid pET—Host E. coli BL21 (DE3) pLysS with SLPA (4) was added to 1.5 ml of 2xYT (25 μg / ml kanamycin, 25 μg / ml chloramphenicol) liquid medium. The cells were cultured at 37 ° C for 16 hours. Next, the culture solution was added to a test tube containing 12 ml of 2xYT (25 μg / ml kanamycin, 25 μg / ml chloramphenicol) and cultured at 37 ° C for 16 hours. Next, it was added to 21 fermenters containing 1.21 of 2xYT (25 μg / ml kanamycin, 25 μg / ml chloramphenicol), and at 37 ° C, 0D600 = 0.5 to 0.7 ( Shimadzu UV-160)). In this case, IPTG (final concentration ImM) was added to induce protein expression, and the cells were further cultured for 4 hours and harvested (8500 rpm, 30 minutes, 4 ° C) to obtain cells. The obtained cells were stored at 120 ° C.

Gently thaw the cells stored at 20 ° C on ice, suspend in Lysis buffer (5 OmM Tris-HCl, 300 mM NaCl, l OmM imidazole) and sonicate on ice. (Out put 3.5, Duty 60% (TOMY UD201)) was performed 20 times for 2 minutes each with a cooling time of 1 minute. The resulting et the disrupted cells was centrifuged (10,000 r _P m, 40 min, 4 ° C) have row operations, and the precipitate was collected.

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. Was. 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.

Example 3.

<Construction of SELP gene>

 Four oligonucleotides synthesized by Asahi Techno Glass Co., Ltd. and represented by Sequence Listing 2730 were designed.

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 μg / μl. 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. (See SELP amino acid sequence IJ; Thr Ser [(Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₂ Ala Ser] _n , Sequence Table 33 (when _n = 8)) .

 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

(1) was obtained.

<Construction of SELP (n)>

 Using pUC-SELP (1), 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 μl to obtain an inserted gene sample.

 After 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.

 Confirm the DNA concentration in the transfected gene sample and the vector sample by electrophoresis using a 1.5% agarose gel, mix the transfected gene sample and the vector sample so that the ratio becomes 10: 1, and mix with the mixed solution. An equal volume of Takara Ligation kit ver2 solution I was added, and the cells were ligated with 16 for 1 hour.

After completion of the reaction, 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).

 Using pUC-Link SELP (1), DH5α was transformed and cultured in a 2χΥΤ 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 μl using a microcon to obtain a transfected gene sample.

 After 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.

 Confirm the DNA concentration in the inserted gene sample and the vector sample by electrophoresis using 1.5% agarose gel, mix the inserted gene sample and the vector sample so that the force becomes 10: 1, and make an equivalent volume of the mixed solution. Takara Ligation kit ver2 solution I was added and allowed to bind at 16 ° C for 1 hour.

After completion of the reaction, 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. PC orchid 10

 twenty five

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. Then

By performing DNA sequencing and confirming the sequence, a plasmid pUC-Link SELP (2) was obtained.

 Enter SELP (2) into pUC-Link SELP (2) and enter pUC—Link SELP (

4) was constructed and SELP (4) was inserted into pUC-Link SELP (4) to construct pUC-Link SELP (8).

Construction of expression vector pET—SELP (n)>

 pUC-SELP8 was digested with BamHI and HindIII. After concentrating the reaction solution to 5 μl 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 μΐ using a microphone-mouth to obtain an inserted gene sample.

 After the expression vector pET30a was digested with restriction enzymes BamHI and HindIII, CIAP was added thereto and treated with alkaline phosphatase. Reaction solution is phenol

: Black mouth form: Mixed solution of isoamyl alcohol (25:24 by volume ratio)

: 1) and extracted and purified. Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to obtain a vector sample.

 Confirm the DNA concentration in the inserted gene sample and the vector sample by electrophoresis using a 1.5% agarose gel. Mix the inserted gene sample and the vector sample so that the ratio becomes 10: 1, and equip with the mixed solution. Of Takara Ligation kit ver2 solution I was added and allowed to bind at 16 ° C for 1 hour.

 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.

. Extract the vector from the culture medium by alkaline SDS method, dissolve in TE A sample was used. After simultaneously digesting the sample with Nhe I and Spe I, the presence and size of the insert DNA was confirmed by electrophoresis, and the sequence was confirmed by DNA sequencing, whereby the expression vector pET-SELP8 was constructed.

Expression of pET-SELP (8)

Plasmid pET— E. coli BL21 (DE3) pLysS with each of SELP (8) and 1.5 ml of 2 x YT (25 μg / ml kanamycin, 25 μg I ml chloramphen-col) liquid medium The cells were cultured at 37 ° C for 16 hours. Next, add 100 μl of the culture solution to a test tube containing 5 ml of 2 X YT (25 g / ml kanamycin, 25 μg / ml chloramphenicol), and add 0D at 37 ° C. ₆ . . = 0.5-0.7. In this case, IPTG (final concentration ImM) was added to induce the expression of SELP8, and 100 μ μ 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.

 After SDS-PAGE, SELP 8 was detected by Western blot using His-Tag antibody (Fig. 3).

 As shown in the figure, a band was observed at 35 kDa in SELP 8. These results demonstrate that the addition of IPTG induces the SLP gene and yields a strain that overexpresses it.

Plasmid pET— E. coli BL21 (DE3) p LysS with each of SELP 8 in 1.5 ml of 2 x YT (25 g / ml kanamycin, 25 g / ml Kuala ramfe-col) liquid medium The cells were cultured at 7 ° C for 16 hours. Next, the culture solution was cultured at 37 ° C. for 16 hours in 12 ml of 2 × YT (25 g / ml kanamycin, 25 μg / ml chloramphenicol) liquid medium. The culture was further transferred to 21 fermenters containing 1 · 2 2xYT (25 g / ml kanamycin, 25 g / ml chloramphenicone), and 37 ° ς at 0D ₆ . . = 0.5-0.7. In this case, IPTG (0.2 mM final concentration) was added to induce protein expression, 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.

 — Slowly thaw the cells stored at 20 ° C on water, suspend in Lysis buffer (5 OmM Tris-HCl, 300 mM NaCl, 10 raM imidazole), and sonicate on water (Out put) 3.5, Duty 60% (TOMY UD201)) was performed 20 times for 2 minutes each with a cooling time of 1 minute. The obtained cell lysate was centrifuged (10000 rpm, 30 minutes, 4 ° C), and the supernatant was recovered.

 Using the obtained supernatant as an added sample, purification by affinity chromatography (flow rate 15 to 2 Oml / h) was performed using a column packed with Ni-NTA agarose beads that had been equilibrated with the same buffer in advance. went. Each eluate was fractionated, and the fraction containing the target protein was confirmed and recovered by SDS-PAGE.

 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.

Example 4.

Construction of SLPF gene>

 Four oligonucleotides synthesized by Asahi Techno Glass Co., Ltd. and shown in Sequence Listings 34 to 37 were designed.

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 μg / μ1. I understand. 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. After mixing equal amounts of each double-stranded DNA, they were bound at 16 ° C for 1 hour using TaKaRa Ligation Kit vero 2 solution I to prepare double-stranded DNA encoding SLPF monomer (SLPF Amino acid sequence of [Thr Ser Thr Gly Arg Gly Asp Ser Pro Ala Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser] _n , see Sequence Listing 40 (when n = 5)).

 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

<Construction of SLPF (n)>

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. In addition, 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 μl to obtain an imported gene sample.

 Confirm the DNA concentration in the transfected gene sample by electrophoresis using a 1.5% agarose gel, and calcine an equivalent amount of Takara Ligation kit ver2 solution I with the transfected gene sample and bind at 16 ° C for 16 hours I let it.

 After 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. Purified reaction solution Ethanol was added, and the resulting precipitate was dissolved in sterilized water to make one vector sample.

 Confirm the DNA concentration in the inserted gene sample and the vector sample by electrophoresis using a 1.5% agarose gel. Mix the inserted gene sample and the vector sample so that the ratio becomes 10: 1. An equal amount of Takara Ligation kit ver2 solution I was added, and the mixture was ligated with 16 for 1 hour.

After completion of the reaction, the competent cell DH5α 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).

Construction of expression vector pET—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.

 After the expression vector pET30a was digested with BamHI and HindIII, CIAP was treated with alkaline phosphatase. The reaction solution is a mixed solution of phenol: chloroform: isoamyl alcohol (25: 24: 1 by weight)

) And extracted and purified. Ethanol was added to the purified reaction solution, and the resulting precipitate was dissolved in sterilized water to prepare a vector sample.

 Confirm the DNA concentration in the transfected gene sample and the vector sample by electrophoresis using a 1.5% agarose gel, and mix the transfected gene sample and the vector sample so that the ratio becomes 10: 1. And an equal amount of Takara Ligation kit ver2 solution I, and allowed to bind at 16 ° C for 1 hour.

 Using the ligation reaction mixture, transform the Combinant Cell DH5a

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.

Expression of pET-SLPF (5)

Plasmid pET— Escherichia coli BLR (DE3) pLysS (Novagen) with SLPF (5) in 1.5 ml of 2 x TY (25 μg / ml kanamycin, 25 μg / ml chloramphenol) liquid The cells were cultured in the medium at 37 ° C for 16 hours. Next, add 100 μl of the culture solution to a test tube containing 5 ml of 2 X TY (25 μg / ml kanamycin, 25 g / ml chloramphenicol), and add 0D at 37 ° C. ₆₀ . = 0.5-0.7. In this case, IPTG (final concentration ImM) was added to induce the expression of SLPF5, and 100 μl 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.

 After SDS-PAGE, SLPF5 was detected by performing an easter blot using His-Tag antibody (Fig. 4).

 As shown in FIG. 4, a band was observed at 23 kDa in SLPF5. From these results, it was demonstrated that the addition of IPTG induced a strain in which the SLPF gene was induced and expressed in large amounts.

Plasmid pET—Host E. coli BLR (DE3) pLysS with SLPF 5 in 1.5 ml of 2xYT (25 μg / ml kanamycin, 25 g / ml Kuala rampunicol) liquid medium at 37 ° C C. The cells were cultured for 16 hours. Next, the culture solution was cultured at 37 ° C. for 16 hours in 5 ml of a 2 × YT (25 μg / ml kanamycin, 25 μg / ml mouth rampunicol) liquid medium. Then, add 250 ml of 2xYT (25 μg / ml kanamycin, 25 g / ml Force [] to 11 Erlenmeyer flasks containing chloramphenicone) and OD ₆ at 37. . = 0.5 to 0.7. In this case, IPTG (final concentration: 0.2 mM) is added to induce protein expression, and the cells are further cultured for 4 hours and collected (8,500 rpra, 30 minutes, 4 ° C) to separate the cells. Obtained. The obtained cells were stored at 120 ° C.

 Slowly thaw the cells stored in step 20 on ice, suspend them in Lysis buffer (5 OmM Tris-HC1, 300 raM NaCl, 10 mM imidazole), and sonicate on ice (Out put 3.5 , Duty 60% (TOMY UD201)) was performed 20 times for 2 minutes each with a cooling time of 1 minute. The obtained cell lysate was centrifuged (10000 rpm, 30 minutes, 4 ° C), and the supernatant was recovered.

 Purification by affinity chromatography (flow rate: 15 to 2 Oml / h) using a force ram packed with Ni-NTA agarose beads previously equilibrated with the same buffer using the resulting supernatant as an added sample Was done. 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 24 to 48 hours, and then lyophilized to obtain a white powder. The yield was 38.8 mg ZL for the 23 kDa protein.

Claims

The scope of the claims

1. At least one protein selected from the group consisting of silkworm silk mouth, wild silkworm silk mouth, elastin, and fibronectin, wherein at least one of the above silkworm silk silk mouth or wild silkworm fiproin is required. Designing a silk or silk-like polymer, synthesizing the smallest unit of the silk or the designed polymer, and selecting the synthesized polymer of the smallest unit from an expression vector including a T7 promoter. At least i expression vector, then incorporating the expression vector into E. coli either BL21 (DE3) pLysS or BLR (DE3) pLysS, A method for producing silk or silk-like protein, characterized in that the method is grown using a medium selected from a composite medium.

 2. The method for producing silk or a recombinant silk-like protein according to claim 1, wherein the cultivation of the Escherichia coli is carried out at a temperature lower by 2 to 7 ° C than the optimal growth temperature of the Escherichia coli.

 3. The method for producing silk or a recombinant silk-like protein according to claim 1, wherein the expression vector is an expression vector selected from expression vectors containing a T71 ac promoter.

 4. The method for producing silk or a recombinant silk-like protein according to claim 3, wherein the expression vector is pET30a.

 5. The method for producing silk or a recombinant silk-like protein according to claim 1, wherein the medium is a TB medium.