JP2003310295A - Method for producing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective method for collecting a protein supporting a biological function in a protein synthesis system. <P>SOLUTION: An examination on materials usable in the protein synthesis based upon information from molecular chaperone is performed, and bases upon the discovery that nanoparticles incorporates the protein synthesized in the protein synthesizing system and can be collected. The method allows the coexistence of the nanoparticles comprising a polymer with a hydrophobic property, in the protein synthesizing system and incorporates the protein or a peptide synthesized in the synthesizing system. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a means for recovering a protein having a physiological function in a protein synthesis system using nanoparticles. More specifically, the present invention relates to a method for producing a protein, which comprises a step of incorporating nanoparticles made of a hydrophobic polymer in a protein synthesis system and incorporating a protein or peptide synthesized by the protein synthesis system into the particles.

[0002]

2. Description of the Related Art The main research subject of genetic engineering technology is rapidly expanding from gene structure analysis to gene function analysis. Proteins in cells do not function independently, but a wide variety of protein factors, nucleic acids,
It is considered that they proceed under the coordinated interaction of low molecular species and cell membrane components, and the biological functions are performed as the sum of them. One of the central tasks of the post-genome project is to analyze the relationship between the structure and function of these various individual protein factors as a complex. The results obtained here will provide extremely important knowledge to a wide range of fields from basic biology including structural biology and biochemistry to the development and production of pharmaceuticals as applications.

Means for synthesizing heterologous proteins using genetic engineering techniques have been almost established and widely used in E. coli, Bacillus subtilis, yeast, animal cells, insect cells, etc. as hosts, interferon, human growth hormone, blood coagulation. Factor, CS
F, HSA, etc. have been put to practical use. However, once intracellular protein synthesis is performed, it passes through the cell membrane,
Many proteins are not extracellularly secreted, and some of them are denatured as inclusion bodies encapsulated in the host.

On the other hand, as a method for carrying out a protein synthesis reaction that efficiently progresses in cells in vitro, for example, components including ribosome, which is a protein translation device provided in cells, are extracted from an organism, and the extract is used as the extract. Translation template,
A research on a cell-free protein synthesis method in which a protein is synthesized in vitro by adding an amino acid serving as a substrate, an energy source, various ions, a buffer solution, and other effective factors has been actively conducted (JP-A-6- 98790, JP-A-6-22
5783, JP-A-7-194, JP-A-9-291, and JP-A-7-147992).

Escherichia coli, wheat germ, rabbit reticulocyte and the like are used for the preparation of the reaction system for cell-free protein synthesis, that is, the extract of cell or biological tissue for protein synthesis used in the cell-free protein synthesis system. Has been. Since the cell-free protein synthesis system retains performance comparable to that of living cells in terms of peptide synthesis rate and accuracy of translation reaction, it has the advantage of not requiring complicated chemical reaction steps or complicated cell culture steps. The practical system has been developed.

With the development of this cell-free protein synthesis system, it is becoming possible to more easily synthesize the protein and confirm the function of the obtained protein.

A protein exerts its biological function for the first time in the presence of a three-dimensional structure in which a polypeptide is folded, and a group of proteins called molecular chaperones assists in vivo. As a result, in the cell-free protein synthesis system, the substance exhibiting the folding function is absent, and in many cases, disorder having aggregation is reduced, and thus the protein having the function cannot be recovered in many cases. In addition, even in a protein synthesis system utilizing a cell system, it may not be possible to recover a functional protein because it is denatured as an inclusion body encapsulated in the host, or aggregates and denatures after secretion. Many.

[0008]

An object of the present invention is to provide an effective method for recovering a protein having a biological function in a protein synthesis system.

[0009]

[Means for Solving the Problems] The present inventor examined substances that can be used in protein synthesis systems based on knowledge from molecular chaperones. As a result, it is possible to recover proteins bearing biological functions by allowing the nanoparticles to incorporate the protein expressed in the protein synthesis system or the denatured protein and then elute from the nanoparticles. That is, the present invention has been completed. That is, the present invention includes the steps of "1. Coexisting nanoparticles composed of a hydrophobic polymer in a protein synthesis system and incorporating a protein or peptide synthesized by the protein synthesis system (hereinafter referred to as protein or simply protein) into the particles. 2. A method for producing a protein, which comprises: 2. The method according to item 1, wherein the nanoparticles have a particle size of 20 to 30 nm 3. The method according to item 2, wherein the nanoparticles are cholesterol-substituted pullulan 4. Protein The synthesis system synthesizes a heterologous protein or the like using a gene recombination technique in a host, and the heterologous protein or the like secreted from the host or an inclusion body encapsulated in the host (inclusion)
(1) Incorporation into nanoparticles from the body)
The manufacturing method according to any one of 1. 5. The method according to item 4, wherein the host is selected from animal cells, insect cells, yeast, Bacillus subtilis, and Escherichia coli. 6. 6. The production method according to the above 4 or 5, wherein the denatured protein or the like in the inclusion body is solubilized and then coexisted with the nanoparticles. 7. The protein synthesis system is a cell-free protein synthesis system (1)
The manufacturing method according to any one of 1 to 3. 8. 8. The production method according to the above 7, wherein the cell-free protein synthesis system utilizes a plant seed-derived embryo extract. 9. 9. The production method according to item 8 above, wherein the plant seed is selected from wheat, barley, rice, corn and spinach. 10. 8. The production method according to the above 7, wherein the cell-free protein synthesis system is E. coli lysate. It consists of.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Nanoparticles comprising the hydrophobized polymer of the present invention are known. For example, it is disclosed in WO00 / 12564 (polysaccharide containing high-purity hydrophobic group and method for producing the same). According to it, the first stage reaction has 12 to 5 carbon atoms.
0 hydroxyl group-containing hydrocarbon or sterol, and OCN-
A diisocyanate compound represented by R1-NCO (wherein R1 is a hydrocarbon group having 1 to 50 carbon atoms) is reacted to give a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms. A molecularly reacted hydrophobic compound containing an isocyanate group is produced. In the second-step reaction, the isocyanate group-containing hydrophobic compound obtained in the first-step reaction is further reacted with the polysaccharide, and the hydrophobic group has 12 carbon atoms.
A hydrophobic group-containing polysaccharide containing -50 hydrocarbon or steryl groups is prepared. The reaction product of the second step reaction can be purified with a ketone solvent to produce a highly pure hydrophobic group-containing polysaccharide. Polysaccharides that can be used include
It is one or more selected from the group consisting of pullulan, amylopectin, amylose, dextran, hydroxyethyl cellulose, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan, and water-soluble cellulose.

Of these, preferred nanoparticles are cholesterol-substituted pullulan (CHP, pullulan having a molecular weight of 108,000, and 1 to 10 cholesterols, preferably 1 to several cholesterols per 100 monosaccharides). The properties of the hydrophobized polymer can be changed by changing the cholesterol substitution amount depending on the size of the protein and the degree of hydrophobicity. In order to control the hydrophobicity, it is also suitable to introduce an alkyl group having 10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms. The nanoparticles used in the present invention have a particle size of 10-40 nm, preferably 20-30 nm. Nanoparticles are already widely commercially available, and these commercially available products can be widely used in the present invention.

[0012] In the cell-free protein synthesis system, nanoparticles are made to coexist in the phase in which mRNA is present. For example, mR
Add 1-0.01 mg of nanoparticles to about 1-1000 μg of NA. However, this addition amount can be changed at any time in consideration of the ratio with the protein production amount and the incorporation efficiency.
In the cell protein synthesis system, if it is a secretory type, it is brought into contact with the nanoparticles as it is or in the presence of a chemical modifier.
Further, if non-secreted or aggregated in cells, the cells are disrupted, the aggregates and the like are solubilized with a chemical denaturing agent, and then contacted with nanoparticles. The appropriate amount to be used is appropriately determined by experimental repetition depending on the amount and molecular weight of the target protein and the like. Generally, the weight of the target protein and the like:
Nanoparticle weight = 1: 0.1-10, preferably 1: 1-5.

The protein synthesizing system of the present invention is intended for protein synthesizing means and natural protein synthesizing means to which genetic engineering technology is widely applied, and the synthesized protein or the like is denatured due to aggregation or the like, or cell It means all that is capable of denaturation-refolding of proteins and the like, such as those encapsulated inside.

A typical system of the present invention is the synthesis of proteins and the like by a system transformed by a gene recombination technique using a host known per se such as Escherichia coli, yeast, Bacillus subtilis, insect cells, animal cells and plant cells. Is.

For the transformation, a means known per se is widely applied. For example, a replicon is used to transform a host using a plasmid, a chromosome, a virus or the like. As a more preferable system, if the stability of the gene is considered,
Although it is an integration method into chromosomes, it is simply the use of an autonomous replication system utilizing an extranuclear gene. The vector is selected according to the type of host selected, and has a gene sequence for expression and a gene sequence carrying information on replication and control as its components.

Representative of suitable hosts are bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis. Bacillus sub
tilis) cells; fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CH.
O, COS, HeLa, C127, 3T3, BHK, 2
93 and Bows melanoma cells; and plant cells and the like.

Vectors include those derived from chromosomes, episomes and viruses, such as bacterial plasmids,
Bacteriophage origin, transposon origin, yeast episome origin, insertion element origin, yeast chromosomal element origin such as baculovirus, papovavirus,
Vectors derived from viruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviruses, and vectors combining them, such as plasmids and vectors derived from genetic elements of bacteriophage, such as cosmids and There are phagemids and the like.

The transformant is cultivated by selecting the optimum conditions for the culturing conditions of each host known per se. Thus,
When the target protein or the like is secreted into the culture medium of the transformant by culturing, the nanoparticles of the present invention are allowed to exist in the medium, and the protein or the like is incorporated into the nanoparticles, which is then recovered and purified. By doing so, proteins and the like can be obtained. When a protein or the like is produced in the transformant cells (not secreted) or encapsulated in the cells (aggregated state), the cells are first lysed and the protein or the like can be treated with a chemical denaturant. The protein or the like can be obtained by solubilizing and then bringing the protein or the like into contact with the nanoparticles to incorporate the protein or the like into the nanoparticles, and then collecting and purifying the protein.

The cell-free protein synthesis system means that components including ribosome, which is a protein translation device, represented by Escherichia coli lysate and wheat germ extract are extracted from an organism,
A system for synthesizing a protein in vitro by adding a translation template, an amino acid serving as a substrate, an energy source, various ions, a buffer, and other effective factors to this extract. As an alternative to the wheat germ extract, plant seeds, for example, germs of barley, rice, corn, spinach and the like can be prepared as raw materials. As the synthesis condition, a widely known method may be applied as it is.

The wheat germ extract of the cell-free protein synthesis system is
For example, it is prepared by the method disclosed in WO00 / 68412. Other cell-free protein synthesis systems can be prepared by the methods disclosed below. JP-A-6-98790, JP-A-6-225783, JP-A-7-19
4, JP-A-9-291, JP-A-7-147992).

The method of using the nanoparticles of the present invention may be applied to a batch method known as a cell-free protein synthesis method, or may be an amino acid or energy as in the known continuous cell-free protein synthesis system of Sprin et al. It may be applied to a synthetic method of continuously supplying a source (hereinafter referred to as a continuous supply synthetic method). In the batch method, the reaction may be stopped if the protein synthesis is performed for a long time, so by using the latter continuous feed synthesis method, the reaction can be maintained for a long time,
Further efficiency improvement is possible. When the protein is synthesized by the continuous feeding synthetic method, the dialysis method can be used in combination. For example, in the ultrafiltration membrane dialysis synthesis method in which the germ extract of the present invention is used as a dialyzing solution and a mixed solution containing an energy source and an amino acid is used as a dialyzing solution, it is possible to continuously prepare a large amount of protein. is there. Here, examples of the energy source include ATP, GTP, creatine phosphate and the like, and examples of the amino acid include 20 types of L-type amino acids. Specifically, it may be performed according to the method disclosed in JP-A-2000-236896. Others The present invention relates to the multi-layer method disclosed in the examples (Waken Yaku Co., Ltd., Invitro Tech Co., Ltd.).
The method using / PG-MATE ^(TM) is simple and suitable. In either case, it is sufficient to make the nanoparticles coexist in the protein synthesis phase of the cell-free synthesis system.

As described above, the protein and the like incorporated into the nanoparticles in the protein synthesis system are separated into nanoparticles, and the dissociating agent or eluent is separated under the physiological conditions under stabilization and physiological conditions according to the individual characteristics of the protein. It is possible to separate the nanoparticles from the nanoparticles by adding them appropriately, and then to purify them appropriately by ion exchanger, gel filtration, hydrophobic chromatography, etc. based on the properties of individual proteins.

[0023]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the following examples should be regarded as an aid for obtaining a concrete recognition of the present invention, and the scope of the present invention is as follows. It is not limited in any way.

[0024]

Example 1 Drugs and Kits Used An E. coli lysate kit was used as an in vitro transcription / translation system. This kit was commercially available (Promega kit: E.coli T7 S30 System for
Circular DNA). (Product description) Using this product, transcription / translation of a DNA base sequence cloned in a plasmid containing a T promoter or a lambda vector can be easily performed. This extract contains T7 RNA polymerase for transcription and all components required for translation. Therefore, all that is required is a cloned DNA with a T7 promoter and a liposome binding site.

As a sample of a vector for protein expression,
Commercially available GFP (Green fluoresce
(TAKARA GFP, BFP vector: pQBI T7 (0.5 μg / m
l)). (Commodity description) pQBIT7 is a copypector for E. coli having a gene encoding rsGFP. The rsGFP gene is efficiently expressed in E. coli producing T7 RNA polymerase by the T7 promoter in the vector.

Using the above cell-free protein synthesis system and plasmid, the effect of adding the nanoparticles of the present invention was examined. The nanoparticles are CHP [cholesterol-substituted pullulan (molecular weight
1 cholesterol per 100 monosaccharides in 108,000 pullulan
~ 2 substitutions)] was used.

(Study on effect of CHP on cell free protein synthesis system derived from E. coli) Amino acid m
ixture 20 μl, S30 Premix without amino acid
80 μl and 80 μl of S30 Extract Circular were placed in this order in a PCR tube and stirred by up side down. This was divided into 4 wells of 40 μl each in 96 wells. Add 50 μl MilloQ-Water or CHP to the sample solution
Solution 50 μl (8.0 mg / ml CHP 40 μl + 10 μl
MilloQ-Water) or CD solution 50μl (CD 8μ
1 + MilloQ-Water (42 μl) was added and the mixture was gently pipetted. Finally, pQBI T7 template DNA is added to these 4 wells.
8 μl each was added. Immediately after addition, 37 ℃ PG-MAT
Incubated in E ^(TM) . Five minutes after the addition, the fluorescence intensity was measured by a multiplate reader.
152.When the change in fluorescence intensity almost stops changing.
5 mM Mechyl-β-cyclodextrin solution was added to all the solutions, and the change in fluorescence intensity was examined.

Results When CHP was mixed in the GFP expression system, no increase in fluorescence intensity was confirmed at all, irrespective of the change over time. On the other hand, G
An increase in fluorescence intensity could be confirmed when CHP was not mixed in the FP expression system. The increase in fluorescence intensity reached saturation at around 400 minutes. Where Mechyl-β-cycl
When odextrin was added, a large increase in fluorescence intensity could be confirmed in the CHP mixed system. After that, CD was added 2 times and 3 times, but almost no change was confirmed. From this experiment, it seems that CHP certainly traps GFP in the folding process. Although the fluorescence intensity increased when CD was added, it is certain that the inclusion of CD will have some influence on the GFP synthesis system or GFP itself. The results are shown in Fig. 1.

[0029]

Example 2 Instead of the E. coli lysate kit of Example 1, a wheat germ extract (Wakken Yakuhin Co., Ltd., Invitro Tech Co., Ltd.) (trade name: PROTIOSS ^(TM) ) was used and nanoparticles containing nanoparticles were used. The cell-free protein synthesis was performed by the multi-layer method and tested.

1) Preparation of mRNA Solution Containing Nanoparticles 15 μg of mRNA prepared in advance was dispensed into an Eppendorf, concentrated and dried by ethanol precipitation. Buffer # 1 and Buffer # 2 from PROTIOS ^(TM) to Buffer Pre M
ix was prepared as follows, and 10 μl was added to the mRNA pellet to dissolve it.

Using nanoparticles 8 mg / ml solution, 4 mg / m
l, 2 mg / ml was prepared in MilliQ water. 30 μl of each concentration of the nanoparticle solution was added to the previously prepared mRNA solution to finally prepare a 40 μl nanoparticle-containing mRNA solution.

2) Preparation of reaction solution containing nanoparticles Particle reaction solution containing components other than mRNA solution
Prepared from ^(TM) PG-MATE ^{(TM) as} follows.

To the mRNA solution containing nanoparticles prepared previously, 20
The reaction solution was prepared by adding μl each. This gives the final concentration
A reaction solution containing 4,2,1 mg / ml nanoparticles was prepared.

3) Preparation of Buffer Mix with Nanoparticles Buffer # 1 and Buff attached to PROTIOS ^(TM) PG-MATE ^(TM)
Prepare 2x Buffer Mix from er # 2 as follows and add equal volume of nanoparticle solution (8,4,2 mg / ml) to final concentration.
Buffer Mix containing 4,2,1 mg / ml nanoparticles was prepared.

4) Multilayer reaction 96 μm flat bottom plate containing 250 μl of Buffer M containing nanoparticles
Add ix, overlay 50 μl of each corresponding reaction solution on the lower layer, and use PG-MATE ^(TM) PROTIOS ^(TM) to
Reacted at 3 ° C for 17 hours.

5) Chemicals used and kit PG-MATE ^(TM) (Waken Yaku Co., Ltd., Invitro Tech Co., Ltd.) (Explanation) Since the upper lid is set at 1 ° C. higher than the bottom, evaporation due to the reaction It is suppressed as much as possible. Vector DNA expressing insoluble protein pEU (Ω) T7 GST SHP pEU (Ω) T7 His ER pEU GFP (Description) (pEU vector (PROTEIOS ^(TM) Plasmid set) ((Waken Yakuhin Co., Ltd., Invitro Tech ( Is a vector suitable for plant translation system.) RNase inhibitor (Toyobo) Thermo T7 RNA polymerase (Toyobo) G-25 Microspin G-25 Columns
: Code No.:27-5325-01 (Amersham Pharmacia Biotech Co., Ltd.)) Nanoparticles: CHP (108-1.09) containing 5% acetone (Hayashibara) Purchased CHP is not used as it is, but it is completely used for MilliQ-water. Dissolve and dialyze against MilliQ water for 4 days,
Then, white solid was obtained by freeze-drying. This was used for the experiment. Methyl-b-CD (Tokyo Kasei)

6) Treatment method Preparation of CHP solution A white solid of CHP was weighed in a target amount and uniformly dispersed in MilliQ water by heating and stirring. This was sonicated at 40 W for 10 minutes to produce a colorless liquid. afterwards
It was autoclaved at 121 ° C. for 20 minutes, filtered through a 0.22 mM filter to obtain a CHP sample solution. Preparation of CD Solution Methyl-b-CD was weighed in a target amount and dissolved in MilliQ water. This is autoclaved at 121 ℃ for 20 minutes and 0.22m
After filtering with an M filter, a CD solution was prepared.

(Experiment 1) Synthesis of GFP-expressing m-RNA using wheat germ system in the presence of CHP PG-MATE ^{(TM) from} Wakken Co., Ltd., Invitro Tech Co., Ltd.
Synthesis was performed according to the instruction manual. MilliQ-water (hereinafter dd
H20) 94.5 ml, 10 x TB solution 15 ml, 25 mM NTPs solution 15 m
Collect l, into an Eppendorf tube and put this on the up side.
Mixed by doing down. Then RNase inhibitor
1.5 ml of (120 U / ml) was added to the above sample tube. Further, add 9 ml of T7 RNA polymerase (TT7, 50U / ml).
In addition, the solution was mixed by up side down so that the solution became uniform, and immediately put on ice. Finally DNA solution (1 mg / ml)
Was added to the sample tube above and mixed by up side down and left at 37 ° C for 4 hours. Total reaction solution is 150
ml. All operations used gloves that hate mixing RNase, and experiments were carried out on ice to prevent enzyme inactivation. Purification of m-RNA Waken Yaku Co., Ltd., Invitro Tech Co., Ltd. PG-MATE
Purification was performed according to the instruction manual of ^(TM) . The G-25 microspin column was set in a 1.5 ml microtube and centrifuged at 3000 rpm (or 735 G) for 1 minute to remove the storage solution. Buffer mix [Prepared using the solution attached to PG-MATE ^{(TM) of} Waken Yaku Co., Ltd., Invitro Tech Co., Ltd. (Bu
ffer # 1 1.07ml Buffer # 2 1.25ml MilliQ-Water
A mixture of 7.68 ml; hereinafter referred to as Buffer mix] was added to a 200 ml microspin column, allowed to stand for 1 minute, then centrifuged at 3000 rpm for 2 minutes, and then the eluate was removed. This operation was repeated three times with Buffer mix, and the column was completely replaced with Buffer mix. Centrifuge the transferred reaction solution synthesized above at 12000 rpm for 2 minutes and add the supernatant after centrifugation to the microspin column prepared above.
Centrifugation was performed at 3000 rpm for 2 minutes. Synthesis of m-RNA, Confirmation of Purity The purified m-RNA was quantified by measuring the absorbance at UV 260 nm. The purity was confirmed by performing agarose gel electrophoresis. (The amount of transcribed m-RNA is the template
Since it is larger than the amount of DNA etc., the absorbance can be calculated as reflecting the concentration of the produced m-RNA.) Protein synthesis by the baking soda method Buffer mix [1C-Buffer mix preparation: Buffer # 1 64.
2 ml Buffer # 2 75.0 ml CHP solution 120 ml ddH ₂ O 22
Preparation of 0.8 ml, 2-Buffer mix: Buffer # 1 1.07 ml Buf
fer # 2 1.25 ml ddH ₂ O 7.68 ml] was prepared. Next Reac
I made a tion mix. ddH ₂ O 9.0ml, Buffer # 2 10ml,
Creatine kinase (10 mg / ml) 8.5ml, RNase inhib
itor (400U / ml) 5.0ml, Wheat germ extract 50ml, m
-Add RNA (0.3-0.4 mg / ml) 167.5 ml reaction mi
I made an xture. 25 buffer mix in a 96-well plate
0 ml was added. Then 50 ml of reaction mix slowly Buffe
Add to the bottom of rmix to form a layer. As it is PG-MATE
^(TM) at 26 ° C for 16 to 24 hours. Confirmation of protein synthesis The concentration of GFP was quantified by measuring the fluorescence intensity from the already prepared calibration curve.

Results In the case where CHP was mixed in the IN VITRO protein synthesis system, the fluorescence intensity of GFP decreased in a solution having a high CHP concentration. Moreover, when each concentration of CD was added to this solution, an increase in fluorescence intensity was confirmed. Regarding the tendency of the increase of the fluorescence intensity, the fluorescence intensity was increased in the solution having a low CHP concentration as compared with the solution having a high concentration. When the CHP concentration was kept constant and each concentration of CD was added, the CD concentration dependency could be confirmed. That is
It was confirmed that the fluorescence intensity increased more in the one with a high CD concentration. Moreover, no change in fluorescence intensity could be confirmed in the sample to which CD was not added at all. Although the fluorescence intensity increased with the increase of the CD concentration, the fluorescence intensity was less than that in the case where CHP was not mixed (NONE SAMPLE).
(Table 1)

[Table 1]

FIG. 2 shows 10% acrylamide SDS PAGE, 25
The electrophoresis result by mA, 100 min is shown. For those that do not contain nanoparticles, synthetic proteins (GST-SHP and HAHisER) can be confirmed strongly (No. 1 & 5), but due to the increase in concentration of nanoparticles, synthetic proteins cannot be confirmed (No. 4 & 8). This means that the nanoparticles trapped the synthesized protein. Figure 3 shows 15% acrylamide SDS PAGE,
It is a diagram of GFP and DHFR showing the results of electrophoresis at 25 mA for 100 min, but the same tendency can be confirmed.

[0041]

[Example 3] An experiment was carried out using an inclusion body that was sometimes produced by genetically engineering E. coli.
The protein used was a recombinant form of the serine protease (mouse BSSP 4 (Prosemin)) present in the cerebellum of mice, which was an enzyme that had not been successfully solubilized and refolded by the conventional methods. Serine protease (mouse BSSP 4 (Prosem
in)) recombinant body (50-100 mg) produced when the recombinant was expressed in Escherichia coli.
The mixture was added to GuHCl solution and stirred. Next, it was completely dispersed using a probe type sonicator. This solution at 20 ℃ 3500 rp
The mixture was centrifuged for 20 minutes, and the supernatant solution was collected. This was used as the solubilization solution for the inclusion body.

500 m of this inclusion body solution
3.5 ml of buffer solution was added to l, diluted 8 times, left at room temperature for 1 hour, and then centrifuged at 20 ° C. and 3500 rpm for 20 minutes to collect a supernatant. When the supernatant was checked by Western blotting, it was found that the target protein was not solubilized at all (Fig. 4, lane c).
On the other hand, 2.66 mg in 500 ml of Inclusion Body Solution
3.5 ml of / ml CHP solution was added and diluted to 8 times for solubilization. Leave it for 1 hour at room temperature, then at 20 ℃ 3500 rpm
After centrifugation for 20 minutes, the supernatant was collected. When the supernatant was checked by Western blotting as described above, it was found that the target protein was solubilized (Fig. 4, lane b). It can be said that by forming a complex with CHP, aggregation is suppressed and the protein is solubilized as a result. Since this protein has a so-called His-Tag, it can be purified on a His-Trap gel. When the solution complexed with CHP was allowed to interact with a His-Trap gel and examined by Western blotting, the protein was detected in the supernatant and was hardly adsorbed on the gel. On the other hand, 4 ml of CHP complex solution
71.3 mM Hydroxyporopyl b-Cyclodextrin (HP-b-
When 1 ml of the CD) solution was added and the solution in which the microparticles were disintegrated (left at room temperature for 40 minutes) was allowed to interact with the His-Trap gel, almost all proteins were adsorbed to the gel. From this, it is understood that the protein can be adsorbed to the His-Trap gel only after it is released into the bulk along with the collapse of the CHP microparticles. In addition, add excess imidazole to add His-Tr
It was revealed that the protein was recovered from the ap gel ((FIG. 4, lane d). Furthermore, it was also found that the purified protein had an enzymatic activity (added Enterokinase to the obtained sample). After overnight activation at 0 ° C., the activity was measured).

As described above, it was revealed that the CHP-cyclodextrin system effectively functions for solubilization and refolding of protease, which was difficult to solubilize in the conventional absence of urea.

Reference Example A wheat germ extract is prepared as follows. Plant seeds are first crushed. The crushing is performed, for example, by adding seeds to the mill at a rate of 50 g to 1 kg per minute and gently at a rotation speed of 5,000 to 10,000 rpm. The crushed seeds are sorted by particle size. A sieve is simply used for this classification, but there is no particular limitation as long as it is a particle size classification method that can be used by those skilled in the art. When using a sieve, those which pass a sieve mesh size of 1.0 mm and do not pass 0.45 mm are collected. Preferably pass through a sieve mesh size of 1.0 mm and 0.71
Those that do not pass mm are collected. The crude germ fraction is collected by this treatment. 1.0 mm for this crude germ fraction
The rind components of the following sizes are also contaminated, and it is necessary to remove the rind components. For this removal, the fractionation based on the weight difference is preferably performed. Since the outer skin component is light, it flies by wind selection, for example. Other sorting based on weight difference is not limited as long as it is available to those skilled in the art. The removal process is performed by a screening process using wind on a sieve having a sieve mesh size of 0.5 mm or 0.45 mm (wind selection). 0.45mm-1.0m by going through this process
m, preferably 0.71 mm to 0.85 mm in size, the crude germ fraction is obtained. Next, this crude embryo fraction is more preferably further fractionated based on the difference in specific gravity. One mode for that purpose is sorting based on the difference in buoyancy. Flotation is generally recommended for sorting based on the difference in buoyancy. Flotation is to collect the substance that floats in the supernatant fraction in a short time after the crude embryo fraction is soaked in water or an aqueous solution, and select it from the substance that precipitates. In addition, preferably, after being floated, the outer skin component and dust that are contaminated are first precipitated by vibrating, and then collected, and then collected. The flotation is preferably performed repeatedly, but is not particularly limited. More preferably, it is repeated 2 to 3 times. Flotation is performed with water or an aqueous solution containing no organic solvent. The aqueous solution is not particularly limited as long as it does not reduce the protein synthesizing ability of the embryo, and only the embryo floats on the water surface and impurities such as outer skin components can sink. The water or aqueous solution to be used is preferably at a low temperature, preferably at a temperature at which the embryo does not start its activity. A more preferred temperature is about 5 ° C or lower. For example, ice cold water is preferably used. The water may be tap water, or may be distilled water or water obtained by further deionizing distilled water. Further, a protease inhibitor may be added to water or an aqueous solution and used. After the fractionation based on the particle size, the weight difference and the specific gravity difference as described above, the components remaining on the surface portion of the crude germ fraction are fractionated by the contact treatment with water. One means of contact treatment with water is, for example, washing or friction treatment with water. By this treatment, for example, the residual endosperm component is more easily dissolved. As the contact treatment with water, for example, the crude germ fraction is subjected to a washing step with a washing solution containing no organic solvent. For cleaning, it is effective to add physical impact (physical contact treatment) such as rubbing, collision, and stirring. It is preferable to perform the washing sufficiently while exchanging the washing liquid until the washing liquid becomes substantially cloudless. Water or an aqueous solution is used as the cleaning liquid. For example, the crude embryo fraction obtained by flotation may be wrapped in gauze or the like, and washed by rubbing in the washing solution while exchanging the washing solution. In addition, for example, after suspending the crude embryo fraction in the washing solution and applying a physical shock such as stirring,
The crude germ fraction and the cloudy washing solution may be separated by a known separation means such as filtration. At this time, the washing operation and the separation operation can be performed in the same container. The aqueous solution is not particularly limited as long as it does not reduce the protein synthesizing ability of embryo. The washing liquid is preferably at a low temperature, preferably at a temperature at which the embryo does not start its activity. A more preferred temperature is about 5 ° C or lower. For example, ice cold water is preferably used. The water is preferably distilled water or deionized water or sterilized water. The thus-washed crude embryo fraction may optionally be further sonicated with detergent and / or formycin 5'-phosphate (Formycin 5'-phospho).
ate) in an aqueous solution. The ultrasonic treatment can be used according to a known method, and the treatment time is 10 minutes or less and several minutes is sufficient, but it varies depending on the treatment amount.
The ultrasonic treatment is preferably repeated, but is not particularly limited. More preferably, it is repeated 2 to 3 times.
The use of the surfactant is also in accordance with known means. For example, as the surfactant, Nonidet P-40 (NP-4
0) or IGEPAL CA-630 (code I3021 manufactured by Sigma) can be used, and for example, the concentration is 0.01% to 0.9%, preferably 0.03% to.
The content may be 0.7%, more preferably 0.05% to 0.5%. Surfactant and / or formycin 5 '
-Phosphate (Formycin 5'-phos
In order to remove the surfactant and / or the formycin 5′-phosphate (Formycin 5′-phosphate) after the ultrasonic treatment with an aqueous solution containing the same, it is preferable to wash with water or an aqueous solution not containing them. . The aqueous solution used for the treatment is not particularly limited as long as it does not reduce the protein synthesizing ability of the embryo. The water or aqueous solution is preferably at a low temperature, preferably at a temperature below which the embryo does not start its activity. A more preferred temperature is about 5 ° C or lower. For example, ice cold water is preferably used. The water is preferably distilled water or deionized water or sterilized water. The embryo fraction recovered by such treatment is treated with an organic solvent by a conventional method,
It is possible to obtain a purified product with a clean appearance that is almost the same as the germ material obtained by visual selection. Thus, this purified product was used as the raw material for the germ extract. The germ extract is prepared from the above raw materials by a known conventional method. For example, (Erickson,
A. H. et al. , (1996) Meth. in
Enzymol. , 96, 38-50).

[0045]

INDUSTRIAL APPLICABILITY The present invention provides means for synthesizing proteins using nanoparticles. The provided means is extremely useful as a means for preparing a large amount of protein in a cell-free system and a cell system, and a method for preparing an insoluble protein.

[Brief description of drawings]

FIG. 1 GFP in vitro protein synthesis using E. coli lysate. In the figure, GFP1 and GFP2 are controls without CHP addition, and CHP1 and CHP2 are each CHP 2.0 mg / ml addition,
This is an example of addition of CHP 8.0 mg / ml. None1 and None2 are examples in which CHP was added at 2.0 mg / ml and CHP was added at 8.0 mg / ml, but CD was not added thereafter. , GFP1, GF
For P2, CHP1, and CHP2, CD (6.1 mM, 1.52 m at 400 minutes)
M, 0.38 mM, 0.024 mM, 0.095 mM) was added.

FIG. 2 shows the relationship between GST-SHP and HAHisER in cell-free protein synthesis and the amount of nanoparticles. Line M is control molecular weight, Lines 1 to 4 are GST-SHP and 0,1,2,4 mg / ml of CHP (nanoparticles), and Lines 5 to 8 are HAHisE.
At R, CHP (nanoparticles) was added at 0, 1, 2, and 4 mg / ml, respectively.

FIG. 3 shows the relationship between GFP and DHFR confirmation in cell-free protein synthesis and the amount of nanoparticles. Line M is a control molecular weight, line 0 is no protein and no CHP (nanoparticles) added, and lines 1 to 4 are GFP with 0 CHP (nanoparticles) each.
1,2,4 mg / ml and lines 5-8 were added with 0,1,2,4 mg / ml of CHP (nanoparticles) in DHFR.

FIG. 4 is a diagram showing the unwinding of CPC-based infusion body (serine protease) expressed in Escherichia coli. In the figure, line a is a control protein, b is CHP added, c is CHP-free, and d is CH.
Each case in which excess imidazole is added after P disintegration is shown.

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Claims (10)

[Claims] 1. A method comprising coexisting nanoparticles comprising a hydrophobized polymer in a protein synthesis system and incorporating a protein or peptide synthesized by the protein synthesis system (hereinafter referred to as protein etc. or simply protein) into the particles. And a method for producing a protein. 2. The nanoparticles have a particle size of 20 to 30 nm.
Manufacturing method. 3. The method according to claim 2, wherein the nanoparticles are cholesterol-substituted pullulan. 4. The protein synthesis system synthesizes a heterologous protein or the like using a gene recombination technique in a host, and the heterologous protein secreted from the host or an inclusion body encapsulated in the host (in
The method according to any one of claims 1 to 3, which is incorporation from a clusion body) into nanoparticles. 5. The method according to claim 4, wherein the host is selected from animal cells, insect cells, yeast, Bacillus subtilis, and Escherichia coli. 6. The method according to claim 4, wherein the denatured protein or the like in the inclusion body is solubilized and then coexisted with the nanoparticles. 7. The production method according to claim 1, wherein the protein synthesis system is a cell-free protein synthesis system. 8. The production method according to claim 7, wherein the cell-free protein synthesis system utilizes a plant seed-derived embryo extract. 9. The method according to claim 8, wherein the plant seed is selected from wheat, barley, rice, corn and spinach. 10. The production method according to claim 7, wherein the cell-free protein synthesis system is Escherichia coli lysate.