JP2003342292A - Method for controlling protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling an embedded protein (including peptide) by photic stimulation, used as a controlling means for a physiological function of the protein (including the peptide) by utilizing a nano-particle, so that the means for reversibly controlling the physiological function of the protein, or the like, is provided more simply than ever by utilizing the nano-particle. <P>SOLUTION: This method for controlling the embedded protein (including the peptide) by the photic stimulation includes a process in which the nano- particle comprising a hydrophilic polymer is modified with a photoresponsive compound (compound causing a structural change by the photic stimulation) to change the hydrophilic polymer into a hydrophobic polymer, and then the target protein (including the peptide) is embedded in the nano-particle comprising the formed hydrophobic polymer. The method is developed on a fact that a function homologous to a molecular chaperone function to the target protein is realized by utilizing control of dynamic association of an associating molecule which responds to the photic stimulation, after utilization of the photic stimulation as a means for controlling a reversible change between hydrophilicity and hydrophobicicity is investigated. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a means for controlling physiological functions of proteins using nanoparticles. For more information,
The hydrophilic polymer is modified with a photo-responsive compound (a compound that can change the structure by photostimulation to control the hydrophilicity-hydrophobicity), and the target protein (peptide Embedded protein (including peptides) by photostimulation, which comprises a step of incorporating
Control method. More specifically, the present invention relates to achieving at least one of the following functions for embedded proteins (including peptides) incorporated into nanoparticles using this control means. 1) Transport of protein (including peptide) in vivo, 2) Storage and stabilization of protein (including peptide), 3) Purification of protein (including peptide), 4) Control of enzyme / substrate reactivity, 5) Control of antigen / antibody reaction.

[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.

In connection with the creation of artificially functional proteins by genetic engineering and the functional analysis of novel proteins in post-genome research, research on protein folding control and recovery of target proteins from intracellular inclusion bodies is highly important. . A molecular chaperone that controls protein folding in the living body is effective in vitro, but it is difficult to obtain it, so development of a general method is desired. The denatured protein solubilized by the denaturing agent is dialyzed (S.
M. West, JB Chaudhuri, JA Howell, Biotechnol
Ogy and Bioengineering, 1998, 57 (5), 590-599] and the like dilute the denaturing agent to some extent, but its efficiency is poor.

Up to now, a surfactant [Shobha Tandon, Paul M.
Horowits, The Journalof Biological Chemistry, 198
6, 261 (33), 15615-15681] (Shobha Tandon, Paul MH
orowits, The Journal of Biological Chemistry, 198
7, 262 (10), 4486-4491) (Gustavo Zardeneta, Paul M.
Horowits, The Journal of Biological Chemistry, 19
92, 267 (9), 5811-5816], sucrose (P. Valax, G. G.
eorgiou, in protein folding, G. Georgiou, Ed., ACS
Symposium series, Washington DC, 1991, 470, pp9
7], cyclodextrin (N. Karuppiah, A. Sharma, B
iochem. Biophys. Res. Commun., 1995, 211, 60-6
6], trifluoroethanol [K. Shiraki, K. Nishik
awa, Y. Goto, J. Mol. Biol., 1995, 245, 180-194],
(Patrizia Polverino de Laureto, Martina Donadi, El
ena Scaramella, Erica Frare, AngeloFontana, Biochi
micaet Biophysica Acta, 2001, 1548, 29-37], L-arginine (H. Lilie, E. Schwarz, R. Rudolph, Curr. o.
pin.Biotechnol., 1999, 9, 497-501] (J. Buchner,
R. Rudolph, Bio / Technology, 1991, 9, 157-162] (Ur
ich Brinkman, Johannes Buchner, Tra Pastan, Proc. N
atl. Acdd. Sci. USA, 1992, 89, 3075-3079] (F. Pec
orari, ACTissot, A. Plucktun, J. Mol. Biol., 19
99, 285, 1831-1843) (Kouhei Tsumoto, Katsushishi Sh
inoki, Hidemasa Kondo, Makoto Uchikawa, Takeo Juj
i, Izumi Kumagai, Journal of Immunological Methods,
1998, 219, 119-129], Proline (Fan-Guo Meng, Yong
-Doo Park, Hai-Meng Zhou, The International Journa
l of Biochemistry and Cell Biology, 2001, 33, 701-7
09], Glycerol 〔Fan-Guo Meng, Yong-Doo Park, H
ai-MengZhou, The International Journal of Biochemi
stry and Cell Biology, 2001, 33, 701-709] and PEO (Jeffrey L. Cleland, Theodore W.
Randolph, The Journal of Biological Chemistry, 199
2, 267 (5), 3147-3153) 〔Jeffrey L. Cleland, Cheste
rHedgepeth, Daniel IC Wang, The Journal of Biol
ogical Chemistry, 1992, 267 (19), 13327-13334], polyamino acid (Jeffrey L. Cleland, Daniel I. Wang, in
Biocatalistdesign for stability and specificity,
M. Himmel, Ed., ACS Symposium Series, Washington D.
C., 1993, pp 151-166) (Jeffrey L. Cleland, in Pr
otein foliding invivo and in vitro, Jeffrey L. Cle
land, Ed., ACS Symposium Series, Washington DC,
1993, 526, pp 1], heparin (Fan-Guo Meng, Yong-Do
o Park, Hai-Meng Zhou, The International Journal of
Biochemistry and Cell Biology, 2001, 33, 701-70
Water-soluble polymers such as [9] have been reported.

Gellman et al. [Surfactant and cyclodextrin [David Rozema, Samuel H. Gellman, J. Am. Che.
m. Soc. 1995, 117, 2373-2374) 〔David Rozema, Samue
l H. Gellman, Biochemistry, 1996, 35, 15760-15771)
〔David L. Daugherty, David Rozame, Peter E. Hanso
n, Samuel H. Gellman, The Journal of Bioligical Che
mistry, 1998, 273 (51), 33961-33971] has been reported as a two-step system similar to the molecular chaperone. In this system, cycloamylose [Sachiko Machida, Set
suko Ogawa, Shi Xiaohua, Takeshi Takada, Kazutoshi
Fujii, Kiyoshi Hayashi, FEBS Letters, 2000, 486,13
1-135] has also been reported to have the same effect as cyclodextrin.

[0006] The present inventor has found that a hydrophobized polysaccharide [K. Akiyoshi,
S. Degichi, H. Tajima, T. Nishikawa, J. Sunamoto,
Macromolecules, 1997, 30, 857-861] (K. Akiyoshi,
J. Sunamoto, Supermolecularscience, 1996, 3, 157-16
3) 〔T. Nishikawa, K. Akiyoshi, J. Sunamoto, Macrom
olecules, 1997, 27, 7654-7659) (T. Nishikawa, K.
Akiyoshi, J. Sunamoto, Journal of American Chemistr
y Society, 1996, 118, 6110-6115) (K. Akiyoshi, T.
Nishikawa, S. Shichibe, J. Sunamoto, Chem. Lett.,
1995, 707-708] have been shown to function as a protein host, and to have a function similar to that of a molecular chaperone by using cyclodextrin [Kazunari Akiyoshi, Yoshihiro Sasaki,
Junzo Sunamoto, Bioconjugate Chemistry, 1999,10
(3), 321-324].

[0007]

An object of the present invention is to provide means for controlling physiological functions of proteins (including peptides) using nanoparticles. That is, it is to provide a simpler means for reversibly controlling physiological functions of proteins and the like using nanoparticles.

[0008]

Means for Solving the Problems As a result of various studies on the properties of nanoparticles, the present inventor studied the use of photostimulation as a means for controlling the reversible changes in hydrophilicity and hydrophobicity, It has been found that a function equivalent to the molecular chaperone function for proteins can be achieved by utilizing the dynamic association control of associative molecules that respond to light stimulus, and completed the present invention. That is, the present invention provides "1. Formation of an amphiphilic polymer obtained by modifying a hydrophilic polymer with a photoresponsive compound (a compound capable of controlling a hydrophilic-hydrophobic property by causing a structural change by photostimulation). 2. A method for controlling an embedded protein (including peptide) by photostimulation, which comprises a step of incorporating a target protein (including peptide) into the nanoparticle 2. The nanoparticles have a particle size of 50-100 nm, The control method of the preceding clause 1. 3. The control method of the preceding clause 2, wherein the nanoparticles are polysaccharide pullulan 4. The preceding clause 1-wherein the photoresponsive compound is a spiropyran group
The control method according to any one of 3 above. 5. The control method according to any one of items 1 to 4 above, wherein the control is performed by photostimulation, and thereby the refolding of the protein is controlled. 6. 6. The control method according to any one of items 1 to 5 above, which achieves at least one of the following functions for the embedded protein (including peptide) incorporated into the nanoparticles by control. 1) Transport of protein (including peptide) in vivo, 2) Storage and stabilization of protein (including peptide), 3) Purification of protein (including peptide), 4) Control of enzyme / substrate reactivity, 5) Control of antigen / antibody reaction. 7. A preparation containing protein (including peptide) embedded nanoparticles prepared by the control method according to any one of 1 to 5 above. 8. A method for producing the preparation according to the preceding paragraph 7. It consists of.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Methods for preparing the nanoparticles of the present invention are widely 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-step reaction is a hydroxyl group-containing hydrocarbon or sterol having 12-50 carbon atoms and OCN-R1-NC.
O (In the formula, R1 is a hydrocarbon group having 1 to 50 carbon atoms.)
By reacting with a diisocyanate compound represented by the following formula to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms has reacted. In the second-step reaction, the isocyanate group-containing hydrophobic compound obtained in the first-step reaction is further reacted with a polysaccharide to contain a hydrocarbon group having 12 to 50 carbon atoms or a steryl group as a hydrophobic group. To produce a hydrophobic group-containing polysaccharide. The reaction product of this second-step reaction can be purified with a ketone solvent to produce a high-purity hydrophobic group-containing polysaccharide.

The modification with the photoresponsive compound of the present invention (a compound which undergoes a structural change by photostimulation) is carried out by the above-mentioned carbon number 12-
Instead of 50 hydroxyl group-containing hydrocarbons or sterols that have reacted one molecule, an isocyanate group-containing hydrophobic compound,
A compound that causes a reversible conversion of hydrophilicity / hydrophobicity or a large structural change accompanying photochromism is used. The representative compound is spiropyran shown in the examples of the present invention, but is not limited thereto. For example, an azobenzene group and a triphenylmethane group are exemplified.

Hydrophilic polysaccharides that can be used include pullulan, amylopectin, amylose, dextran,
It is one or more selected from the group consisting of hydroxyethyl cellulose, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan and water-soluble cellulose.

In the synthesis of a photoresponsive compound-substituted hydrophilic polysaccharide, when pullulan is used as a hydrophilic polysaccharide, 1-20 photoresponsive compounds per 100 monosaccharides, preferably 1- 10 pieces, more preferably 1-
5 substitutions) are illustrated. The properties of the resulting hydrophobized polymer can be changed by changing the substitution amounts of the photoresponsive compound and cholesterol or hydrocarbon depending on the size and the degree of hydrophobicity of the protein. In order to control the hydrophobicity, it is also suitable to introduce a small amount of an alkyl group having 10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms, in addition to the photoresponsive compound. The preparation of the combination can be achieved by confirming the particle size, photoresponsiveness (sensitivity), solubilization property and the like by experimental repetition.

The nanoparticles used in the present invention have a particle size of 50-100.
nm.

The target protein (including peptide) is incorporated into the nanoparticles by bringing the nanoparticles into contact with the target protein in a denatured state or an unfolded state.
In the embedding method using the cell-free protein synthesis system, mR
Coexist in the phase where NA exists. For example, 1-0.01 mg of nanoparticles is added to about 1-1000 μg of mRNA. However, this added amount is a ratio with the protein production amount,
It can be changed at any time considering the uptake 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. In addition, if non-secretory type or aggregation in cells occurs, after destroying the cells and solubilizing the aggregates with a chemical denaturant,
Contact 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, but in general, a ratio of weight of the target protein, etc.:nanoparticle weight = 1: 0.1-10 is preferable. Is 1: 1-5.

The protein synthesizing system of the present invention is intended for a protein synthesizing means and a 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 possible denaturation-refolding of proteins etc. such as those encapsulated inside.

The 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 include bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis. Bacillussubt
ilis) cells; fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf
9 (Spodoptera Sf9) cells; animal cells such as CHO, C
OS, HeLa, C127, 3T3, BHK, 293 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 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. When a protein or the like is produced in the transformant cell (non-secreted state), it is encapsulated in the cell (aggregated state),
Alternatively, in the already-folded state, the cells are first lysed and / or the protein or the like is solubilized with a chemical denaturant, and then the nanoparticles are brought into contact with the protein or the like to incorporate the protein or the like. Collect.

As described above, the protein and the like incorporated into the nanoparticles are prepared according to the purpose of use by separating the nanoparticles and stabilizing under physiological conditions and physiological conditions according to the individual properties of the protein. . In other words, the embedded proteins are released by changing the properties and structure of nanoparticles by photostimulation,
Regains physiological activity by refolding. This control means is used, for example, to control the following proteins and the like. Suitable proteins include known TPA, IFN (α, γ
Etc.), CSF (M-, GM-, etc.), blood coagulation factors (eg, VI
II, IX, factor XIII, etc.), but are not limited thereto.

1) In vivo delivery of proteins (including peptides) This relates to so-called targeting therapy. The target protein is embedded in the particles, and the nanoparticles embedded with the protein or the like are administered in vivo by oral or injection, and the particles are moved to target tissues, target cells, cancer cells, etc., and after arriving at the target site, Change the particle structure by light stimulation and release the embedded proteins etc.,
It is to exert biological activity after refolding. In this way, the inactivation or side effects of the active protein during its transfer in vivo can be controlled. Conveniently, it is orally administered to activate proteins etc. at specific sites in the intestine,
Then, subsequent irradiation with light achieves activation at the target site in the intestine. As an injectable drug, the surface of the nanoparticles is modified in consideration of the affinity with the target tissue / cell, and nanoparticles such as monoclonal antibodies are used to accumulate the protein-embedded nanoparticles at the target site and then photostimulate. Change the particle structure, release the embedded protein and the like, and exert biological activity after refolding. In this way, the inactivation or side effects of the active protein during its transfer in vivo can be controlled.

2) Preservation / stabilization of proteins (including peptides) In the folded state, preferable results are obtained as a means for stabilizing proteins having poor stability. There are various pharmaceuticals, cosmetics, foods, and the like, which are mainly composed of proteins and the like. If the target protein, etc. undergoes irreversible denaturation / aggregation if left unattended, trap the target protein, etc. in the nanoparticles, and release the target protein, etc. by errand or light stimulation to achieve the desired physiological function. Let it work.

3) Purification of proteins (including peptides) It is effective when the target protein or the like is produced by genetic engineering, and when it is not secreted extracellularly after synthesis of the protein or the like and becomes an inclusivity body. . Lyse the cells, denature the target protein with a denaturing agent, and incorporate into nanoparticles.
It can be recovered and then subjected to light stimulation under appropriate physiological conditions to change the structure of the nanoparticles and refold the target protein and the like to recover. In the cell-free protein synthesis means or secretory intracellular protein synthesis means, nanoparticles are allowed to coexist in the synthesis system, and the synthesized proteins and the like are continuously incorporated into the nanoparticles, and then the nanoparticles are It can be separated and recovered, and subjected to light stimulation under appropriate physiological conditions to change the structure of the nanoparticles, and refold and recover the target protein and the like.

4) Enzyme / Substrate Reactivity Control If the enzyme or substrate is embedded in the nanoparticles, it is possible to control the enzymatic reaction. The enzyme or substrate embedded in the nanoparticles is in an inactive state, and the crossing with the partner is controlled. For example, when it is commercialized as an integrated preparation as a reagent, the nanoparticles change the structure only when they are stimulated by light to release the target enzyme or substrate for embedding, and as a result, make the system where the enzyme reaction can start for the first time. If this is done, the efficiency and simplification of the formulation can be achieved. Also, in order to ensure the storage stability of the enzyme, the enzyme is embedded in the nanoparticles,
It is also possible to release the target enzyme from the nanoparticles by photostimulation and place it in the reaction system.

5) Control of antigen-antibody reaction If the antigen or antibody is embedded in the nanoparticles,
It is possible to control the antibody reaction. In a reagent or medicine using an antigen-antibody reaction, it is also possible to release the target antibody or antigen from the nanoparticles by photostimulation and put them in the reaction system.

[0027]

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.

[0028]

Example 1 Using pullulan, which is a hydrophilic polysaccharide, spiropyran-substituted pullulan (SpP) having a spiropyran group introduced therein was synthesized.

(Sample) Acetone (Wako pure chemical industry. Ltd., special grade) 1- (β-Carboxyethyl) -3 ', 3'-dimethyl-6-nitrospiro (io
dorine-2 ', 2' [2H-1] benzopyran (Spi-COOH) (Environmental Chemistry Center, Ltd.) N, N'-dicyclocarbodiimide (DCC) (Peptide instutute.
Inc.) 4-Dimethylaminopyridine (DMAP) (Wako pure chemical
industry. Ltd., special grade) Dimethyl Sulfoxide (DMSO), dehydrated (Wako pure ch
emical industry. Ltd. Organic synthesis) Pullulan (Hayashibara biochemical laboratories. In
c.)

(Synthesis of SpP) The synthetic scheme and structural formula of SpP are shown below. In the formula, R is a spiropyran group, which is a compound whose structure is changed by irradiation with ultraviolet light (UV) / visible light (VIS) or heat (chemical formula 1). This compound was exposed to UV irradiation in various organic solvents to give the cis-form (closed ring type, S-type).
conversion from the piro type) to the trans form occurs, and at the same time, the nonionic to zwitterionic state (open ring type, Mer type). It has a strong absorption in the visible region, is in a metastable state due to the molecular structure of the merocyanine type dye, and when heat is applied, the Spir
o Return to type. In addition, it can be returned to the Spiro type by stimulating with light of a wavelength in the Mer type absorption band. Such a property is called photochromism.

[0031]

[Chemical 1]

Pullulan (Mw = 108,000) was dried under reduced pressure at 70 ° C. overnight. This was dried under reduced pressure at 70 ° C overnight and frame dr
1.00g (glucoseunit 6.18mmol) in the eggplant type flask
Weighed, 20 ml of Dehydrated DMSO was added thereto, and stirred for 2 hours at room temperature under nitrogen flow to dissolve. In another well-dried eggplant-shaped flask, carboxylic acid derivative of spiropyran (Spi-COOH) (run1,2, 587mg, 1.54mmol, run3,1174m
g, 3.08mmol), DCC (run1,2, 318mg, 1.54mmol, run3 6
(36 mg, 3.08 mmol) was weighed, 10 ml of Dehydrated DMSO was added, and the mixture was stirred under a nitrogen stream at room temperature for 2 hours.

After adding 30 mg of DMAP to the pullulan solution, the spiropyran solution was added, and the mixture was stirred at 35 ° C. for 40 hours under a nitrogen stream. The color of the solution was deep purple. After the reaction was completed, the reaction solution was mixed with excess acetone and purified by reprecipitation. The obtained compound was a light purple solid. The IR spectra (Shimazu FT-I
R 8100S), 1HNMR (JOEL 400MHz), and elemental analysis. (Yield: run1 85.5%, run2 85.0%, run3-a43.2%,
run3-b 47.2%)

From the above results, hydrophilic polysaccharide pullulan was obtained.
Spiropyran-substituted pullulan (SpP) containing 0.4, 1.4, 2.8 and 6.8 spiropyran groups per 100 monosaccharides (Spiropyra
nbearing pullulan) (SpP 0.4, 1.4, 2.6, 6.8) was successfully synthesized. Hereinafter, SpP in which X spiropyrans are introduced per 100 monosaccharides is referred to as SpPX.

[0035]

Example 2 Association Behavior of Spiropyran-Substituted Pullulan (SpP) The photochromism and association behavior of the SpP synthesized in Example 1 in an aqueous solution were examined.

(Sample) 2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic
acid (HEPES) (Wako purechemical industry. Ltd.) Hydrochloride (HCl) (Wako pure chemical industry.Lt
d., For precision analysis) Sodium chloride (NaCl) (Wako pure chemical industr
y. Ltd.) Spiropyran bearing pullulan (SpP 0.4, 1.4, 2.6, 6.
8) (synthesized in Example 1)

(Preparation of Solution) The light purple solid of SpP was heated and stirred in a buffer (100 mM HEPES, pH 7.5) in the dark.
The solution obtained by dissolving at 50 ° C and removing the impurities was filtered with a filter (Sterileacro disk 25, Gelman
(science, pore size: 1.2 μm, 0.45 μm, 0.2 μm), and the mixture was allowed to stand at room temperature.

[SEC-MALS (Multi angle laser light scc
atering)] SEC-MALS and Rapid MALS were measured. SEC-
MALS is the component separated by SEC, which is detected by RI and by MALS, and by analyzing the light scattering intensity at 18 angles for each concentration corresponding to the elution time, Mw ( Weight average molecular weight) and Rw
(Weight average radius) can be measured. In RapidMALS, a guard column (pre-column) is used as the column, and it is possible to analyze in a short time (measurement time of about several minutes) by a method that creates a concentration gradient of the sample while minimizing the interaction with the column. .

(Evaluation of Association Behavior by Rapid MALS, DLS) On Association Behavior of SpP in Aqueous Solution Rapid MAL
The examination was conducted by S method and DLS measurement. For Rapid MALS measurement, TOSOH CCDP dualpump, TOSOH RI-8010 RI detecto
r, Wyatt tech.co.DAWN-E MALS detector system, column is TOSOHSWXL guard column, flow rate is 1.0 ml / min, eluent is 100 mM HEPES, 100 mM NaCl, pH
7.5 was used. A sample was prepared by dissolving a light purple solid of SpP1.4 in an eluent by heating and stirring at 50 ° C. for 30 min in a dark place, and then allowing it to cool to room temperature while shielded from light. The light stimulus is 8W UV lamp (254nm) (UVP,
8 watt handheld model UVM-18), VIS irradiation 8W White
It was performed using a light lamp (UVP, 8 watt handheld model UVM-18). The concentration of the measurement sample is 0.25-2.5mg / ml, inj
ection volume should be 10 so that the peak size is appropriate.
-100 μl. When applying the sample to the column, it was filtered using a filter (0.45 μm). The refractive index increment dn / dc of the sample is calculated by Wyatttech. Co. Optilab DSP in
The value measured by the terferometric refractometer was used.

For dynamic light scattering (DLS) measurement, Otsuka Elec
tronics Co., Ltd., DLS-700 was used. The wavelength of the light source is 63
A 3 nm He-Ne laser was used at a temperature of 25.0 ± 0.2 ° C.
The sample was a light purple solid of SpP1.4, 50 in the dark.
Buffer solution (100mM HEPES, pH
It was prepared by dissolving it in 7.5) and then allowing it to cool to room temperature while shielding it from light. The resulting solution is a filter (0.45 μm)
Filtered. The light stimulus is based on 8W UV lamp (254n
m) (UVP, 8 watt handheld model UVM-18), VIS irradiation 8
W White lightlamp (UVP, 8 watt handheld model UVM-1
8).

Preparation of SpP Aqueous Solution Solubility of SpP (0.4, 1.4, 2.8, 6.8) with various substitution ratios in water was 0.4-2.8, which was dissolved by slightly heating and stirring at a few mg / ml. However, 6.8 was hardly dissolved. Since the raw material pullulan is soluble in water, it is considered that this is because the solubility of sprupyran was low in water with a high substitution rate because of its hydrophobicity.

Photochromism of SpP Regarding photochromism shown in an aqueous solution of SpP1.4, UV
-Investigated by tracing the VIS absorption spectrum.
UVspectr equipped with Peltier temperature controller for UV measurement
An ophotometer (Hitachi, U-3300) was used. Light stimulation is U
8V UV lamp (UVP, 8 watthandheld model UVM-1
8), VIS irradiation 8W White light lamp (UVP, 8 watt hand
held model UVM-18). Upon sample preparation, the color of the solution was pale red and a UV spectrum was obtained with λmax at 515 nm (Mer I type). When this is irradiated with UV, λmax
Wavelength shifted to 535 nm (Mer II type). Also, MerI
When the solution was irradiated with visible light, the pale red color of the solution rapidly disappeared, and a closed-ring UV spectrum was obtained (Spiro type). From this, it was found that SpP exhibits photochromism that moves between three states. In summary, the following correlations were confirmed for the photochromism of SpP in aqueous solution.

First, at the time of sample preparation, spiropyran was ring-opened (MerI), which was stable for several hours in the dark at 25 ° C. If this is irradiated with VIS, S within 10 minutes
Structural change to piro type occurs (1). And this Spir
o Heating the structure rapidly returned to MerI (2). This is reverse photochromism. When Spiro type was left in the dark, it returned to MerI in about 180 minutes (3).
This suggests that SpP is the most stable species of MerI in the dark at 25 ° C. UV irradiation of Spiro type 1
It became MerII in about 0 minutes (4). When this was irradiated with VIS, it took 240 minutes to return to the Spiro type (5). Me
UV irradiation of rI turned MerII in about 5 minutes (6), and when heated, it quickly returned to MerI (7). MerII has a longer wavelength shift of λmax than MerI, and the structural change rate of (5) is very slow. (Spiropyran ring-opening type has a It significantly inhibits structural changes.
This is because the ring-opening type has a planar structure, and the spiro type has a three-dimensional structure.), Suggesting that the spiropyran molecules are associated with each other by stacking.

Association behavior of SpP Rapid MALS for SpP1.4 during sample preparation (MerI)
From the analysis results of, Mw = 1.54 × 106 (g / mol), Mw / Mn = 1.64 ±
It was found that 0.17 and Rw = 79.2 (nm). Since the raw material of the main chain, pullulan, was Mw = 1.08 × 10 5 (g / mol), it was suggested that 10 or more molecules of SpP were associated in the aqueous solution. CHP aggregate has a molecular weight of about 450,000 and has 4 associations.
It can be seen from the fact that a considerably large aggregate is formed in comparison with the above. The aggregate was stable at room temperature and in the dark (the spiropyran molecule was also stable).

VIS irradiation on MerI (0, 1, 5, 10, 30, 60, 9
At 0 min) (1), the aggregate of SpP1.4 had a slightly smaller molecular weight and particle size, and formed aggregates of about 10 and several molecules. At this time, the density of the aggregates is slightly higher. When this Spiro type is left in the dark at room temperature (0-180 min), it gradually becomes M
It changed to erI (3), but the aggregate was hardly changed at this time. Next, UV irradiation of the Spiro-type seeds by VIS irradiation for 10 min (4) caused the aggregate to increase in molecular weight and particle size at a peak of 10-30 min. Although there was variation among the data, the maximum molecular weight was about 4 million (the number of SpP associations was about 40). After this, the aggregate gradually became smaller. Next, UV irradiation of MerI (6),
As in the case of (4), the aggregates become a little larger,
Although it became smaller after that, the change was not so large as in (4). Then, VIS irradiation (10 min) is applied to MerI to turn it into Spiro, and then UV irradiation (10 min) is applied to MerI.
When II was added and then VIS irradiation was performed (5), the aggregate having a molecular weight of about 3.5 million at first became gradually smaller, and the molecular weight became about 1.7 million in about 240 minutes. This is in agreement with the result that the conversion from the association structure (MerII) to the Spiro structure due to stacking of spiropyran molecules is slow. The DLS results suggest that it behaves similarly to RapidMALS. The higher the concentration, the larger the SpP particles appear to be.

Summarizing the above results, it can be seen that 10 to several tens of molecules of SpP are aggregated to form hydrogel nanoparticles, and the reversible change of association behavior is caused by photostimulation. It is considered that the increase or decrease in size of the aggregate due to UV irradiation is due to a change in the properties of spiropyran in the crosslinked region.

[0047]

[Example 3] The purpose of this study was to improve the protein refolding efficiency using SpP, and to obtain the knowledge about the interaction between SpP and the protein obtained therefrom. CS was used as a model enzyme. As a control, the system of hydrophobized polysaccharide-cyclodextrin was also examined.

(Sample) Acethyl-CoA (Wako pure chemical industry. Ltd., for biochemistry) Choresterol bearing pullulan 108-1.2 (CHP108-1.2) (Sy
nthesized in this labo.) Dimethyl sulfoxide (DMSO) (Wako pure chemical indus
try. Ltd., special grade) 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) (Wako pu
(re chemical industry.Ltd., for SH group determination) (±) Dithiothreitol (DTT) (Wako pure chemical indust
ry. Ltd., SH group oxidation prevention) Ethylenediamine-N, N, N ', N'-tetraacetic acid, disodi
um salt, dihydrate (EDTA Na) (Wako pure chemical in
dustry. Ltd.) Guanidine hydrochloride (GuHCl) (Wako pure chemical
industry. Ltd., Biochemistry) Hydrochloride (HCl) (Wako pure chemical industry. L
td., For precision analysis) Hydroxypropyl-β-cyclodexitrin (HP-β-CD) (Nippon Shokuhin Kako Co., Ltd.) Oxaloacetic acid (Wako pure chemical industry.Lt
d.) Phenol (Wako pure chemical industry. Ltd., special grade) Sodium chloride (NaCl) (Wako pure chemical industr
y. Ltd.) Spiropyran bearing pullulan1.4 (SpP1.4) (Synthesize
d in this labo.) Sulfonic acid (Wako pure chemical industry. Ltd.,
For precision analysis) Tris [hydroxymethyl] aminomethane (Trizma base) (Sigm
a chemical co.) Tris [hydroxymethyl] aminomethane hydrochloride (Triz
ma) (Sigma chemical co.) The above purchased reagents were used as they were without purification. Throughout this experiment, as a buffer, 150 mM Tris-HC
1, 0.75 mM EDTA, pH 7.6 was used.

(Preparation of SpP Solution and CHP Solution) The SpP solution is S
30m of pP1.4 light purple solid against buffer in the dark
In order to dissolve impurities by heating and stirring (50 ° C), remove the obtained aqueous solution with a filter (Sterileacro disk 2
5, Gelman science, pore size: 1.2 μm, 0.45 μm, 0.2
(μm), and the mixture was allowed to stand at room temperature in the dark to prepare.

[Citrate Synthase (CS)] CS is an enzyme at the initial stage for introducing a product produced by glycolysis and fatty acid decomposition into the TCA cycle, and catalyzes a reaction of synthesizing citric acid from acetyl-CoA and oxaloacetic acid. To do. The reaction is acetyl
-Aldol condensation of the carbanion of the methyl group of CoA with oxaloacetate. CS is a homodimeric protein and its subunits are inactive. Also, in the subunit
It has 5 cysteine residues but no disulfide bonds are formed. As a model protein in the study of quaternary structure, CS has been studied in detail for its physicochemical properties, amino acid primary sequence, three-dimensional structure, active site, substrate binding site, and the like.

(CS solution preparation) CS (From porcine heart, M
Wsubunit = 50,000) Solution is a buf suspension purchased from Roche
It was prepared by dissolving it in fer. The concentration was quantified by measuring the absorbance at 280 nm (ε280 nm = 1.75 ml · cm / mg). In addition, the denaturing solution of CS is
1.0mg / ml CS, 6M GuHCl, 40mM DTT buffer
It was obtained by incubating at 25 ° C. for 1 h.

(Activity measurement) The enzyme activity of CS was measured by a substrate solution (23 μM, 19 μg / ml Acetyl-CoA, 0.5 mM, 66 μg / ml, Ox).
ialoacetic acid, 0.12mM, 48μg / ml, DNTB) 0.76ml and CS
After rapidly mixing 15 μl of the solution and stirring for about 5 s, the increase in the concentration of mercaptide ion generated in the following reaction was monitored by measuring the absorbance at 412 nm. Although the absorbance increases with the first-order slope, the activity recovery rate was estimated by comparing this slope with that of the natural state.

(Refolding experiment) CS is 6M GuHCl
It denatures to a state close to a random coil in the solution. At this time, it has no enzyme activity. DTT is a reducing agent that suppresses unproductive disulfide formation of cysteine residues that CS does not form disulfide bonds. This CS denaturation solution is refolded by diluting it 50 times with buffer, various concentrations of SpP1.4 solution, and CHP solution (1.0 mg / ml).
The enzyme activity (after addition) was measured. Further, as a control, a refolding experiment was carried out using a carboxylic acid derivative of pullulan or spiropyran, which is a raw material of SpP. In the system using SpP, VIS (Whitelight) irradiation and UV (254 nm) irradiation were performed to examine the effect of the conformation of spiropyran on the refolding of CS. For light stimulation, UV irradiation is 8W UV lamp (UVP, 8 watt ha
ndheld model UVM-18), VIS irradiation 8W White light lamp
(UVP, 8 watt handheld model UVM-18). In the system using CHP, the CS denaturing solution was diluted with the CHP solution, allowed to stand for 30 minutes, and then HP-β-CD (40 mM) was added to measure the enzyme activity.

(Refolding control of chemically modified CS) Chaperone action / conformation dependence of SpP In examining the effect of SpP on CS refolding, the effect of SpP on CS in the natural state was examined first. Spiro's is irradiated with VIS for 10 min and then mixed with CS solution, MerI is shaded, MerII is UV for 10 min.
After irradiation, they were mixed, and then irradiation was continued. Since there is no change in CS activity in the presence or absence of SpP, SpP nanoparticles do not interact with CS in the natural state or, even if there is an interaction such as conjugation, the enzyme activity does not increase. It has been suggested that does not affect. (Table 1) Table 1 Sample Ralative enzyme activity No denaturation Naitve CS 1.00 SpP1.4 (Spiro) 2.0mg / ml 0.99 ± 0.02 (Mer) 0.98 ± 0.05 Pullulan 1.01 ± 0.02 After GuHCl denaturetion and dilution ^* No additive 0.34 ± 0.02 Pullulan 0.37 ± 0.04 Spi-COOH (Spiro) 0.32 ± 0.06 Spi-COOH (Mer) 0.34 ± 0.05 SpP1.4 (Spiro) 1.0 mg / ml 0.53 ± 0.01 (Mer) 1.0 mg / ml 0.10 ± 0.03 (Spiro → Mer) 1.0 mg / ml 0.58 ± 0.04 (Mer → Spiro) 1.0mg / ml 0.81 ± 0.01 * Modified CS solution ([CS] = 1.0mg / ml, 6M GuHCl, 400mM D
TT) was diluted 50 times with various solutions. Buffer solution: 150mM T
ris-HCl, 0.75 mM EDTA, pH 7.6.

The effect of SpP on CS refolding was examined. Refolding experiments were performed on two conformations of Spiro and MerI.
The system used was a denaturing solution with a CS concentration of 1.0 mg / ml and a dilution ratio of 50.
Double the SpP concentration is 1.0 mg / ml. Spiro was irradiated with visible light (10 min) after preparing the SpP solution, and continued to be irradiated with visible light even after mixing with the modified CS solution. For the MerI type, the test was performed under light-shielding conditions.
In addition, pullulan (1.0 mg / ml) and Spi-COOH (87.7 μM, 1.0 mg / ml)
The chaperone activity of the SpP1.4 spiropyran unit (the same concentration as ml) was also evaluated.

No chaperone activity was found in pullulan or Spi-COOH. In this system, spontaneous refolding was about 30%, and about 70% of CS could not carry out normal folding or assembly, and are presumed to have aggregated. In the system using SpP, the maximum activity recovery was MerI, which was about 60%. Next to that, Spiro
Was about 45%. The recovery rates of these two activities were slower than those of spontaneous activity, suggesting an interaction. In addition, since the activity recovery rate in the SpP system was slower than that of the spontaneous activity, the interaction between the SpP nanoparticles and the CS refolding intermediate was suggested. (Table 1,
(Fig. 2)

Next, with the progress of folding,
We examined how chaperone action changes by changing the nano-space properties of these SpP nanoparticles by photostimulation (Fig. 2). It was a Spiro at the beginning of refolding, and immediately shielded from light and gradually became M.
About 35% of the cases with erI were spontaneous, while about 55% showed a large recovery of activity compared to 45% of Spiro → Spiro. It was almost the same as Spiro → Spiro that it was left for 30 minutes with Spiro and then shielded from light.

Regarding the one that was MerI in the early stage of refolding, it was 65 when it was immediately changed to Spiro.
% Was almost the same as MerI → MerI. 30 meters at Mer I
In the case of using Spiro after a while, a very large activity recovery was observed, which was about 80%.

[0059]

INDUSTRIAL APPLICABILITY In this study, the chaperone action of SpP in the refolding system of CS in a chemically denatured state was examined. As a result, 1) provide a denatured protein with a nanospace suitable for folding, and 2) spontaneously perform complexation / release, and 2) create a "nanospace suitable for folding" in a nanospace that undergoes folding by photostimulation. It has been found that it functions as an artificial molecular chaperone with the property that it can be modified (Fig. 3). That is, it has been clarified that the affinity with a protein can be controlled by light stimulation to promote folding. It is expected that this system will be able to provide an environment suitable for various proteins depending on the degree and interval of light stimulation. In addition, it is expected to be applied to the system of refolding from intracellular inclusions and in vitro translation system.

[Brief description of drawings]

FIG. 1 shows the photochromism of SpP1.4 (UV / VIS absorption spectrum change).

FIG. 2 shows time-dependent changes in SpP-based molecular chaperone action (time-dependent recovery of activity).

FIG. 3 shows a mechanism of action of a photoresponsive artificial molecule chaperone.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 47/48 A61P 35/00 A61P 35/00 C12N 9/00 // C12N 9/00 C12P 21/02 C C12P 21/02 A61K 37/02 F-term (reference) 4B050 CC02 CC03 GG04 KK15 LL01 4B064 AG01 CA19 CC24 DA01 4C076 AA65 AA95 BB11 CC27 EE30H FF21 GG21 4C084 AA03 AA11 BA44 DA19 DA22 DA24 DC15 DC13 MA05 DC20 MA16 DC05 MA20 DC05 MA16 DC20 MA05 DC20 AA20 BA53 DA89 FA84

Claims (8)

[Claims] 1. A nanoparticle formed by modifying the hydrophilic polymer with a photoresponsive compound (a compound capable of controlling the hydrophilicity-hydrophobicity by causing a structural change by photostimulation) to form the resulting amphiphilic polymer. A method for controlling an embedded protein (including peptide) by photostimulation, which comprises a step of incorporating a target protein (including peptide) into the. 2. The control method according to claim 1, wherein the nanoparticles have a particle size of 50-100 nm. 3. The control method according to claim 2, wherein the nanoparticles are polysaccharide pullulan. 4. The control method according to claim 1, wherein the photoresponsive compound is a spiropyran group. 5. The control method according to any one of claims 1 to 4, wherein the control is performed by photostimulation, and thereby the refolding of the protein is controlled. 6. The control method according to claim 1, which achieves at least one of the following functions for the embedded protein (including peptide) incorporated into the nanoparticles by control. 1) Transport of protein (including peptide) in vivo, 2) Storage and stabilization of protein (including peptide), 3) Purification of protein (including peptide), 4) Control of enzyme / substrate reactivity, 5) Control of antigen / antibody reaction. 7. A preparation containing protein (including peptide) embedded nanoparticles prepared by the control method according to any one of claims 1 to 5. 8. A method for producing the formulation of claim 7.