WO2003097672A1 - Method for controlling protein - Google Patents

Method for controlling protein Download PDF

Info

Publication number
WO2003097672A1
WO2003097672A1 PCT/JP2003/006257 JP0306257W WO03097672A1 WO 2003097672 A1 WO2003097672 A1 WO 2003097672A1 JP 0306257 W JP0306257 W JP 0306257W WO 03097672 A1 WO03097672 A1 WO 03097672A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
nanoparticles
proteins
spp
control
Prior art date
Application number
PCT/JP2003/006257
Other languages
French (fr)
Japanese (ja)
Inventor
Kazunari Akiyoshi
Original Assignee
Japan Science And Technology Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2002147147A external-priority patent/JP4100499B2/en
Application filed by Japan Science And Technology Corporation filed Critical Japan Science And Technology Corporation
Publication of WO2003097672A1 publication Critical patent/WO2003097672A1/en

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier

Definitions

  • the present invention relates to a means for controlling a physiological function of a protein using nanoparticles. More specifically, a hydrophilic polymer is modified with a photo-responsive compound (a compound that changes its structure by photostimulation and can control hydrophilicity / hydrophobicity), and the target is added to the resulting nanoparticles formed by the amphiphilic polymer.
  • the present invention relates to a method for controlling an embedded protein (including a peptide) by photostimulation, which comprises a step of incorporating a protein (including a peptide). More specifically, the present invention relates to achieving at least one of the following functions for an embedded protein (including a peptide) incorporated in a nanoparticle using the control means.
  • surfactants have been used as additives to suppress aggregation during dilution of denaturants, such as surfactants L Shobha Tandon, Paul M. Horowits, The Journal of Biological Chemistry, 1986, 261 (33), 15615-15681] [Shobha Tandon, Paul M. Horowits, The Journal of Biological Chemistry, 1987, 262 (10), 4486-4491] [Gustavo Zardeneta, Paul M. Horowits, The Journal of Biological Chemistry, 1992, 267 (9), 581 5816], sucrose [P. Valax, G. Georgiou, in protein folding, G.
  • the present inventor has proposed a hydrophobic polysaccharide in which several wt% of hydrophobic groups are grafted into a hydrophilic polysaccharide (K. Akiyoshi, S. Degichi, H. Tajima, T. Nishikawa, J. Sun recitation to K. Akiyoshi, J. Sunamoto, Supermolecular science, 1996, 3, 157-163] [T. Ni shikawa, K. Akiyoshi, J. Sunamoto, Macromolecules, 1997] , 27, 7654-7659] [T. Nishikawa, K. Akiyoshi, J. Sunamoto, Journal of American Chemistry Society, 1996, 118, 6110-6115] [K.
  • An object of the present invention is to produce proteins (including peptides) using nanoparticles.
  • the purpose is to provide means for controlling physical functions. In other words, it is to provide a simpler means for reversibly controlling physiological functions such as proteins using nanoparticles. (Means for solving the problem)
  • the present inventor has considered using photostimulation as a means for controlling the reversible change in hydrophilicity and hydrophobicity.
  • the present inventors have found that a function equivalent to a molecular chaperone function for proteins can be achieved by using the control of dynamic association of molecules, and completed the present invention.
  • the hydrophilic polymer is modified with a photo-responsive compound (a compound that changes its structure by photostimulation and can control hydrophilic-hydrophobic properties), and the target protein is contained in the nanoparticles formed by the resulting amphiphilic polymer.
  • a method for controlling an embedded protein (including a peptide) by light stimulation which comprises a step of incorporating a peptide (including a peptide) ''
  • control method according to any one of the above items 1 to 4, wherein the control is performed by light stimulation, whereby the refolding of the protein is controlled.
  • control achieves at least one of the following functions with respect to the embedded protein (including peptide) incorporated in the nanoparticles. 1) In vivo transport of proteins (including peptides),
  • a preparation containing a protein (including a peptide) -embedded nanoparticle prepared by the control method according to any one of 1 to 5 above.
  • FIG. 1 is a diagram showing the photochromism (change of UV and VIS absorption spectrum) of SpPl.4.
  • FIG. 2 is a graph showing the time-dependent change in the molecular chaperone action of the SpP system (time-dependent change in activity recovery).
  • FIG. 3 is a diagram showing an action mechanism of a photoresponsive artificial molecular chaperone.
  • the second-stage reaction comprises the isocyanate group-containing hydrophobic compound obtained in the first-stage reaction and Tanuka.
  • the reaction product of this second-step reaction can be purified with a ketone-based solvent to produce a high-purity polysaccharide containing a hydrophobic group.
  • the modification with the photoresponsive compound of the present invention is carried out in place of the above-mentioned hydrophobic compound having a hydroxyl group-containing hydrocarbon or a sterol having 12 to 50 carbon atoms, which is reacted with one molecule of sterol.
  • hydrophobic compound having a hydroxyl group-containing hydrocarbon or a sterol having 12 to 50 carbon atoms, which is reacted with one molecule of sterol.
  • compounds that cause reversible conversion of hydrophilicity and hydrophobicity and large structural changes accompanying photomism are used.
  • a typical compound is spiropyran shown in Examples of the present invention, but is not limited thereto.
  • an azobenzene group and a triphenylmethane group are exemplified.
  • Hydrophilic polysaccharides that can be used include pullulan, amylopectin, mime, mime, mime, microbial growth factor, microbial growth factor, fungal growth factor, microbial growth factor, fungal growth factor, fungal growth factor, fungal growth factor, fungal growth factor, fungal growth factor, fungal growth factor, fungal growth factor, fungal growth factor, fungal growth factor, cellulose, and at least one selected from the group consisting of xyloglucan and water-soluble cellulose.
  • the photoresponsive compound-substituted hydrophilic polysaccharide when pullulan is used as the hydrophilic polysaccharide, 1 to 20 photoresponsive compounds per 100 monosaccharides per 100 monosaccharides in pullulan having a molecular weight of 108,000 are preferably used. 1-10, more preferably 1-5).
  • the properties of the resulting hydrophobized polymer can be changed by changing the substitution amount of the photoresponsive compound and cholesterol or hydrocarbon depending on the size and hydrophobicity of the protein.
  • the nanoparticles used in the present invention have a particle size of 50-100 nm.
  • Incorporation of the target protein (including peptide) into the nanoparticle is carried out by bringing the denatured or unfolded target protein into contact with the nanoparticle.
  • coexistence is made in the phase where mRNA is present.
  • 1-0.01 mg of nanoparticles is added to about 1-1000 g of mRNA.
  • this amount can be changed at any time in consideration of the ratio to the amount of protein produced and the uptake efficiency.
  • the cell protein synthesis system if it is a secretory type, it is brought into contact with nanoparticles as it is or in the presence of a chemical denaturant.
  • the protein synthesis system of the present invention covers protein synthesis means and natural protein synthesis means that widely apply genetic engineering techniques, and the synthesized protein or the like is denatured by aggregation or the like, or It means everything that can be denatured and refolded, such as those in an encapsulated state.
  • a typical system of the present invention is a system for synthesizing a protein or the like by a system transformed by a gene recombination technique using a known host such as Escherichia coli, yeast, Bacillus subtilis, insect cells, animal cells, and plant cells. It is.
  • Transformation can be performed by a widely known means, for example, by using a plasmid, chromosome, virus, or the like as a replicon to transform a host.
  • a more preferable system is a method of integration into a chromosome if gene stability is taken into consideration, but a simpler method is the use of an autonomous replication system using an extranuclear gene.
  • Vectors are selected according to the type of host selected, and The current gene sequence of interest and the gene sequence carrying information on replication and control are the constituent elements.
  • bacterial cells such as streptococci, butterflies 3 ⁇ 4ih (staphylococci), bacteria (E. coli Streptomyces) and (Bacilus ussubti).
  • Fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeL a, C127, 3T3, BHK: and 293 Bows melanoma cells; and plant cells.
  • Vectors include chromosomal, episomal, and viral vectors, such as bacterial plasmids, pacteriophage, transposons, yeast episomal, import elements, yeast chromosomal elements, such as baculovirus, Vectors from vinoles, such as papovaviruses, e.g., SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors combining them, e.g., the genetics of plasmids and pateriophages And cosmids and phagemids.
  • viral vectors such as bacterial plasmids, pacteriophage, transposons, yeast episomal, import elements, yeast chromosomal elements, such as baculovirus
  • Vectors from vinoles such as papovaviruses, e.g., SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and
  • the transformant is cultured under conditions that are optimal for the culture conditions of each host known per se.
  • the nanoparticle of the present invention is present in the medium, and the protein or the like is incorporated into the nanoparticle.
  • proteins and the like are produced in the cells of the transformant (non-secreted state), encapsulated in the cells (aggregated state), or already folded, the cells are first lysed and Solubilize Z or protein with a chemical denaturant, After being brought into contact with the particles to incorporate proteins and the like into the nanoparticles, the proteins are recovered.
  • proteins and the like incorporated into the nanoparticles are separated from the nanoparticles, and are prepared according to the intended use under stabilization and physiological conditions according to the properties of each protein.
  • the embedded proteins and the like are released due to the change in the properties and structure of the nanoparticle upon light stimulation, and regain physiological activity by refolding.
  • This control means is used for controlling, for example, the following proteins.
  • suitable proteins include known TPA, IFN (hi, ⁇ , etc.), CSF ( ⁇ -, GM-, etc.), and blood coagulation factors (eg, factors VIII, IX, XIII, etc.). It is not limited to.
  • the target protein is embedded in the particles, and the nanoparticles such as proteins are administered orally or by injection into the living body, and the particles are transferred to target tissues, target cells, cancer cells, etc., and after arriving at the target site, It changes the particle structure by light stimulation, releases embedded proteins, etc., and exerts biological activity after refolding. In this way, inactivation or side effects of the active protein during in vivo transfer can be controlled.
  • oral administration is performed to activate proteins and the like at a specific site in the intestine, and subsequent activation of light at the target site in the intestine is achieved.c.
  • the surface of the particles is modified in consideration of the affinity for the target tissue / cells, and the nanoparticles embedded in proteins or the like are accumulated at the target site by affinity, for example, monoclonal antibodies, and then the particle structure is stimulated by light. It changes the release, releasing the embedded proteins, etc., and exerting its biological activity after refolding. In this way, inactivation or side effects of the active protein during in vivo transfer can be controlled. 2) Preservation and stabilization of proteins (including peptides)
  • the target protein or the like is produced by genetic engineering, and when the protein or the like is in the form of an inclusion body without being extracellularly secreted after synthesis.
  • the cells are lysed and the target protein or the like is denatured with a denaturant, taken up in the nanoparticles, recovered, and then subjected to light stimulation under appropriate physiological conditions to change the structure of the nanoparticles, thereby converting the target protein or the like. It can be recovered by refolding.
  • nanoparticles are made to coexist in the synthesis system, and the synthesized proteins and the like are continuously incorporated into the nanoparticles, and then the nanoparticles Can be separated and recovered, and under appropriate physiological conditions, a light stimulus can be applied to change the structure of the nanoparticles, and the target protein can be recovered by refolding.
  • the enzyme reaction can be controlled until use.
  • the enzyme or substrate embedded in the nanoparticle is in an inactive state, and its crossing with the partner is controlled.
  • a product is commercialized as an integrated preparation as a reagent, the structure of the nanoparticle will not The efficiency and simplification of the preparation can be achieved by changing the system to release the enzyme or substrate for embedding, so that the enzyme reaction can be started for the first time.
  • the antigen or antibody is embedded in the nanoparticles, it is possible to control the antigen-antibody reaction.
  • the target antibody or antigen can be released from the nanoparticles by light stimulation before use and placed in a reaction system.
  • Spiropyran-substituted pullulan (SpP) with spiropyran group was synthesized using pullulan, a hydrophilic polysaccharide.
  • Acetone (Wako pure chemical industry. Ltd., special grade)
  • DMAP 4-Dimethylaminopyridine
  • R is a spiropyran group, which is a compound whose structure is changed by irradiation with ultraviolet light (UV) or visible light (VIS) or heat (I-Dani 1).
  • UV ultraviolet light
  • VIS visible light
  • I-Dani 1 heat
  • This compound is converted from its cis form (ring-closed type, Spiro type) to trans form by UV irradiation in various organic solvents, and at the same time, it changes from nonionic to zwitterionic state (ring-opened type, Mer type). .
  • It has strong absorption in the visible region, is metastable due to the molecular structure of the merocyanine-type dye, and returns to Spiro-type when heated. In addition, it returns to Spiro type by stimulating with wavelength light of Mer type absorption band. This property is called photochromism.
  • Spiropyran carboxylic acid derivative (Spi-COOH) (runl, 2,587 mg, 1.54 mmol, run3, 1174 mg, 3.08 mmol), DCC (runl, 2, 318 mg, 1) 54 mmol, run3 636 mg, 3.08 mmol) were weighed, 10 ml of Dehydrated DMSO was added, and the mixture was stirred at room temperature under a nitrogen stream for 2 hours.
  • SpP Spiropyran-substituted pullulan
  • the light purple solid of SpP was dissolved in a buffer (100 mM HEPES, ⁇ 7.5) by heating and stirring (50 ° C) in a dark place, and an aqueous solution filter (Sterileacrodisk 25, Gelman (science, pore size: 1.2 m, 0.45 ⁇ m, 0.2 ⁇ m) and allowed to stand at room temperature.
  • a buffer 100 mM HEPES, ⁇ 7.5
  • an aqueous solution filter (Sterileacrodisk 25, Gelman (science, pore size: 1.2 m, 0.45 ⁇ m, 0.2 ⁇ m)
  • LSEC-MALS Multi angle laser light sccatering
  • SEC-MALS and Rapid MALS were measured.
  • SEC-MALS is an SEC Mw (weight average molecular weight) of each component by analyzing the light scattering intensity at 18 angles for each concentration corresponding to the elution time by detecting the components separated by RI and MALS And (weight average radius) can be measured.
  • RapidMAS uses a guard column (pre-column) as a column to minimize the interaction with the column and to create a sample concentration gradient, which enables analysis in a short time (about several minutes of measurement time). Can be done.
  • the association behavior of SpP in aqueous solution was studied by Rapid MALS method and DLS measurement.
  • Rapid MALS measurement a system consisting of T0S0H CCDP dualpump, T0S0H RI-8010 RI detector, Wyatt tech.co. DAWN-E MALS detector was used, the column was a T0S0HSWXL guard column, the flow rate was 1.0 ml / min, The eluent used was 100 mM HEPES, 100 mM NaCl, pH 7.5. The sample was prepared by dissolving the light purple solid of SpPl.
  • the photostimulation was performed by UV irradiation using an 8 W UV lamp (254 nm) (UVP, 8 watt handheld model, picture 18) and VIS irradiation using an 8 W White 1 ightlamp (UVP, 8 watt handheld model UVM-18).
  • the concentration of the measurement sample was 0.25-2.5 rag / ml, and the injection volume was set at 10-100 ⁇ 1 to obtain an appropriate peak size.
  • the sample was filtered using a filter (0.45 ⁇ ).
  • the refractive index increment dn / dc of the sample was measured using a Wyatttech. Co. Optilab DSP interferometric refractometer.
  • DLS-700 was used for dynamic light scattering (DLS) measurement.
  • the wavelength of the light source is a He-Ne laser with a wavelength of 633 nm, and the temperature is 25.0 ⁇ 0.2 ° C I went in.
  • the sample was prepared by dissolving the light purple solid of SpPl. 4 in a buffer solution (100 MMHEPES, pH 7.5) by heating and stirring at 50 ° C for 30 minutes in the place, and then allowed to cool to room temperature while protected from light It was prepared by doing.
  • the obtained solution was filtered using a filter (0.45 ⁇ m).
  • the photostimulation was performed by UV irradiation using an 8 W UV lamp (254nra) (UVP, 8 watt handheld model UVM-18) and VIS irradiation using an 8 W White lightlamp (UVP, 8 watt handheld model UVM-18).
  • the photochromism of SpPl. 4 in aqueous solution was examined by following the UV'VIS absorption spectrum.
  • a UV spectrophotometer (Hitachi, U-3300) equipped with a Peltier type temperature controller was used for UV measurement.
  • the photostimulation was performed using an 8 W UV lamp (UVP, 8 watt handheld model UVM-18) for UV irradiation and an 8 W white light lamp (UVP, 8 watt handheld model UVM-18) for VIS irradiation.
  • spiropyran was ring-opened (Mer I) at the time of sample preparation and was stable for several hours in the dark at 25 ° C.
  • the structure changes to Spiro type within 10 minutes (1).
  • the Spiro structure was heated, it quickly returned to Mer I (2). This is reverse photochromism.
  • the Spiro type was left in place, it returned to Mer I in about 180 minutes (3). This suggests that SpI is the most stable species of Mer I in the dark at 25 ° C.
  • the Spiro type was irradiated with UV, it became Mer II in about 10 minutes (4).
  • this was irradiated with VIS it returned to the Spiro type in about 240 minutes (5).
  • the purpose of this study was to improve the refolding efficiency of proteins using SpP and to obtain information on the interaction between SpP and proteins obtained therefrom.
  • CS was used as a model enzyme.
  • the hydrophobized polysaccharide-cyclodextrin system was also examined.
  • Acethyl—oA for Wako pure chemical industry. Ltd. 7
  • D SO Dimethyl sulfoxide
  • Ethyl enediamine-N N, N ', N'-tetraacetic acid, di sodium salt, dihydrate (EDTA Na) (Wako pure chemical industry. Ltd.)
  • Guanidine hydrochloride (GuHCl) (Wako pure chemical industry. Ltd., for biochemistry)
  • HP- ⁇ -CD Hydroxypropyl- ⁇ -cyclodexitrin
  • Oxaloacetic acid (Wako pure chemical industry. Ltd.)
  • Phenol (Wako pure chemical industry. Ltd., special grade) Sodium chloride (NaCl) (Wako pure chemical industry. Ltd.) Spiropyran bearing pul lulanl. 4 (SpPl.) (Synthesized in this labo.)
  • Tris [hydroxymethyl] aminoraethane Tri zma base (Sigma chemi cal co.)
  • Tris [hydroxymethyl] aminomethane hydrochloride (Trizma) (Sigma chemical co.)
  • the SpP solution is obtained by dissolving the light purple solid of SpP1.4 in a dark place by heating and stirring (50 ° C) for 30 minutes in a buffer, and removing the resulting aqueous solution with a finole letter (Sterileacroix). It was prepared by filtration using sk 25, Gelman science, pore size: 1.2 / zm, 0.45 m, 0.2 ⁇ m) and leaving it to room temperature in the dark. [Citrate Synthase (CS)]
  • CS is the first-stage enzyme that guides the process of glycolysis and the breakdown of fatty acids to the TCA cycle, catalyzing the reaction of cuenic acid synthesis from acetyl-CoA and oxa-mouth acetic acid.
  • the reaction is an aldono-condensation of the carbodione of the methyl group of acetyl-CoA with oxa mouth acetic acid.
  • CS is a homodimeric protein and the subunit has no activity. It also has five cysteine residues in the subunit, but disulphide No bond has been formed. CS has been studied in detail as a model protein in studies of quaternary structure, including its physicochemical properties, primary sequence of amino acids, three-dimensional structure, active site, and substrate binding site. (CS solution preparation)
  • a denaturing solution of CS was obtained by dissolving in a buffer so that the concentration of CS was 1.0 mg / ml CS, 6 M GuHCl, and 40 mM DTT, and incubating at 25 ° C. for 1 hour.
  • CS changes to almost a random coil state in 6M GuHCl solution. At this time, it has no enzymatic activity.
  • DTT is a reducing agent for CS that suppresses the non-productive disulfide formation of cysteine residues that do not form disulfide bonds. Buffer this CS denaturing solution with various concentrations Refolding was performed by diluting 50-fold with SpPl.4 solution and CHP solution (1.0 mg / ml), and enzyme activity was measured after the start of dilution (after addition of CD in CHP system). As a control, a refolding experiment was performed using a carboxylic acid derivative of pullulanspiropyropyran, which is a raw material of SpP.
  • UV irradiation was performed using an 8 W UV lamp (UVP, 8 watt handheld model rain-18), and VIS irradiation was performed using an 8 W white light lamp (UVP, 8 watt handheld model UVM-18).
  • CHP the CS denaturing solution was diluted with the CHP solution, allowed to stand for 30 minutes, and then the enzyme activity was measured after adding HP-j3-CD (40 mM).
  • the effect of SpP on CS refolding was examined. Refolding experiments were performed on two conformations, Spiro and Mer I.
  • the system used was a denaturing solution with a CS concentration of 1.0 mg / ml, a dilution factor of 50 and a SpP concentration of 1.0 mg / ml.
  • Spiro was irradiated with visible light (10 minutes) after preparing the SpP solution, and continued to be irradiated after mixing with the denatured CS solution.
  • the Merl type the measurement was performed under light shielding conditions.
  • the nano space that performs folding by photostimulation can function as an artificial molecular chaperone with the property that it can be made into an environment more suitable for folding (Fig. 3) ).
  • the affinity with the protein can be controlled by light stimulation to promote folding.
  • This system is expected to be able to provide an appropriate environment for various proteins depending on the degree and interval of light stimulation. It is also expected to be applied to intracellular inclusions for refolding systems and in vitro translation systems.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Medical Informatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A method for controlling an embeded protein (including peptide) by photic stimulation, characterized in that it comprises a step of modifying nano particles comprising a hydrophilic polymer with a light-respondent compound (a compound undergoing the change of its structure by photic stimulation), to convert it to a hydrophobic material, and incorporating an objective protein (including peptide) to the inside of the resulting nano particles comprising a hydrophobic polymer. The method is based on the finding that, with respect to the utilization of a photic stimulation as a means for controlling the reversible change between hydrophilic and hydrophobic substances, the use of the dynamic association control of an associative molecule responding to the photic stimulation allows the achievement of a function similar to the molecular chaperone function to a protein. The method provides a means improved in the simplicity and easiness for use in controlling reversibly a physiological function of a protein (including peptide) or the like by the use of nano particles.

Description

明 細 書 蛋白質の制御方法 本出願は、 参照によりここに援用されるところの、 日本特許出願番号 2002-147147及ぴ米国仮出願番号 60/382173からの優先権を請求する。 技術分野  DESCRIPTION PROCESS FOR CONTROLLING PROTEINS This application claims priority from Japanese Patent Application No. 2002-147147 and US Provisional Application No. 60/382173, which are hereby incorporated by reference. Technical field
本発明は、 ナノ粒子を利用した蛋白質の生理的機能の制御手段に関す る。 詳しくは、 親水性高分子を光応答性化合物 （光刺激により構造変化 をおこし親水性 -疎水性を制御し得る化合物） で修飾し、得られた両親性 高分子の形成するナノ粒子内に目的蛋白質 （ペプチドを含む） を取り込 む工程を含むことを特徴とする光刺激による包埋蛋白質 （ぺプチドを含 む） の制御方法に関する。 さらに詳しくは、 この制御手段を利用レたナ ノ粒子内に取り込まれた包埋蛋白質 （ペプチドを含む） について以下の 少なくとも一の機能を達成することに関する。  The present invention relates to a means for controlling a physiological function of a protein using nanoparticles. More specifically, a hydrophilic polymer is modified with a photo-responsive compound (a compound that changes its structure by photostimulation and can control hydrophilicity / hydrophobicity), and the target is added to the resulting nanoparticles formed by the amphiphilic polymer. The present invention relates to a method for controlling an embedded protein (including a peptide) by photostimulation, which comprises a step of incorporating a protein (including a peptide). More specifically, the present invention relates to achieving at least one of the following functions for an embedded protein (including a peptide) incorporated in a nanoparticle using the control means.
1 ) 蛋白質 （ペプチドを含む） の生体内運搬、  1) In vivo transport of proteins (including peptides),
2 ) 蛋白質 （ペプチドを含む） の保存 ·安定化、  2) Preservation and stabilization of proteins (including peptides)
3 ) 蛋白質 （ペプチドを含む） の精製、  3) Purification of proteins (including peptides)
4 ) 酵素 ·基質反応性の制御、  4) Enzyme and substrate reactivity control,
5 ) 抗原，抗体反応の制御。 背景技術  5) Control of antigen and antibody reactions. Background art
遺伝子工学技術の研究課題の中心が遺伝子構造解析から遺伝子機能解 析へと急速に展開してきている。 細胞内における蛋白質はそれが単独で 機能している訳ではなく、 多種多様な蛋白質因子、 核酸、 低分子種や細 胞膜成分等の協調した相互作用のもとで進行し、 さらにそれらの総和と して生物学的機能が営まれているものと考えられている。 ボストゲノム 計画の中心課題の一つは、 これらの多種多様な個々の蛋白質因子につい て、それらの複合体としての構造と機能との関係を解析することである。 ここから得られる成果は、 構造生物学や生化学を含む基礎生物学から応 用としての医薬の開発 ·生産に至る広い分野に極めて重要な知見を提供 することになろう。 The focus of genetic engineering research is rapidly evolving from gene structure analysis to gene function analysis. Proteins in cells do not function by themselves, but they contain a wide variety of protein factors, nucleic acids, It is thought that they proceed under the coordinated interaction of the alveolar components, etc., and that the biological function is performed as a sum of them. One of the core tasks of the Bost Genome Project is to analyze the relationship between the structure and function of these various individual protein factors as a complex. The results obtained will provide crucial knowledge in a wide range of fields from basic biology, including structural biology and biochemistry, to the development and production of pharmaceuticals for applications.
遺伝子操作による人工機能蛋白質の創生や、 ボストゲノム研究におけ る新規蛋白質の機能解析に関連して、 蛋白質のフォールデイング制御 · 細胞内封入体からの目的蛋白質の回収に関する研究の重要性は高い。 生 体において蛋白質のフォールディングを制御している分子シャペロンは in vitroにおいても有効だが、 入手が困難なことから一般的な手法の開 発が望まれている。変性剤によって可溶化した変性蛋白質は、透析〔S. M. West, J. B. Chaudhuri, J. A. Howel l, Biotechnology and Bioengineering, 1998, 57 (5)， 590-599〕などにより変性剤を希釈することである程度のフ オールディングが成されるがその効率は悪い。  In relation to the creation of artificially functional proteins by genetic manipulation and the functional analysis of new proteins in the Bost genome research, the importance of research on protein folding control and recovery of target proteins from intracellular inclusions is high. Although molecular chaperones that regulate protein folding in organisms are effective in vitro, they are difficult to obtain, and the development of general methods is desired. The denatured protein solubilized by the denaturant can be reduced to some extent by diluting the denaturant by dialysis [SM West, JB Chaudhuri, JA Howell, Biotechnology and Bioengineering, 1998, 57 (5), 590-599]. Ding is done, but its efficiency is poor.
これまでに変性剤希釈の際、 凝集を抑制する添加剤として、 界面活性 剤 L Shobha Tandon, Paul M. Horowits, The Journalof Biological Chemi stry, 1986， 261 (33) , 15615-15681〕 〔 Shobha Tandon, Paul M. Horowits, The Journal of Biological Chemistry, 1987, 262 (10) , 4486-4491 ] [ GustavoZardeneta, Paul M. Horowits, The Journal of Biological Chemi stry, 1992, 267 (9)， 581ト 5816〕、スクロース〔P. Valax, G. Georgiou, in protein folding , G. Georgiou, Ed. , ACS Symposium series, Washington D. ， 1991, 470, pp97〕、 シクロデキス ト リ ン [N. Karuppiah, A. Sharma, Biochem. Biophys. Res. Commun. , 1995， 211， 60-66〕、 トリフルォロエタノール 〔Κ· Shiraki, K. Nishikawa, Y. Goto, J. Mol. Biol. , 1995, 245, 180 - 194〕 、 [PatriziaPolverino de Laureto, Martina Donadi, Jilena Scaramella, Erica Frare, AngeloFontana, Biochimica et Biophysica Acta, 2001, 1548, 29-37〕、 いアルギニン [H. Lilie, E. Schwarz, R. Rudolph, Curr. opin. Biotechnol. , 1999， 9, 497-501] 〔 J. Buchner, R. Rudolph, Bio/Technology, 1991， 9， 157 - 162〕 [Urich Brinkman, Johannes Buchner, Tra Pastan, Proc. Natl. Acdd. Sci. USA, 1992， 89, 3075-3079〕 [F. Pecorari, A. C. Tissot, A. Plucktun, J. Mol. Biol. , 1999, 285, 1831-1843] [Kouhei Tsumoto, Katsutoshi ShinoKi, Hidemasa Kondo, Makoto Uchikawa, Takeo Juji, Izumi Kumagai, Journal of Immunological Methods, 1998， 219， 119-129〕、 プ 口 リ ン〔Fan - Guo Meng, Yong-Doo Park, Hai-Meng Zhou, The Internationa丄 Journal of Biochemistry andCell Biology, 2001, 33， 70ト 709〕、 グリ セ ロ ー ル 〔 Fan - Guo Meng, Yong-Doo Park, Hai - MengZhou, The International Journal of Biochemistry and Ce丄丄 Biology, 2001, 33， 701-709〕のような低分子化合物や PE0〔Jeffrey L. Cleland, Theodore W. Randolph, The Journal ofBiological Chemistry, 1992， 267(5), 3147-3153] [Jeffrey L. Cleland, ChesterHedgepeth, Daniel I. C. Wang, The Journal of Biological Chemistry, 1992, 267(19), 13327—13334〕、 ポ リ ア ミ ノ 酸 〔 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 Protein foliding invivo and in vitro, Jeffrey L. し丄 eland, Ed.， ACS Symposium Series, Washington D. C. , 1993, 526, pp l〕、 へ ノ リ ン〔Fan - Guo Meng, Yong-Doo Park, Hai-Meng Zhou, Thelnternational Journal of Biochemistry and Cell Biology, 2001, 33, 701-709] のよ うな水溶性高分子が報告されている。 Gellman らは界面活性剤とシクロデキス ト リ ン 〔 David Rozema, Samuel H. Gel lman, J. Am. Chem. Soc. 1995, 117, 2373-2374〕 [David Rozema, Samuel H. Gellman, Biochemistry, 1996， 35, 15760-15771〕 〔 David L. Daugherty, David Rozame, Peter E. Hanson, Samuel H. Gellman, The Journal of Bioligical Chemistry, 1998， 273 (51)， 33961-33971) を用いた分子シャぺ口ン類似の two-step systemを報告し ている。 この系においてシクロアミロース [Sachiko Machida, Setsuko Ogawa, Shi Xiaohua, Takeshi Takada, Kazutoshi Fuji i, Kiyoshi Hayashi, FEBS Letters, 2000, 486, 131-135〕 もシクロデキス トリンと同様の効果 を示す報告もある。 So far, surfactants have been used as additives to suppress aggregation during dilution of denaturants, such as surfactants L Shobha Tandon, Paul M. Horowits, The Journal of Biological Chemistry, 1986, 261 (33), 15615-15681] [Shobha Tandon, Paul M. Horowits, The Journal of Biological Chemistry, 1987, 262 (10), 4486-4491] [Gustavo Zardeneta, Paul M. Horowits, The Journal of Biological Chemistry, 1992, 267 (9), 581 5816], sucrose [P. Valax, G. Georgiou, in protein folding, G. Georgiou, Ed., ACS Symposium series, Washington D., 1991, 470, pp97], cyclodextrin [N. Karuppiah, A. Sharma, Biochem. Biophys. Res. Commun., 1995, 211, 60-66], trifluoroethanol [Κ Shiraki, K. Nishikawa, Y. Goto, J. Mol. Biol., 1995, 245, 180-194), [Patrizia Polverino de Laureto, Martina Donadi, Jilena Scaramella, Erica Frare, Angelo Fontaña, Biochimica et Biophysica Acta, 2001, 1548, 29-37], Arginine [H Lilie, E. Schwarz, R. Rudolph, Curr. Opin. Biotechnol., 1999, 9, 497-501] [J. Buchner, R. Rudolph, Bio / Technology, 1991, 9, 157-162] [Urich Brinkman Natl. Acdd. Sci. USA, 1992, 89, 3075-3079] [F. Pecorari, AC Tissot, A. Plucktun, J. Mol. Biol., 1999, 285, 1831- 1843] [Kouhei Tsumoto, Katsutoshi ShinoKi, Hidemasa Kondo, Makoto Uchikawa, Takeo Juji, Izumi Kumagai, Journal of Immunological Methods, 1998, 219, 119-129], Rin Puguchi (Fan-Guo Meng, Yong-Doo Park, Hai-Meng Zhou, The Internationalization Journal of Biochemistry and Cell Biology, 2001, 33, 70, 709), Glycerol (Fan-Guo Meng, Yong-Doo Park, Hai-MengZhou, The International Journal of Biochemistry and Ce丄 丄 Biology, 2001, 33 701-709) and PE0 [Jeffrey L. Cleland, Theodore W. Randolph, The Journal of Biological Chemistry, 1992, 267 (5), 3147-3153] [Jeffrey L. Cleland, Chester Hedgepeth, Daniel IC Wang , The Journal of Biological Chemistry, 1992, 267 (19), 13327-13334), and polyamino acid (Jeffrey L. Cleland, Daniel I. Wang, in Biocatalistdesign for stability and specificity, M. Himmel, Ed., ACS Symposium Series, Washington DC, 1993, pp 151-166] [Jeffrey L. Cleland, in Protein foliding invivo and in vitro, Jeffrey L. shu eland, Ed., ACS Symposium Series, Washington DC, 1993, 526, pp l], and water-soluble polymers such as phenol (Fan-Guo Meng, Yong-Doo Park, Hai-Meng Zhou, Thelnternational Journal of Biochemistry and Cell Biology, 2001, 33, 701-709). I have. Gellman et al. Added surfactants and cyclodextrins [David Rozema, Samuel H. Gellman, J. Am. Chem. Soc. 1995, 117, 2373-2374] [David Rozema, Samuel H. Gellman, Biochemistry, 1996, 35, 15760-15771] [David L. Daugherty, David Rozame, Peter E. Hanson, Samuel H. Gellman, The Journal of Bioligical Chemistry, 1998, 273 (51), 33961-33971) They report a similar two-step system. In this system, cycloamylose [Sachiko Machida, Setsuko Ogawa, Shi Xiaohua, Takeshi Takada, Kazutoshi Fujii, Kiyoshi Hayashi, FEBS Letters, 2000, 486, 131-135] has been reported to show the same effect as cyclodextrin.
本発明者は、親水性の多糖類に数 wt %の疎水基をグラフト状に導入し た疎水ィ匕多糖 (K. Akiyoshi, S. Degichi, H. Tajima, T. Nishikawa, J. Sun誦 to, Mac進 olecules, 1997, 30, 857-861 〕 [ K. Akiyoshi, J. Sunamoto, Supermolecular science, 1996， 3， 157 - 163〕 〔T. Ni shikawa, K. Akiyoshi, J. Sunamoto, Macromolecules, 1997, 27, 7654-7659〕 〔T. Nishikawa, K. Akiyoshi, J. Sunamoto, Journal of American Chemi stry Society, 1996， 118, 6110 - 6115〕 [ K. Akiyoshi, T. Nishikawa, S. Shichibe, J. Sunamoto, Chem. Lett. , 1995， 707-708〕 からなるナノ微 粒子が蛋白質のホス トとして機能すること、 そしてシクロデキストリン を用いることで分子シャペロン類似の機能を有することを明らかにして さ た し Kazunari Akiyoshi, Yoshihiro Sasaki, Junzo Sunamoto, Bioconjugate Chemi stry, 1999, 10 (3) ， 321-324] 0 発明の開示 The present inventor has proposed a hydrophobic polysaccharide in which several wt% of hydrophobic groups are grafted into a hydrophilic polysaccharide (K. Akiyoshi, S. Degichi, H. Tajima, T. Nishikawa, J. Sun recitation to K. Akiyoshi, J. Sunamoto, Supermolecular science, 1996, 3, 157-163] [T. Ni shikawa, K. Akiyoshi, J. Sunamoto, Macromolecules, 1997] , 27, 7654-7659] [T. Nishikawa, K. Akiyoshi, J. Sunamoto, Journal of American Chemistry Society, 1996, 118, 6110-6115] [K. Akiyoshi, T. Nishikawa, S. Shichibe, J. Sunamoto, Chem. Lett., 1995, 707-708] that the nanoparticle functions as a protein host and that it has a function similar to a molecular chaperone by using cyclodextrin. Kazunari Akiyoshi, Yoshihiro Sasaki, Junzo Sunamoto, Bioconjugate Chemistry, 1999, 10 (3), 321-324] 0 Disclosure of the Invention
(発明が解決しようとする課題）  (Problems to be solved by the invention)
本発明の課題は、 ナノ粒子を利用した蛋白質 （ペプチドを含む） の生 理的機能の制御手段を提供することである。 つまり、 ナノ粒子を利用し て、 蛋白質等の生理機能を可逆的に制御するためのより簡便な手段を提 供することである。 (課題を解決するための手段） An object of the present invention is to produce proteins (including peptides) using nanoparticles. The purpose is to provide means for controlling physical functions. In other words, it is to provide a simpler means for reversibly controlling physiological functions such as proteins using nanoparticles. (Means for solving the problem)
本発明者は、 ナノ粒子の性状について種々検討の結果、 親水性と疎水 性の可逆的変化を制御するための手段として光刺激を利用することを検 討し、 この光刺激により応答する会合性分子の動的会合制御を利用すれ ば、 蛋白質に対する分子シャペロン機能と同等の機能が達成できること を見出し本発明を完成した。  As a result of various studies on the properties of nanoparticles, the present inventor has considered using photostimulation as a means for controlling the reversible change in hydrophilicity and hydrophobicity. The present inventors have found that a function equivalent to a molecular chaperone function for proteins can be achieved by using the control of dynamic association of molecules, and completed the present invention.
つまり、 本発明は、  That is, the present invention
厂  Factory
1 . 親水性高分子を光応答性化合物 （光刺激により構造変化をおこし 親水性-疎水性を制御し得る化合物） で修飾し、得られた両親性高分子の 形成するナノ粒子内に目的蛋白質 （ペプチドを含む） を取り込む工程を 含むことを特徴とする光刺激による包埋蛋白質 （ペプチドを含む） の制 御方法 '  1. The hydrophilic polymer is modified with a photo-responsive compound (a compound that changes its structure by photostimulation and can control hydrophilic-hydrophobic properties), and the target protein is contained in the nanoparticles formed by the resulting amphiphilic polymer. A method for controlling an embedded protein (including a peptide) by light stimulation, which comprises a step of incorporating a peptide (including a peptide) ''
2 . ナノ粒子が、 粒径 50-100nmである、 前項 1の制御方法。  2. The control method according to 1 above, wherein the nanoparticles have a particle size of 50-100 nm.
3 . ナノ粒子が、 多糖プルランである、 前項 2の制御方法。  3. The control method according to the above item 2, wherein the nanoparticles are polysaccharide pullulan.
4 . 光応答性化合物が、スピロピラン基である前項 1 - 3の何れか一に 記載の制御方法。  4. The control method according to any one of the above items 1 to 3, wherein the photoresponsive compound is a spiropyran group.
5 . 制御が、 光刺激によっておこなわれ、 これにより蛋白質のリフォ ールディングが制御される前項 1 - 4の何れか一に記載の制御方法。  5. The control method according to any one of the above items 1 to 4, wherein the control is performed by light stimulation, whereby the refolding of the protein is controlled.
6 . 制御によって、 ナノ粒子内に取り込まれた包埋蛋白質 （ペプチド を含む）について以下の少なくとも一の機能を達成する前項 1 - 5の何れ か一に記載の制御方法。 1 ) 蛋白質 （ペプチドを含む） の生体内運搬、 6. The control method according to any one of the preceding items 1 to 5, wherein the control achieves at least one of the following functions with respect to the embedded protein (including peptide) incorporated in the nanoparticles. 1) In vivo transport of proteins (including peptides),
2) 蛋白質 （ペプチドを含む） の保存 ·安定化、  2) Storage and stabilization of proteins (including peptides)
3) 蛋白質 （ペプチドを含む） の精製、  3) Purification of proteins (including peptides)
4) 酵素 ·基質反応性の制御、  4) Control of enzyme and substrate reactivity,
5) 抗原 ·抗体反応の制御。  5) Control of antigen-antibody reaction.
7. 前項 1-5の何れか一に記載の制御方法で調製される蛋白質（ぺプ チドを含む） 包埋ナノ粒子を含有する製剤。  7. A preparation containing a protein (including a peptide) -embedded nanoparticle prepared by the control method according to any one of 1 to 5 above.
8. 前項 7の製剤の製造方法。」  8. Manufacturing method of the preparation of the preceding clause 7. "
からなる。 図面の簡単な説明  Consists of BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 SpPl.4のフォトクロミズム （UV · VIS吸収スぺク トル変化） を示した図である。  FIG. 1 is a diagram showing the photochromism (change of UV and VIS absorption spectrum) of SpPl.4.
第 2図は、 SpP 系の分子シャペロン作用の経時変化 （活性回復の経時 変化） を示した図である。  FIG. 2 is a graph showing the time-dependent change in the molecular chaperone action of the SpP system (time-dependent change in activity recovery).
第 3図は、光応答性人工分子シャペロンの作用機構を示した図である。 発明を実施するための最良の形態  FIG. 3 is a diagram showing an action mechanism of a photoresponsive artificial molecular chaperone. BEST MODE FOR CARRYING OUT THE INVENTION
本発明のナノ粒子の調製法は広く公知である。 例えば WO O 0/ 1 2 5 64 (高純度疎水性基含有多糖類およびその製造方法）に開示がある。 それによると、第 1段階反応は、炭素数 1 2-5 0の水酸基含有炭化水素 またはステロールと、 OCN- R1 - NCO (式中、 R1 は炭素数 1 - 50 の炭化水素基である。）で表されるジィソシアナ一ト化合物とを反応させ て、炭素数 1 2-5 0の水酸基含有炭化水素またはステロールが 1分子反 応したイソシアナ一ト基含有疎水性化合物を製造する。第 2段階反応は、 前記第 1段階反応で得られたィソシアナート基含有疎水性化合物と多糠 類とをさらに反応させて、疎水性基として炭素数 1 2 - 5 0の炭化水素基 またはステリル基を含有する疎水性基含有多糖類を製造する。 この第 2 段階反応の反応生成物をケトン系溶媒で精製して高純度疎水性基含有多 糖類の製造が可能である。 Methods for preparing the nanoparticles of the present invention are widely known. For example, it is disclosed in WO 0/125656 (high-purity hydrophobic group-containing polysaccharide and a method for producing the same). According to the first step reaction, the hydroxyl-containing hydrocarbon or sterol having 12-50 carbon atoms and OCN-R1-NCO (where R1 is a hydrocarbon group having 1-50 carbon atoms). Is reacted with a diisocyanate compound represented by the formula (1) 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. The second-stage reaction comprises the isocyanate group-containing hydrophobic compound obtained in the first-stage reaction and Tanuka. To produce a hydrophobic group-containing polysaccharide containing a hydrocarbon group or a steryl group having 12 to 50 carbon atoms as a hydrophobic group. The reaction product of this second-step reaction can be purified with a ketone-based solvent to produce a high-purity polysaccharide containing a hydrophobic group.
本発明の光応答性化合物 （光刺激により構造変化をおこす化合物） で の修飾は、上記炭素数 1 2 - 5 0の水酸基含有炭化水素またはステロール が 1分子反応したィソシアナート基含有疎水性化合物の代わりに、 フォ トク口ミズムに伴って親水性♦疎水性の可逆変換や大きな構造変化を引 起す化合物が利用される。 その代表的な化合物は本発明の実施例で示し たスピロピランであるがこれに限定されない。 例えば、 ァゾベンゼン基 やトリフエニルメタン基が例示される。  The modification with the photoresponsive compound of the present invention (the compound that undergoes a structural change by photostimulation) is carried out in place of the above-mentioned hydrophobic compound having a hydroxyl group-containing hydrocarbon or a sterol having 12 to 50 carbon atoms, which is reacted with one molecule of sterol. In particular, compounds that cause reversible conversion of hydrophilicity and hydrophobicity and large structural changes accompanying photomism are used. A typical compound is spiropyran shown in Examples of the present invention, but is not limited thereto. For example, an azobenzene group and a triphenylmethane group are exemplified.
使用されうる親水性の多糖類としては、 プルラン、 アミロぺクチン、 了ミロース、 デキス トラン、 ヒ ドロキシェチルセノレロース、 ヒ ドロキシ ェチ /レデキス トラン、 マンナン、 レバン、 ィヌ リン、 キチン、 キ トサン、 キシログルカンおよび水溶性セルロースからなる群より選択される 1種 以上である。  Hydrophilic polysaccharides that can be used include pullulan, amylopectin, mirose, dextran, hydroxyxetilsenorelose, hydroxyeti / redextran, mannan, leban, inulin, chitin, chitosan. And at least one selected from the group consisting of xyloglucan and water-soluble cellulose.
光応答性化合物置換親水性多糖類の合成は、 親水性多糖類としてプル ランを利用した場合、 分子量 108， 000のプルランに 100単糖あたり光応 答性化合物が 1 - 2 0個、好ましくは 1 - 1 0個、 より好ましくは 1 - 5個 置換） が例示される。 得られる疎水化高分子の性状は、 蛋白質のサイズ や疎水性の程度により、 光応答性化合物及ぴコレステロール又炭化水素 の置換量を換え変更可能である。 疎水性をコントロールするためには、 光応答性化合物に加えて、 炭素数.1 0 - 3 0、 好ましくは炭素数 1 2 - 2 0程度のアルキル基を少量導入することも好適である。組合せの調製は、 粒子径、光応答性（感受性）、可溶化性等を実験的繰り返しにより確認し、 達成可能である。 本発明で使用するナノ粒子は、 粒径 50- lOOnmである。 In the synthesis of the photoresponsive compound-substituted hydrophilic polysaccharide, when pullulan is used as the hydrophilic polysaccharide, 1 to 20 photoresponsive compounds per 100 monosaccharides per 100 monosaccharides in pullulan having a molecular weight of 108,000 are preferably used. 1-10, more preferably 1-5). The properties of the resulting hydrophobized polymer can be changed by changing the substitution amount of the photoresponsive compound and cholesterol or hydrocarbon depending on the size and hydrophobicity of the protein. In order to control the hydrophobicity, it is also suitable to introduce a small amount of an alkyl group having about 0.10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms, in addition to the photoresponsive compound. Preparation of the combination can be achieved by confirming the particle size, photoresponsiveness (sensitivity), solubilization, and the like by repeated experiments. The nanoparticles used in the present invention have a particle size of 50-100 nm.
ナノ粒子への目的蛋白質 （ペプチドを含む） の取り込みは、 目的蛋白 質の変性状態あるいは未フォールディング状態のものとナノ粒子を接触 させ行う。 無細胞蛋白質合成系を利用した包埋法においては、 m R N A の存在する相に共存させる。例えば、 m R N Aの約 1 - 1 0 0 0 gに対 して 1 - 0 . 0 1 m gのナノ粒子を添加する。 但し、 この添加量は、 蛋白 産生量との比率、 取込み効率を考慮し、 随時変更可能である。 細胞蛋白 質合成系においては、 分泌型であればそのまま或は化学的変性剤との共 存下でナノ粒子と接触させる。 また、 非分泌型或は細胞内での凝集体化 がおこるものであれば、 細胞を破壊し、 凝集体等を化学的変性剤で可溶 化した後、ナノ粒子と接触させる。その使用適量は、対象蛋白質等の量 · 分子量によって適宜実験的繰り返しにより決定されるが、 一般的には、 対象蛋白質等の重量：ナノ粒子重量 = 1 : 0 . 1 - 1 0の比率で、 好適に は 1 ： 1—5である。  Incorporation of the target protein (including peptide) into the nanoparticle is carried out by bringing the denatured or unfolded target protein into contact with the nanoparticle. In the embedding method using the cell-free protein synthesis system, coexistence is made in the phase where mRNA is present. For example, 1-0.01 mg of nanoparticles is added to about 1-1000 g of mRNA. However, this amount can be changed at any time in consideration of the ratio to the amount of protein produced and the uptake efficiency. In the cell protein synthesis system, if it is a secretory type, it is brought into contact with nanoparticles as it is or in the presence of a chemical denaturant. If non-secretory or aggregates occur in the cells, destroy the cells, solubilize the aggregates and the like with a chemical denaturant, and then contact the nanoparticles. The appropriate amount to be used is determined by repeating experiments as appropriate according to the amount and molecular weight of the target protein, etc., but in general, the weight of the target protein, etc .: the weight of the nanoparticle = 1: 0.1-10, Preferably it is 1: 1-5.
本発明の蛋白質合成系とは、 広く遺伝子工学技術を応用した蛋白質合 成手段及び天然の蛋白質合成手段をも対象とし、 合成される蛋白質等が 凝集等により変性してしまう、 或は細胞内に封入された状態にあるもの など、 蛋白質等の変性-リフォールディングが可能な全てを意味する。 本発明の典型的な系は、大腸菌、酵母、枯草菌、昆虫細胞、動物細胞、 植物細胞等の自体公知の宿主を利用し遺伝子組換え技術によつて形質転 換した系による蛋白質等の合成である。  The protein synthesis system of the present invention covers protein synthesis means and natural protein synthesis means that widely apply genetic engineering techniques, and the synthesized protein or the like is denatured by aggregation or the like, or It means everything that can be denatured and refolded, such as those in an encapsulated state. A typical system of the present invention is a system for synthesizing a protein or the like by a system transformed by a gene recombination technique using a known host such as Escherichia coli, yeast, Bacillus subtilis, insect cells, animal cells, and plant cells. It is.
形質転換は、 自体公知の手段が広く応用され、 例えばレブリコンとし て、 プラスミ ド、 染色体、 ウィルス等を利用して宿主の形質転換が行わ れる。 より好ましい系としては、 遺伝子の安定性を考慮するならば、 染 色体内への組込み法であるが、 簡便には核外遺伝子を利用した自律複製 系の利用である。 ベクターは、 選択した宿主の種類により選別され、 発 現目的の遺伝子配列と複製そして制御に関する情報を担持した遺伝子配 列とを構成要素とする。 Transformation can be performed by a widely known means, for example, by using a plasmid, chromosome, virus, or the like as a replicon to transform a host. A more preferable system is a method of integration into a chromosome if gene stability is taken into consideration, but a simpler method is the use of an autonomous replication system using an extranuclear gene. Vectors are selected according to the type of host selected, and The current gene sequence of interest and the gene sequence carrying information on replication and control are the constituent elements.
適当な宿主の代表的なものには、 細菌細胞、 例えば連鎖球菌属 (streptococci)、 ブトク珠 ¾ih (staphylococci)、 大 菌 (E. coli ストレプトミセス属菌（Strept omvces)およひ (Bacil丄 ussubti丄 isノ 細胞；真菌細胞、例えば酵母細胞およびァスペルギルス属（Aspergillus) 細胞；昆虫細胞、 例えばドロソフイラ S 2 (DrosophilaS2) およびスポ ドプテラ S f 9 (SpodopteraSf9)細胞；動物細胞例えば CHO、 C O S、 H e L a、 C 1 2 7、 3 T 3、 BHK：、 2 9 3ぉょぴボウズ （Bows) メ ラノーマ細胞；ならびに植物細胞等がある。  Representative of suitable hosts include bacterial cells, such as streptococci, butterflies ¾ih (staphylococci), bacteria (E. coli Streptomyces) and (Bacilus ussubti). Fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeL a, C127, 3T3, BHK: and 293 Bows melanoma cells; and plant cells.
ベクターには、 染色体、 ェピソ一ムおよびウィルス由来のベクター、 例えば細菌プラスミ ド由来、 パクテリオファージ由来、 トランスポゾン 由来、 酵母ェピソ一ム由来、 揷入エレメント由来、 酵母染色体エレメン ト由来、 例えばバキュロウィルス、 パポバウィルス、 例えば S V 4 0、 ワクシニアウィルス、 アデノウィルス、 鶏痘ウィルス、 仮性狂犬病ウイ ルスおよびレトロウィルス等のウイノレス由来のベクタ一、 ならびにそれ らを組み合わせたベクター、 例えばプラスミ ドおよびパクテリオファー ジの遺伝学的エレメント由来のベクター、 例えばコスミ ドおよぴファー ジミ ド等がある。  Vectors include chromosomal, episomal, and viral vectors, such as bacterial plasmids, pacteriophage, transposons, yeast episomal, import elements, yeast chromosomal elements, such as baculovirus, Vectors from vinoles, such as papovaviruses, e.g., SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors combining them, e.g., the genetics of plasmids and pateriophages And cosmids and phagemids.
形質転換体は、 自体公知の各々の宿主の培養条件に最適な条件を選択 して培養される。 かく して、 培養により 目的とする蛋白質等が形質転換 体の培養培地中に分泌される場合は、 該培地中に本発明のナノ粒子を存 在させナノ粒子中に蛋白質等を取込む。 蛋白質等が形質転換体の細胞内 に生成される場合 （分泌されない状態）、 細胞内に封入された状態 （凝集 体化した状態）、或は既にフォールディングされた状態では、まず細胞を 溶解し及び Z若しくは蛋白質等を化学変性剤で可溶化し、 次いで、 ナノ 粒子と接触させてナノ粒子中に蛋白質等を取込ませた後、 これを回収す る。 The transformant is cultured under conditions that are optimal for the culture 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 the culturing, the nanoparticle of the present invention is present in the medium, and the protein or the like is incorporated into the nanoparticle. When proteins and the like are produced in the cells of the transformant (non-secreted state), encapsulated in the cells (aggregated state), or already folded, the cells are first lysed and Solubilize Z or protein with a chemical denaturant, After being brought into contact with the particles to incorporate proteins and the like into the nanoparticles, the proteins are recovered.
以上のようにして、 ナノ粒子に取り込まれた蛋白質等は、 ナノ粒子を 分離し、 蛋白質個々の性状に応じた安定化及び生理的条件下で、 その使 用目的に応じて調製される。 つまり包埋された蛋白質等が、 光刺激でナ ノ粒子の性状 ·構造が変化することで、 遊離され、 リフォールデイング により生理活性を取り戻す。 この制御手段は、 例えば以下のような蛋白 質等の制御に利用される。好適な蛋白質としては、公知の TPA、 IFN (ひ、 γ等）、 CSF (Μ -、 GM-等）、 血液凝固因子 （例えば、 第 VIII、 IX、 XIII 因子等） が例示されるが、 これに限定されない。  As described above, proteins and the like incorporated into the nanoparticles are separated from the nanoparticles, and are prepared according to the intended use under stabilization and physiological conditions according to the properties of each protein. In other words, the embedded proteins and the like are released due to the change in the properties and structure of the nanoparticle upon light stimulation, and regain physiological activity by refolding. This control means is used for controlling, for example, the following proteins. Examples of suitable proteins include known TPA, IFN (hi, γ, etc.), CSF (Μ-, GM-, etc.), and blood coagulation factors (eg, factors VIII, IX, XIII, etc.). It is not limited to.
1 ) 蛋白質 （ペプチドを含む） の生体内運搬 1) In vivo delivery of proteins (including peptides)
これは、 所謂、 ターゲティング治療に関する。 目的蛋白質を、 粒子内 に包埋し、 経口或は注射によって生体内に蛋白質等包埋ナノ粒子を投与 し、 粒子を目標組織、 目標細胞、 癌細胞等に移動させ、 目標部位に到着 後、 光刺激により粒子構造を変化させ、 包埋蛋白質等を遊離させ、 リフ オールディング後に生物活性を発揮させるというものである。 この方法 で、 活性蛋白質の生体内移動中の失活或は副作用が制御可能である。 簡 便には、 腸内の特別の部位での蛋白質等の活性化のために経口投与し、 そしてそれに続く光照射で腸内のターゲット部位での活性化を達成する c 注射薬としては、 ナノ粒子の表面を目標組織 ·細胞との親和性を考慮し た修飾を施し、 例えばモノクローナル抗体等、 その親和性により 目標部 位に蛋白質等包埋ナノ粒子を集積させ、 その後光刺激により粒子構造を 変化させ、 包埋蛋白質等を遊離させ、 リフォールデイング後に生物活性 を発揮させるというものである。 この方法で、 活性蛋白質の生体内移動 中の失活或は副作用が制御可能である。 2 ) 蛋白質 （ペプチドを含む） の保存，安定化 This relates to so-called targeting therapy. The target protein is embedded in the particles, and the nanoparticles such as proteins are administered orally or by injection into the living body, and the particles are transferred to target tissues, target cells, cancer cells, etc., and after arriving at the target site, It changes the particle structure by light stimulation, releases embedded proteins, etc., and exerts biological activity after refolding. In this way, inactivation or side effects of the active protein during in vivo transfer can be controlled. Conveniently, oral administration is performed to activate proteins and the like at a specific site in the intestine, and subsequent activation of light at the target site in the intestine is achieved.c. The surface of the particles is modified in consideration of the affinity for the target tissue / cells, and the nanoparticles embedded in proteins or the like are accumulated at the target site by affinity, for example, monoclonal antibodies, and then the particle structure is stimulated by light. It changes the release, releasing the embedded proteins, etc., and exerting its biological activity after refolding. In this way, inactivation or side effects of the active protein during in vivo transfer can be controlled. 2) Preservation and stabilization of proteins (including peptides)
フォールディングの状態で、 安定性に乏しい蛋白質等の安定化のため の手段としても好ましい結果を導く。蛋白質等を主成分とする製剤には、 医薬品、 化粧品、 食品等様々のものがある。 目的とする蛋白質等が、 放 置すれば不可逆的に変性 ·凝集を起こす場合、 本ナノ粒子の中に目的蛋 白質等を閉じ込め、 用時、 光刺激で目的蛋白質等を遊離させ、 目的の生 理学的機能を発揮させる。 3 ) 蛋白質 （ペプチドを含む） の精製  In the state of folding, a favorable result can be obtained as a means for stabilizing poorly stable proteins and the like. There are various types of preparations containing protein as a main component, such as pharmaceuticals, cosmetics, and foods. If the target protein, etc., undergoes irreversible denaturation / aggregation when left alone, the target protein, etc., is confined in the present nanoparticles, and at the time of use, the target protein, etc. is released by light stimulation to produce the target protein, etc. Exercise physical function. 3) Purification of proteins (including peptides)
目的の蛋白質等が遺伝子工学的に生産される場合、蛋白質等の合成後、 細胞外分泌されず封入体の状態になる場合に有効である。 細胞を溶解し 変性剤で目的蛋白質等を変性させ、 ナノ粒子中に取込み、 回収し、 その 後、 適当な生理的条件下で光刺激を与えてナノ粒子の構造を変化させ、 目的蛋白質等をリフォールデイングさせて回収することができる。 無細 胞蛋白質合成手段或は分泌系の細胞内蛋白質合成手段にあっては、 合成 系にナノ粒子を共存させ、 合成されてくる蛋白質等をナノ粒子内に連続 的に取込み、 その後、 ナノ粒子を分離回収して、 適当な生理的条件下で 光刺激を与えてナノ粒子の構造を変化させ、 目的蛋白質等をリフォール デイングさせて回収することができる。  It is effective when the target protein or the like is produced by genetic engineering, and when the protein or the like is in the form of an inclusion body without being extracellularly secreted after synthesis. The cells are lysed and the target protein or the like is denatured with a denaturant, taken up in the nanoparticles, recovered, and then subjected to light stimulation under appropriate physiological conditions to change the structure of the nanoparticles, thereby converting the target protein or the like. It can be recovered by refolding. In cell-free protein synthesis means or secretory intracellular protein synthesis means, nanoparticles are made to coexist in the synthesis system, and the synthesized proteins and the like are continuously incorporated into the nanoparticles, and then the nanoparticles Can be separated and recovered, and under appropriate physiological conditions, a light stimulus can be applied to change the structure of the nanoparticles, and the target protein can be recovered by refolding.
4 ) 酵素 ·基質反応性の制御 4) Control of enzyme and substrate reactivity
酵素又は基質を、 ナノ粒子内に包埋させておけば、 酵素反応の制御が 用時まで可能である。 ナノ粒子内に包埋された酵素又は基質は、 不活性 状態として、 相手側との交差が制御される。 例えば、 試薬として一体化 製剤として製品化した場合に、 光刺激を受けて初めてナノ粒子が構造を 変化させ、 包埋目的酵素又は基質を遊離させ、 その結果、 酵素反応が初 めて開始できる系にしておけば、 製剤の効率化 ·簡略化が達成できる。 また、 酵素の保存安定性の確保のためにナノ粒子内に酵素を包埋してお き、 用時光刺激で目的酵素をナノ粒子から遊離させて反応系におくこと も可能である。 If the enzyme or substrate is embedded in the nanoparticles, the enzyme reaction can be controlled until use. The enzyme or substrate embedded in the nanoparticle is in an inactive state, and its crossing with the partner is controlled. For example, if a product is commercialized as an integrated preparation as a reagent, the structure of the nanoparticle will not The efficiency and simplification of the preparation can be achieved by changing the system to release the enzyme or substrate for embedding, so that the enzyme reaction can be started for the first time. In addition, it is also possible to embed the enzyme in nanoparticles to ensure the storage stability of the enzyme, and to release the target enzyme from the nanoparticles by light stimulation before use and put it in the reaction system.
5 ) 抗原 ·抗体反応の制御 5) Control of antigen-antibody reaction
抗原又は抗体を、 ナノ粒子内に包埋しておけば、 抗原 ·抗体反応の制 御が可能である。 抗原抗体反応を利用した試薬、 医薬において、 用時光 刺激で目的抗体又は抗原をナノ粒子から遊離させて反応系におくことも 可能である。  If the antigen or antibody is embedded in the nanoparticles, it is possible to control the antigen-antibody reaction. In a reagent or a medicine utilizing an antigen-antibody reaction, the target antibody or antigen can be released from the nanoparticles by light stimulation before use and placed in a reaction system.
(実施例） (Example)
以下、 本発明を実施例によりさらに具体的に説明するが、 下記の実施 例は本発明についての具体的認識を得る一助とみなすべきものであり、 本発明の範囲は下記の実施例により何ら限定されるものではない。  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be regarded as helping to obtain specific recognition of the present invention, and the scope of the present invention is not limited by the following examples. It is not something to be done.
(実施例 1 ) (Example 1)
親水性の多糖類であるプルランを使い、 これにスピロピラン基を導入 したスピロピラン置換プルラン（SpP)を合成した。  Spiropyran-substituted pullulan (SpP) with spiropyran group was synthesized using pullulan, a hydrophilic polysaccharide.
(試料） (Sample)
Acetone (Wako pure chemical industry. Ltd.， 特級)  Acetone (Wako pure chemical industry. Ltd., special grade)
1- ( β -Carboxyethyl) -3'， 3， - dimethyl- 6- nitrospiro (iodorine-2'， 2， [2H-l] benzopyran (Spi - C00H) ( (株）環境化学センター） , N' -dicyclocarbodi imide (DCC) (Peptide instutute. Inc. ) 1- (β-Carboxyethyl) -3 ', 3, -dimethyl-6-nitrospiro (iodorine-2', 2, [2H-l] benzopyran (Spi-C00H) (Environmental Chemistry Center) , N'-dicyclocarbodi imide (DCC) (Peptide instutute. Inc.)
4-Dimethylaminopyridine (DMAP) (Wako pure chemical industry. Ltd. , 特級）  4-Dimethylaminopyridine (DMAP) (Wako pure chemical industry. Ltd., Special grade)
Dimethyl Sulfoxide (DMSO) , dehydrated (Wako pure chemical industry. Ltd. 有機合成用）  Dimethyl Sulfoxide (DMSO), dehydrated (Wako pure chemical industry. Ltd. for organic synthesis)
Pullulan (Hayashibara biochemical laboratories. Inc. )  Pullulan (Hayashibara biochemical laboratories. Inc.)
(SpPの合成） (Synthesis of SpP)
SpP の合成スキーム及び構造式を以下に示す。 式中、 Rで示したのが スピロピラン基で、 紫外光 （UV) ·可視光 （VIS) の照射や熱によって構 造を変化させる化合物である（ィ匕 1 )。この化合物は種々の有機溶媒中で、 UV照射により、 シス体（閉環型、 Spiro型）からトランス体への変換が起 こり、 同時にノニオン性から両性イオン状態（開環型、 Mer 型）となる。 これは可視領域に強い吸収を持ち、 メロシアニン型色素の分子構造で準 安定状態とされ、 熱を加える事により Spiro型に戻る。 また、 Mer型の 吸収帯の波長光で刺激する事によっても Spiro型に戻る。 このような性 質をフォ卜クロミズム (photochromi sm)と言う。  The synthesis 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) or visible light (VIS) or heat (I-Dani 1). This compound is converted from its cis form (ring-closed type, Spiro type) to trans form by UV irradiation in various organic solvents, and at the same time, it changes from nonionic to zwitterionic state (ring-opened type, Mer type). . It has strong absorption in the visible region, is metastable due to the molecular structure of the merocyanine-type dye, and returns to Spiro-type when heated. In addition, it returns to Spiro type by stimulating with wavelength light of Mer type absorption band. This property is called photochromism.
(化 1 ) (Formula 1)
Figure imgf000016_0001
Figure imgf000016_0001
プルラン （Mw=108，000) をー晚 70°Cで減圧乾燥させた。 これを 70°Cで ー晚減圧乾燥させ、 frame dryしたナス型フラスコに 1. OOg (glucoseunit 6. 18mraol) 秤取し、 そこへ Dehydrated DMS0を 20mlカロえ、 窒素気流下、 常温で 2時間撹拌を行い溶解させた。 Pullulan (Mw = 108,000) was dried under reduced pressure at -70 ° C. This was dried at 70 ° C under reduced pressure and weighed in a frame-dried eggplant-shaped flask. 1. OOg (glucoseunit 6.18mraol) was weighed, and 20 ml of Dehydrated DMS0 was caloried there. The mixture was stirred at room temperature for 2 hours under a nitrogen stream. And dissolved.
別のよく乾燥させたナス型フラスコにスピロピランのカルボン酸誘導 体 (Spi-COOH) (runl, 2, 587mg, 1. 54mmol、 run3, 1174mg, 3. 08mmol)、 DCC (runl, 2, 318mg, 1. 54mmol、 run3 636mg, 3. 08mmol)を秤取し、 DehydratedDMSOを 10ml加え、 窒素気流下、 室温で 2時間撹拌した。  Spiropyran carboxylic acid derivative (Spi-COOH) (runl, 2,587 mg, 1.54 mmol, run3, 1174 mg, 3.08 mmol), DCC (runl, 2, 318 mg, 1) 54 mmol, run3 636 mg, 3.08 mmol) were weighed, 10 ml of Dehydrated DMSO was added, and the mixture was stirred at room temperature under a nitrogen stream for 2 hours.
プルラン溶液に腹 APを 30mg加えた後、 スピロピラン溶液を加え、 窒 素気流下、 35°Cで 40時間撹拌した。 溶液の色は濃い紫色であった。 反応 終了後、 反応溶液を過剰のアセ トンに対して混合し、 再沈殿による精製 を行った。 得られた化合物は薄紫色の固体であった。 構造の確認及ぴ置 換率の決定は IRスぺク トル（ShimazuFT-IR 8100S)、lHNMR (JOEL 400MHz)、 元素分析によって行なった。（収率： runl 85. 5%, run2 85. 0°ん run3-a43. 2%, 進 3— b 47. 2%) 以上の結果より、 親水性の多糖プルランに 100単糖あたり 0. 4、 1. 4、 2. 8、 6. 8 個のスピロ ピラン基を導入したスピロ ピラン置換プルラン (SpP) (Spiropyranbearing pullulan) (SpP 0. 4, 1. 4， 2. 6， 6. 8)の合 成に成功した。 以下、 100単糖あたり X個のスピロピランが導入された SpPを SpPXと呼ぶ。 After 30 mg of belly AP was added to the pullulan solution, a 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 dark purple. After completion of the reaction, the reaction solution was mixed with an excess of acetone and purified by reprecipitation. The obtained compound was a light purple solid. Confirmation of the structure and determination of the replacement ratio were performed by IR spectrum (ShimazuFT-IR 8100S), lHNMR (JOEL 400 MHz), and elemental analysis. (Yield: runl 85.5%, run2 85.0 °, run3-a43. 2%, hex 3—b 47.2%) Based on the above results, Spiropyran-substituted pullulan (SpP) (Spiropyranbearing pullulan) (0.4, 1.4, 2.8, 6.8 spiropyran groups per 100 monosaccharides) was added to hydrophilic polysaccharide pullulan (Spiropyranbearing pullulan) The synthesis of SpP 0.4, 1.4, 2.6, 6.8) was successful. Hereinafter, SpP into which X spiropyrans are introduced per 100 monosaccharides is referred to as SpPX.
(実施例 2 ) (Example 2)
スピロピラン置換プルラン（SpP)の会合挙動  Association behavior of spiropyran-substituted pullulan (SpP)
実施例 1で合成した SpPの水溶液中におけるフォトクロミズムゃ会合 挙動について検討した。  The photochromism- ゃ association behavior of the SpP synthesized in Example 1 in an aqueous solution was examined.
(試料） (Sample)
2- [4 - (2-Hydroxyethyl) - 1 - piperazinylj etnanesulf onicacid (ΗΕΡΕύノ ( Wako puxechemica丄 industry. Ltd. )  2- [4-(2-Hydroxyethyl)-1-piperazinylj etnanesulfonic acid (ΗΕΡΕύ ノ (Wako puxechemica 丄 industry. Ltd.)
Hydrochloride (HC1) (Wako pure chemical industry. Ltd.，精密分析用) Hydrochloride (HC1) (Wako pure chemical industry. Ltd., for precision analysis)
Sodium chloride (NaCl) ( Wako pure chemical industry. Ltd. )Sodium chloride (NaCl) (Wako pure chemical industry. Ltd.)
Spiropyran bearing pul lulan (SpP 0. 4, 1. 4， 2. 6， 6· 8) (実施例 1で 合成） Spiropyran bearing pul lulan (SpP 0.4, 1.4, 2.6, 6.8) (synthesized in Example 1)
(溶液の調製）  (Preparation of solution)
SpP の薄紫色固体を暗所において、 buffer (100mM HEPES、 ρΗ7· 5)に対 して加熱撹拌（50°C)することで溶解させ、 不純物を取り除くため得られ た水溶液 フィルター (Sterileacrodisk 25, Gelman science, pore size : 1. 2 m, 0. 45 μ m, 0. 2 μ m)を用いて濾過し、 常温まで放置した。  The light purple solid of SpP was dissolved in a buffer (100 mM HEPES, ρΗ7.5) by heating and stirring (50 ° C) in a dark place, and an aqueous solution filter (Sterileacrodisk 25, Gelman (science, pore size: 1.2 m, 0.45 μm, 0.2 μm) and allowed to stand at room temperature.
LSEC-MALS (Multi angle laser l ight sccatering ] LSEC-MALS (Multi angle laser light sccatering)
SEC-MALS及び Rapid MALSの測定をした。 SEC- MALS とは、 SECによつ て分離された成分について、 RIによる検出と MALSによる検出を行う事 で、 溶出時間に対応した各濃度について、 18角度の光散乱強度を解析す ることによって、 各成分の Mw (重量平均分子量） および、 (重量平均 半径） を測定する事が出来る。 RapidMAS ではカラムにガードカラム（プ レカラム）を用いる事で、カラムとの相互作用を最小限に抑えつつ、サン プルの濃度勾配を作り出す方法で、 短時間 （測定時間数分程度） で解析 することが出来る。 SEC-MALS and Rapid MALS were measured. SEC-MALS is an SEC Mw (weight average molecular weight) of each component by analyzing the light scattering intensity at 18 angles for each concentration corresponding to the elution time by detecting the components separated by RI and MALS And (weight average radius) can be measured. RapidMAS uses a guard column (pre-column) as a column to minimize the interaction with the column and to create a sample concentration gradient, which enables analysis in a short time (about several minutes of measurement time). Can be done.
(Rapid MALS, DLSによる会合挙動の評価） (Evaluation of association behavior by Rapid MALS, DLS)
SpPの水溶液中における会合挙動について Rapid MALS法や DLS測定よ つて検討を行なった。 Rapid MALS測定には、 T0S0H CCDP dualpump、 T0S0H RI-8010 RI detector, Wyatt tech. co. DAWN - E MALS detector カ らな るシステムを用い、 カラムは T0S0HSWXLのガードカラム、 流速は 1. 0ml/ 分、 溶離液は lOOmM HEPES、 lOOmM NaCl、 pH7. 5 を用いた。 サンプルは SpPl. 4の薄紫色固体を、 暗所において、 50°C、 30分間の加熱撹拌をする 事で溶離液に溶解させ、 その後遮光したまま室温まで放冷することで調 製した。 光刺激は、 UV照射を 8WUV lamp (254nm) (UVP, 8 watt handheld model 画- 18)、 VIS照射を 8 W White 1 ightlamp (UVP, 8 watt handheld model UVM-18)を用いて行った。測定サンプルの濃度は、 0. 25-2. 5rag/ml, injectionvolumeは、 適当なピークの大きさになるように 10- 100 μ 1 で 行った。 サンプルをカラムにアプライする際に、 フィルター（0. 45 μ ηι) を用いて濾過した。 サンプルの屈折率増分 dn/dc は、 Wyatttech. co. Opti lab DSP interf erometric refractometer によって測疋した値を用 いた。  The association behavior of SpP in aqueous solution was studied by Rapid MALS method and DLS measurement. For Rapid MALS measurement, a system consisting of T0S0H CCDP dualpump, T0S0H RI-8010 RI detector, Wyatt tech.co. DAWN-E MALS detector was used, the column was a T0S0HSWXL guard column, the flow rate was 1.0 ml / min, The eluent used was 100 mM HEPES, 100 mM NaCl, pH 7.5. The sample was prepared by dissolving the light purple solid of SpPl. 4 in the eluent by heating and stirring at 50 ° C for 30 minutes in the dark, and then allowing it to cool to room temperature while protected from light. The photostimulation was performed by UV irradiation using an 8 W UV lamp (254 nm) (UVP, 8 watt handheld model, picture 18) and VIS irradiation using an 8 W White 1 ightlamp (UVP, 8 watt handheld model UVM-18). The concentration of the measurement sample was 0.25-2.5 rag / ml, and the injection volume was set at 10-100 μ1 to obtain an appropriate peak size. When applying the sample to the column, the sample was filtered using a filter (0.45 μηι). The refractive index increment dn / dc of the sample was measured using a Wyatttech. Co. Optilab DSP interferometric refractometer.
動的光散乱 (DLS) 測定には、 Otsuka Electronics Co. , Ltd. , DLS-700 を用いた。光源の波長は 633nmの He- Neレーザーで、温度は 25. 0± 0. 2°C で行った。 サンプルは SpPl. 4の薄紫色固体を、 喑所において、 50°C、 30 分間の加熱撹拌をする事で緩衝液（lOOmMHEPES, pH7. 5)に溶解させ、その 後遮光したまま室温まで放冷することで調製した。 得られた溶液はフィ ルター（0. 45 μ m)を用いて濾過した。 光刺激は、 UV 照射を 8WUV lamp (254nra) (UVP, 8 watt handheld model UVM- 18)、 VIS照射を 8 W White lightlamp (UVP, 8 watt handheld model UVM- 18)を用いて行った。 Otsuka Electronics Co., Ltd., DLS-700 was used for dynamic light scattering (DLS) measurement. The wavelength of the light source is a He-Ne laser with a wavelength of 633 nm, and the temperature is 25.0 ± 0.2 ° C I went in. The sample was prepared by dissolving the light purple solid of SpPl. 4 in a buffer solution (100 MMHEPES, pH 7.5) by heating and stirring at 50 ° C for 30 minutes in the place, and then allowed to cool to room temperature while protected from light It was prepared by doing. The obtained solution was filtered using a filter (0.45 μm). The photostimulation was performed by UV irradiation using an 8 W UV lamp (254nra) (UVP, 8 watt handheld model UVM-18) and VIS irradiation using an 8 W White lightlamp (UVP, 8 watt handheld model UVM-18).
(SpP水溶液の調製） (Preparation of SpP aqueous solution)
種々の置換率の SpP (0. 4、 1. 4、 2. 8、 6. 8) の水への溶解性は、 0. 4-2. 8 のものは数 mg/ml のとき少しの加熱撹拌することで溶解し、 6. 8 のもの はほとんど溶解しなかった。 原料プルランは水に溶解することから、 こ れは、 スピロピランの疎水性のため置換率の高いものでは、 水への溶解 性が低くなったと考えられる。 (SpPのフォ トクロミズム）  The solubility of SpP (0.4, 1.4, 2.8, 6.8) with various substitution rates in water is 0.4-2.8 with little heating at several mg / ml. It was dissolved by stirring, and 6.8 was hardly dissolved. Since the raw material pullulan dissolves in water, it is considered that this is due to the hydrophobicity of spiropyran, which means that if it had a high substitution rate, its solubility in water was low. (Photochromism of SpP)
SpPl. 4の水溶液中で示すフォ トクロミズムについて、 UV ' VIS吸収スぺ ク トルを追跡することによって検討した。 UV測定にはペルチヱ式温度制 御装置を装備した UVspectrophotometer (Hitachi, U-3300)を用いた。 光刺激は、 UV照射を 8W UV lamp (UVP, 8 watthandheld model UVM- 18)、 VIS照射を 8W White l ight lamp (UVP, 8 watt handheld modelUVM- 18) を用いて行なった。  The photochromism of SpPl. 4 in aqueous solution was examined by following the UV'VIS absorption spectrum. A UV spectrophotometer (Hitachi, U-3300) equipped with a Peltier type temperature controller was used for UV measurement. The photostimulation was performed using an 8 W UV lamp (UVP, 8 watt handheld model UVM-18) for UV irradiation and an 8 W white light lamp (UVP, 8 watt handheld model UVM-18) for VIS irradiation.
サンプル調製時、 溶液の色は薄い赤色で、 515nmに； maxを持つ UVス ぺク トルが得られた（Mer I型）。 これに UV照射すると λ maxが 535nmに 長波長シフトした（Mer I I型）。 また、 Mer Iに可視光照射すると、 溶液 の薄い赤色は速やかに消失し、閉環型の UVスぺク トルが得られた（Spiro 型）。 これより、 SpPは 3種の状態を行き来するフォトクロミズムを示す ことがわかった。 これをまとめると、 水溶液中における SpPのフォトク 口ミズムは次のような相関が確認された。 At the time of sample preparation, the color of the solution was light red and a UV spectrum with a maximum at 515 nm was obtained (Mer I type). When this was irradiated with UV light, the λ max shifted to a long wavelength of 535 nm (Mer II type). When Mer I was irradiated with visible light, the light red color of the solution quickly disappeared, and a closed UV spectrum was obtained (Spiro type). From this, SpP shows photochromism that moves between three states I understand. Summarizing this, the following correlations were confirmed between the photochromism of SpP in aqueous solution.
まず、 サンプル調製時においてスピロピランは開環型（Mer I )であり、 これは 25°C暗所において数時間安定であった。 これに VIS照射すると、 10分以内に Spiro型への構造変化が起こる （1 )。 そして、 この Spiro 構造を加熱すると速やかに Mer Iに戻った （2 )。 これは逆フォトクロミ ズムである。 また、 Spiro 型を喑所において放置すると、 180 分間程で Mer Iに戻った （3 )。 これにより、 SpPは 25°C暗所において、 Mer Iが最 も安定な種であることが示唆される。 Spiro型に UV照射すると 10分間 程で Mer I Iになった （4 )。 これに VIS照射するとこれは 240分間程か かって Spiro型へ戻った（ 5 )。 Mer Iに UV照射することでも 5分間程で Mer I Iになり （6 )、これを加熱すると、速やかに Mer Iへ戻った（ 7 )。 Mer I Iは、 Mer Iに比べ； L maxが長波長シフトしていることや、 （ 5 ) の 構造変化の速度が非常に遅いこと （スピロピランの開環型は分子同士が スタック構造をとるとき、 スピロ型への構造変化を著しく阻害する。 こ れは、開環型は平面構造であり、スピロ型は立体構造であるためである） から、 スピロピラン分子同士のスタッキングによる会合が起こっている ことが示唆される。 (SpPの会合挙動）  First, spiropyran was ring-opened (Mer I) at the time of sample preparation and was stable for several hours in the dark at 25 ° C. When irradiated with VIS, the structure changes to Spiro type within 10 minutes (1). When the Spiro structure was heated, it quickly returned to Mer I (2). This is reverse photochromism. When the Spiro type was left in place, it returned to Mer I in about 180 minutes (3). This suggests that SpI is the most stable species of Mer I in the dark at 25 ° C. When the Spiro type was irradiated with UV, it became Mer II in about 10 minutes (4). When this was irradiated with VIS, it returned to the Spiro type in about 240 minutes (5). Even when UV irradiation was performed on Mer I, it became Mer II in about 5 minutes (6), and when this was heated, it quickly returned to Mer I (7). Mer II is different from Mer I; L max has a long wavelength shift, and the structural change rate of (5) is very slow. (The ring-opened form of spiropyran has This significantly inhibits the structural change to the spiro type, because the ring-opened type has a planar structure and the spiro type has a three-dimensional structure. It is suggested. (Association behavior of SpP)
サンプル調製時（Mer I )の SpPl. 4についての Rapid MALSの解析結果か ら、 Mw=l . 54 X 106 (g/mol) , Mw/Mn=l . 64 ± 0. 17、 R =79. 2 (nm)であること がわかった。 主鎖となる原料のプルランは Mw=l. 08 X 105 (g/mol)である ことから、 SpPは水溶液中において 10数分子が会合していることが示唆 された。 CHP集合体が分子量約 45万、 会合数が 4ほどであることから比 ベると、かなり大きな会合体を形成していることがわかる。また、室温、 暗所においてこの会合体は安定であった（スピロピラン分子も安定）。From the results of Rapid MALS analysis of SpPl. 4 at the time of sample preparation (Mer I), Mw = l. 54 x 106 (g / mol), Mw / Mn = l. 64 ± 0.17, R = 79. 2 (nm). Since the pullulan as the main chain material had Mw = l.08 × 105 (g / mol), it was suggested that a dozen or more molecules of SpP were associated in the aqueous solution. Compared to the molecular weight of the CHP aggregate of about 450,000 and the number of associations being about 4, it can be seen that the CHP aggregates form fairly large aggregates. Also, at room temperature, This aggregate was stable in the dark (the spiropyran molecule was also stable).
Mer Iに VIS照射 （0, 1， 5, 10， 30， 60， 90分間） したとき （ 1 )、 SpP1. 4の会合体は分子量、粒径は多少小さくなり、 10数分子ほどの会合 体を形成している。 このとき会合体の密度は多少大きくなつている。 こ の Spiro型を室温、暗所に放置（0-180分間）すると除々に Mer Iへと変化 するが （3 )、 このとき会合体はほとんど変化しなかった。 次に 10分間 の VIS照射により Spiro型にした種に UV照射すると（4 )、会合体は 10-30 分間をピークにして分子量、 粒径ともに大きくなつた。 データ間にばら つきはあるが、 最大で分子量約 400万 （SpPの会合数は約 40) ほどであ つた。 このあと、 除々に会合体は小さくなつていつた。 次に Mer lに UV 照射すると（6 )、 （4 )のときと同様に会合体は多少大きくなつていき、 その後小さくなつていつたが、 （4 ) ほど大きな変化は見られなかった。 そして、 Mer Iに VIS照射（10分間）することで Spiroにしてから、 UV照 射（10分間）することで Mer I Iにして、 その後 VIS照射すると （ 5 )、 は じめ分子量 350万ほどであった会合体は、除々に小さくなっていき、 240 分間ほどで分子量約 170万ほどになった。 これは、 スピロビラン分子同 士のスタツキングによる会合構造（Mer I I )カゝら Spiro 構造への変換が 遅いという結果とも一致している。 DLSによる結果より、 RapidMALSによ るものと同様の挙動を示していることが示唆される。 濃度が高くなると SpP微粒子は多少大きくなるようである。 When Mer I was exposed to VIS (0, 1, 5, 10, 30, 60, and 90 minutes) (1), the molecular weight and particle size of the aggregate of SpP1.4 were slightly reduced, and the aggregate of about 10 or more molecules Is formed. At this time, the density of the aggregate is somewhat larger. When this Spiro type was left in the dark at room temperature (0-180 minutes), it gradually changed to Mer I (3), but the aggregate hardly changed at this time. Next, when UV irradiation was applied to the seeds that had been made into Spiro type by VIS irradiation for 10 minutes (4), the aggregate increased in both molecular weight and particle size with a peak at 10-30 minutes. Although the data varied, the maximum molecular weight was about 4 million (the number of SpP associations was about 40). After this, the association gradually became smaller. Next, when Merl was irradiated with UV light (6), the aggregates became slightly larger and then smaller as in (4), but did not show as large a change as (4). Then, Mer I was made into Spiro by VIS irradiation (10 minutes), UV irradiation (10 minutes) was made Mer II, and then VIS irradiation (5), the initial molecular weight was about 3.5 million. The associated aggregate gradually became smaller and reached a molecular weight of about 1.7 million in about 240 minutes. This is consistent with the result that the conversion of spirovirane molecules into the aggregate structure (Mer II) by the stacking is slow. The results from DLS suggest that the behavior is similar to that by RapidMALS. SpP microparticles appear to be somewhat larger at higher concentrations.
以上の結果をまとめると、 SpPは 10数分子から数 10分子が集まって ヒ ドロゲルナノ微粒子を形成し、 光刺激によって可逆的な会合挙動の変 化をすることがわかる。 UV照射によって会合体が大き'くなったり小さく なったりするのは、 架橋領域のスピロピランの性質が変化することによ るものであると考えられる。 (実施例 3 ) Summarizing the above results, it is found that SpP forms hydrogel nanoparticles by assembling dozens or dozens of molecules, and changes the reversible association behavior by light stimulation. It is considered that the size of the aggregate becomes larger or smaller due to UV irradiation because the property of spiropyran in the crosslinked region changes. (Example 3)
SpP を用いて、 蛋白質のリフォールデイング効率を向上させること、 またそこから得られる SpP と蛋白質の相互作用に関する知見を得ること を目的として検討した。 モデル酵素としては CSを用いた。 コントロール として疎水化多糖-シクロデキス トリンのシステムについても検討を行 なった。  The purpose of this study was to improve the refolding efficiency of proteins using SpP and to obtain information on the interaction between SpP and proteins obtained therefrom. CS was used as a model enzyme. As a control, the hydrophobized polysaccharide-cyclodextrin system was also examined.
(試料） (Sample)
Acethyl—し oA (Wako pure chemical industry. Ltd. 7 生ィ匕'子用) Acethyl—oA (for Wako pure chemical industry. Ltd. 7
Choresterol bearing pul lulanl08-l. 2 (CHP108-1. 2) (Synthesized in this labo. )  Choresterol bearing pul lulanl08-l. 2 (CHP108-1.2) (Synthesized in this labo.)
Dimethyl sulfoxide (D SO) (Wako pure chemical industry. Ltd. , 特 級）  Dimethyl sulfoxide (D SO) (Wako pure chemical industry. Ltd., special grade)
5， 5' -Dithiobi s (2-nitrobenzoic acid) (DTNB) (Wako pure chemical industry. Ltd.，SH基定量用）  5, 5'-Dithiobi s (2-nitrobenzoic acid) (DTNB) (Wako pure chemical industry. Ltd., for SH group determination)
( ± ) Dithiothreitol (DTT) (Wako pure chemi cal industry, Ltd. , SH 基酸化防止用）  (±) Dithiothreitol (DTT) (Wako pure chemi cal industry, Ltd., for SH base oxidation prevention)
Ethyl enediamine-N; N， N'， N' - tetraacetic acid, di sodium salt, dihydrate (EDTAゲ Na) (Wako pure chemical industry. Ltd. ) Ethyl enediamine-N ; N, N ', N'-tetraacetic acid, di sodium salt, dihydrate (EDTA Na) (Wako pure chemical industry. Ltd.)
Guanidine hydrochloride (GuHCl) (Wako pure chemical industry. Ltd. , 生化学用）  Guanidine hydrochloride (GuHCl) (Wako pure chemical industry. Ltd., for biochemistry)
Hydrochloride (HC1) (Wako pure chemical industry. Ltd. , 精密分析 用）  Hydrochloride (HC1) (Wako pure chemical industry. Ltd., for precision analysis)
Hydroxypropyl- β -cyclodexitrin (HP- β - CD) (曰本食品ィ匕ェ（株） ) Oxaloacet ic acid (Wako pure chemical industry. Ltd. )  Hydroxypropyl-β-cyclodexitrin (HP-β-CD) (Sakura Honten Food Co., Ltd.) Oxaloacetic acid (Wako pure chemical industry. Ltd.)
Phenol (Wako pure chemical industry. Ltd.， 特級) Sodium chloride (NaCl) (Wako pure chemical industry. Ltd. ) Spiropyran bearing pul lulanl. 4 (SpPl. ) (Synthesized in this labo. ) Phenol (Wako pure chemical industry. Ltd., special grade) Sodium chloride (NaCl) (Wako pure chemical industry. Ltd.) Spiropyran bearing pul lulanl. 4 (SpPl.) (Synthesized in this labo.)
Sulfonic acid (Wako pure chemical industry. Ltd. , 精密分析用) Tri s [hydroxymethyl] aminoraethane (Tri zma base) (Sigma chemi cal co. )  Sulfonic acid (Wako pure chemical industry. Ltd., for precision analysis) Tris [hydroxymethyl] aminoraethane (Tri zma base) (Sigma chemi cal co.)
Tris [hydroxymethyl] aminomethane hydrochloride (Trizma) (Sigma chemical co. )  Tris [hydroxymethyl] aminomethane hydrochloride (Trizma) (Sigma chemical co.)
以上の試薬の中で購入したものは精製せずそのまま用いた。 また本実 験を通して buffer として、 150mM Tri s - HC1、 0. 75ra EDTA、 pH7. 6 を用 いた。  Among the above reagents, those purchased were used without purification. Throughout this experiment, 150 mM Tris-HC1, 0.75ra EDTA, pH 7.6 was used as a buffer.
( SpP溶液、 CHP溶液の調製） (Preparation of SpP solution and CHP solution)
SpP溶液は、 SpP1. 4の薄紫色固体を暗所において、 bufferに対して 30 分間加熱撹拌（50°C)することで溶解させ、 不純物を取り除くため得られ た水溶液をフイノレター（St eri leacro i sk 25, Gelman science, pore size : 1. 2 /z m, 0. 45 m, 0. 2 μ m)を用いて濾過し、 暗所下、 常温まで放 置することで調製した。 [Citrate Synthase (CS) ]  The SpP solution is obtained by dissolving the light purple solid of SpP1.4 in a dark place by heating and stirring (50 ° C) for 30 minutes in a buffer, and removing the resulting aqueous solution with a finole letter (Sterileacroix). It was prepared by filtration using sk 25, Gelman science, pore size: 1.2 / zm, 0.45 m, 0.2 μm) and leaving it to room temperature in the dark. [Citrate Synthase (CS)]
CSは、解糖および脂肪酸の分解によって生じたを TCAサイクルへ導く ための初発段階の酵素で、 ァセチル- CoAとォキサ口酢酸からクェン酸を 合成する反応を触媒する。反応はァセチル -CoAのメチル基のカルボア二 オンとォキサ口酢酸のアルドーノレ縮合である。  CS is the first-stage enzyme that guides the process of glycolysis and the breakdown of fatty acids to the TCA cycle, catalyzing the reaction of cuenic acid synthesis from acetyl-CoA and oxa-mouth acetic acid. The reaction is an aldono-condensation of the carbodione of the methyl group of acetyl-CoA with oxa mouth acetic acid.
CSはホモ二量体の蛋白質であり、 サブユニットは活性を持たない。 ま た、 サブユニッ ト内に 5つのシスティン残基を有するが、 ジスルフイ ド 結合は形成されていない。 CSは 4次構造の研究におけるモデル蛋白質と してその物理化学的特性、 アミノ酸の一次配列、 立体構造、 活性点、 基 質結合部位などが詳細に検討されてきた。 (CS溶液調製） CS is a homodimeric protein and the subunit has no activity. It also has five cysteine residues in the subunit, but disulphide No bond has been formed. CS has been studied in detail as a model protein in studies of quaternary structure, including its physicochemical properties, primary sequence of amino acids, three-dimensional structure, active site, and substrate binding site. (CS solution preparation)
CS (From porcine heart, MWsubunit=50, 000)溶液は Rocheカ ら購入し た懸濁液を bufferに対して溶解させることで調製した。 濃度の定量は、 280nm における吸光度を測定する こ と によって行なっ た （ ε 280nm=l.75ml · cm/mg 。  CS (From porcine heart, MWsubunit = 50,000) solution was prepared by dissolving the suspension purchased from Roche in buffer. The concentration was quantified by measuring the absorbance at 280 nm (ε 280 nm = 1.75 mlcm / mg).
また、 CSの変性溶液は、 1.0mg/ml CS、 6M GuHCl、 40mM DTTになるよ うに bufferに溶解させ、 25°Cで 1時間ィンキュベーションすることによ つて得た。  A denaturing solution of CS was obtained by dissolving in a buffer so that the concentration of CS was 1.0 mg / ml CS, 6 M GuHCl, and 40 mM DTT, and incubating at 25 ° C. for 1 hour.
(活性測定） (Activity measurement)
CSの酵素活性の測定は基質溶液（23μ Μ、19μ g/ml Acetyl_CoA、0.5mM、 66 μ g/ml, Oxialoacetic acid, 0.12mM、 48 μ g/ml, DNTB) 0.76ml と CS 溶液 15μ 1 を速やかに混合し、 5秒間程撹拌した後、 下記の反応で生じ るメルカプチドイオンの濃度増加を 412nmの吸光度を追跡した。 吸光度 がー次の傾きによって増加するが、 この傾きを天然状態のそれと比較す ることで活性回復率を見積もった。 To measure the enzymatic activity of CS, 0.76 ml of substrate solution (23 μΜ, 19 μg / ml Acetyl_CoA, 0.5 mM, 66 μg / ml, Oxialoacetic acid, 0.12 mM, 48 μg / ml, DNTB) and 15 μl of CS solution were used. After mixing immediately and stirring for about 5 seconds, the absorbance at 412 nm was monitored for an increase in the concentration of mercaptide ions generated by the following reaction. The absorbance increases with the next slope, and the rate of activity recovery was estimated by comparing this slope with that of the native state.
(リフォールディング実験） (Refolding experiment)
CSは 6M GuHCl溶液中においてほぼランダムコイルに近い状態まで変 性する。 このとき酵素活性は有さない。 DTTは CSの持つジスルフィ ド結 合を形成していないシスティン残基の非生産的なジスルフィ ド形成を抑 制するための還元剤である。 この CS変性溶液を buffer、 種々の濃度の SpPl.4溶液、 および CHP溶液（1. Omg/ml)で 50倍に希釈することでリフ オールディングを行わせ、希釈開始後（CHP系では CD添加後）の酵素活性 を測定した。 また、 コントロールとして SpPの原料であるプルランゃス ピロピランのカルボン酸誘導体を用いてリフォールディング実験を行な つた。 SpPを用いた系では、 スピロピランのコンフオメーションが CSの リフォールディングに与える効果を検討するために VlS(Whitelight)照 射や UV(254nm)照射を行った。 光刺激には、 UV照射を 8W UV lamp (UVP, 8 watt handheld model雨 - 18)、 VIS照射を 8W White light lamp (UVP, 8 watt handheld model UVM-18)を用いて行なった。 CHPを用いた系では、 CS 変性溶液を CHP 溶液で希釈した後 30 分間放置し、 その後 HP-j3 -CD (40mM)を添加してからの酵素活性を測定した。 CS changes to almost a random coil state in 6M GuHCl solution. At this time, it has no enzymatic activity. DTT is a reducing agent for CS that suppresses the non-productive disulfide formation of cysteine residues that do not form disulfide bonds. Buffer this CS denaturing solution with various concentrations Refolding was performed by diluting 50-fold with SpPl.4 solution and CHP solution (1.0 mg / ml), and enzyme activity was measured after the start of dilution (after addition of CD in CHP system). As a control, a refolding experiment was performed using a carboxylic acid derivative of pullulanspiropyropyran, which is a raw material of SpP. In the system using SpP, VlS (Whitelight) irradiation and UV (254 nm) irradiation were performed to examine the effect of spiropyran conformation on CS refolding. UV irradiation was performed using an 8 W UV lamp (UVP, 8 watt handheld model rain-18), and VIS irradiation was performed using an 8 W 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 the enzyme activity was measured after adding HP-j3-CD (40 mM).
(化学変性 CSのリフォールディング制御) (Refolding control of chemically modified CS)
SpPのシャペロン作用 'コンフオメーション依存性  SpP chaperone action 'conformation dependence
SpPが CSのリフォールディングに与える影響を検討するにあたって、 まず天然状態の CSに SpPが与える影響について検討した。 Spiroのも のは 10分間の VIS照射をしてから CS溶液と混合し、 Mer lは遮光、 Mer I Iは 10分間の UV照射をしてから混合し、 その後も照射を続けたもの である。 SpP存在下、非存在下における CSの活性の変化がないことから、 SpPナノ微粒子は、 天然状態の CS と相互作用しない、 または、 複合化の ような相互作用があつたとしても、 酵素活性には影響を与えないという ことが示唆された。 （表 1 )  In examining the effect of SpP on CS refolding, we first examined the effect of SpP on native CS. Spiro was irradiated with VIS for 10 minutes and then mixed with the CS solution, Merl was shielded from light, MerII was irradiated with UV for 10 minutes, mixed, and then irradiated. Since there is no change in CS activity in the presence or absence of SpP, SpP nanoparticles do not interact with CS in the native state, or even when there is an interaction such as complexation, SpP nanoparticles have a negative effect on enzyme activity. Has no effect. (table 1 )
sample Relative enzyme activity ho denaturation Naitve CS 1.00 sample Relative enzyme activity ho denaturation Naitve CS 1.00
SpPl.4(Spiro) 2. Omg/ml 0.99 + 0.02  SpPl.4 (Spiro) 2.Omg / ml 0.99 + 0.02
(Mer) 0.98±0· 05  (Mer) 0.98 ± 005
Pullulan 1.01±0.02  Pullulan 1.01 ± 0.02
After GuHCl denaturetion and dilution*After GuHCl denaturetion and dilution *
No additive 0.34±0.02 No additive 0.34 ± 0.02
Pullulan 0.37 + 0.04  Pullulan 0.37 + 0.04
Spi-COOH (Spiro) 0.32±0.06  Spi-COOH (Spiro) 0.32 ± 0.06
Spi-COOH (Mer) 0.34±0.05  Spi-COOH (Mer) 0.34 ± 0.05
SpPl.4 (Spiro) l. Omg/ml 0.53±0.01  SpPl.4 (Spiro) l.Omg / ml 0.53 ± 0.01
(Mer) 1. Omg/ml 0.10±0.03  (Mer) 1.Omg / ml 0.10 ± 0.03
(Spiro→Mer) 1. Omg/ml 0.58±0.04  (Spiro → Mer) 1.Omg / ml 0.58 ± 0.04
(Mer→Spiro) 1. Omg/ml 0.81±0.01  (Mer → Spiro) 1.Omg / ml 0.81 ± 0.01
* 変性化 CS 溶液 （[CS]=1. Omg/ml, 6M GuHCl, 400mM DTT) は種々の 溶液で 50倍希釈された。緩衝液 ： 150mMTris-HCl, 0.75mM EDTA, pH7.6. * Denatured CS solution ([CS] = 1. Omg / ml, 6M GuHCl, 400mM DTT) was diluted 50-fold with various solutions. Buffer: 150 mM Tris-HCl, 0.75 mM EDTA, pH 7.6.
CSのリフォールディングにおける SpPの効果について検討した。 Spiro、 Mer Iの 2つのコンフオメーシヨンについてリフォールディング実験を 行なった。用いた系は、変性溶液の CS濃度 1.0mg/ml、希釈倍率は 50倍、 SpP濃度は 1.0mg/mlである。 Spiroは SpP溶液調製後、 可視光照射（10 分間）し、 変性 CS溶液と混合後も可視光照射し続けた。 Mer l型では、遮 光条件下で行った。 また、 プルラン（1. Omg/ml)や Spi- C00H(87.7 M、 1. Omg/mlの SpPl.4のスピロピランュニットと同濃度）のシャペロン活个生 についても評価した。 プルランや Spi- C00Hにシャぺ口ン活性は見られなかった。この系にお いて自発的なリフォールデイングは 30%ほどであり、 70%ほどの CSは正 常なフォールディングゃアセンブリーを成しえず、 凝集したものと推察 される。 SpP を用いた系では、 最大活性回復を示したのが Mer Iであり 60%ほどであった。 それに次いで、 Spiroが 45%ほどであった。 この二 つの活性回復速度は、 自発的なものと比べて遅く、 相互作用が示唆され る結果となった。 また、 SpP 系においては活性回復速度が自発的なもの に比べ遅いことから SpPナノ微粒子と CSのリフォールディング中間体と の相互作用が示唆された。 （表 1、 図 2 ) The effect of SpP on CS refolding was examined. Refolding experiments were performed on two conformations, Spiro and Mer I. The system used was a denaturing solution with a CS concentration of 1.0 mg / ml, a dilution factor of 50 and a SpP concentration of 1.0 mg / ml. Spiro was irradiated with visible light (10 minutes) after preparing the SpP solution, and continued to be irradiated after mixing with the denatured CS solution. For the Merl type, the measurement was performed under light shielding conditions. Active chaperones of pullulan (1.0 mg / ml) and Spi-C00H (87.7 M, same concentration as spiropyranunitite of 1.0 mg / ml SpPl.4) were also evaluated. No pull-out activity was observed in pullulan or Spi-C00H. In this system, spontaneous refolding is about 30%, and about 70% of CS is not able to form a normal folding-assembly, and is presumed to be aggregated. In the system using SpP, the highest activity was recovered by Mer I, which was about 60%. This was followed by Spiro at about 45%. The recovery rates of these two activities were slower than the spontaneous ones, suggesting an interaction. In the SpP system, the activity recovery rate was slower than the spontaneous one, suggesting the interaction between SpP nanoparticles and the CS refolding intermediate. (Table 1, Figure 2)
次に、 フォールディングの経過に伴って、 この SpPナノ微粒子のナノ 空間の性質を光刺激することによって変化させることで、 シャペロン作 用がどのように変化するかについて検討した（図 2 )。  Next, we examined how the behavior of the chaperone changes by changing the properties of the nanospace of the SpP nanoparticles by photostimulation as the folding progresses (Fig. 2).
リフォールディング初期に Spiroであったもので、 すぐに遮光し除々 に Mer Iにしたものでは自発的なものが 35%ほどであるのに対して 55% ほどであり、 Spiro→Spiro の 45%に比べて大きな活性回復を示した。 Spiroで 30分間ほど放置してから遮光したのは Spiro→Spiro とほぼ同 様であった。  In the early stage of refolding, Spiro was immediately shaded and gradually changed to Mer I, while spontaneous one was about 35%, whereas spontaneous one was about 55%, and 45% of Spiro → Spiro It showed a large recovery of activity. It was almost the same as Spiro → Spiro after having been left on Spiro for about 30 minutes and then shielded from light.
リフォールデイング初期に Mer Iであったものについてであるが、 す ぐに Spiroにしたものでは 65%ほどで Mer I→Mer I とほぼ同様であった c Mer Iで 30分間ほどおいてから Spiroにしたものでは非常に大きな活性 回復が見られ、 80%ほどであった。 産業上の利用可能性 Although for those were Mer I to refolding initial, from at about 30 minutes c Mer I were substantially the same as the Mer I → Mer I in about 65% obtained by the Spiro to immediately in Spiro The results showed a very large recovery of activity, about 80%. Industrial applicability
本検討では、化学変性状態にある CSのリフォールディングの系におけ る、 SpPのシャペロン作用について検討した。 その結果、  In this study, we examined the chaperone action of SpP in the refolding system of chemically denatured CS. as a result,
1)変性蛋白質にフォールディングに適したナノ空間を提供するとともに、 複合化 ·放出を自発的に行なう 1) While providing denatured protein with nano space suitable for folding, Combination and spontaneous release
2)光刺激することによってフォ一ルディングを行なうナノ空間を、"より フォールディングに適した環境"にすることが可能であるといった特性 を持つ人工分子シャペロンとして機能することが見出された （図 3 )。 すなわち、 蛋白質との親和性を光刺激により制御し、 フォールディン グを促進しえることが明らかになった。この系では光刺激の程度 ·間隔な どによって種々の蛋白質に対して適した環境を提供することが可能とな ることが期待される。 また、 細胞内封入体からリフォールデイングの系 や、 生体外翻訳系などへの応用が期待される。  2) It has been found that the nano space that performs folding by photostimulation can function as an artificial molecular chaperone with the property that it can be made into an environment more suitable for folding (Fig. 3) ). In other words, it became clear that the affinity with the protein can be controlled by light stimulation to promote folding. This system is expected to be able to provide an appropriate environment for various proteins depending on the degree and interval of light stimulation. It is also expected to be applied to intracellular inclusions for refolding systems and in vitro translation systems.

Claims

請 求 の 範 囲 The scope of the claims
1 . 親水性高分子を光応答性化合物 （光刺激により構造変化をおこし 親水性 -疎水性を制御しえる化合物） で修飾し、得られた両親性高分子の 形成するナノ粒子内に目的蛋白質 （ペプチドを含む） を取り込む工程を 含むことを特徴とする光刺激による包埋蛋白質 （ペプチドを含む） の制 御方法。 1. The hydrophilic polymer is modified with a photoresponsive compound (a compound that changes its structure by photostimulation and controls hydrophilicity / hydrophobicity), and the target protein is contained in the nanoparticles formed by the amphiphilic polymer. A method for controlling an embedded protein (including a peptide) by photostimulation, which comprises a step of taking up (including a peptide).
2 . ナノ粒子が、粒径 50- lOOnmである、請求の範囲第 1項に記載の制 御方法。 2. The control method according to claim 1, wherein the nanoparticles have a particle size of 50-100 nm.
3 . ナノ粒子が、 多糖プルランである、 請求の範囲第 2項に記載の制 御方法。 3. The control method according to claim 2, wherein the nanoparticles are polysaccharide pullulan.
4 . 光応答性化合物が、 スピロピラン基である請求の範囲第 1項から 第 3項の何れか一に記載の制御方法。 4. The control method according to any one of claims 1 to 3, wherein the photoresponsive compound is a spiropyran group.
5 . 制御が、 光刺激によって行われ、 これにより蛋白質のリフォール ディングが制御される請求の範囲第 1項から第 4項の何れか一に記載の 制御方法。 5. The control method according to any one of claims 1 to 4, wherein the control is performed by light stimulation, whereby the protein refolding is controlled.
6 . 制御によって、 ナノ粒子内に取り込まれた包埋蛋白質 （ペプチド を含む） について以下の少なくとも一の機能を達成する請求の範囲第 1 項から第 5項の何れか一に記載の制御方法。 6. The control method according to any one of claims 1 to 5, wherein the control achieves at least one of the following functions for an embedded protein (including a peptide) incorporated in the nanoparticle.
1 ) 蛋白質 （ペプチドを含む） の生体内運搬、 2) 蛋白質 （ペプチドを含む） の保存 ·安定化、 1) In vivo transport of proteins (including peptides), 2) Storage and stabilization of proteins (including peptides)
3) 蛋白質 （ペプチドを含む） の精製、  3) Purification of proteins (including peptides)
4) 酵素，基質反応性の制御、  4) Enzyme and substrate reactivity control,
5) 抗原 ·抗体反応の制御。  5) Control of antigen-antibody reaction.
7. 請求の範囲第 1項から第 5項の何れか一に記載の制御方法で調製 される蛋白質 （ペプチドを含む） 包埋ナノ粒子を含有する製剤。 7. A preparation containing proteins (including peptides) embedded nanoparticles prepared by the control method according to any one of claims 1 to 5.
8. 請求の範囲第 7項の製剤の製造方法。 8. A method for producing the preparation according to claim 7.
PCT/JP2003/006257 2002-05-21 2003-05-20 Method for controlling protein WO2003097672A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US38217302P 2002-05-21 2002-05-21
US60/382,173 2002-05-21
JP2002147147A JP4100499B2 (en) 2002-05-22 2002-05-22 Protein control method
JP2002-147147 2002-05-22

Publications (1)

Publication Number Publication Date
WO2003097672A1 true WO2003097672A1 (en) 2003-11-27

Family

ID=29552333

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/006257 WO2003097672A1 (en) 2002-05-21 2003-05-20 Method for controlling protein

Country Status (1)

Country Link
WO (1) WO2003097672A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019515893A (en) * 2016-03-31 2019-06-13 シールド ティーエックス （ユーケー） リミテッド Method for producing ferric maltol composition from ligand modified and ligand coated ferric hydroxide

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5354187A (en) * 1976-10-28 1978-05-17 Ajinomoto Co Inc Modified spiropyran carrier
WO2000012564A1 (en) * 1998-08-31 2000-03-09 Nof Corporation High-purity polysaccharide containing hydrophobic groups and process for producing the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5354187A (en) * 1976-10-28 1978-05-17 Ajinomoto Co Inc Modified spiropyran carrier
WO2000012564A1 (en) * 1998-08-31 2000-03-09 Nof Corporation High-purity polysaccharide containing hydrophobic groups and process for producing the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Fumio HAMADA et al:, "Photochromic molecular recognition of beta-cyclodextrin bearing spiropyran moiety for organic quests", J.Chem.Soc., Perkin Trans.2, 1996, vol. 12, pages 2567 to 2570m full text *
Itamar Willner et al:, "Reversible photoregulation of the activities of proteins", Reactive Polymers, 1993, Vol. 21, pages 177 to 186, full text *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019515893A (en) * 2016-03-31 2019-06-13 シールド ティーエックス （ユーケー） リミテッド Method for producing ferric maltol composition from ligand modified and ligand coated ferric hydroxide
JP7033545B2 (en) 2016-03-31 2022-03-10 シールド ティーエックス （ユーケー） リミテッド Methods for Producing Maltol Ferric Ferric Compositions from Ligand-Modified and Ligand-Coated Ferric Hydroxide

Similar Documents

Publication Publication Date Title
Carta et al. Solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in aqueous solutions at various pHs and NaCl concentrations
Zhang et al. Characterization of soy β-conglycinin–dextran conjugate prepared by Maillard reaction in crowded liquid system
Shenoy et al. Stability of crystalline proteins
Sawada et al. Cyclodextrin-responsive nanogel as an artificial chaperone for horseradish peroxidase
Nomura et al. Protein refolding assisted by self‐assembled nanogels as novel artificial molecular chaperone
CN102575244B (en) Dissociation method and dissociation agent for avidin and biotin
Lopez-Tobar et al. Stability of the disulfide bond in cystine adsorbed on silver and gold nanoparticles as evidenced by SERS data
Carlson et al. Chemically controlled self-assembly of protein nanorings
Nomura et al. Thermoresponsive controlled association of protein with a dynamic nanogel of hydrophobized polysaccharide and cyclodextrin: heat shock protein-like activity of artificial molecular chaperone
Kameta et al. Soft nanotube hydrogels functioning as artificial chaperones
US20220227838A1 (en) Covalent modification of biological macromolecules
US8569463B2 (en) Method of covalently modifying proteins with organic molecules to prevent aggregation
US20150246132A1 (en) Non-covalent, self-organzing hydrogel matrix for biotechnological applications
Taheri-Kafrani et al. Structure–function relationship of β-lactoglobulin in the presence of dodecyltrimethyl ammonium bromide
Mollmann et al. Displacement of adsorbed insulin by Tween 80 monitored using total internal reflection fluorescence and ellipsometry
Norioka et al. Biomolecularly stimuli-responsive tetra-poly (ethylene glycol) that undergoes sol–gel transition in response to a target biomolecule
EP0471125B1 (en) Modified protease, method of producing the same and cosmetic compositions containing the modified protease
MXPA01011561A (en) Rapid dehydration of proteins.
Matsuhira et al. Entropy-Driven Supramolecular Ring-Opening Polymerization of a Cyclic Hemoglobin Monomer for Constructing a Hemoglobin–PEG Alternating Polymer with Structural Regularity
Friedman Protein crosslinking: biochemical and molecular aspects
Zhang et al. Site-selective glycosylation of hemoglobin on Cys β93
Gao et al. Thiolated human serum albumin cross-linked dextran hydrogels as a macroscale delivery system
Mahajan et al. A glutathione-based hydrogel and its site-selective interactions with water
US9770420B2 (en) Method for producing a polyglycerol nanogel for the encapsulation and release of biologically active substances
WO2003097672A1 (en) Method for controlling protein

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)