CN1729319A - Apparatus and method for storing proteins - Google Patents
Apparatus and method for storing proteins Download PDFInfo
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- CN1729319A CN1729319A CNA2003801072399A CN200380107239A CN1729319A CN 1729319 A CN1729319 A CN 1729319A CN A2003801072399 A CNA2003801072399 A CN A2003801072399A CN 200380107239 A CN200380107239 A CN 200380107239A CN 1729319 A CN1729319 A CN 1729319A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D1/00—Treatment of filament-forming or like material
- D01D1/02—Preparation of spinning solutions
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
- D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
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Abstract
The present invention provides an apparatus for the storage of a protein (20) comprising a first compartment (30) for storing the protein (20) and a second compartment (40) for storing an alkaline buffer (50), the second compartment (40) being in fluid communication with the first compartment (30) is described. In one preferential embodiment of the invention, the alkaline buffer comprises calcium ions which encourage the gelling of the protein (20). A method for the storage of a protein (20) is also described. The method comprises: a first step of placing the protein in a first storage compartment (30); a second step of exposing the protein (20) to an alkaline buffer (50); and a third step of maintaining the protein (20) in the alkaline atmosphere in the first storage compartment (30).
Description
Technical field
The present invention relates to the apparatus and method of storage protein.
Background technology
A problem relevant with long-term storage of proteins is that they are passed in time and lose biological nature owing to molecular degradation.The method of having developed storage protein in the prior art is to address this problem.
For example, Japanese patent application JP-A-63-092671 (Kanebo) has introduced a kind of method of storage protein, and silk quality albumen or collagen are dissolved in the aqueous solution.Hydrolase is added in the solution, add chelating agent then.The pH value of regulating mixture is 4.5～7.5, usefulness have about 1um or more the filter of aperture filter this solution.Final drying solution obtains average degree of polymerization at 200～600 polymer.
However, hope can store it under the nature of protein, does not need enzyme to handle or handle with chelating agent.
In fact, the method for known several storage proteins.Natural silk is the very thin glossiness filament that silkworm (Bombyx mori) and other invertebrate produce from the protein dope or the storing of storage.Compare with the synthetic polymer that is commonly used to produce material, some advantage of natural silk, their performance are not subjected to protein slurry in the medium-term and long-term influence that stores of biological body of gland substantially.For example, the intensity of some spider traction fiber and toughness can surpass the most tough and the strongest artificial fibre-Kevlar
TMFiber.Spider silk also has high heat endurance.Many natural silks are biodegradable also, does not continue to stay in the environment.They are callable, can only use water as solvent High-efficient Production under low-pressure low-temperature technology.Natural spinning technique is noticeable, because the aqueous solution of protein is converted into tough and tensile and highly insoluble material.The spinning technique of spider and silkworm is briefly described in Vollrath and Knight are published in the article " Liquid crystalline spinning of spider silk " of Nature (2001,410 29 phases of volume, 541-548 page or leaf).The author points out to store their protein slurry and slurries are spun into learning knowledge the behavior of strong silk from spider and silkworm.The principle that the natural spinning of spider summarized in article comprises interpolation H ion and potassium ion, removes sodium ion when spinning slurries downwards by spin duct.
Article " Biological Liquid Crystal the Elastomers " (Trans.Phil.R.Soc.B.357 that writes at Knight and Vollrath, 155-163 (2002)) in, the author points out that spider silk fibroin and fibroin are combined to form the following fact of the main foundation of tough sex pilus: main histone, the fibroin in the silkworm and the spider silk fibroin in the spider of silk are repetition bipolarity (ABAB type) block copolymers, here A represents hydrophobic section, and B is not too hydrophobic part.
Find that there are two states in spider silk fibroin and fibroin: the firstth, safe storing state, very Chang protein chain is folded into rodlike molecule short rather than compression.Fibroin under first kind of state and spider silk fibroin have main random secondary structure that twine and/or spiral.Second kind of state is the solid state of main β crystallization secondary structure, and this second state is the nano-fiber composite material that contains the long nanofiber of the about 5nm of a large amount of diameters.Nanofiber is parallel to the major axis orientation of tough sex pilus substantially, is considered to contain whole or most of β type crystalline solid.The about 5nm of width of β type crystalline solid is parallel to the nanofiber long axis direction and arranges.Littler crystalline solid content is few, and more random material forms the matrix between the nanofiber.
At first, storing state is a metastable state.Think that it is transformed into the second nanofiber attitude and comprises the cohesion of molecule and the variation of secondary structure conformation.Think that conformation change occurs in great majority and repeats hydrophobic section in the bipolarity block copolymer, it becomes β crystalline texture from random coil/α screw type.Under field conditions (factors), at least five factors participate in cohesion and conformation change formation toughness fiber: add potassium ion in the reduction of pH value, the protein, remove sodium ion from protein, dry out and use mechanical strain to form silk.In addition, polyhydroxy secretion and surfactant can promote to change.At the last part of spider spinning pipeline, the thin radial ridges with low-surface-energy helps lend some impetus to protein slurry and is transformed into solid-state silk.In case short nanofiber forms, can be used as further cohesion of seed initiation and protein conformation and change, strengthened the total speed that changes like this.
If shift out to non-processor the first storing state solution of fibroin or spider silk fibroin, they are transformed into insoluble β crystalline state immediately, form insoluble floccule or gelinite so prematurely, can not be extruded or be woven into useful silk.Transformation can be very fast, finished in 1-3 days usually.This is a problem when extruding or spinning from fibroin thread.The bacterial degradation that becomes the peptide of conformation or the easy weak point that changes of crystallization by cut-out protein plays a part certain to this transformation in the glass tube.Perhaps by removing the protectiveness territory that stops cohesion or conformation change, proteolysis can promote this transformation.Make seed by the material that is transformed into the β crystalline state and can promote that also natural silk albumen or spidroin solution are transformed into insoluble attitude.Natural silk is dissolved in chaotropic agent, in lithium bromide, lithium rhodanate, sodium sulfocyanate, calcium chloride, calcium nitrate, when chaotropic agent is separated when removing, the formation that the fibroin of preparation regeneration and spidroin solution also experience floccule and similar gels gradually.When attempting to spin or also have a problem during extruded material from regenerated silk.
Find first storing state at the rear portion of silkworm body of gland and the similar A district of mesozone and spider.In these districts of body of gland separately, (20～40%w/v) are stored protein with very high concentration.In the spider, protein stores with high viscosity liquid crystal state solation, and it exists in the major part of first, second and the 3rd limb of sericterium pipe.In the silkworm, protein is stored in the rear portion and the mesozone of sericterium of silkworm in the middle of newly moult final with gel state, but in carrier pipe protein just by be transformed into before spinning colloidal sol (than the proparea than forward part).Transformation from the gel state to colloidal sol afterwards expands to the gland path backward can make middle and mobile forward than the material of back zone storage, can pull down in the front portion of pipe (tip part) like this and be stretched into silk.
Summary of the invention
The object of the invention provides the apparatus and method of storage protein.
These and other objects of the present invention are realized with second storehouse that stores ealkaline buffer in first storehouse that comprises storage protein by the device that storage protein is provided.Preferably implement among the embodiment in the present invention, the second storehouse splendid attire contains the ealkaline buffer of calcium chloride.Second storehouse is connected with the first storehouse fluid (as liquid or gas).The protein that is stored in first storehouse is alkalescence and contains calcium ion, and it is than the untreated spider silk fibroin that directly shifts out from organism or fibroin or that spider silk or silk are dissolved in the solution for preparing the chaotropic agent is more stable under this condition.Thereby, form by the decomposition of the morphogenetic β sheet-form of the β of protein or prematurity and to be delayed greatly.
Among the embodiment of invention, from following alkali, select ealkaline buffer: ammoniacal liquor, Ammoniom-Acetate, ammonium formate, lemon acid amide, Tri(Hydroxymethyl) Amino Methane Hydrochloride (tris/HCL), HEPES, PIPES, sodium carbonate, potash, sodium phosphate, potassium phosphate or their mixture.
In one embodiment, except that ealkaline buffer, sodium azide is added in the protein.Perhaps, can add benzene thiocarbamide, Cymag or potassium cyanide.
In one embodiment, in ealkaline buffer, also add the calcium ion of 100-700mM, preferably calcium chloride.Under these environment, ealkaline buffer can not contain carbonic acid or phosphate ion.
When calcium ion is added into ealkaline buffer, cause concentrated silk protein solution or their homologue gel.The gel that this method produces is more stable than the collosol state of silk protein solution.Before extruding, spinning or other form silk, the gel state that this method causes can convert dissolved colloidal state again to, can realize the transformation of gel state to dissolved colloidal state with the dialysis solution dialysis that contains 20-100mM vinyl diamino four acetic acid (regulating the pH value with ammoniacal liquor or sodium hydroxide solution is 7.0-7.8).Calcium ion is removed in this processing.Perhaps, remove calcium ion with the deionized water dialysis.In arbitrary example, polyethylene glycol or another water-soluble polymer can be added in the dialysate by the concentration of reverse osmosis with maintenance or increase protein.
Form by low-surface-energy material or apply to the inner wall surface of small part first storage warehouse in a preferred embodiment with the material of low-surface-energy, as polytetrafluoroethylene (PTFE), silane, nylon, polyethylene or Merlon, they also help the β sheet prematurity that stops protein and form.
In device, ealkaline buffer is gaseous state or solution form.If use with gaseous form, it can be applied directly to the surface of protein solution or allow to enter by porous or semi permeable surface diffusion into the surface.If ealkaline buffer uses with the solution form, it can be separated from protein by porous or pellicle, or the buffer stream that separates is applied in the protein solution stream, derives from the protein solution surface then.If ealkaline buffer uses with gaseous form, it can directly diffuse into protein solution makes it be alkalescence.If by porous or pellicle ealkaline buffer is separated from protein, buffer that may contain and calcium ion can diffuse into protein solution by described film then.
At another embodiment of invention, when helping to keep protein at collosol state, protein directly mixes with alkaline solution.Calcium ion can be introduced directly in the alkaline solution.
In the invention preferred embodiment, sodium azide is added into pH be in 7.4 the buffer so that ultimate density greater than 0.0001M.
The protein that stores can be by dissecting the native protein that animal obtains, or genetic engineering obtain again in conjunction with the regenerated silk solution of albumen or dissolving silkworm or silk fiber (being removed by dialysis afterwards) preparation in chaotropic agent (chaotropic agent), or the mixture of above-mentioned albumen or albumen analog.Found that the present invention is being useful aspect the silk of storage of fibroin or spider silk fibroin or their homologue or regeneration and/or the spider silk fibroin.
Usually, the protein of storage is repetition bipolarity block copolymerization protein or the protein analogue that contains charged group and prepare by chemical synthesis or genetic engineering.
Realize goal of the invention by the method that storage protein is provided, comprise that the first step puts into first storage warehouse with protein.In second step, protein is exposed to ealkaline buffer (suggestion contains calcium ion and sodium azide) a period of time.In the 3rd step, protein is maintained in the alkaline environment in first storage warehouse.
Silk protein solution or the analog and the regenerated silk solution of long term storage are provided.
Description of drawings
Fig. 1 illustrates the schematic diagram of first embodiment of the device that is fit to storage protein.
Fig. 2 illustrates the schematic diagram of second embodiment of the device that is fit to storage protein.
Fig. 3 illustrates ealkaline buffer and azides ion to the influence in solution storage time of concentrated natural fibroin.
Detailed description of the Invention
Fig. 1 is the schematic diagram that first embodiment of the device 10 that is fit to storage protein 20 is described.Device 10 has the protein storage compartment 30 of placing protein 20.Protein storage compartment 30 is connected with alkali storage compartment 40 by managing 35.Alkali storage compartment 40 stores alkaline solution 50.Protein storage compartment 30 is fit to part or all basically inwalls and makes or apply low-surface-energy material with the material of low-surface-energy, as polytetrafluoroethylene (PTFE), and polyethylene or Merlon.
In a preferred embodiment, except that ealkaline buffer, also in the protein 20 of first storage warehouse, add sodium azide.In another embodiment, benzene thiocarbamide, Cymag or potassium cyanide can be added in the protein 20.
In another embodiment of invention, as shown in Figure 2, by pellicle or perforated membrane 60, protein storage compartment 30 can be separated with alkali storage compartment 40.Pellicle or perforated membrane 60 allow ion by be stored in the pH value of the protein 20 in the protein storage compartment 30 with change.Using under the condition of pellicle, polyethylene glycol can be added in the ealkaline buffer greater than 70%w/v, to remove the water of the protein solution in the protein storage compartment by the method for reverse osmosis.Under these environment, the molecular weight of employed polyethylene glycol must be greater than the molecular weight of pellicle obstruction.Can use water miscible and have other polymer of enough big size to pass through dialyser to prevent them.
Like this, protein 20 in first storehouse 30, handle a period of time short as 1 minute, but suggestion at least 20 minutes can prevent immature cohesion.Processing time is according to the amount of protein 20, initial pH value, temperature and be exposed to that the diffusion of the surface area of protein 20 in the ealkaline buffer, subtracting property buffer reaches distance that all proteins 20 passed through and the buffer capacity of protein 20 and ealkaline buffer is determined.
In preferred inventive embodiments, protein 20 and alkaline buffer solution, greater than 7.4, Ammoniom-Acetate or ammonium formate that concentration is equal to or greater than 0.1M mix as the pH value.In the preferred embodiment of invention, alkaline buffer solution contains the calcium ion of 100-700mM, the sodium azide greater than 0.0001.
Promote the suitable ability of the different alkaline solutions that protein 20 is stable to estimate in two ways: first, in 4 time-of-weeks, the protein solution droplet that concentrates with different alkaline buffer solution dialysis is determined the gel or the collosol state of protein solution at regular intervals.General Result as shown in Figure 3.The second, determine the validity of this trend with a spot of spinning fibre that contacts the protein solution of different time with ealkaline buffer.In the second approach, use the universal bionical device for spinning of describing among the PCT application WO-A-01/38614, list this in list of references here.With after saturated vapour atmosphere that the 1M Dilute Ammonia Solution obtains contacted for 1 week, the protein 20 of storage still can be woven into silk, fiber or filament with the test shows of bionical device for spinning.
These apparatus and method not only can be used to the storing natural and the protein of combination again, and it also can be used to the fibroin and the spidroin solution of store regenerated, by dissolving in the more suitable solution that contains chaotropic agent (hydrogen bond rupture) of silk that these albumen make.The example of this chaotropic agent is 50: saturated lithium bromide of 50v/v and straight alcohol mixture.
Embodiment 1: the storage time prolongs
In the following example, the storage time of the spidroin solution that concentrates that obtains for the protein solution that contains fibroin that obtains from the sericterium of silkworm (Bombyx mori), regeneration silkworm (Bombyx mori) solution or from the main excision gland of Nephila spider is extended.
The first step, protein solution are sent in the dialysis bag (MWCO5-8kDa), are that the buffer of 7.8 Ammoniom-Acetates is concentrated 4 ℃ of following reverse osmosis 1 hour with containing 20%w/vPEG (MW15-20kDa) and 0.1mM pH value.Sodium azide can be added into and make ultimate density in the dialysate is that 0.001mM is to prevent bacterial growth.
The gel that obtains can store at least 4 weeks down at 4 ℃.
Extrude form goods with alternate manner before, can make the gel that obtains be transformed into colloidal sol again with distilled water and water-soluble 100mM vinyl diamines four acetum dialysis.
Embodiment 2: fibroin is stored in proof in the Bombyx mori silkworm body as gel before the spinning.
The silkworm Bombyx mori that dissects different periods of expansion under binocular microscope is to estimate rear portion, centre and the anterior fibroin state than the rear section of silk gland.The body of gland of removing rapidly from the silkworm body is sent to the Ringer's mixture (pH=7.8) of silkworm to observe.The material of body of gland inner chamber shows that it initially exists as colloidal sol in all stages, is stored initial period up to the cocoon spinning as gel afterwards in to the last several days of intermediate state before the cocoon spinning begins.Whether by crosscut pipe cutting (anterior forward part), observing has the fibroin drip to go out, and only shows that before spinning and in the spinning, there is the marker space in fibroin with dissolved colloidal state.In case spinning begins, along with the sericterium size disappears in spinning, colloidal sol forms to demonstrate to expand by silk backward gradually and drips.This explanation gel forms safety is stored silk is that necessary and colloidal sol form and spinning dripped to flow downward be necessary.
Embodiment 3: add and remove the influence of calcium ion to the sol/gel attitude of storing natural fibroin
Contain 10mM sodium azide and 100mM EDTA with 1ml, the high-load of ammonia spirit that pH=7.8 concentrates or the 100mM Ammoniom-Acetate buffer of acetic acid dilution mesozone obtains the fibroin droplet solution.The 1M calcium chloride that adds 1 volume is in the fibroin droplet solution of 1 volume or with the dialysis of 500nM calcium chloride solution, this solution of gel materialization rapidly.With distilled water or (pH=7.8) dialysis of 100mM Ammoniom-Acetate buffer or with the 100mM Ammoniom-Acetate buffer (pH=7.8) that contains 500mMEDTA dialysis fast, protein can be got back to dissolved colloidal state.This explanation adds calcium ion can cause the sol/gel conversion, removes the calcium ion gel and is convertible into colloidal sol.Store a couple of days even a few week under gelation condition after, the transformation that calcium causes still demonstrates invertibity.
Embodiment 4 calcium ions add or remove the influence that the natural silk protein solution is stored
As the adding 1M calcium chloride that embodiment 3 describes, the fibroin solution that can obtain concentrating, and gelation.Tested gel and can be stabilized time of storage at a certain state, it can transform back into colloidal sol to shift out calcium ion.Gel is placed different time at 4 ℃, and the preparation gel sample is used 100mM Ammoniom-Acetate buffer (pH=7.8) dialysis of 500mM EDTA then immediately.These observation reports show: after storing at least 4 weeks, calcium-fibroin gel can be changed into colloidal sol by safety.The other EDTA of formation adding on the contrary, under 4 ℃, only can store 5-8 days, before also can not be transformed into the polymeric material of colloidal sol again at the colloidal sol that forms in the presence of the calcium ion.Dissolve degumed silk in the 9.6M lithium bromide, the regenerated silk solution that contains 100mM lithium, 20%w/v polyethylene glycol (mean molecule quantity 15Kda) dialysis preparation obtains similar result at the synthetic solvent of the dialysis bag (Spectropor 10Kda) of low molecular weight blocking-up.With the gel phase ratio that obtains by gelation natural silk albumen, has low relatively rigidity by the gel that in solution, adds the calcium ion preparation as mentioned above.
Claims (32)
1, a kind of device that is used for storage protein (20) comprises: second storehouse (40) of first storehouse (30) of storage protein (20) and storage ealkaline buffer (50), second storehouse (40) are connected with first storehouse (30) fluid.
2, device according to claim 1, wherein ealkaline buffer (50) contains calcium ion.
3, device according to claim 1 and 2, wherein ealkaline buffer is selected from the group of the ealkaline buffer that comprises that ammonia spirit, Ammoniom-Acetate, ammonium formate, Tri(Hydroxymethyl) Amino Methane Hydrochloride, HEPES, PIPES, sodium carbonate, potash, sodium phosphate, potassium phosphate or their mixture are formed.
4, require described device according to arbitrary aforesaid right, wherein ealkaline buffer (50) contains the calcium ion of 50-700mM.
5, require described device according to arbitrary aforesaid right, wherein ealkaline buffer (50) also contains sodium azide.
6, require described device according to arbitrary aforesaid right, wherein the part of the inner wall surface of at least the first storehouse (30) is formed or is coated with the material of low-surface-energy by the material of low-surface-energy.
7, require described device according to arbitrary aforesaid right, wherein ealkaline buffer (50) exists with gaseous state.
8,, wherein use dialyser (60) that ealkaline buffer (50) is separated with protein (20) according to the arbitrary described device of claim 1 to 6.
9, require described device according to arbitrary aforesaid right, wherein protein (20) mixes with alkaline solution.
10, require described device according to arbitrary aforesaid right, wherein protein (20) mixes with at least a or multiple alkaline buffer salt.
11, device according to claim 10, wherein at least a or multiple alkaline buffer salt also contains sodium azide, phenylthiourea, Cymag or potassium cyanide.
12, according to arbitrary described device in the claim 9 to 11, wherein alkaline solution is oxyammonia, Ammoniom-Acetate, ammonium formate, lemon acid amide, Tri(Hydroxymethyl) Amino Methane Hydrochloride, PIPES, HEPES, sodium carbonate, potash, sodium phosphate, potassium phosphate buffer agent or the pH value of the 0.1M mixture greater than 7.4 buffer.
13, require described device according to arbitrary aforesaid right, wherein the pH value of ealkaline buffer (50) is greater than 7.4.
14, require described device according to arbitrary aforesaid right, wherein the concentration of ealkaline buffer (50) or combination buffer is equal to or greater than 0.025M.
15, require described device according to arbitrary aforesaid right, wherein protein (20) is natural, regeneration or the protein of recombinant, the mixture of native protein, the mixture of regenerated protein or the mixture of recombinant protein.
16, require described device according to arbitrary aforesaid right, wherein protein (20) is fibroin or spider silk fibroin or their thing of the same clan.
17, require described device according to arbitrary aforesaid right, wherein protein (20) is repetition bipolarity block copolymerization protein or the protein homologue that contains charged group and with the protein of chemical synthesis or genetic engineering preparation.
18, the method for a kind of storage protein (20) comprising:
-the first step: protein is put into first storage warehouse (30);
-the second step: make protein (20) be exposed to ealkaline buffer (50);
Three steps of-Di: the protein (20) in first storage warehouse (30) is remained in the alkaline environment.
19, method according to claim 18, wherein protein (20) remained in first storage warehouse (30) at least one minute.
20, according to claim 18 or 19 described methods, wherein ealkaline buffer (50) contains calcium ion.
21, according to arbitrary described method in the claim 18 to 20, wherein ealkaline buffer (50) is selected from the group of the ealkaline buffer of being made up of ammonia spirit, Ammoniom-Acetate, ammonium formate, lemon acid amide, Tri(Hydroxymethyl) Amino Methane Hydrochloride, HEPES, PIPES, sodium carbonate, potash, sodium phosphate, potassium phosphate or their mixture.
22, according to arbitrary described method in the claim 18 to 21, wherein ealkaline buffer contains the calcium ion of 50-700mM.
23, according to arbitrary described method in the claim 18 to 22, wherein ealkaline buffer (50) exists with gaseous state.
24, according to arbitrary described method in arbitrary claim 18 to 23, wherein at least a alkaline buffer salt is added in the protein (20).
25, method according to claim 24, its neutral and alkali buffer salt also contains sodium azide, phenylthiourea, Cymag or potassium cyanide.
26,, wherein use dialyser (60) to make buffer (50) and Separation of Proteins according to arbitrary described method in the claim 18 to 25.
27, according to arbitrary described method in the claim 18 to 26, wherein the pH value of ealkaline buffer (50) is greater than 7.4.
28, according to arbitrary described method in the claim 18 to 27, wherein (50) are preceding mixes storing for protein (20) and alkaline solution.
29, according to arbitrary described method in the claim 18 to 28, wherein the concentration of ealkaline buffer (50) or combination buffer is equal to or greater than 0.025M.
30, according to arbitrary described method in the claim 18 to 29, wherein protein (20) is natural, regeneration or the mixture of the mixture of the protein of combination again, native protein, regenerated protein or the mixture of conjugated protein again.
31, according to the arbitrary described method of claim 18 to 30, wherein protein (20) is fibroin or spider silk fibroin or their thing of the same clan.
32, according to the arbitrary described method of claim 18 to 31, wherein protein (20) is repetition bipolarity block copolymerization protein or the protein homologue that contains charged group and with the protein of chemical synthesis or genetic engineering preparation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0230102.6 | 2002-12-23 | ||
| GBGB0230102.6A GB0230102D0 (en) | 2002-12-23 | 2002-12-23 | Apparatus and method for storing proteins |
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| CN1729319A true CN1729319A (en) | 2006-02-01 |
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| US (1) | US20060289327A1 (en) |
| EP (1) | EP1576212A1 (en) |
| JP (1) | JP2006511722A (en) |
| KR (1) | KR20050084455A (en) |
| CN (1) | CN1729319A (en) |
| AU (1) | AU2003293981A1 (en) |
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| CA (1) | CA2511183A1 (en) |
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| CN102083477A (en) * | 2008-04-30 | 2011-06-01 | 奥索克斯有限公司 | An implantable material and a method for the preparation thereof |
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| US7148343B2 (en) * | 2001-10-12 | 2006-12-12 | Gentra Systems, Inc. | Compositions and methods for using a solid support to purify RNA |
| KR20070097430A (en) | 2004-11-05 | 2007-10-04 | 퀴아젠 노쓰 아메리칸 홀딩즈, 인크. | Compositions and methods for purifying nucleic acids from stabilization reagents |
| WO2012145594A2 (en) * | 2011-04-20 | 2012-10-26 | Trustees Of Tufts College | Molded regenerated silk geometries using temperature control and mechanical processing |
| JP2017170082A (en) * | 2016-03-25 | 2017-09-28 | 優一郎 新崎 | Brush bristle material and brush |
| US11142553B2 (en) | 2016-11-29 | 2021-10-12 | Spiber Inc. | Protein composition, method for producing same and method for improving heat stability |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH08295697A (en) * | 1995-04-26 | 1996-11-12 | Kanebo Ltd | Production of aqueous solution of silk fibroin at high concentration |
| JP4194291B2 (en) * | 2001-04-17 | 2008-12-10 | 興和株式会社 | Process for producing undegraded silk fibroin aqueous solution and skin care agent containing the same |
| GB0126118D0 (en) * | 2001-10-31 | 2002-01-02 | Vollrath Friedrich W L | Precursor feedstock for forming filaments |
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2002
- 2002-12-23 GB GBGB0230102.6A patent/GB0230102D0/en not_active Ceased
-
2003
- 2003-12-23 JP JP2004561426A patent/JP2006511722A/en not_active Withdrawn
- 2003-12-23 BR BR0317651-7A patent/BR0317651A/en not_active IP Right Cessation
- 2003-12-23 EA EA200501144A patent/EA008044B1/en not_active IP Right Cessation
- 2003-12-23 WO PCT/EP2003/014786 patent/WO2004057068A1/en not_active Application Discontinuation
- 2003-12-23 NZ NZ541236A patent/NZ541236A/en unknown
- 2003-12-23 US US10/540,042 patent/US20060289327A1/en not_active Abandoned
- 2003-12-23 KR KR1020057011708A patent/KR20050084455A/en not_active Application Discontinuation
- 2003-12-23 MX MXPA05006953A patent/MXPA05006953A/en unknown
- 2003-12-23 CN CNA2003801072399A patent/CN1729319A/en active Pending
- 2003-12-23 CA CA002511183A patent/CA2511183A1/en not_active Abandoned
- 2003-12-23 EP EP03789394A patent/EP1576212A1/en not_active Withdrawn
- 2003-12-23 AU AU2003293981A patent/AU2003293981A1/en not_active Abandoned
-
2005
- 2005-06-21 IL IL169331A patent/IL169331A0/en unknown
- 2005-06-22 ZA ZA200505071A patent/ZA200505071B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102083477A (en) * | 2008-04-30 | 2011-06-01 | 奥索克斯有限公司 | An implantable material and a method for the preparation thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| NZ541236A (en) | 2007-05-31 |
| EP1576212A1 (en) | 2005-09-21 |
| GB0230102D0 (en) | 2003-01-29 |
| JP2006511722A (en) | 2006-04-06 |
| BR0317651A (en) | 2005-12-06 |
| US20060289327A1 (en) | 2006-12-28 |
| KR20050084455A (en) | 2005-08-26 |
| EA200501144A1 (en) | 2006-04-28 |
| AU2003293981A1 (en) | 2004-07-14 |
| ZA200505071B (en) | 2006-11-29 |
| IL169331A0 (en) | 2009-02-11 |
| MXPA05006953A (en) | 2006-02-22 |
| CA2511183A1 (en) | 2004-07-08 |
| EA008044B1 (en) | 2007-02-27 |
| WO2004057068A1 (en) | 2004-07-08 |
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