KR20050084455A - Apparatus and method for storing proteins - Google Patents

Abstract

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

Apparatus and method for storing proteins {APPARATUS AND METHOD FOR STORING PROTEINS}

The present invention relates to apparatus and methods for the storage of proteins.

One of the problems associated with long-term storage of proteins is that over time, molecules degrade and lose their biological properties. In order to solve this problem, prior art regarding protein storage method has been developed.

For example, Japanese Patent Application JP-A-63-092671 (Kanebo) describes a method for storing proteins in which fibrin or collagen is dissolved in an aqueous solution. The hydrolase is added to the solution after the chelating agent. The pH of the mixture is adjusted to 4.5 to 7.5 and the solution is dried to yield a polymer having an average degree of polymerization in the range of 200-600.

However, it would be desirable to be able to store proteins in nature without enzyme treatment and chelating agent treatment.

Several methods are known for storing proteins. Natural silk is a glossy filament produced by silkworm Bombyx mori and other protein species of stored protein dope or feedstock. The silk provides an advantage compared to synthetic polymers currently used to make materials and their properties are substantially unaffected by long term storage of protein dope in the secretory organs. For example, the tensile strength and toughness of certain webs outweighs the tensile strength and toughness of Kevlar ^™ fiber, the toughest and most durable artificial fiber. Cobweb silk also has stability to high temperatures. Many silks are also biodegradable and do not persist in the environment. They are regenerated and are produced by very effective low pressure and low temperature treatments using only water as solvent. The natural weaving process is noteworthy in that the protein in the aqueous solution is converted into a tough and very insoluble substance. The process of weaving spiders and silkworms that produce silk threads is described in Vollrath and Knight, "Liquid crystalline spinning of spider silk", Nature, vol. 410, 29 March 2001, pages 541-8. The authors noted that spiders and silkworms store their protein dope molecules and extrude them into strong yarns. The article observed evidence that the spider's natural weaving mechanism involves the addition of hydrogen ions and potassium ions and the removal of sodium ions as the textile dope passes through the textile tube.

Knight and Vollrath, "Biological Liquid Crystal Elastomers", Trans. Phil. R. Soc. In B. 357, 155-163 (2002), the authors note that the combination of velcroin and fibroin protein forms a tough yarn, which indicates that the major protein structures of silk, silkworm fibroin, and spider velcroin are of the ABAB type of kin It is pointed out that it depends on the fact that it is a repeatable block copolymer.

Speed-in and fibroin are found in two states. The first state is a safe storage state, in which very long protein chains are folded and are thought to be short, somewhat dense stick-like molecules. In the first state the fibroin or speedoin protein has an overwhelmingly random coil and / or helical secondary structure. The second state is the solid state and has a predominantly secondary structure of beta crystals. This second state is a nanomicrofiber composite containing a high packing portion of very long nanomicrofibers of about 5 nm in diameter. Nanomicrofibers are thought to contain substantially all or most of the beta crystals that are directed substantially parallel to the long axis of the tough yarn. The beta crystals have a width of about 5 nm and are arranged substantially parallel to the long axis of the nanomicrofibers. Lesser amounts of crystals and more disordered materials are believed to form a matrix between nanomicrofibers.

The first storage state is metastable. The transition to the second nanomicrofiber state is believed to be involved in the aggregation of molecules and the morphology of secondary structures. Morphological changes are believed to occur in the most hydrophobic block types of repeating block copolymers that transform from 'random coil' / alpha helices into beta crystalline structures. At least five factors are believed to be involved in aggregation and morphological changes that form tough yarns in vivo: a decrease in pH; Addition of potassium ions to the protein; Removal of sodium ions from proteins; Removal of water and the application of mechanical strain to the formed thread. The conversion can also be facilitated by the secretion of polyols and surfactants. Sophisticated longitudinal ridges with low surface energy in the final part of the spider's inner tube layer will facilitate the conversion of protein dope to solid threads. Once short nanomicrofibers are formed, they can act as seeds to initiate further aggregation and morphology of proteins, thereby improving the overall rate of change.

If the solution of the first stored state of fibroin or speedloin is not treated, they spontaneously deform into insoluble beta crystals, forming an immature insoluble floc or gel that cannot be extruded or woven into a useful yarn. The deformation is so fast that it takes 1-3 days to complete. This is a problem when extruding or weaving yarns from silk proteins. Bacterial proteolysis is responsible for some of these modifications in vivo by cleaving proteins into short peptides. Alternatively, proteolysis can promote the modification by removing protective regions that inhibit aggregation or morphology. The modification of the natural fibroin or velcroin solution into an insoluble form can be facilitated by sparging the already modified material in beta state. Freezing or mechanical shearing of the natural solution will result in transformation to beta crystalline state. Regenerated fibroin and velcroin solutions prepared by decomposing silk yarn in a chaotropic agent such as lithium bromide, lithium thiocyanate, sodium thiocyanate, calcium chloride, calcium nitrate are prepared from the disorder. A similar formation of flocs or gels generally occurs when I am removed by dialysis. This is a problem when weaving or extruding material from recycled silk.

The first state of storage is found in the posterior and middle portions of the silk gland and the pseudo-A-zone of spiders. Proteins in each of these glands are stored at surprisingly high concentrations (2--40% w / v). For spiders, the protein is believed to be stored as high mucus liquid crystals that persist through most of the first, second and third legs of the silk gland. In the case of silkworms, the protein is stored as a gel in the back and middle portions of the silk glands of the newly infested final instar silkworms but is transformed into a sol in the tube (front part) just before the protein is threaded. Subsequently, the gel-to-sol modification can propagate the secretory pathway backwards, allowing the material stored in the middle and posterior portions to flow forward, reducing the front (end) filaments of the tube.

1 shows a schematic of a first embodiment of a device suitable for protein storage.

2 shows a schematic of a second embodiment of a device suitable for protein storage.

FIG. 3 is a result showing the effect of storage time of alkaline buffer and sodium azide on concentrated natural fibroin solution.

Summary of the Invention

It is an object of the present invention to provide an apparatus and method for protein storage.

It is an object of the present invention to provide an apparatus for the storage of proteins comprising a first compartment for protein storage and a second compartment for storage of alkaline buffer. In a preferred embodiment of the invention, the second compartment contains an alkaline buffer containing calcium chloride. The second compartment is in communication with the first compartment and the fluid (ie liquid or gas). The protein stored in the first compartment is in an alkaline state containing calcium ions, which is considerably more than that produced by decomposing spiders or silkworms in an untreated velcroin or fibroin solution or disordered preparation removed directly from the secretory organs. Stable. Therefore, degradation or early formation of the beta-sheet form that forms the beta state of the protein is very delayed.

In one embodiment of the invention, the alkaline buffer is ammonia, ammonium acetate, ammonium formate, ammonium citrate, tris / HCl, HEPES, PIPES, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate or mixtures thereof It is selected from the alkali group which consists of.

In an embodiment, sodium azide is added to the protein in addition to the alkaline buffer. Alternatively, phenyl thiourea, sodium cyanide or potassium cyanide can be added.

In another embodiment, 100-700 mM calcium ion is added to the alkaline buffer, preferably as chloride. Under such circumstances, the alkaline buffer may not contain carbonate or phosphate ions.

When calcium ions are added to alkaline buffers, they result in these analogs to concentrated solutions or gels of silk proteins. The gel produced in this way is much more stable than a sol solution of silk protein. The gel state derived in this way can be converted back to the sol state before forming the extrusion, textile or silk. This can be done by dialysis against a dialysis solution containing 20-100 mM ethylenediamine tetraacetic acid (EDTA) solution adjusted to pH 7.0-7.8 using ammonia or sodium hydroxide solution. This treatment removes calcium ions. Alternatively, calcium ions can be removed by dialysis against ion-free water. In either case, polyethylene glycol or other water soluble polymer may be added to maintain or increase protein concentration by reverse osmosis.

In a preferred embodiment, at least a portion of the inner surface of the first storage compartment wall is low, such as polytetrafluoroethylene (PTFE), silane, nylon, polyethylene or polycarbonate, which helps prevent premature formation of the beta sheet form of the protein. It is formed or coated from a material having surface energy.

In the device, the alkali buffer is in gaseous form or in solution. If the buffer is used in gaseous form, the buffer may be applied directly to the surface or the surface of the protein solution or may be diffused through a porous or semipermeable surface. If an alkaline buffer is used as the solution, the buffer can be separated from the protein by a porous or semipermeable membrane or a separate flow of buffer solution can be applied to the flow of the protein solution and, as a result, off the surface of the protein solution. If the alkaline buffer is in gaseous form, the buffer diffuses directly into the protein solution, making the protein solution alkaline. When the alkaline buffer is separated from the protein by a porous or semipermeable membrane, both the buffer and the Calsum ions can diffuse through the membrane into the protein solution.

In another embodiment of the invention, the protein is mixed directly with the alkaline solution, which allows the protein to remain in the sol state. Calcium ions can be added directly to the alkaline solution.

In a preferred embodiment, sodium azide is added to a buffer having a pH greater than 7.4 so that the final concentration exceeds 0.0001M.

The stored protein is, for example, a natural protein obtained by animal incision or a recombinant protein obtained by genetic engineering or a regenerated silk solution prepared by decomposing silkworm or silk fibers in a disordered preparation which is subsequently removed by dialysis or as described above. It can be a mixture of one protein or protein analog. The present invention has been found to be useful in the storage of regenerated solutions of fibroin or velcroin protein or its homologues or fibroin and / or velcroin.

Generally, stored proteins are repetitive amphiphilic block copolymer proteins or protein analogs that have a charge and are prepared by chemical synthesis or genetic engineering.

It is an object of the present invention to provide a method of storing a protein comprising the first step of placing the protein in a first storage compartment. In a second step, the protein is exposed to alkaline buffer (preferably containing calcium ions and sodium azide) for a period of time. In a third step, the protein is maintained in the alkaline environment of the first storage compartment.

This provides long term stock solutions and regenerated silk solutions for silk proteins or their analogs.

Detailed description of the invention

1 is a schematic diagram illustrating a first embodiment of an apparatus 10 suitable for storing protein 20. The device 10 has a protein storage compartment 30 in which the protein 20 is placed. The protein storage compartment 30 is connected to the alkaline storage compartment 40 by a pipe or tube 35. The alkali storage compartment 40 stores an alkaline solution 50. The protein storage compartment 30 has some or substantially all of the inner wall formed or coated from a material having low surface energy, such as polytetrafluoroethylene (PTFE), polyethylene or polycarbonate.

The protein 20 in the protein storage compartment 30 may be a natural protein obtained by animal dissection. Such natural proteins include, but are not limited to, phosphoproteins obtained from the main ampullate gland of spiders of the genus Nephila or fibroin proteins obtained from Bombyx mori or other species of silkworms. The present invention is also applicable to recombinant proteins obtained by homologs or genetic engineering of these proteins. The present invention is also applicable to regenerated silk solutions prepared by dissolving silk in solutions containing disordered preparations. The present invention is also applicable to the storage of any protein or protein analog that is a repeating amphiphilic block copolymer and contains charges.

In the alkaline storage compartment 40, several different types of alkaline solutions can be used. For example, the alkali may be ammonia / acetic acid, ammonium acetate, ammonium / formic acid or ammonium formate. These buffers are volatile and create an alkaline environment in the protein storage compartment 30. Tris / HCl, HEPES or PIPES can be used instead but these buffers are not volatile. In one embodiment of the invention, the alkali buffer is selected from the group consisting of ammonia, ammonium acetate, ammonium formate and ammonium citrate buffer. Potassium phosphate and potassium carbonate are also suitable. In a preferred embodiment, the alkaline buffer contains 100-700 mM calcium ions, preferably calcium ions added in the form of calcium chloride.

In a preferred embodiment, in addition to the alkaline buffer, sodium azide is also added to the protein 20 in the first storage compartment. In another embodiment, phenyl thiourea, sodium cyanide or potassium cyanide is added to the protein 20.

In another embodiment of the invention, the protein storage compartment 30 is separated from the alkaline storage compartment 40 by a semipermeable membrane or a porous membrane 60 as shown in FIG. The semipermeable membrane or porous membrane 60 allows ions to pass through to change the pH of the protein 20 stored in the protein storage compartment 30. If a semipermeable membrane is used, polyethylene glycol can be added up to 70% w / v of alkaline buffer to remove water from the protein solution in the protein storage compartment by reverse osmosis. Under these circumstances, the polyethylene glycol molecular weight used should be at least the cleavage molecular weight of the semipermeable membrane. Other polymers can be used in this way as long as they are water soluble and of sufficient size to prevent the polymer from passing through the osmotic membrane.

Thus, the protein 20 can be prevented from premature coagulation by treating it in the first compartment for as little as 1 minute, preferably at least 20 minutes. This time is determined by the amount of protein 20, the initial pH value, the temperature, the surface area of the protein 20 exposed to the alkaline buffer, the distance required for the alkaline buffer to spread to reach all the protein 20 and the protein 20 and It depends on the buffering capacity of the alkaline buffer.

In a preferred embodiment, the protein 20 is mixed with an alkaline buffer such as ammonium acetate or ammonium formate having a pH higher than 7.4 and a concentration equal to or greater than 0.1M. In a preferred embodiment, the alkaline buffer solution contains between 100 and 700 mM calcium ions and more than 0.0001 M sodium azide.

The suitability of different alkaline solutions to improve the stability of the protein 20 is assessed in two ways: First, a small drop of concentrated protein solution is dialyzed against different alkaline buffer solutions for a period of up to four weeks, and occasionally The gel or sol state is measured. General results are shown in FIG. 3. Second, the feasibility of this method can be confirmed by storing them for different periods of time in contact with alkaline buffer and then spinning the fibers from a small amount of protein solution. In a second method, a general type of biomimetic textile device is disclosed in PCT Application No. WO-A-01 / 38614, which is incorporated herein by reference. Experiments with biomimetic weaving devices have shown that stored protein 20 in contact with air saturated with steam derived from 1M ammonium hydroxide can produce yarn, fibers or filaments after one week. If sodium azide is added to the protein 20, the storage period can be increased.

The apparatus and method are used to store natural and recombinant proteins as well as regenerated solutions of fibroin and velcroin prepared by degrading silk produced from these proteins in a suitable solution containing a disordered agent (breaking hydrogen bonds). It can also be used to store. One example of such a disordering agent is a 50:50 v / v mixture of saturated lithium bromide and anhydrous alcohol.

Example 1 Extension of Storage Time

In the examples below, silkworm protein obtained from the silk gland of Bombyx mori, regenerated Bombyx mori fibroin solution or concentrated speedloin obtained from the main ampoulate gland of Nephila spider Extends for protein solutions, including solutions.

In the first step, the protein solution was transferred to a dialysis bag (MWCO 5-8 kDa) and 4 by reverse osmosis against a solution containing 20% w / w PEG (MW 15-20 kDa) and 0.1 mM ammonium acetate buffer at pH 7.8. Concentrated at 5 ° C. for 5 hours.

The protein was gelled for 1 hour at 4 ° C. by dialysis against a solution containing 500 mM calcium chloride solution and 0.1 mM ammonium acetate at pH 7.8. Sodium azide can be added to the dialysate to a final concentration of 0.001 mM to prevent bacterial growth.

The resulting gel can be stored at 4 ° C. for at least 4 weeks.

The resulting gel can be converted back to the sol by dialysis against distilled water or 100 mM aqueous ethylene diamine tetraacetic acid solution before extrusion or other desired product is formed.

Example 2 Verification of Fibroin Stored in Gels before Weaving in Bombyx mori Silkworms

The status of the fibroin in the posterior, middle and posterior part (secretory gland) of the front part silk gland of Bombyx mori silkworms was evaluated by incision under binocular microscopy at different stages of silkworm development. The gland was quickly removed from the silkworm and transferred to silkworm ringer solution (pH 7.8) for observation. The material of the gland lumen was initially present as a sol at all stages until the final instar a few days before the start of cocoon weaving and then stored as a gel until cocoon weaving and during the initiation of cocoon weaving. By cutting the tube (front part) and observing the fibroin dope flowing out, it was demonstrated that the fibroin was present as a sol in this part before and during weaving. Once weaving began, sol formation was shown to proliferate backwards through the silk dope as the size of the silk glands decreased during weaving. This proved that gel formation is essential for the safe storage of silk and that sol formation is essential for the flow of silk dope in a tube for weaving.

Example 3: Effect of Addition and Removal of Calcium Ion on the Sol / Gel State of Stored Natural Fibroin

Fibroin dope solution was obtained by diluting the contents of the middle portion of the secretory gland using 1 ml of 100 mM ammonium acetate buffer containing 10 mM sodium azide and concentrated ammonia solution or 100 mM EDTA adjusted to pH 7.8 by acetic acid. These solutions can be rapidly gelatinized by adding one volume of 1M calcium chloride to one volume of fibroin dope solution or by dialysis against an aqueous 500 mM calcium chloride solution. The protein can be returned to the sol state by dialysis against distilled water or 100 mM ammonium acetate buffer (pH 7.8) or can be returned to the sol state more quickly by dialysis against 100 mM ammonium acetate buffer (pH 7.8) containing 500 mM EDTA. . This suggests that sol / gel changes can be induced by the addition of calcium ions and that the gel can be returned back to the sol by removing the calcium ions again. The calcium-induced changes were shown to be reversible after several days of storage.

Example 4 Effect of Calcium Ion Addition or Removal on Storage of Natural Fibroin Solution

A concentrated fibroin solution was obtained and gelled by the addition of 1M calcium chloride as described in Example 3. The period in which the gel was stored in a stable state and changed to sol by removal of calcium ions was examined. To this end, sol samples stored for different periods at 4 ° C. were taken out and immediately dialyzed against 100 mM ammonium acetate buffer (pH 7.8) containing 500 mM EDTA. These observations showed that the calcium-fibroin gel can be safely transformed into a sol after a storage period of at least 4 weeks. In contrast, the sol formed in the absence of calcium ions could only be stored for 5-8 days at 4 ° C. before forming a polymeric material that could not be converted to the sol by the addition of additional EDTA. Dialysis bags were prepared for a solution containing 100 mM lithium, 20% w / v polyethylene glycol (nominal molecular weight 15 KDa), using a regenerated silk solution prepared by decomposing the degreasing silk in 9.6 mM lithium bromide. Similar results were obtained by dialysis of the resulting solution at a low molecular weight of Spectropor 10KDa). Gels prepared by adding calcium ions to this solution as described above were significantly less hard than gels obtained by gelling natural fibroin.

Claims (32)

A first compartment 30 for storing the protein 20 and a second compartment 40 for storing the alkaline buffer 50, the second compartment 40 being a protein in fluid communication with the first compartment 30; 20) storage device. The device of claim 1, wherein the alkali buffer contains 50 calcium ions. The method according to claim 1 or 2, wherein the alkaline buffer 50 is ammonia solution, ammonium acetate, ammonium formate, tris / HCl, HEPES, PIPES, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate or mixtures thereof. Apparatus selected from the group of alkali buffers comprising a. The device according to any one of claims 1 to 3, wherein the alkali buffer contains 50-700 mM calcium ions. 5. The device of claim 1, wherein the alkaline buffer further comprises sodium azide. 6. 6. The device according to any one of the preceding claims, wherein at least a portion of the inner wall surface of the first compartment (30) is produced or coated from a material having low surface energy. The device according to claim 1, wherein the alkaline buffer is in gaseous form. The device according to any one of the preceding claims, wherein the alkaline buffer (50) is separated from the protein (20) by a dialysis membrane (60). The device of claim 1, wherein the protein (20) is mixed with an alkaline solution. 10. The device of any one of the preceding claims, wherein the protein (20) is mixed with at least one alkali buffer salt or salt. The device of claim 10, wherein the at least one alkali buffer salt or salt also contains sodium azide, phenyl thiourea, sodium cyanide or potassium cyanide. The method of claim 9, wherein the alkaline solution is 0.1 mM ammonium hydroxide, ammonium acetate, ammonium formate, ammonium citrate, tris / HCl, PIPES, HEPES, sodium carbonate, potassium carbonate, sodium Phosphate, potassium phosphate buffer or a mixture of these buffers having a pH greater than 7.4. 13. Apparatus according to any one of the preceding claims, wherein the pH of the alkaline buffer (50) is greater than 7.4. The device according to claim 1, wherein the concentration of the alkaline buffer or the mixed buffer is equal to or greater than 0.025M. 15. The device according to any one of the preceding claims, wherein said protein (20) is a natural, regenerated or recombinant protein, a mixture of natural proteins, a mixture of regenerated proteins or a mixture of recombinant proteins. 16. The device according to any one of claims 1 to 15, wherein said protein (20) is fibroin or speedoline or its homologues. 17. The device according to any one of claims 1 to 16, wherein said protein (20) is a repeating amphiphilic block copolymer protein or protein analog containing charge and produced by chemical synthesis or genetic engineering. A first step of placing the protein in the first storage compartment 30; A second step of exposing the protein 20 to alkaline buffer 50; And A third step of maintaining the protein (20) in an alkaline environment in the first storage compartment (30). 19. The method of claim 18, wherein the duration for maintaining the protein (20) in the first storage compartment (30) is at least 1 minute. 20. The method of claim 18 or 19, wherein the alkaline buffer (50) contains calcium ions. 21. The method according to any one of claims 18 to 20, wherein the alkaline buffer 50 is ammonia solution, ammonium acetate, ammonium formate, tris / HCl, HEPES, PIPES, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate or And a buffer of alkali buffers comprising a mixture of these buffers. 22. The method of any one of claims 18 to 21, wherein the alkaline buffer contains 500-700 mM calcium ions. 23. The method of any one of claims 18 to 22, wherein the alkaline buffer (50) is in gaseous form. 24. The method of any one of claims 18 to 23, wherein at least one alkaline buffer salt is added to the protein (20). The method of claim 24, wherein the alkali buffer salt also contains sodium azide, phenyl thiourea, sodium cyanide or potassium cyanide. 26. The method of any of claims 18-25, wherein the alkaline buffer (50) is separated from the protein by a dialysis membrane (60). 27. The method of any one of claims 18 to 26, wherein the pH of the alkaline buffer (50) is greater than 7.4. 28. The method of any of claims 18 to 27, wherein the protein (20) is mixed with an alkaline solution prior to storage. 29. The method of any one of claims 18-28, wherein the concentration of the alkaline buffer or the mixed buffer is greater than or equal to 0.025M. 30. The method of any one of claims 18 to 29, wherein the protein (20) is a natural, regenerated or recombinant protein, a mixture of natural proteins, a mixture of regenerated proteins or a mixture of recombinant proteins. 31. The method of any one of claims 18-30, wherein said protein (20) is fibroin or speedoline or an analog thereof. 32. The method of any one of claims 18-31, wherein the protein (20) is a repeating amphiphilic block copolymer protein or protein analog containing charges and prepared by chemical synthesis or genetic engineering.