JP2006511722A - Protein storage apparatus and method - Google Patents

Abstract

A protein (20) storage device comprising a first compartment (30) for storing protein (20) and a second compartment (40) for storing alkaline buffer (50), wherein the second compartment A storage device is disclosed in which (40) is in fluid communication with the first compartment (30). In a preferential embodiment of the invention, the alkaline buffer comprises calcium ions that promote the gelation of protein (20). A method for storing protein (20) is also disclosed. The method includes a first step of placing a protein in a first storage compartment (30), a second step of exposing the protein (20) to an alkaline buffer (50), and a first storage compartment (30 ) To maintain the protein (20) in an alkaline atmosphere.

Description

  The present invention relates to protein storage devices and methods.

  One problem with long-term storage of proteins is that proteins lose their biological properties as molecules degrade over time. In order to overcome this problem, prior art methods for storing proteins have been developed.

  For example, JP-A 63-92671 (Kanebo) teaches a protein storage method in which fibroin or collagen is dissolved in an aqueous solution. To this solution is added a hydrolase and then a chelating agent. The pH of the mixture is adjusted to the range of 4.5 to 7.5, and the solution is filtered through a filter having pores of about 1 μm or less. Finally, when the solution is dried, a polymer having an average degree of polymerization of 200 to 600 is obtained.

  However, it is desirable that proteins can be stored in their natural state without enzymatic treatment or treatment with a chelating agent.

  In nature, several methods for storing proteins are known. Natural silks are delicate, glossy filaments that silkworm Bombyx mori and other invertebrate species create from stored protein dopes or feedstocks. Silks are superior to the synthetic polymers currently used in the manufacture of materials, and their properties appear to be substantially unaffected by long-term storage of protein dope in the gland of the organism . For example, the tensile strength and toughness of certain dragline silks may exceed that of Kevlar ™ fiber, the toughest and strongest man-made fiber. Spider bookmark yarn has high thermal stability. Many silks are also biodegradable and will not remain in the natural environment. They are recyclable and are manufactured in a very efficient low pressure low temperature process using only water as a solvent. The natural spinning process is notable for converting aqueous protein solutions into tough, highly insoluble materials. The process by which spiders and silkworms produce silk is outlined in a paper by Vollath and Knight “Liquid crystalline spinning of spider silk”, Nature, 410, March 29, 2001, pages 541-8. The authors point out that spiders and silkworms should learn wisdom from how to store their protein-doped molecules and extrude them as strong threads. In this paper, spider's natural spinning mechanism scrutinizes the evidence that the spinning dope passes through the spinning tube with the addition of hydrogen and potassium ions and removal of sodium ions.

  A paper by Knight and Volllath "Biological Liquid Crystal Elastomers", Trans. Phil. R. Soc. B. 357, 155-163 (2002), the authors indicate that the spidroin and fibroin protein aggregates form tough threads that are the main structural proteins of silk, namely silkworm fibroin and spiders. Indicates that it depends on the fact that it is an ABAB-type amphiphilic repetitive block copolymer where A represents a hydrophobic block and B represents a less hydrophobic block. .

  Spidroin and fibron are found in two states. The first state is a safe storage state, in which a very long protein chain is folded into a short, fairly dense rod-like molecule. The fibroin or spidroin in this first state has mainly a random coil and / or a helical secondary structure. The second state is a solid state mainly having a β crystalline secondary structure. This second state is a nanofibril composite comprising a highly filled portion of very long nanofibrils with a diameter of about 5 nm. Nanofibrils are oriented substantially parallel to the long axis of the tough yarn and are believed to contain all or most of the fine β crystals. Fine β crystals have a width of about 5 nm and are arranged substantially parallel to the long axis of the nanofibril. A small amount of less crystalline, less disordered material is believed to form a matrix between the nanofibrils.

  The first storage state is metastable. This conversion to the second nanofibrillar state is thought to involve both molecular aggregation and change in secondary structure conformation. Conformational changes are believed to occur in the most hydrophobic of the block species transitioning from the “random coil” / α helical structure to the β crystalline structure of the repeating block copolymer. Aggregation and conformational changes that form tough threads in vivo include pH reduction, addition of potassium ions to proteins, removal of sodium ions from proteins, loss of water, and threads formed It is considered that at least five factors for applying mechanical force to the slab are involved. Furthermore, this conversion can be facilitated by the secretion of polyols and surfactants. Fine longitudinal ridges with low surface energy at the end of the spider tube lining may help to facilitate the conversion of protein dope into solid yarn. Once short nanofibrils are formed, they may become seeds and initiate subsequent protein aggregation and conformational changes, thereby increasing the overall rate of change.

  The first storage state solution of fibroin or spidroin spontaneously transitions to an insoluble β crystalline state when left untreated to form an insoluble floc or gel that cannot be extruded or spun into useful yarns. To do. This transition usually takes 1-3 days to complete, but can be significantly faster. This becomes a problem when extruding or spinning yarns from silk proteins. Proteolysis by bacteria may play a role in this transfer in vitro. This degradation cleaves the protein into short peptides, making conformational changes or crystallization easier. Alternatively, proteolysis may facilitate this transfer by removing protection domains that prevent aggregation or conformational changes. The transition of a natural fibroin or spidroin solution to an insoluble form can also be facilitated by seeding material that has already transitioned to the β state. A transition to the β-crystalline state can also be obtained by freezing or mechanically shearing the natural solution. A regenerated fibroin or spidroin solution in which silks are dissolved in a chaotropic agent such as lithium bromide, lithium thiocyanate, sodium thiocyanate, calcium chloride, or calcium nitrate is also gradually removed by dialysis. Form. This is also a problem when trying to spin or extrude material from recycled silk.

  The first storage state has been found in the posterior and middle parts of silkworm glands and the similar A region of spiders. In these areas of each gland, proteins are stored at significantly higher concentrations (20-40% w / v). In spiders, the protein is thought to be stored as a very high viscosity liquid crystal sol throughout most of the first, second, and third limbs of the silk gland duct. In silkworms, the protein is stored as a gel in the posterior and middle part of the freshly molted old silkworm silk gland, but the sol is inside the tube (in front of the anterior silk gland) just before the protein is spun. To metastasize. The gel-to-sol transition then propagates backward in the secretory pathway, allowing forward flow of material stored in the middle and rear, which is stretched into filaments at the front (end) of the tube become able to.

  It is an object of the present invention to provide a protein storage device and method.

  These and other objects of the present invention are solved by providing a protein storage device comprising a first compartment for storing protein and a second compartment for storing alkaline buffer. In a preferred embodiment of the invention, the second compartment contains an alkaline buffer containing calcium chloride. The second compartment is in fluid communication with the first compartment (ie, liquid or gas). Therefore, the protein stored in the first compartment is in an alkaline state containing calcium ions. Under these conditions, the protein is significantly stabilized compared to an untreated spidroin or fibroin solution taken directly from the gland of the organism or prepared by dissolving spider or silkworm silk in a chaotropic agent. Therefore, degradation or premature formation of the β-sheet type, ie the formation of the β state of the protein, is significantly 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 these Selected from the group of alkalis consisting of a mixture.

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

  Furthermore, in one embodiment, 100-700 mM calcium ions are added to the alkaline buffer, preferably as chloride. Under these circumstances, the alkaline buffer may not contain carbonate or phosphate ions.

  When calcium ions are added to an alkaline buffer, they gel a highly concentrated solution of silk protein or the like. The gel thus produced is significantly more stable than the silk protein sol solution. The gel state thus induced can be converted back to a sol state before the silk is extruded, spun, or otherwise formed. This can be achieved by dialysis against a dialysate containing 20-100 mM ethylenediaminetetraacetic acid (EDTA) solution adjusted to pH 7.0-7.8 with ammonia or sodium hydroxide solution. This treatment removes calcium ions. Alternatively, calcium ions can be removed by dialysis against deionized water. In either case, polyethylene glycol or another water soluble polymer can be added to the dialysate to maintain or increase the protein concentration by reverse dialysis.

  In a preferred embodiment, at least a portion of the wall interior surface of the first storage compartment is formed of a low surface energy material such as polytetrafluoroethylene (PTFE), silane, nylon, polyethylene, or polycarbonate, or It has been applied. This also helps to prevent premature formation of the β-sheet form of the protein.

  In this device, the alkaline buffer is gaseous or solution. If alkaline buffer is used in gaseous form, it can be applied directly to one or more surfaces of the protein solution. Alternatively, it can diffuse into the protein solution through a porous or semi-permeable surface. When an alkaline buffer is used as a solution, it can be separated from the protein by a porous membrane or a semipermeable membrane. Alternatively, the buffer may act as a separate stream on the protein solution stream and then be directed away from the protein surface. If the alkaline buffer is gaseous, it can be diffused directly into the protein solution to make it alkaline. When alkaline buffer is separated from protein by a porous or semi-permeable membrane, both the buffer and any calcium ions that may be contained may diffuse through the membrane into the protein solution. it can.

  In another embodiment of the invention, the protein is mixed directly with the alkaline solution. This helps keep the protein in a sol state. Calcium ions can be added directly to the alkaline solution.

  In a preferred embodiment of the invention, sodium azide is added to the buffer such that the pH is above 7.4 and the final concentration is above 0.0001M.

  The stored protein can be, for example, a natural protein obtained by animal demolition, a recombinant protein obtained by genetic engineering, or a regenerated silk solution in which silkworms or silk fibers are dissolved in a chaotropic agent (which is later removed by dialysis). Or a mixture of the above proteins or protein analogs. The present invention has been found to be useful for the storage of fibroin or spidroin protein, or equivalents thereof, or a regenerated solution of fibroin and / or spidroin.

  More generally, the stored protein is a repetitive amphiphilic block copolymer protein or protein analog. Both contain charged groups and are prepared by chemical synthesis or genetic engineering.

  The object of the present invention is also solved by providing a protein storage method comprising a first step of placing a protein in a first storage compartment. In the second step, the protein is exposed to an alkaline buffer (preferably containing calcium ions and sodium azide) for a period of time. In the third step, the protein is retained in the alkaline environment of the first storage compartment.

  The present invention provides long-term storage solutions of silk proteins or analogs thereof and regenerated silk solutions.

  FIG. 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 that contains protein 20. The protein storage compartment 30 is connected to the alkali storage compartment 40 by a pipe or tube 35. An alkaline solution 50 is stored in the alkaline storage compartment 40. Preferably, part or substantially all of the inner wall of the protein storage compartment 30 is made from or coated with a low surface energy material such as polytetrafluoroethylene (PTFE), polyethylene, or polycarbonate.

  The protein 20 in the protein storage compartment 30 can be, for example, a natural protein obtained by animal demolition. Examples of such natural proteins include, but are not limited to, spidroin protein obtained from the major ampule of the Nephila spider, or fibroin protein obtained from Brombyx mori or other silkworm species. Is mentioned. The present invention can also be applied to the equivalents of these proteins or recombinant proteins obtained by genetic engineering. Furthermore, the present invention can also be applied to a regenerated silk solution in which silk yarns are dissolved in a solution containing a chaotropic agent. More generally, it is believed that the present invention is a repeating amphiphilic block copolymer and can be applied to the storage of any protein or protein analog containing a charged group. However, these materials do not indicate the limits of the present invention.

  In the alkaline storage compartment 40, several different types of alkaline solutions can be used. For example, the alkali can be ammonia / acetic acid, ammonium acetate, ammonium / formic acid, or ammonium formate. These buffers are volatile and form an alkaline atmosphere within 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 present invention, the alkaline buffer is selected from the group of alkalis consisting of ammonia, ammonium acetate, ammonium formate, and ammonium citrate buffer. Also suitable are potassium phosphate and potassium carbonate. In a preferred embodiment of the invention, the alkaline buffer contains 100-700 mM calcium ions. Preferably it is added in the form of calcium chloride.

  In a preferred embodiment, in addition to the alkaline buffer, further sodium azide is added to the protein 20 in the first storage compartment. In another embodiment of the invention, phenylthiourea, sodium cyanide or potassium cyanide is added to protein 20.

  In another embodiment of the invention shown in FIG. 2, the protein storage compartment 30 is separated from the alkaline storage compartment 40 by a semi-permeable or porous membrane 60. The semipermeable membrane or porous membrane 60 can permeate ions and 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 to alkaline buffer up to 70% w / v to remove water from the protein solution in the protein storage compartment by reverse dialysis. In these environments, the molecular weight of the polyethylene glycol used must be higher than the fractionated molecular weight of the semipermeable membrane. Other polymers can also be used in this way provided they are water soluble and large enough not to pass through the dialysis membrane.

  Thus, premature aggregation of the protein 20 can be prevented by treating in the first compartment 30 for a time as short as 1 minute, but preferably for at least 20 minutes. This time includes the amount of protein 20, its initial pH value, temperature, the surface area of protein 20 exposed to alkaline buffer, the distance it takes for the alkaline buffer to diffuse and reach all proteins 20, and protein 20 and It depends on the buffer capacity of the alkaline buffer.

  In a preferred embodiment of the invention, protein 20 is mixed with an alkaline buffer such as ammonium acetate or ammonium formate having a pH above 7.4 and a concentration of 0.1 M or higher. In a preferred embodiment of the invention, the alkaline buffer comprises 100-700 mM calcium ions and greater than 0.0001 M sodium azide.

  The suitability of various alkaline solutions that increase the stability of protein 20 was evaluated in two ways. First, a small drop of high concentration protein solution was dialyzed against various alkaline buffers for up to 4 weeks, and the gel or sol state of the protein solution was measured at intervals. A representative result is shown in FIG. Secondly, a small amount of protein solution was stored in contact with alkaline buffer at various times and then validated by attempting to spin fibers from this solution. In this second method, a general type biomimetic spinning device described in WO 01/38614 was used. The teachings of this publication are incorporated herein by reference. According to the test using this biomimetic spinning device, the protein 20 stored in contact with the steam-saturated atmosphere from the 1M ammonium hydroxide solution is still spun after 1 week. It was found that can be formed. The addition of sodium azide to protein 20 can increase this storage time.

  This apparatus and method can be used not only for storage of natural and recombinant proteins, but also for storage of regenerated solutions of fibroin and spidroin. These solutions have been prepared by dissolving silks made from these proteins in a suitable solution containing a chaotropic agent. An example of such a chaotropic agent is a 50:50 v / v mixture of saturated lithium bromide and absolute ethanol.

In the following examples, a protein solution containing silkworm protein obtained from Bombyx mori silk gland, a regenerated Bombyx mori fibroin solution, or a high concentration spirodoin solution obtained from a Nephila spider's main limb sac gland , Extended storage time.

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

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

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

  The resulting gel can be reconverted to a sol by dialysis against distilled water or 100 mM aqueous ethylenediaminetetraacetic acid before the target is extruded or otherwise shaped.

Demonstration that fibroin is stored as a gel before spinning in Bombyx mori silkworms Fibroin status in the posterior, middle, and posterior (gland) silk glands of Bombyx mori silkworms Stages were evaluated by dissection under a binocular microscope. Glands that were quickly removed from silkworms were transferred to silkworm Ringer's solution (pH 7.8) and observed. The material in the lumen of the gland is initially present as a sol at all stages until the end of 2-3 days before the start of spun spinning, and then until the initial stage of sputum spinning, and during this spinning, It appears to be stored as a gel. By cutting the tube (front part of the front part) in cross-section and observing whether the fibroin dope flows out, it was demonstrated that fibroin was present as a sol in this part immediately before and during spinning. Once spinning has begun, it appears that the sol formation gradually propagates back through the silk dope as the size of the silk gland during spinning decreases. This demonstrates that gel formation was essential for the stable storage of silk and that sol formation was essential for the silk dope to flow through the tube and be spun.

Effect of calcium ion addition and removal on the sol / gel state of stored natural fibroin 10 mg sodium azide and 100 mM EDTA adjusted to pH 7.8 with concentrated ammonia solution or acetic acid in the central gland content of the gland A solution of fibroin dope was obtained by dilution with 1 ml of 100 mM ammonium acetate buffer. This solution could be rapidly gelled by adding 1 volume of 1 M calcium chloride to 1 volume of this fibroin dope solution or by dialysis against a 500 nM aqueous calcium chloride solution. The protein is solated by dialysis against distilled water or 100 mM ammonium acetate buffer (pH 7.8) or more rapidly by dialysis against 100 mM ammonium acetate buffer (pH 7.8) containing 500 mM EDTA. I was able to return to. This indicates that the addition of calcium ions can induce a sol / gel transition and that the gel can be returned to the sol state again by removing calcium ions. This calcium-induced transition was reversible even after storage in the gel state for days or practically weeks.

Effect of calcium ion addition or removal on storage of natural fibroin solution A concentrated fibroin solution was prepared and gelled by adding 1 M calcium chloride as described in Example 3. The length of time that the gel could be stably stored in a state that could be converted to a sol by removal of calcium ions was tested. To do this, samples of gels placed at 4 ° C. for various lengths of time were taken and immediately dialyzed against 100 mM ammonium acetate buffer (pH 7.8) containing 500 mM EDTA. These observations show that the calcium-fibroin gel can be safely transferred to the sol after storage for at least 4 weeks. In contrast, a sol formed in the absence of calcium ions can only be stored at 4 ° C. for 5-8 days, and then forms a polymeric material that cannot be converted to a sol even when EDTA is added. did. Similar results were obtained by dissolving degammed silk in 9.6 M lithium bromide and placing the resulting solution in a low molecular weight cut dialysis bag (Spectropoor 10 KDa), 100 mM lithium, 20% w / v polyethylene glycol. It was also obtained using a regenerated silk solution prepared by dialysis against a solution containing (nominal molecular weight 15 KDa). However, the gel prepared by adding calcium ions to the solution as described above was significantly less rigid than that obtained by gelling natural fibroin.

1 shows a schematic diagram of a first embodiment of an apparatus suitable for protein storage. FIG. Figure 2 shows a schematic view of a second embodiment of an apparatus suitable for protein storage. FIG. 3 shows the results of the effects of alkaline buffer and sodium azide on the number of storages of high concentration natural fibroin solution.

Claims (32)

  A protein (20) storage device comprising a first compartment (30) for storing protein (20) and a second compartment (40) for storing alkaline buffer (50), wherein said second compartment A storage device wherein the compartment (40) is in fluid communication with the first compartment (30).   The apparatus according to claim 1, wherein the alkaline buffer (50) contains calcium ions.   The alkaline buffer (50) is an alkaline buffer comprising an ammonia solution, ammonium acetate, ammonium formate, Tris / HCl, HEPES, PIPES, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, or mixtures thereof. The device according to claim 1 or 2, selected from the group.   The device according to any one of the preceding claims, wherein the alkaline buffer (50) contains 50-700 mM calcium ions.   A device according to any one of the preceding claims, wherein the alkaline buffer (50) also contains sodium azide.   The device according to any one of claims 1 to 5, wherein at least a part of the inner wall surface of the first compartment (30) is made of or coated with a material having a low surface energy. .   The device according to any one of the preceding claims, wherein the alkaline buffer (50) is gaseous.   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 according to any one of the preceding claims, wherein the protein (20) is mixed with an alkaline solution.   10. Apparatus according to any one of the preceding claims, wherein the protein (20) is mixed with at least one alkaline buffer salt.   The apparatus of claim 10, wherein the at least one alkaline buffer salt also contains sodium azide, phenylthiourea, sodium cyanide, or potassium cyanide.   The alkaline solution has a pH higher than 7.4, 0.1M ammonium hydroxide, ammonium acetate, ammonium formate, ammonium citrate, Tris / HCl, PIPES, HEPES, sodium carbonate, potassium carbonate, sodium phosphate, phosphorus 12. The device according to any one of claims 9 to 11, which is a potassium acid buffer or a mixture of these buffers.   The device according to any one of the preceding claims, wherein the pH of the alkaline buffer (50) is higher than 7.4.   The device according to any one of claims 1 to 13, wherein the concentration of the alkaline buffer (50) or the mixed buffer is 0.025M or more.   15. Apparatus according to any one of the preceding claims, wherein the protein (20) is a natural protein, a regenerated protein or a recombinant protein, a mixture of natural proteins, a mixture of regenerated proteins or a mixture of recombinant proteins.   16. A device according to any one of the preceding claims, wherein the protein (20) is fibroin or spidroin, or an equivalent thereof.   17. The protein (20) according to any one of claims 1 to 16, wherein the protein (20) is a repetitive amphiphilic block copolymer protein or protein analogue comprising a charged group and prepared by chemical synthesis or genetic engineering. The device described in 1. Placing the protein in the first storage compartment (30);
A second step of exposing said protein (20) to alkaline buffer (50);
A protein (20) storage method comprising: a third step of maintaining the protein (20) in an alkaline atmosphere in the first storage compartment (30).   19. The method of claim 18, wherein the time for retaining the protein (20) in the first storage compartment (30) is at least 1 minute.   20. A method according to claim 18 or 19, wherein the alkaline buffer (50) contains calcium ions.   The alkaline buffer (50) is an ammonia solution, ammonium acetate, ammonium formate, ammonium citrate, Tris / HCl, HEPES, PIPES, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, or a mixture of these buffers. 21. A method according to any one of claims 18 to 20 selected from the group of alkaline buffers consisting of   The method according to any one of claims 18 to 21, wherein the alkaline buffer contains 50 to 700 mM of calcium ions.   23. A method according to any one of claims 18 to 22, wherein the alkaline buffer (50) is gaseous.   24. A method according to any one of claims 18 to 23, wherein at least one alkaline buffer salt is added to the protein (20).   25. The method of claim 24, wherein the alkaline buffer salt also contains sodium azide, phenylthiourea, sodium cyanide, or potassium cyanide.   26. A method according to any one of claims 18 to 25, wherein the alkaline buffer (50) is separated from the protein by a dialysis membrane (60).   27. A method according to any one of claims 18 to 26, wherein the pH of the alkaline buffer (50) is higher than 7.4.   28. A method according to any one of claims 18 to 27, wherein the protein (20) is mixed with an alkaline solution (50) prior to storage.   29. A method according to any one of claims 18 to 28, wherein the concentration of the alkaline buffer (50) or the mixed buffer is 0.025M or more.   30. A method according to any one of claims 18 to 29, wherein the protein (20) is a natural protein, a regenerated protein or a recombinant protein, a mixture of natural proteins, a mixture of regenerated proteins or a mixture of recombinant proteins.   31. A method according to any one of claims 18 to 30, wherein the protein (20) is fibroin or spidroin, or an equivalent thereof. 32. Any one of claims 18-31, wherein the protein (20) is a repetitive amphiphilic block copolymer protein or protein analogue comprising a charged group and prepared by chemical synthesis or genetic engineering. The method described in 1.

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