EA008044B1 - 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

The scope of the invention

The present invention relates to a storage device and method for storing proteins.

Prior art

One of the problems associated with long-term storage of proteins is that they lose their biological properties over time, as the molecule degrades. Various methods for storing proteins have already been proposed to solve this problem.

For example, Japanese Patent Application DR-A-63-092671 (Kaieo) proposes a method for storing proteins, according to which fibroin or collagen is dissolved in an aqueous solution. A hydrolysis enzyme is added to the solution, followed by a chelating additive. The pH of the mixture is set in the range from 4.5 to 7.5 and the solution is passed through a filter having pores of about 1 μm or less. Finally, the solution is dried to obtain a polymer with an average degree of polymerization in the range of 200-600.

However, it would be desirable to store the protein in its natural state without treatment with an enzyme or without treatment with a chelate additive.

In nature, there are various ways of storing proteins. Natural silk consists of thin, shiny yarns produced by the mulberry silkworm Vulbop top and other invertebrates from the accumulated protein dope or from raw materials. These silk threads are often superior to synthetic polymers that are currently used for the manufacture of materials, and their properties mainly remain unchanged during prolonged storage of protein dope in the glands of the body. For example, the tensile strength and toughness of dragline silks produced by some spiders may exceed the similar characteristics of the Keulier ™ (Kevlar) material, which is the most durable fiber currently manufactured by the industry. Such dragline silk also has high heat resistance. Many silks are also biodegradable and cannot withstand environmental influences. They are recoverable and can be obtained using a highly efficient process at low pressure and low temperature, using only water as a solvent. The natural spinning process is remarkable in that the aqueous protein solution turns into a strong and insoluble material. The method by which spiders and silkworms produce silk threads is described in the article Yb. 410, 29 Magsy 2001, Radek 541-8. The authors pay attention to how spiders and silkworms store the molecules of the protein dope and extrude strong strands from them. The article analyzes evidence that the natural mechanism of spinning by spiders involves the addition of hydrogen ions and potassium ions and the removal of sodium ions when the spinning solution passes through the spinning path (channel).

In the article Kp1dN1 apb Uo11ga111 Vu1odyu1 Ys. | Shb Sguk1a1 E1a81oteg (Biological liquid crystal elastomers), Tgapk. PY1. I. 8os. C. 357, 155-163 (2002), the authors indicate that the combination of the spidroin and fibroin proteins forms strong strands, since the main structural proteins of silk, silkworm fibroins and spider speedroins are amphiphilic repeating block copolymers of the ABAB type, where A represents a hydrophobic block, and B is a less hydrophobic block.

Spidroin and fibroin have two conditions. The first state is a state of safe storage in which it is believed that extremely long protein chains coagulate into short, fairly compact molecules in the form of rods. Proteins of fibroin or spidroin in this first state have a predominantly random helical and / or helical secondary structure. The second state is a solid state with a predominantly beta crystalline secondary structure. In this second state, a nanofibrillar composite is formed that contains a well-packed fraction of very long nanofibrils (thin fibers) having a diameter of about 5 nm. Nanofibrils are oriented mainly parallel to the long axis of the strong filament and probably contain all or most of the beta crystallites. Beta crystallites have a width of about 5 nm and are located mainly parallel to the long axis of the nanofibrils. It is believed that smaller amounts of less crystalline and more disordered material form a matrix between nanofibrils.

The first storage state is metastable. It is believed that its transformation into the second state of nanofibrils causes both aggregation of molecules and a change in the conformation of the secondary structure. It is believed that a change in conformation occurs in the most hydrophobic block types of the repeating block copolymer, which is converted from a random helical / alpha helical structure to a beta crystalline structure. It is believed that at least 5 factors are involved in the transition of aggregation and conformation, resulting in the formation of strong filaments, namely a decrease in pH; adding potassium ions to protein; removal of sodium ions from protein; water loss and application of mechanical stress to the resulting threads. In addition, the conversion (conversion) can be accelerated due to the secretion of fields and surfactants. Thin longitudinal protrusions with low surface energy in the department of the final part of the spinning path in spiders can

- 1 008044 to facilitate the conversion of the protein dope to a solid filament. After the formation of short nanofibrils, they can act as seeds, initiating further aggregation and changes in protein conformation, resulting in accelerated full rate of transition.

If you leave the solutions of fibroin or speedroin in the first storage state without treatment, then they spontaneously transform into an insoluble beta crystalline state, as a result of which insoluble flocculates (Pos5) or gels form prematurely, from which useful filaments cannot be extruded or extended. This conversion can be unusually fast and usually takes 1-3 days to complete. This creates a problem when extruding or pulling filaments from silk proteins. Bacterial proteolysis (protein cleavage) can take part in this ίη νίΐΓΟ transformation by cutting proteins into shorter peptides, which are more prone to conformational changes or crystallization. Alternatively, proteolysis may facilitate conversion by removing protective domains that inhibit aggregation or conformational change. The conversion of a natural solution of fibroin or spidroin into an insoluble form can also be accelerated by the introduction of seed using material that has already gone into a beta state. Freezing or mechanical cutting of natural solutions can also lead to conversion to beta crystalline state. Regenerated solutions of fibroin and speedroin prepared by dissolving silk threads in a release agent, such as lithium bromide, lithium thiocyanate, sodium thiocyanate, calcium chloride, calcium nitrate, also gradually undergo a similar formation of flocculates or gel when the release agent is removed by dialysis. This also creates a problem when it is desired to spin or extrude material from regenerated silks.

It was found that the first state of storage is maintained in the posterior and middle glands of silkworms and corresponds to the A zone for spiders. In these areas of the corresponding glands, the protein is stored at surprisingly high concentrations (20-40% by weight / volume). It is believed that spiders store the protein in the form of a liquid crystalline sol of high viscosity, which is stored in the first, second, and most of the third branch of the silk gland tract. Silkworms of the final age stage after a new molt store the protein in the form of a gel in the back and middle sections of the silk gland, however, it is converted to sol in the tract (in the back of the back section) shortly before spinning. After the gel is transformed into sol, it moves back down into the secretion tract and causes the material stored in the middle and rear sections to flow forward downward, so that it can be pulled into the thread in the front (distal) part of the tract.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a storage device and a method for storing proteins.

This and other objectives of the present invention have been achieved by providing a protein storage device that comprises a first protein storage chamber and a second alkaline buffer storage chamber. According to a preferred embodiment of the present invention, the second chamber contains an alkaline buffer solution containing calcium chloride. The second chamber is in fluid (i.e., liquid or gaseous) communication with the first chamber. Thus, the protein stored in the first chamber is in an alkaline state and contains calcium ions, and in this state it is much more stable than untreated solutions of spidroin or fibroin directly extracted from the glands of organisms or prepared by dissolving spider silk or silkworm in uncoupling agent. Due to this, the decay or premature formation of a beta leaf form significantly slows down, with the beta state of the protein.

According to a first embodiment of the present invention, the alkaline buffer solution is selected from the group of alkalis which includes ammonia, ammonium acetate, ammonium formate, ammonium citrate, Tgb / HCT HEPE8, ,8, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, and also mixtures thereof.

In one embodiment, sodium azide is introduced into the protein in addition to an alkaline buffer solution. Alternatively, phenylthiourea, sodium cyanide or potassium cyanide may be added.

In one embodiment, 100-700 mM calcium ions are added to the alkaline buffer solution, preferably in the form of chloride. Under such circumstances, the alkaline buffer solution may not contain carbonate or phosphate ions.

When calcium ions are added to the alkaline buffer solution, they cause the concentrated solution of silk proteins or their analogues to turn into a gel. The gel obtained in this way is much more stable than solutions of the sol state of silk proteins. Forced (induced) in this way, the gel state can be converted back to the sol state before extrusion, spinning, or other shaping of silk. This can be achieved by dialysis using a dialysis solution containing 20-100 mm ethylene diamine tetraacetic acid solution (ΕΌΤΑ), adjusting the pH to 7.0-7.8 using an ammonia solution or a guide

- 2 008044 sodium oxide. This treatment removes calcium ions. Alternatively, calcium ions can be removed by dialysis using ion-free water. In either case, polyethylene glycol or another water-soluble polymer must be added to the dialysant to maintain or increase the protein concentration by reverse dialysis.

According to a preferred embodiment, at least part of the inner surface of the walls of the first storage chamber is formed or coated with a material with low surface energy, such as polytetrafluoroethylene (PTEE), silane, nylon, polyethylene or polycarbonate, which also helps to prevent premature beta sheet formation protein.

In the apparatus, the alkaline buffer solution has a gaseous form or a solution form. If it is used in gaseous form, it can flow directly to the surface or surfaces of the protein solution or can diffuse into them through a porous or semi-permeable surface. If an alkaline buffer solution is used, it can be separated from the protein using a porous or semi-permeable membrane, or a separate stream of the buffer solution can enter the stream of the protein solution and then be diverted from the surface of the protein solution. If the alkaline buffer solution is used in gaseous form, it can diffuse directly into the protein solution to make it alkaline. If the alkaline buffer solution is separated from the protein by a porous or semi-permeable membrane, then both the buffer solution and the calcium ions it may contain can diffuse through the membrane into the protein solution.

In accordance with another embodiment of the present invention, the protein is directly mixed with an alkaline solution, as this helps to keep the protein in a sol state. Calcium ions can be introduced directly into the alkaline solution.

In accordance with a preferred embodiment of the present invention, sodium azide is introduced into a buffer solution with a pH above 7.4 to obtain a final concentration above 0.0001 M.

The stored protein can be either a natural protein obtained, for example, by dismembering an animal, or a recombinant protein obtained by genetic engineering, or a regenerated silk solution prepared by dissolving a silkworm or silk fibers in a release agent, which is then removed using dialysis, or a mixture of the above proteins or protein analogues. The present invention may find use in the storage of fibroin or speedroin proteins, or their homologues or regenerated solutions of fibroin or speedroin.

Generally speaking, stored proteins are repeating amphiphilic block copolymer proteins or protein analogues that contain charged groups and which are prepared by chemical synthesis or genetic engineering.

The objective of the present invention was also solved by creating a method of storing protein, and this method includes a first step of placing the protein in the first storage chamber. During the second operation, the protein is exposed with an alkaline buffer solution (which mainly contains calcium ions and sodium azide) for a certain period of time. During the third operation, the protein is stored in an alkaline environment in the first storage chamber.

This allows you to create a solution for long-term storage of silk proteins or their analogues and regenerated silk solutions.

Brief Description of the Drawings

In FIG. 1 shows a diagram of a first embodiment of a device suitable for storing proteins.

In FIG. 2 shows a diagram of a second embodiment of a device suitable for storing proteins.

In FIG. Figure 3 shows the effects of alkaline buffer solution and sodium azide on the storage time of concentrated solutions of natural fibroin.

DETAILED DESCRIPTION OF THE INVENTION

In FIG. 1 shows a diagram of a first embodiment of a device 10 suitable for storing protein 20. The device 10 has a protein storage chamber 30 in which the protein 20 is placed. The protein storage chamber 30 is connected via a pipe or tube 35 to the alkali storage chamber 40. An alkaline solution 50 is stored in the alkali storage chamber 40. The protein storage chamber 30 advantageously has a portion of the inner walls or substantially all of the inner walls that are made of or coated with a material with low surface energy such as polytetrafluoroethylene (PTEE), polyethylene or polycarbonate .

The protein 20 in the protein storage chamber 30 may be a natural protein, which is obtained, for example, by dismembering an animal. Examples of such natural proteins include, but are not limited to, a speedroin-type protein obtained from the large ampullary (salivary) gland of spiders of the genus No. L1A or a fibroin-type protein obtained from the silkworm Vhoshuh shop or from other varieties of silkworms. The present invention is also applicable to homologues of these proteins or to recombinant proteins obtained by genetic engineering.

- 3 008044 RII. The present invention is also applicable to regenerated silk solutions prepared by dissolving silks in solutions that contain uncoupling agents. Generally speaking, it can be believed that the present invention can be used to store any proteins or protein analogues that are repeating amphiphilic block copolymers and which contain charged groups.

In the alkali storage chamber 40, a wide variety of types of alkaline solution can be used. For example, as an alkali, a mixture of ammonia with acetic acid, ammonium acetate, a mixture of ammonia with formic acid, or ammonium formate can be used. These buffer solutions are volatile and create an alkaline atmosphere in the protein storage chamber 30. Instead, TI8 / HC1, HEPE8, or ΡΙΡΕ8 can be used, however, these buffer solutions are not volatile. According to a first embodiment of the present invention, the alkaline buffer solution is selected from the group of alkalis which includes buffer solutions of ammonia, ammonium acetate, ammonium formate and ammonium citrate. Potassium phosphate and potassium carbonate may also be used. In accordance with a preferred embodiment of the present invention, the alkaline buffer solution contains 100-700 mm calcium ions, mainly in the form of calcium chloride.

In a preferred embodiment, sodium azide is also introduced into protein 20 in a first storage chamber in addition to an alkaline buffer solution. According to an alternative embodiment of the present invention, phenylthiourea, sodium cyanide or potassium cyanide are added to protein 20.

In accordance with another embodiment of the present invention shown in FIG. 2, the protein storage chamber 30 is separated from the alkali storage chamber 40 by a semi-permeable or porous membrane 60. The semi-permeable or porous membrane 60 allows ions to pass through to change the pH of the protein 20 stored in the protein storage chamber 30. In the case of using a semi-permeable membrane, polyethylene glycol can be introduced into the alkaline buffer solution at a concentration of up to 70% by weight / volume in order to remove water from the protein solution in the protein storage chamber using reverse dialysis. Under these circumstances, the molecular weight of the polyethylene glycol used must exceed the molecular weight of the cutoff of the semipermeable membrane. Other polymers can be used in this way, provided that they dissolve in water and are of sufficient size to prevent them from passing through the dialysis membrane.

Thus, protein 20 can be protected from premature coagulation by treatment in the first chamber 30 for a period of 1 minute, and preferably at least 20 minutes. This time period depends on the amount of protein 20, its initial pH, temperature, the surface area of protein 20 exposed to the alkaline buffer solution, the distance that the alkaline buffer solution must diffuse to reach all proteins 20, and the buffer capacity protein 20 and alkaline buffer solutions.

In accordance with a preferred embodiment of the present invention, protein 20 is mixed with an alkaline buffer solution, such as ammonium acetate or ammonium formate, having a pH above 7.4 and a concentration of 0.1 M or higher. According to a preferred embodiment of the present invention, the alkaline buffer solution contains from 100 to 700 mM calcium ions and an excess of 0.0001 M sodium azide.

The suitability of various alkaline solutions to increase the stability of protein 20 has been evaluated in two ways. First of all, small drops of concentrated protein solutions were dialyzed using various alkaline buffer solutions for up to four weeks, and the state of the gel or sol solution of the protein was determined at intervals. Typical results are shown in FIG. 3. Secondly, the validity of this approach was confirmed by an attempt to draw fibers from small volumes of a protein solution, after its storage for various time durations in contact with alkaline buffer solutions. In this case, a biomimetic spinning device was used as described in PCT publication νθ-Α-01/38614. Tests using a biomimetic spinning device showed that protein 20, which was stored in contact with an atmosphere saturated with 1M ammonium hydroxide solution pairs, can still be used to spin and produce a yarn, filament or fiber after one week of storage. The duration of storage time can be increased if sodium azide is added to protein 20.

The device and method can be used not only for storing natural and recombinant proteins, they can also be used for storing regenerated solutions of fibroin and speedroin, which were prepared by dissolving silks made from these proteins in an appropriate solution containing uncoupling (breaking the hydrogen bond ) agent. As one example of such a release agent, a 50:50 mixture by volume of saturated lithium bromide and absolute ethanol can be given.

Example 1. The increase in storage time.

- 4 008044

In the given example, the storage time of protein solutions that contain silkworm protein obtained from the silk glands of the silkworm Voshuh Shop, a regenerated solution of fibroin Woshuh Shop, or a concentrated solution of spidroin obtained from large ampullary glands of spiders No. pyla has been increased.

During the first operation, the protein solution is transferred into a dialysis bag (M ^ CO 5-8 kDa) and concentrated using reverse dialysis using a solution that contains 20% by weight / volume of REP (M \ U 15-20 kDa) and 0, 1 mm buffer ammonium acetate with a pH of 7.8, for 5 hours at 4 ° C.

The protein is gelled using a solution that contains a solution of 500 mM calcium chloride and 0.1 mM ammonium acetate buffer with a pH of 7.8, for 1 h at 4 ° C. Sodium azide can be added to the dialysant to a final concentration of 0.001 mM to prevent bacterial growth.

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

The resulting gel can be converted back to sol by using distilled water or an aqueous solution of 100 mM ethylenediaminetetraacetic acid, before extrusion or other formation of the object.

Example 2. Demonstration that fibroin is stored as a gel before spinning in the silkworm Voshuh top.

The state of fibroin in the posterior, middle parts and in the posterior (glandular) part of the anterior part of the silk gland of the silkworm Vulbopus was studied by dissecting the animal under a binocular microscope at different stages of the development of the silkworm. The glands that were quickly removed from the silkworm were transferred to the Qtidet silkworm solution (pH 7.8) for observation. The material in the lumen of the gland is initially present in the form of a sol at all stages, until the final age stage a few days before the cocoon spinning begins, after which this material is stored in gel form, before the cocoon spinning, and during the initial stage of cocoon spinning. By cutting the path (in the front of the front section) across and observing the outflow of the fibroin spinning solution, it was found that the fibroin is present as a sol in this section, immediately before and during spinning. It seems that after the beginning of spinning, the formation of sol gradually spreads back through the silk spinning solution as the size of the silk gland decreases during spinning. This shows that gel formation is essential for the safe storage of silk, and sol formation is essential for the flow of silk dope through the spinning path.

Example 3. The effect of adding and removing calcium ions on the sol / gel state of stored natural fibroin

Spinning solutions of fibroin were obtained by dissolving the combined content of the middle part of the gland using 1 ml of 100 mM ammonium acetate buffer containing 10 mM sodium azide and 100 mM, adjusting the pH to 7.8 using a concentrated solution of ammonia or acetic acid. These solutions can be quickly gelled by adding one volume of 1M calcium chloride to one volume of fibroin spinning solution, or by dialysis using aqueous solutions of 500 nM calcium chloride. The protein can be returned to the sol state by dialysis using distilled water or 100 mM ammonium acetate buffer solution (pH 7.8), or more quickly by dialysis using 100 mM ammonium acetate buffer solution (pH 7.8), containing 500 mM ΕΌΤΑ. This suggests that the sol / gel transition can be caused by the addition of calcium ions, and the gel can be returned to the sol state by removing calcium ions. It appears that the calcium-induced transition is reversible after storage for several days or even weeks in a gel state.

Example 4. The effect of adding or removing calcium ion on the storage of solutions of natural fibroin.

A concentrated solution of fibroin was obtained, which was gelled by adding 1M calcium chloride, as described in Example 3. The length of time during which the gel could be stored stably in a state that could be returned to the sol state by removing calcium ions was investigated. . To do this, gel samples were taken at 4 ° C for various time intervals and dialyzed using 100 mM ammonium acetate buffer solution (pH 7.8) containing 500 mM. These studies show that calcium-fibroin gel can be reliably converted to sol after storage for at least four weeks. In contrast, a sol formed in the absence of calcium ions can only be stored for 5-8 days at 4 ° C, after which a polymerizable material is formed from it, which cannot be converted to sol even with the addition of ΕΌΤΑ. Similar results were obtained using regenerated silk solution prepared by dissolving dehumidified silk in 9.6 M lithium bromide and dialysing the resulting solution in a dialysis bag with a low molecular weight cut (8 less than 1 torot of 10 kDa), using a solution that contains 100 mM lithium, 20 % by weight / volume of polyethylene glycol (nominal molecular weight 15 kDa). However, the gel obtained by adding, as before, calcium ions to this solution was significantly less rigid than the gel obtained from natural fibroin.

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Claims (32)

CLAIM 1. A device for storing protein, which contains a first chamber for storing protein and a second chamber for storing alkaline buffer solution, and the second chamber has fluid communication with the first chamber. 2. The device according to claim 1, in which the alkaline buffer solution contains calcium ions. 3. The device according to claim 1 or 2, in which the alkaline buffer solution is selected from the group consisting of solutions of ammonia, ammonium acetate, ammonium formate, 1P8 / HC1. HEPE8, P1RE8, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, as well as mixtures thereof. 4. The device according to any one of claims 1 to 3, in which the alkaline buffer solution contains 50-700 mm calcium ions. 5. The device according to any one of claims 1 to 4, in which the alkaline buffer solution also contains sodium azide. 6. The device according to any one of claims 1 to 5, in which at least a portion of the surface of the inner walls of the first chamber is formed or coated with a material with low surface energy. 7. The device according to any one of claims 1 to 6, in which the alkaline buffer solution is in gaseous form. 8. The device according to any one of claims 1 to 6, in which the alkaline buffer solution is separated from the protein by a dialysis membrane. 9. The device according to any one of claims 1 to 8, in which the protein is mixed with an alkaline solution. 10. The device according to any one of claims 1 to 9, in which the protein is mixed with at least one alkaline buffer solution of salt or salts. 11. The device according to claim 10, in which at least one alkaline buffer solution of salt or salts also contains sodium azide, phenylthiourea, sodium cyanide or potassium cyanide. 12. The device according to any one of claims 9 to 11, wherein the alkaline solution is a buffer solution of 0.1 M ammonium hydroxide, ammonium acetate, ammonium formate, ammonium citrate, 1P8 / HC1. ΡΙΡΕ8, HEPE8, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, or a mixture of these buffer solutions, with a pH above 7.4. 13. The device according to any one of claims 1 to 12, in which the pH of the alkaline buffer solution exceeds 7.4. 14. The device according to any one of claims 1 to 13, in which the concentration of the alkaline buffer solution or the combined buffer solutions is equal to or greater than 0.025M. 15. The device according to any one of claims 1 to 14, in which the protein 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, in which the protein is fibroin, or speedroin, or their homologue. 17. The device according to any one of claims 1 to 16, in which the proteins are repeating amphiphilic block copolymer proteins or analogs of proteins that contain charged groups and which are prepared using chemical synthesis or genetic engineering. 18. A method of storing protein, comprising placing the protein in a first storage chamber; exposure to the protein with an alkaline buffer solution and preservation of the protein in an alkaline medium in the first storage chamber. 19. The method according to p, in which the period of time the protein is stored in the first storage chamber is at least one minute. 20. The method according to p. 18 or 19, in which the alkaline buffer solution contains calcium ions. 21. The method according to any one of claims 18 to 20, wherein the alkaline buffer solution is selected from the group consisting of solutions of ammonia, ammonium acetate, ammonium formate, ammonium citrate, TI8 / HC1, HEPE8, P1PE8, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, as well as a mixture of these buffer solutions. 22. The method according to any one of claims 18 to 21, wherein the alkaline buffer solution contains 50-700 mM calcium ions. 23. The method according to any one of claims 18 to 22, wherein the alkaline buffer solution is in gaseous form. 24. The method according to any one of claims 18 to 23, wherein at least one alkaline salt buffer solution is added to the protein. 25. The method according to paragraph 24, in which the alkaline buffer solution of salts also contains sodium azide, phenylthiourea, sodium cyanide or potassium cyanide. 26. The method according to any one of claims 18 to 25, wherein the alkaline buffer solution is separated from the protein using a dialysis membrane. 27. The method according to any one of paragraphs.18-26, in which the pH of the alkaline buffer solution exceeds 7.4. - 6 008044 28. The method according to any one of claims 18 to 27, wherein the protein is mixed with an alkaline solution before storage. 29. The method according to any one of paragraphs. 18-28, in which the concentration of alkaline buffer solution or pooled buffer solutions is equal to or greater than 0.025M. 30. The method according to any one of claims 18 to 29, wherein the protein 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 according to any one of claims 18-30, wherein the protein is fibroin, or speedroin, or a homologue thereof. 32. The method according to any one of claims 18-31, wherein the proteins are repeating amphiphilic block copolymer proteins or protein analogs that contain charged groups and which are prepared by chemical synthesis or genetic engineering.