Preservation method of regenerated silk protein aqueous solution

Abstract

The invention discloses a preservation method of a regenerated silk protein aqueous solution. The method comprises the following steps: adding a cryoprotectant into a freshly prepared regenerated silk protein aqueous solution, pre-freezing to a final temperature at a temperature lower than-40 DEG C, storing for a long time at the final temperature, and thawing and shaking uniformly during use, the concentration of the regenerated silk protein in the regenerated silk protein aqueous solution is 0.1-20% (w / w); the addition amount of the cryoprotectant is 0.5-10% (w / w), and the selected cryoprotectant is a saccharide or a polyhydric alcohol. The fibroin aqueous solution which can be kept for a long time is obtained by quickly freezing the fibroin aqueous solution with a proper concentration to be below the glass transition temperature and adding a proper anti-freezing agent to limit free movement of a protein molecular chain; the problems that a conventional silk protein storage mode is short in storage time, and the silk protein cannot be used after denaturation are solved. The method is simple, high in generalizability and good in repeatability.

Description

Technical Field

[0001] The present invention relates to a method for preserving an aqueous solution of regenerated silk protein, belonging to the technical field of silk protein solution preservation. Background Art

[0002] Silk fibroin has broad application prospects in the biomedical field due to its rich source, excellent mechanical properties, controllable biodegradability, and good biocompatibility, such as nerve conduit membranes, hydrogels, porous scaffolds, bone repair materials, etc. The research on silk fibroin-based biomaterials has always been a hot topic in the field of biological tissue materials.

[0003] The basis for silk fibroin materials to maintain excellent properties is that the raw material of the aqueous solution of silk fibroin can maintain its natural aggregated structure. In some previous studies, the preparation of silk fibroin solution usually involved boiling degumming with an aqueous solution of sodium carbonate / sodium bicarbonate, and further drying to obtain degummed silk fibers. Usually, lithium bromide solution was used to dissolve the degummed silk fibers, and deionized water dialysis was used to remove lithium bromide to obtain a regenerated silk protein aqueous solution, which was concentrated to the target concentration with polyethylene glycol and then stored in a 4°C refrigerator for standby. However, the molecular structure of the regenerated silk protein aqueous solution is also unstable when stored in a 4°C refrigerator, and it is extremely easy to spontaneously transform from an α-helix to a β-sheet structure. The molecules continuously undergo structural changes, which will directly affect the performance of the subsequent prepared materials. Over time, the regenerated silk protein aqueous solution turns into a jelly-like solid and has no utilization value, and this process may occur within 1-2 weeks. Moreover, there are differences in the protein quality attributes of each batch of freshly prepared regenerated silk protein aqueous solutions, and the molecular structure states at each time point during the storage of the regenerated silk protein solution are also different, which poses a major challenge to the structural consistency of raw materials required for experimental research or commercial material preparation. How to obtain a silk protein solution that maintains the natural aggregated structure is the key to researching or developing silk fibroin-based materials.

[0004] There is literature research that the recombinant silk protein aqueous solution is placed in a container and dehydrated with a freeze dryer to obtain recombinant silk protein powder. This method can be stored for a long time. However, when in use, hexafluoroisopropanol (HFIP) is needed to dissolve the silk protein powder for subsequent material preparation steps, the process is complex, the cycle is long, and the organic solvent is not suitable for biocompatible materials. There is also an invention patent (patent application number: 201410118465.7) that freeze-dries silk fibroin to obtain powder, but the success of the low-temperature freeze-drying technology also depends on freeze-drying parameters, the selection of freeze-drying protectants, the control of the freeze-dryer shelf temperature, etc. Otherwise, the freeze-dried product may not be completely redissolved, or may not maintain the aggregated structure of silk fibroin itself, thus affecting the comprehensive performance of the material. Therefore, there is an urgent need to find a simple and general method to preserve the regenerated silk protein aqueous solution to overcome the defects of structural changes or denaturation easily caused by storage at 4°C currently. Summary of the Invention

[0005] The object of the present invention is to provide a method for preserving a regenerated silk fibroin aqueous solution, maintaining the stable natural aggregated structure of silk fibroin, so as to solve the problems of poor consistency and unstable structure of silk fibroin materials.

[0006] To achieve the above object, the present invention provides a method for preserving a regenerated silk fibroin aqueous solution, adding a cryoprotectant to the freshly prepared regenerated silk fibroin aqueous solution, pre-freezing it to a final temperature at a temperature below -40 °C, and storing it for a long time at this final temperature. Just thaw and shake well before use.

[0007] Preferably, the preparation steps of the regenerated silk fibroin aqueous solution include: 1) degumming, 2) washing and drying, 3) dissolving, 4) dialysis, 5) concentration.

[0008] Preferably, the concentration of the regenerated silk fibroin aqueous solution is 0.1-20% (w / w).

[0009] Preferably, the addition amount of the cryoprotectant is 0-10% (w / w); the cryoprotectant is sugars (such as sucrose, trehalose, etc.) and / or polyols (such as glycerol, etc.).

[0010] Preferably, the temperature condition for pre-freezing is -176 to -40 °C;

[0011] Preferably, the pre-freezing time is 0.5 h to 72 h;

[0012] Preferably, the temperature condition for long-term storage is -80 to -20 °C.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) By quickly freezing an aqueous solution of silk fibroin with an appropriate concentration below the glass transition temperature and adding a suitable antifreeze, the present invention restricts the free movement of protein molecular chains, obtaining an aqueous solution of silk fibroin that can be stored for a long time; solving the problem that the previous silk fibroin preservation method has a short storage time and cannot be used once it is denatured; this method is simple, highly popularizable, and has good repeatability;

[0015] (2) The preservation method of the present invention does not require freeze-drying equipment and waiting for the drying time. Only a common laboratory low-temperature freezer can be used to store the recombinant silk fibroin for more than 3 months, which can meet the experimental cycles of various researches, release resources and manpower, and save experimental costs; when using the frozen silk fibroin solution, no extra steps are required and it can be used after thawing. Description of the Drawings

[0016] Figure 1 It is the compression curve of the frozen regenerated silk fibroin-based hydrogel.

[0017] Figure 2 It is the compression curve of the freeze-dried regenerated silk fibroin-based hydrogel. Specific Embodiments

[0018] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0019] Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0020] Unless otherwise specified, the percentages in the following embodiments are all mass percentages.

[0021] The preparation method of the silk fibroin aqueous solution in the following embodiments is as follows:

[0022] Step 1): Immerse natural mulberry cocoons in a 5 wt% sodium carbonate solution and boil for 30 min, repeat twice for degumming, and then dry to obtain degummed silk.

[0023] Step 2): Dissolve the degummed silk obtained in Step 1) in a 9.7 mol / L lithium bromide solution at a mass ratio of 1:10 in a 60 °C water bath and heat for 1 hour.

[0024] Step 3): After cooling the regenerated silk fibroin solution obtained in Step 2) to room temperature (25 °C), place it in a dialysis bag with a molecular weight cut-off of 14,000 and dialyze in deionized water for 72 hours, replacing the deionized water every 8 hours.

[0025] Step 4): After dialysis in Step 3), place it in a 30 wt% polyethylene glycol aqueous solution with a molecular weight of 9,000 - 10,000 and dialyze for 24 hours for concentration, and prepare the obtained regenerated silk fibroin stock solution into a regenerated silk fibroin (RSF) aqueous solution with the required concentration.

[0026] Example 1:

[0027] A method for preserving a regenerated silk fibroin aqueous solution, the method comprising the following steps:

[0028] After obtaining a silk fibroin aqueous solution with a concentration of 5 wt%, the silk fibroin aqueous solution was dispensed into cryotube containers and then pre-frozen in a -80°C refrigerator for more than 48 h, and stored in the -80°C refrigerator for a long time. When needed, an appropriate amount of the frozen silk fibroin aqueous solution was taken out and thawed at room temperature, and it could be directly used for material preparation and other experiments. The experimental results showed that the storage time of the silk fibroin aqueous solution stored by freezing could be up to more than 3 months, which was more than 3 times the storage period under a 4°C refrigerator. The silk fibroin aqueous solution stored at low temperature for 90 days was used to prepare a silk fibroin hydrogel by enzyme-catalyzed crosslinking with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), and there was no significant difference in the compressive modulus of the hydrogel prepared from the silk fibroin aqueous solution and the fresh silk fibroin aqueous solution, as Figure 1 shown.

[0029] Example 2:

[0030] A method for storing a regenerated silk fibroin aqueous solution, the method comprising the following steps:

[0031] After obtaining a silk fibroin aqueous solution with a concentration of 10 wt%, sucrose was added to make its final concentration 2 wt%. The silk fibroin aqueous solution was dispensed into cryotube containers and then pre-frozen in a -80°C refrigerator for more than 72 h, and stored in the -80°C refrigerator for a long time. When needed, an appropriate amount of the frozen silk fibroin aqueous solution was taken out and thawed at room temperature, and it could be directly used for material preparation and other experiments. The experimental results showed that there was no difference in the compressive modulus of the silk fibroin hydrogel prepared by enzyme-catalyzed crosslinking of the long-term frozen silk fibroin solution with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and the hydrogel prepared from the fresh silk fibroin aqueous solution, as Figure 2 shown.

[0032] Example 3:

[0033] A method for storing a silk fibroin aqueous solution, the method comprising the following steps:

[0034] After obtaining a silk fibroin aqueous solution with a concentration of 0.1 wt%, trehalose was added to make its final concentration 8 wt%. The silk fibroin aqueous solution was dispensed into cryotube containers and then pre-frozen in a -40°C refrigerator for more than 48 h. It was stored in the -80°C refrigerator for a long time. When needed, an appropriate amount of the frozen silk fibroin aqueous solution was taken out and thawed at room temperature, and it could be directly used for material preparation and other experiments.

[0035] Example 4:

[0036] A method for storing a silk fibroin aqueous solution, the method comprising the following steps:

[0037] After obtaining an aqueous silk fibroin solution with a concentration of 20 wt%, mannitol was added to make its final concentration 4 wt%. The aqueous silk fibroin solution was aliquoted into cryotube containers and then pre-frozen in liquid nitrogen for more than 0.5 h. It was stored long-term in a -70 °C refrigerator. When needed, an appropriate amount of the frozen silk fibroin solution was taken out and thawed at room temperature, and it could be directly used for material preparation or other experiments.

[0038] Example 5:

[0039] A method for storing an aqueous silk fibroin solution, the method comprising the following steps:

[0040] After obtaining an aqueous silk fibroin solution with a concentration of 5 wt%, the cryoprotectant sucrose was added to make its final concentration 6 wt%. The aqueous silk fibroin solution was aliquoted into cryotube containers and then pre-frozen in a -70 °C refrigerator for more than 72 h. It was stored long-term in a -20 °C refrigerator. When needed, an appropriate amount of the frozen silk fibroin solution was taken out and thawed at room temperature, and it could be directly used for material preparation and other experiments.

[0041] As described above, the above are only the preferred embodiments of the present invention, and do not impose any formal or substantial limitations on the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preserving a regenerated silk protein aqueous solution, characterized in that: include: A cryoprotectant is added to the freshly prepared regenerated silk protein aqueous solution, and the solution is pre-frozen to a final temperature at a temperature below -40°C, and stored at the final temperature for a long time. When using, the solution can be thawed and shaken well.

2. The storage method according to claim 1, characterized in that: The concentration of the regenerated silk protein aqueous solution is 0.1-20 wt %.

3. The storage method according to claim 1, characterized in that: The addition amount of the cryoprotectant is 0-10wt%; the cryoprotectant is sugar and / or polyol.

4. The storage method according to claim 1, characterized in that: The pre-freezing temperature condition is -176 to -40°C.

5. The storage method according to claim 1, characterized in that: The pre-freezing time is 0.5h to 72h.

6. The storage method according to claim 1, characterized in that: The temperature condition for long-term cryopreservation is -80 to -20°C.

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