Method for purifying recombinant silk fibroin expressed by pichia pastoris

Abstract

The invention discloses a method for purifying recombinant silk fibroin expressed by pichia pastoris, which is characterized in that recombinant silk fibroin fermentation liquor expressed by fermentation induction of pichia pastoris engineering bacteria is used as a raw material, and the purity and the yield of target protein are effectively improved by constructing a tandem chromatographic column system, designing a purification process and controlling process parameters and conditions. The purity of the purified recombinant silk fibroin freeze-dried powder is greater than 95%, and the endotoxin content is less than 2EU / mg.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a purification method for recombinant silk fibroin expressed by Pichia pastoris. Background Technology

[0002] Silk fibroin has excellent cell adhesion properties and can effectively promote cell proliferation, differentiation and migration. It is a highly promising biomaterial that has been applied in the fields of biomedicine, medical devices and daily cosmetic products.

[0003] Currently, silk fibroin is mainly extracted from silkworm silk. This method is highly dependent on the raw materials and has high extraction costs, limiting the widespread application of silk fibroin in various fields. However, with the development of synthetic biology, the expression and preparation technology of recombinant silk fibroin can effectively solve the problems of low extraction efficiency and high cost of traditional silk fibroin. However, purification methods have become a new problem for researchers in the preparation of recombinant silk fibroin. CN202310770727.7 discloses a method for obtaining recombinant humanized silk fibroin in a Pichia pastoris expression system. In this scheme, the purification process of recombinant humanized silk fibroin mainly adopts nickel column chromatography. However, this purification method cannot remove endotoxins and non-protein impurities, resulting in the failure to meet the demand for high-purity, low-endotoxin recombinant humanized silk fibroin products. Therefore, there is an urgent need for a purification method that can obtain recombinant silk fibroin with high purity, low endotoxin, and low impurity content. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a purification method for recombinant silk fibroin expressed in Pichia pastoris. This method, through the construction of a tandem chromatography column system, the design of a purification process, and the control of process parameters and conditions, effectively separates the target protein from endotoxins, impurities, etc., thereby improving the purity and yield of the target protein.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for purifying recombinant silk fibroin expressed by Pichia pastoris, characterized by comprising the following steps:

[0006] Step 1: After centrifuging the recombinant silk fibroin fermentation broth expressed by Pichia pastoris engineered strains, collect the supernatant and filter it to obtain the filtrate.

[0007] Step 2: Perform ultrafiltration on the filtrate from Step 1, control the conductivity to <2.0 mS / cm, concentrate and collect the concentrate, adjust the pH of the concentrate to 5.3-5.8, and obtain the treated concentrate;

[0008] Step 3: Rinse the tandem chromatography column with equilibration buffer; the tandem chromatography column is obtained by connecting a strong cation exchange chromatography column first and a weak anion exchange chromatography column second in series.

[0009] Step 4: Load the concentrated solution processed in Step 2 onto the tandem chromatography column of Step 3. After loading, first rinse the weak anion exchange chromatography column with washing buffer, then rinse the weak anion exchange chromatography column with equilibration buffer, control the conductivity of the cross-linked solution to <32mS / cm, and finally rinse the weak anion exchange chromatography column with elution buffer and collect the target protein eluent.

[0010] Step 5: The target protein eluent collected in Step 4 is concentrated by ultrafiltration and then freeze-dried to obtain recombinant silk fibroin freeze-dried powder.

[0011] The purification method for recombinant silk fibroin expressed by Pichia pastoris described above is characterized in that the ultrafiltration treatment in steps two and five uses a membrane ultrafiltration system with a molecular weight cutoff of ≤3kDa.

[0012] The purification method for recombinant silk fibroin expressed by Pichia pastoris is characterized in that the amino acid sequence of the recombinant silk fibroin is shown in SEQ ID NO:1.

[0013] The purification method for recombinant silk fibroin expressed by Pichia pastoris is characterized in that, in step three, the strong cation exchange chromatography column is a chromatography column packed with SP packing material, and the weak anion exchange chromatography column is a chromatography column packed with DEAE packing material.

[0014] The purification method for recombinant silk fibroin expressed by Pichia pastoris is characterized in that the SP packing material is selected from any one of SP Bestarose FF, SP Bestarose HF and Diamond SP; and the DEAE packing material is selected from any one of DEAE Bestarose FF, DEAE Bestarose HF and Diamond DEAE.

[0015] The purification method for recombinant silk fibroin expressed by Pichia pastoris described above is characterized in that the equilibration buffer in steps three and four is a 5-10 mmol citrate-sodium citrate buffer with a pH of 5.3-5.8.

[0016] The purification method for recombinant silk fibroin expressed by Pichia pastoris is characterized in that the washing buffer in step four is a citrate-sodium citrate buffer containing 400-500 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 5-10 mmol of citrate-sodium citrate buffer and its pH is 5.0-5.5.

[0017] The purification method for recombinant silk fibroin expressed by Pichia pastoris is characterized in that the elution buffer in step four is a citrate-sodium citrate buffer containing 270-350 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 5-10 mmol of citrate-sodium citrate buffer and its pH is 6.5-7.0.

[0018] The purification method for recombinant silk fibroin expressed by Pichia pastoris described above is characterized in that, in step five, ultrafiltration is performed until the conductivity of the retentate is less than 0.1 mS / cm.

[0019] The above-mentioned purification method for recombinant silk fibroin expressed by Pichia pastoris is characterized in that the lyophilized powder obtained by freeze-drying in step five has a purity greater than 95%, an endotoxin content of less than 2 EU / mg, and a target protein yield of greater than 0.8 g / L per liter of fermentation broth.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. This invention constructs a chromatography column system by connecting a strong cation exchange chromatography column and a weak anion exchange chromatography column in series. A purification process is designed and process parameters and conditions are controlled. Through the synergistic three-step purification process of washing, equilibration, and elution using the tandem chromatography column system, combined with precise control of key parameters, the target protein is accurately separated from endotoxins and impurities. This achieves efficient separation and large-scale preparation of recombinant silk fibroin, improving the purity and yield of the target protein. The recombinant silk fibroin lyophilized powder prepared by this invention has a purity greater than 95%, an endotoxin content of less than 2 EU / mg, a target protein yield greater than 0.8 g / L per liter of fermentation broth, and high biosafety.

[0022] 2. In the tandem chromatography column system constructed by the strong cation exchange column and the weak anion exchange column designed in this invention, the strong cation exchange packing material can preferentially adsorb most of the endotoxins and some impurities, reducing the competitive interference of the weak anion column. At the same time, by controlling the conductivity and pH of the loading solution, the target protein is captured to the maximum extent by the weak anion packing material in the tandem chromatography column system during the loading process, thereby improving the yield of the target protein.

[0023] 3. In the purification process of this invention, the weak anion exchange chromatography column is first rinsed with washing buffer to remove impurities, and then the chromatography column is rinsed with equilibration buffer to reduce the ionic strength inside the column, thereby preventing impurities from being co-eluted due to excessive ionic strength inside the column during subsequent elution. Then, the target protein is precisely dissociated by controlling the elution parameters. The three steps work together to reduce the residual amount of non-target protein to below the detection limit, thereby improving the purity and safety of the product.

[0024] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0025] Figure 1 The results of high-performance liquid chromatography (HPLC) purity testing are shown for the recombinant silk fibroin lyophilized powder purified and prepared in Example 1 of this invention.

[0026] Figure 2 This is an SDS-PAGE electrophoresis image of the sample during the chromatographic purification of recombinant silk fibroin in Example 1 of this invention. Lane 1: Protein Marker; Lane 2: Sample to be purified by chromatography (diluted 5-fold); Lane 3: Column pass-through sample obtained after loading the sample onto a tandem chromatography column; Lane 4: Wash solution collected after rinsing the weak anion exchange chromatography column with washing buffer; Lane 5: Sample to be collected after rinsing the weak anion exchange chromatography column with equilibration buffer; Lane 6: Eluent collected after rinsing the weak anion exchange chromatography column with elution buffer (diluted 5-fold); Lane 7: Sample to be collected after rinsing the weak anion exchange chromatography column with 2 mol / L sodium chloride solution; the sample loading volume for lanes 1 to 7 is 5 μL. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0028] The fermentation broth preparation methods in the following examples / comparative examples are as follows:

[0029] This protocol uses Pichia pastoris strain GS115 as the host to express recombinant silk fibroin. The amino acid sequence of the expressed recombinant silk fibroin is a combination of the hexapeptide and octapeptide sequence units selected from the fibroin heavy chain predictor [Bombyx mori] (NCBI Reference Sequence: NP_001106733.1), which is the recombinant silk fibroin. Its amino acid sequence is shown in SEQ ID NO:1. Based on the amino acid sequence and the codon preference of the Pichia pastoris expression system, the corresponding nucleotide sequence was determined. To facilitate subsequent operations, a ctcgag XhoI restriction site was added to the 5' end of the nucleotide sequence, and a stop codon and an EcoRI restriction site were added to the 3' end. The designed nucleotide sequence (i.e., SEQ ID NO:2) was then used for whole-genome synthesis to obtain a synthetic sequence fragment containing the target gene.

[0030] Construction of the expression vector: The synthesized sequence was digested with XhoI / EcoRI restriction endonuclease, the target fragment was recovered, and ligated into the Pichia pastoris vector pPIC9k, which was also digested with XhoI / EcoRI restriction endonuclease.

[0031] For the transformation of expression vectors and the screening of positive recombinant Pichia pastoris (i.e., engineered Pichia pastoris), please refer to the "Pichia pastoris Expression Operation Manual".

[0032] The engineered Pichia pastoris was used as the primary seed and cultured in a 10L fermenter for 18 hours. The cultured cells were then used as the secondary seed and inoculated into a 100L fermenter. During the culture process, recombinant silk fibroin was secreted and expressed. After fermentation, the fermentation broth was collected and removed from the fermenter. The culture medium and specific culture conditions for the primary seed and the culture medium and specific culture conditions for the secondary seed were referred to in the "Pichia pastoris Fermentation Manual".

[0033] The recombinant silk fibroin lyophilized powders obtained in the embodiments and comparative examples of this invention were all tested for purity using the following method: high performance liquid chromatography (HPLC) according to General Chapter 0512 of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV).

[0034] The recombinant silk fibroin lyophilized powders obtained in the embodiments and comparative examples of this invention were all tested using the following bacterial endotoxin detection method: the bacterial endotoxin detection was performed according to Method 1, gel method, in General Chapter 1143 of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV).

[0035] Example 1

[0036] The purification method for recombinant silk fibroin expressed by Pichia pastoris in this embodiment includes the following steps:

[0037] Step 1: After fermentation, the fermentation liquid volume of 76L was transferred to a centrifuge bottle and balanced. The supernatant of 45L was collected by centrifugation in a high-speed centrifuge. The supernatant was then transferred to the storage tank of the 0.45μm hollow fiber microfiltration system. The filter was turned on and the filtrate was collected. During the process, 20L of purified water was added repeatedly to wash the residual liquid in the system. A total of 70L of filtrate was collected.

[0038] Step 2: Transfer the filtrate from Step 1 to the storage tank of the 3kDa spiral wound membrane ultrafiltration system for ultrafiltration. Replace the buffer solution with purified water until the conductivity of the feed solution is 1.93 mS / cm. Concentrate the feed solution and collect 18L of concentrate. Use 10L of purified water to wash away the residue in the system and collect 8.6L of recovered solution, for a total of 26.6L. Add citric acid solution to adjust the pH to 5.6 to obtain the treated concentrate.

[0039] Step 3: Pack columns with SP Bestarose HF packing material and DEAE Bestarose HF packing material respectively, with the SP packing column first and the DEAE packing column second. Connect them in series to the chromatography system through tubing, and then wash the series chromatography columns with 10 mmol of citrate-sodium citrate equilibration buffer at pH 5.5.

[0040] Step 4: Load the concentrated solution obtained in Step 2 into a tandem chromatography column system. The tandem chromatography column system has already undergone the treatment in Step 3. During the later stages of loading, use equilibration buffer to wash away any impurities that are not bound to the packing material within the tandem chromatography column, ensuring thorough removal. After loading, separate the tandem chromatography columns, connecting only the column packed with DEAE packing to the chromatography system. Wash the DEAE column with washing buffer to elute impurities. After washing, wash the DEAE column again with equilibration buffer until the conductivity of the percolation solution is 31 mS / cm. Finally, wash with elution buffer.

[0041] The DEAE column was used for elution, and 17 L of target protein eluent was collected. After the target protein was collected, the DEAE column was washed with 2 mol / L sodium chloride solution to remove residual impurities. The equilibration buffer was 10 mmol of citrate-sodium citrate buffer at pH 5.5. The washing buffer was 498 mmol / L citrate-sodium citrate buffer, and the citrate-sodium citrate buffer in the washing buffer was 10 mmol of citrate-sodium citrate buffer at pH 5.5. The elution buffer was 342 mmol / L citrate-sodium citrate buffer, and the citrate-sodium citrate buffer in the elution buffer was 10 mmol of citrate-sodium citrate buffer at pH 6.9.

[0042] Step 5: Transfer the target protein eluent collected in Step 4 to the storage tank of the 3kDa spiral wound membrane ultrafiltration system for ultrafiltration. Replace the buffer with purified water and ultrafilter until the conductivity of the retentate is 95μS / cm. Continue to concentrate the solution and collect 5.7L of concentrate. Use 5L of purified water to wash away the residue in the system and collect 4.4L of recovery solution. A total of 10.1L of ultrafiltration concentrate is collected. After freeze drying, 63.7g of recombinant silk fibroin lyophilized powder is obtained.

[0043] The purification method in this embodiment yields 0.838 g / L of recombinant silk fibroin lyophilized powder per liter of fermentation broth; the purity of the lyophilized powder sample is determined to be 97.72%; and the endotoxin test, with an endotoxin limit of less than 2 EU / mg, shows that the test results are qualified.

[0044] The high-performance liquid chromatography (HPLC) results of the purity of the recombinant silk fibroin lyophilized powder obtained in this embodiment are as follows: Figure 1As shown, liquid chromatography analysis revealed a single peak of the target protein with an elution time of 19.111 and a relative peak area of ​​97.72%, indicating that the recombinant silk fibroin prepared by the method of this invention has a liquid phase purity of over 97%.

[0045] Figure 2 This is an SDS-PAGE electrophoresis image of the sample during the chromatographic purification of recombinant silk fibroin in this embodiment. Figure 2 As can be seen, the impurities are mainly diffusely distributed proteins with a molecular weight greater than 45 kDa. These proteins were eluted on the weak anion exchange chromatography column using elution buffer (lane 4) and 2 mol / L sodium chloride solution (lane 7), respectively, while the target protein was concentrated and eluted using elution buffer (lane 6). Electrophoresis patterns show that diffusely distributed bands of impurity proteins with a relative molecular mass greater than 45 kDa can be observed in lanes 4 and 7. Comparison of these results with those in lane 2 (the original feed solution) confirms that the tandem chromatography column system constructed in this invention can achieve deep removal of impurity proteins from the feed solution through the complementary effects of different chromatography media. Lane 6 shows a single and clear target protein band, with no diffusely distributed impurity protein bands detected. This result further indicates that: on the one hand, the elution step of this invention can effectively remove impurity proteins that have strong adsorption to the chromatography media; on the other hand, the subsequent equilibration step can reduce the ionic strength of the system, fundamentally avoiding co-elution of impurity proteins and the target protein, ultimately achieving precise dissociation and collection of the target protein through the elution step. Furthermore, no obvious protein bands were observed in lanes 3 and 5. This phenomenon indicates that under the process conditions set in this invention, the chromatography medium has an excellent capture ability for the target protein in the feed solution. At the same time, a large number of non-protein substances and impurities that are not adsorbed by the chromatography medium in the feed solution can be effectively removed during column perforation and equilibration.

[0046] Example 2:

[0047] The purification method for recombinant silk fibroin expressed by Pichia pastoris in this embodiment includes the following steps:

[0048] Step 1: After fermentation, the fermentation liquid volume of 77L was transferred to a centrifuge bottle and balanced. The supernatant of 45L was collected by centrifugation in a high-speed centrifuge. The supernatant was then transferred to the storage tank of the 0.45μm hollow fiber microfiltration system. The filter was turned on and the filtrate was collected. During the process, 20L of purified water was added repeatedly to wash the residual liquid in the system. A total of 71L of filtrate was collected.

[0049] Step 2: Transfer the filtrate from Step 1 to the storage tank of the 3kDa spiral wound membrane ultrafiltration system for ultrafiltration. Replace the buffer solution with purified water and ultrafilter until the conductivity of the feed solution is 1.68mS / cm. Continue to concentrate the feed solution and collect 19L of concentrate. Use 10L of purified water to wash and recover the residue in the system and collect 8.4L of recovered solution, for a total of 27.4L. Add citric acid solution to adjust the pH to 5.3 to obtain the treated concentrate.

[0050] Step 3: Pack columns with SP Bestarose FF packing material and DEAE Bestarose FF packing material respectively, with the SP packing column first and the DEAE packing column second. Connect them in series to the chromatography system through tubing. Wash the series chromatography columns with 5 mmol of citrate-sodium citrate equilibration buffer at pH 5.3.

[0051] Step 4: Load the concentrated solution obtained in Step 2 into a tandem chromatography column system, which has already undergone the treatment in Step 3. During the later stages of loading, use equilibration buffer to wash away any impurities not bound to the packing material within the tandem chromatography column, ensuring thorough removal. After loading, separate the tandem chromatography columns, connecting only the column packed with DEAE packing to the chromatography system. Wash the DEAE column with washing buffer to remove impurities. After washing, wash the DEAE column again with equilibration buffer until the conductivity of the percolation solution reaches 28 mS / cm. Finally, wash with elution buffer.

[0052] The DEAE column was used for elution, and 18 L of target protein eluent was collected. After the target protein was collected, the DEAE column was washed with 2 mol / L sodium chloride solution to remove residual impurities. The equilibration buffer was 5 mmol of citrate-sodium citrate buffer at pH 5.3. The washing buffer was 400 mmol / L citrate-sodium citrate buffer, and the citrate-sodium citrate buffer in the washing buffer was 5 mmol of citrate-sodium citrate buffer at pH 5.0. The elution buffer was 270 mmol / L citrate-sodium citrate buffer, and the citrate-sodium citrate buffer in the elution buffer was 5 mmol of citrate-sodium citrate buffer at pH 6.5.

[0053] Step 5: Transfer the target protein eluent collected in Step 4 to the storage tank of the 3kDa spiral wound membrane ultrafiltration system for ultrafiltration. Replace the buffer with purified water and ultrafilter until the conductivity of the retentate is 87μS / cm. Continue to concentrate the solution and collect 5.9L of concentrate. Use 5L of purified water to wash away the residue in the system and collect 4.3L of recovery solution. A total of 10.2L of ultrafiltration concentrate is collected. After freeze drying, 65.4g of recombinant silk fibroin lyophilized powder is obtained.

[0054] The purification method of this embodiment yields 0.849 g / L of recombinant silk fibroin lyophilized powder per liter of fermentation broth; the purity of the lyophilized powder sample is determined to be 97.1%; and the endotoxin test, with an endotoxin limit of less than 2 EU / mg, shows that the test results are qualified.

[0055] Example 3:

[0056] The purification method for recombinant silk fibroin expressed by Pichia pastoris in this embodiment includes the following steps:

[0057] Step 1: After fermentation, the fermentation liquid volume of 77L was transferred to a centrifuge bottle and balanced. The supernatant of 45L was collected by centrifugation in a high-speed centrifuge. The supernatant was then transferred to the storage tank of the 0.45μm hollow fiber microfiltration system. The filter was turned on and the filtrate was collected. During the process, 20L of purified water was added repeatedly to wash the residual liquid in the system. A total of 70L of filtrate was collected.

[0058] Step 2: Transfer the filtrate from Step 1 to the storage tank of the 1kDa spiral wound membrane ultrafiltration system for ultrafiltration. Replace the buffer solution with purified water until the conductivity of the feed solution is 1.98 mS / cm. Continue to concentrate the feed solution and collect 18.6L of concentrate. Use 10L of purified water to wash away the residue in the system and collect 8.6L of recovered solution, for a total of 27.2L. Add citric acid solution to adjust the pH to 5.8 to obtain the treated concentrate.

[0059] Step 3: Pack columns with Diamond SP packing material and Diamond DEAE packing material respectively, with the SP packing column first and the DEAE packing column second. Connect them in series to the chromatography system through tubing. Wash the series chromatography columns with 10 mmol of citrate-sodium citrate equilibration buffer at pH 5.8.

[0060] Step 4: Load the concentrated solution obtained in Step 2 into a tandem chromatography column system, which has already undergone the treatment in Step 3. During the later stages of loading, use equilibration buffer to wash away any impurities not bound to the packing material within the tandem chromatography column, ensuring thorough removal. After loading, separate the tandem chromatography columns, connecting only the column packed with DEAE packing to the chromatography system. Wash the DEAE column with washing buffer to remove impurities. After washing, wash the DEAE column again with equilibration buffer until the percolation conductivity of the feed solution is 29 mS / cm. Finally, wash with elution buffer.

[0061] The DEAE chromatography column was used for elution, and 18 L of target protein eluent was collected. After the target protein was collected, the DEAE chromatography column was washed with 2 mol / L sodium chloride solution to remove residual impurities. The equilibration buffer was 10 mmol of citrate-sodium citrate buffer at pH 5.8. The washing buffer was 500 mmol / L citrate-sodium citrate buffer, and the citrate-sodium citrate buffer in the washing buffer was 10 mmol of citrate-sodium citrate buffer at pH 5.5. The elution buffer was 350 mmol / L citrate-sodium citrate buffer, and the citrate-sodium citrate buffer in the elution buffer was 10 mmol of citrate-sodium citrate buffer at pH 7.0.

[0062] Step 5: Transfer the target protein eluent collected in Step 4 to the storage tank of the 1kDa spiral wound membrane ultrafiltration system for ultrafiltration. Replace the buffer with purified water and ultrafilter until the conductivity of the retentate is 82μS / cm. Continue to concentrate the solution and collect 5.8L of concentrate. Use 5L of purified water to wash away the residue in the system and collect 4.2L of recovery solution. A total of 10L of ultrafiltration concentrate is collected. After freeze drying, 62.8g of recombinant silk fibroin lyophilized powder is obtained.

[0063] The purification method of this embodiment yields 0.816 g / L of recombinant silk fibroin lyophilized powder per liter of fermentation broth; the purity of the lyophilized powder sample is determined to be 97.8%; and the endotoxin test, with an endotoxin limit of less than 2 EU / mg, shows that the test results are qualified.

[0064] Comparative Example 1

[0065] The only difference between this comparative example and Example 1 is that the filtrate is subjected to ultrafiltration in step two, and the conductivity is controlled to be 2.9 mS / cm.

[0066] By freeze-drying the final liquid, 51.6 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.679 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 92.8%. Endotoxin testing was performed with an endotoxin limit of less than 2 EU / mg, and the test results were qualified.

[0067] In Comparative Example 1, the insufficient ultrafiltration in step two resulted in excessively high conductivity of the feed solution. This reduced the adsorption capacity of the SP-filled chromatography column for impurities in the feed solution and also reduced the adsorption capacity of the DEAE-filled chromatography column for the target protein. Consequently, the eluent of the collected target protein contained too many residual impurities and too little target protein, resulting in a significant reduction in both the yield and purity of the recombinant silk fibroin.

[0068] Comparative Example 2

[0069] The only difference between this comparative example and Example 1 is that the pH of the concentrate was adjusted to 6.7 in step two.

[0070] By freeze-drying the final liquid, 65.3g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.859g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 93.6%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0071] In Comparative Example 2, the pH value of the concentrate was adjusted too high in step two, which caused some impurities and endotoxins to be captured by the chromatography column packed with DEAE packing instead of being captured and removed by the SP packing column. This resulted in the target protein being eluted along with some impurities and endotoxins during the elution process, leading to a significant reduction in the purity of the final target protein and failure to meet the endotoxin limit test.

[0072] Comparative Example 3

[0073] The only difference between this comparative example and Example 1 is that the pH of the concentrate was adjusted to 4.5 in step two.

[0074] By freeze-drying the final liquid, 52.6 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.692 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 97.6%. Endotoxin testing was performed with an endotoxin limit of less than 2 EU / mg, and the test results were qualified.

[0075] In Comparative Example 3, the pH value of the concentrate was adjusted too low in step two, which reduced the adsorption force between the target protein and the chromatography column packed with DEAE packing material, resulting in the loss of some target protein through the column and a significant reduction in the yield of recombinant silk fibroin freeze-dried powder per liter of fermentation broth.

[0076] Comparative Example 4

[0077] The only difference between this comparative example and Example 1 is that in step three, purification was performed using only a chromatography column packed with DEAE packing material.

[0078] By freeze-drying the final liquid, 64.1 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.843 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 90.2%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0079] In Comparative Example 4, only a chromatography column packed with DEAE packing material was used for purification in step three. Some impurities and endotoxins were captured by the chromatography column. During the elution process, the target protein was eluted along with the impurities and endotoxins, resulting in a significant decrease in the purity of the final target protein and failure to meet the endotoxin limit test.

[0080] Comparative Example 5

[0081] The only difference between this comparative example and Example 1 is that the washing buffer in step four is a citrate-sodium citrate buffer containing 590 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 5.5.

[0082] By freeze-drying the final liquid, 59.3g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.780g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 97.4%. Endotoxin testing was performed with an endotoxin limit of less than 2EU / mg, and the test results were qualified.

[0083] In Comparative Example 5, the sodium chloride content in the washing buffer in step four was too high, which caused some of the target proteins to be washed away during the washing process, resulting in a decrease in the yield of recombinant silk fibroin freeze-dried powder per liter of fermentation broth.

[0084] Comparative Example 6

[0085] The only difference between this comparative example and Example 1 is that the washing buffer in step four is a citrate-sodium citrate buffer containing 310 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 5.5.

[0086] By freeze-drying the final liquid, 64.8 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.852 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 93.4%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0087] In Comparative Example 6, the sodium chloride content in the washing buffer in step four was too low, resulting in some impurities and endotoxins not being washed away and remaining on the chromatography column. During subsequent elution, the impurities and endotoxins remaining on the chromatography column were eluted along with the target protein, resulting in a significant reduction in the purity of the final target protein and failure to meet the endotoxin limit test.

[0088] Comparative Example 7

[0089] The only difference between this comparative example and Example 1 is that the washing buffer in step four is a citrate-sodium citrate buffer containing 498 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 6.7.

[0090] By freeze-drying the final liquid, 58.2 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.767 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 97.1%. Endotoxin testing was performed with an endotoxin limit of less than 2 EU / mg, and the test results were qualified.

[0091] In Comparative Example 7, the pH of the washing buffer in step four was too high. During the washing process, some target proteins were washed away along with impurities and endotoxins, resulting in a decrease in the yield of recombinant silk fibroin freeze-dried powder per liter of fermentation broth.

[0092] Comparative Example 8

[0093] The only difference between this comparative example and Example 1 is that the washing buffer in step four is a citrate-sodium citrate buffer containing 498 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 4.3.

[0094] By freeze-drying the final liquid, 64.8 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.852 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 93.4%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0095] In Comparative Example 8, the pH of the washing buffer in step four was too low, causing some impurities and endotoxins to remain on the chromatography column. During subsequent elution, the remaining impurities and endotoxins were eluted along with the target protein, resulting in a significant decrease in the purity of the final target protein and failure to meet the endotoxin limit test.

[0096] Comparative Example 9

[0097] The only difference between this comparative example and Example 1 is that in step four, after rinsing the chromatography column with washing buffer, it is directly rinsed with elution buffer to elute the target protein.

[0098] By freeze-drying the final liquid, 65.1 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.857 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 92.6%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0099] In Comparative Example 9, the chromatography column was first washed with washing buffer in step four. Due to the presence of some sodium chloride residue, the ionic strength of the chromatography column was relatively high. If the elution operation was performed directly, some impurities and endotoxins that were originally firmly bound to the chromatography column would be eluted along with the target protein, resulting in a significant reduction in the purity of the final pure target protein and failure to meet the endotoxin limit test.

[0100] Comparative Example 10

[0101] The only difference between this comparative example and Example 1 is that the elution buffer in step four is a citrate-sodium citrate buffer containing 450 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 6.9.

[0102] By freeze-drying the final liquid, 63.5g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.836g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 92.8%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0103] In Comparative Example 10, the elution buffer in step four contained too much sodium chloride, which caused impurities and endotoxins that were originally firmly bound to the chromatography column to be eluted along with the target protein, resulting in a decrease in the purity of the target protein and failure to meet the endotoxin limit test.

[0104] Comparative Example 11

[0105] The only difference between this comparative example and Example 1 is that the elution buffer in step four is a citrate-sodium citrate buffer containing 190 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 6.9.

[0106] By freeze-drying the final liquid, 53.7 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.707 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 97.7%. Endotoxin testing was performed with an endotoxin limit of less than 2 EU / mg, and the test results were qualified.

[0107] In Comparative Example 11, the sodium chloride content in the elution buffer in step four was too low. During the elution process, some target proteins remained on the chromatography column, resulting in insufficient elution and a decrease in the yield of recombinant silk fibroin per liter of fermentation broth.

[0108] Comparative Example 12

[0109] The only difference between this comparative example and Example 1 is that the elution buffer in step four is a citrate-sodium citrate buffer containing 342 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 5.8.

[0110] By freeze-drying the final liquid, 55.8 g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.734 g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 97.3%. Endotoxin testing was performed with an endotoxin limit of less than 2 EU / mg, and the test results were qualified.

[0111] In Comparative Example 12, the pH of the elution buffer in step four was too low, resulting in insufficient elution of the target protein and a decrease in the yield of recombinant silk fibroin per liter of fermentation broth.

[0112] Comparative Example 13

[0113] The only difference between this comparative example and Example 1 is that the elution buffer in step four is a citrate-sodium citrate buffer containing 342 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 10 mmol and has a pH of 7.8.

[0114] By freeze-drying the final liquid, 66.5g of recombinant silk fibroin freeze-dried powder was obtained, with a yield of 0.853g / L of recombinant silk fibroin freeze-dried powder per liter of fermentation broth. The purity of the freeze-dried powder sample was determined to be 92.3%. However, the endotoxin test, which had an endotoxin limit of less than 2 EU / mg, failed the test.

[0115] In Comparative Example 13, the pH of the elution buffer in step four was too high. Impurities and endotoxins that were originally firmly bound to the chromatography column were eluted along with the target protein during the elution process, resulting in a significant decrease in the purity of the final target protein and failure to pass the endotoxin limit test.

[0116] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for purifying recombinant silk fibroin expressed in Pichia pastoris, characterized in that, Includes the following steps: Step 1: After centrifuging the recombinant silk fibroin fermentation broth expressed by Pichia pastoris engineered strains, collect the supernatant and filter it to obtain the filtrate. Step 2: Perform ultrafiltration on the filtrate from Step 1, control the conductivity to <2.0 mS / cm, concentrate and collect the concentrate, adjust the pH of the concentrate to 5.3-5.8, and obtain the treated concentrate; Step 3: Rinse the tandem chromatography column with equilibration buffer; the tandem chromatography column is obtained by connecting a strong cation exchange chromatography column first and a weak anion exchange chromatography column second in series. Step 4: Load the concentrated solution processed in Step 2 onto the tandem chromatography column of Step 3. After loading, first rinse the weak anion exchange chromatography column with washing buffer, then rinse the weak anion exchange chromatography column with equilibration buffer, control the conductivity of the cross-linked solution to <32mS / cm, and finally rinse the weak anion exchange chromatography column with elution buffer and collect the target protein eluent. Step 5: The target protein eluent collected in Step 4 is concentrated by ultrafiltration and then freeze-dried to obtain recombinant silk fibroin freeze-dried powder.

2. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The ultrafiltration treatments described in steps two and five employ a membrane ultrafiltration system with a molecular weight cutoff of ≤3kDa.

3. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The amino acid sequence of the recombinant silk fibroin is shown in SEQ ID NO:

1.

4. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The strong cation exchange chromatography column mentioned in step three is a chromatography column packed with SP packing material, and the weak anion exchange chromatography column is a chromatography column packed with DEAE packing material.

5. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 4, characterized in that, The SP packing material is selected from any one of SP Bestarose FF, SP Bestarose HF, and Diamond SP; the DEAE packing material is selected from any one of DEAE Bestarose FF, DEAE Bestarose HF, and Diamond DEAE.

6. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The equilibration buffer mentioned in steps three and four is a 5-10 mmol citrate-sodium citrate buffer solution with a pH of 5.3-5.

8.

7. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The washing buffer in step four is a citrate-sodium citrate buffer containing 400-500 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 5-10 mmol of citrate-sodium citrate buffer and its pH is 5.0-5.

5.

8. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The elution buffer in step four is a citrate-sodium citrate buffer containing 270-350 mmol / L sodium chloride, wherein the citrate-sodium citrate buffer is 5-10 mmol of citrate-sodium citrate buffer and its pH is 6.5-7.

0.

9. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, In step five, ultrafiltration is performed until the conductivity of the retentate is less than 0.1 mS / cm.

10. The purification method for recombinant silk fibroin expressed by Pichia pastoris according to claim 1, characterized in that, The freeze-dried powder obtained in step five has a purity greater than 95%, an endotoxin content of less than 2 EU / mg, and a target protein yield of greater than 0.8 g / L per liter of fermentation broth.

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