Purification method of recombinant X VII type collagen expressed by pichia pastoris fermentation

Abstract

The invention discloses a method for purifying recombinant X VII type collagen expressed by pichia pastoris fermentation, and relates to the technical field of protein purification methods. The purification method comprises the following steps: centrifuging a recombinant X VII type collagen fermentation liquid fermented and expressed by pichia pastoris engineering bacteria, filtering the obtained centrifugate, then carrying out ultrafiltration concentration, cooling the obtained ultrafiltration concentrated liquid to 2-10 DEG C, then adding Triton X-114, maintaining the material liquid at 2-10 DEG C, carrying out chromatography, leaching and extraction, carrying out ultrafiltration concentration on the extraction liquid, and then carrying out freeze drying to obtain the recombinant X VII type collagen. The recombinant X VII type collagen freeze-dried powder is obtained. The purification method provided by the invention is simple and convenient in whole operation process, can effectively remove endotoxin, can prepare the high-purity recombinant X VII type collagen freeze-dried powder, and is convenient for industrial large-scale application.

Description

Technical Field

[0001] The invention relates to the technical field of protein purification methods, and in particular to a method for purifying recombinant XVII type collagen expressed by Pichia pastoris fermentation. Background Art

[0002] Type XVII collagen is a transmembrane protein located in the basement membrane region of the epidermis. Its structure consists of an intracellular region, a transmembrane structural region, and an extracellular region. In the structure of type XVII collagen, the transmembrane structural region has a non-helical connection domain, which contains many protease hydrolysis sites, so that under the action of proteases, this region can be hydrolyzed and detached from the cell surface. Due to this unique structural feature, type XVII collagen can exist in the body in the form of transmembrane or detachment, thus having the dual role of cell membrane surface receptor and extracellular matrix component. At the same time, type XVII collagen is also an important component of hemidesmosomes in cells, playing a key role in the interaction between epithelial cells and basement membranes. It participates in regulating the adhesion, separation, development and differentiation of epithelial cells, as well as maintaining the homeostasis of related stem cells in the skin and promoting hair follicle growth. Therefore, type XVII collagen shows great development potential in delaying skin aging and promoting hair follicle growth.

[0003] At present, the extraction and preparation of collagen mainly rely on acid, alkaline hydrolysis or enzymatic hydrolysis. Although these traditional methods can obtain collagen from animal fur, the resulting products have many problems such as poor water solubility, easy to cause pathogenic infection, rejection reaction, etc. Therefore, the introduction of genetic engineering technology has become an effective way to solve this problem. Through genetic engineering technology, human collagen can be recombinantly expressed to obtain products with higher safety and efficiency.

[0004] In the prior art, CN116375847A discloses an industrial purification method for recombinant type XVII collagen, which obtains purified recombinant type XVII collagen by hydrophobic chromatography combined with cation exchange, but the protein purity obtained by the purification method is low and the bacterial endotoxin (hereinafter referred to as "endotoxin") content cannot be fully controlled, affecting the direct use of the product in other fields; CN117466990A discloses a recombinant human type XVII collagen and a preparation method thereof, which expresses recombinant human type XVII collagen in a Corynebacterium glutamicum expression system and purifies it by affinity chromatography, but the purity of the final prepared protein is low, which cannot meet the use standards of raw materials in the field of medical devices and pharmaceuticals, greatly limiting the application of recombinant collagen. Therefore, it is urgently needed to provide a purification method for recombinant type XVII collagen with a simple process flow and effective removal of endotoxins to prepare high-purity recombinant type XVII collagen. Summary of the invention

[0005] The purpose of the present invention is to provide a method for separating and purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris. The method has a simple process flow and can effectively remove impurities such as endotoxins and aggregates, thereby obtaining a recombinant type XVII collagen freeze-dried powder with a purity of ≥95% and an endotoxin content of <0.5EU / mg.

[0006] The scheme discloses a method for purifying recombinant type XVII collagen expressed by a Pichia pastoris engineered bacterium, wherein the recombinant type XVII collagen expressed by the genetically engineered bacterium has 396 amino acids, a molecular weight of 36.53 KDa, and a theoretical isoelectric point of 9.76. For the specific amino acid sequence, see SEQ ID NO.1.

[0007] In order to solve the above technical problems, the purpose of the present invention is achieved through the following technical solutions.

[0008] A method for purifying a fermentation broth of recombinant type XVII collagen expressed by Pichia pastoris fermentation comprises the following steps:

[0009] (1) centrifuging the recombinant type XVII collagen expressed by fermentation of Pichia pastoris engineered bacteria, collecting the centrifuge and filtering;

[0010] (2) ultrafiltration and concentration of the filtered centrifuge obtained in step (1);

[0011] (3) After cooling the obtained ultrafiltration concentrate to 2-10° C., Triton X-114 is added, and the feed liquid temperature is maintained at 2-10° C. and allowed to stand; the content of Triton X-114 in the ultrafiltration concentrate is 10-15 g / L;

[0012] (4) maintaining the ultrafiltration concentrate obtained after standing at 2-10° C., adjusting the pH to 4-4.5, and then adding ion exchange filler for chromatography; collecting the retained ion exchange filler by filtration and rinsing it with purified water, then leaching the ion exchange filler with the washing solution, and collecting the leaching solution; during the elution and leaching process, the temperature of the ion exchange filler is also maintained at 2-10° C.;

[0013] (5) The obtained extract is concentrated by ultrafiltration to collect the retentate, and the retentate is freeze-dried to obtain recombinant type XVII collagen freeze-dried powder.

[0014] Preferably, in step (1), the centrifugal component is a disc centrifuge; in some specific embodiments, the feed rate is 180 L / h, the slag discharge interval is 700 s, and the slag discharge flushing water is 3 s; the filtration is performed using a 0.22 um hollow fiber filter membrane.

[0015] Preferably, in the step (2), ultrafiltration and concentration are performed until the conductivity of the permeate is less than 2 mS / cm. Ultrafiltration and concentration until the conductivity of the permeate is less than 2 mS / cm is conducive to the column hanging of recombinant type XVII collagen. When the conductivity is higher than this range, the protein cannot bind to the ion exchange filler.

[0016] Preferably, in the step (3), the standing time is ≥ 1 h.

[0017] Preferably, in the step (4), the ion exchange filler is a cation exchange filler, and further, the cation exchange filler is one of SP sep life FF and CM sep life FF; the ion exchange filler is added in an amount of not less than 200 g / L in the ultrafiltration concentrate, and a more preferred amount is 200-300 g / L; the specific chromatography method is: adding the ion exchange filler while maintaining the feed liquid at 2-10°C and continuously stirring for not less than 1 hour.

[0018] Preferably, in step (4), the washing liquid is a sodium chloride solution with a concentration of 14.6 g / L-20.5 g / L, and the added amount is not less than 3 times the mass of the ion exchange filler.

[0019] Preferably, in the step (4), the time for the washing liquid to extract the ion exchange filler is not less than 30 minutes.

[0020] Preferably, in the step (4), the amount of purified water used in the elution process is not less than 5 times the mass of the ion exchange filler.

[0021] Preferably, in the step (5), ultrafiltration and concentration are performed until the conductivity of the permeate is less than 100uS / cm. Ultrafiltration and concentration are performed until the conductivity is less than 100uS / cm to ensure the purity of the recombinant type XVII collagen after freeze-drying.

[0022] In some specific embodiments, in steps (2) and (5), 6KD roll filter membrane is used for ultrafiltration and concentration.

[0023] The sequence of the recombinant type XVII collagen of the present invention is shown in SEQ ID NO. 1. The purity of the recombinant type XVII collagen freeze-dried powder obtained by the above purification method is ≥95%, and the endotoxin content is <0.5EU / mg.

[0024] The purification method provided by the present invention has a simple overall operation process, can effectively remove endotoxins without complicated operations, and prepare high-purity recombinant type XVII collagen freeze-dried powder, which simplifies the operation process, reduces the production cost, and is convenient for large-scale industrial application.

[0025] This method uses the fermentation broth of recombinant type XVII collagen expressed by fermentation induction of Pichia pastoris as raw material for the first time, cools the ultrafiltration concentrate and maintains it at 2-10°C, then adds Triton X-114 and stirs it thoroughly, which can ensure that it fully combines with endotoxins, encapsulates endotoxins and other substances, and continues to adjust the pH of the system at low temperature and add ion exchange fillers for chromatography, which can ensure that endotoxins and other impurities encapsulated by Triton X-114 will not combine with the fillers, and improve the efficiency of the ion exchange fillers binding with the target protein, preventing impurities from contaminating the target protein during the binding and extraction process, and finally obtaining high-purity recombinant type XVII collagen. The method for purifying recombinant type XVII collagen and removing endotoxins disclosed in this scheme is simple in process, greatly reduces the cost of industrial production, and is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0027] Figure 1 It is the SDS-PAGE electrophoresis detection diagram of the recombinant XVII type collagen of the embodiment and the comparative example; wherein, lane 1: 1 mg / mL freeze-dried powder sample of embodiment 1; lane 2: 1 mg / mL freeze-dried powder sample of embodiment 2; lane 3: 1 mg / mL freeze-dried sample of comparative example 6; lane 4: 1 mg / mL freeze-dried sample of comparative example 7; lane 5: 1 mg / mL freeze-dried sample of comparative example 10; lane 6: protein marker; the spotting amount of lanes 1 to 6 is 15uL. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The construction and transformation of the expression vector (containing the above gene sequence) and the screening of positive recombinant Pichia pastoris (i.e., recombinant type XVII collagen Pichia pastoris genetically engineered bacteria) all refer to the Pichia Pastoris Expression Operation Manual. The culture medium and specific culture conditions for the primary seed culture, the fermentation medium and specific culture conditions for the fermentation of the recombinant type XVII collagen Pichia pastoris genetically engineered bacteria as the primary seed in the fermenter are all referred to the Pichia Pastoris Fermentation Manual.

[0030] The preparation method of the Pichia fermentation broth containing recombinant type XVII collagen in the embodiment of the present invention is as follows: the designed and artificially synthesized recombinant type XVII collagen nucleotide sequence, the sequence is shown in SEQ ID NO.2, is connected to the plasmid, and the plasmid is extracted, and the Pichia GS115 host bacteria are transformed by electroporation after linearization, and the transformants containing the recombinant collagen target gene are obtained by plate screening, and the obtained transformants are cultured in a BMGY medium shake flask for 24 hours, cultured in a 10L fermentor as a primary seed for 18 hours, and inoculated into a 100L fermentor as a secondary seed for fermentation production, controlling the pH to 5.0, the temperature to 29.0°C, and the dissolved oxygen to 30%, and when the wet weight of the bacteria in the fermenter (100L) reaches 180-200 mg / mL, methanol induction is started, and the fermentation is terminated after 40-50 hours of induction.

[0031] The purity detection method of the recombinant type XVII collagen lyophilized powder in the embodiments and comparative examples of the present invention is: detection according to the non-reduced SDS-polyacrylamide gel electrophoresis method specified in the general rule 0541 electrophoresis method of the "Pharmacopoeia of the People's Republic of China" (Volume 4 of the 2020 edition).

[0032] The bacterial endotoxin detection method of the recombinant type XVII collagen freeze-dried powder in the embodiments of the present invention and the comparative examples is: refer to the general rule 1143 Bacterial Endotoxin Inspection Method 1 Gel Method for bacterial endotoxin detection in the "Pharmacopoeia of the People's Republic of China" (Volume 4 of the 2020 edition).

[0033] Example 1

[0034] (1) After the fermentation is completed, 64 L of the fermentation liquid is taken and centrifuged in a disc centrifuge (feed rate 180 L / h, slag discharge interval 700 s, slag discharge flushing water 3 s), the precipitate is discarded, and the centrifugal supernatant is collected to obtain 51 L of centrifugal supernatant;

[0035] (2) filtering and clarifying the 51 L centrifugal supernatant collected in step (1) through a 0.22 μm hollow fiber filter membrane, adding 10 L of purified water after filtration, and collecting 49 L of filtrate;

[0036] (3) The filtrate obtained in step (2) was ultrafiltered and concentrated using a 6KD spiral membrane ultrafiltration system until the permeate conductivity was 1.5 mS / cm, to obtain 20 L of ultrafiltration concentrate;

[0037] (4) The ultrafiltration concentrate obtained in step (3) was cooled to 6°C, and then 240 g of Triton X-114 was added, and the ultrafiltration concentrate was allowed to stand for 1.5 h while maintaining the temperature of 6°C.

[0038] (5) The ultrafiltration concentrate obtained in step (4) is continued to be maintained at 6° C., the pH value of the ultrafiltration concentrate is adjusted to 4.5 with sodium citrate, 4000 g of ion exchange filler SP sep life FF is added, and the mixture is stirred at 200 RPM for 1 h. The ion exchange filler is collected by sieve filtration, and the filler is rinsed with 28 L of purified water. After elution, the ion exchange filler is extracted with 15 L of 14.6 g / L sodium chloride solution for 30 min, and 14.7 L of an extract of the target protein is obtained by sieve filtration. During the elution and extraction steps, the temperature of the ion exchange filler is always maintained at 6° C.

[0039] (6) The extract obtained in step (5) was ultrafiltered and concentrated through a 6KD spiral membrane ultrafiltration system until the permeate conductivity was 68uS / cm, and 2.0L of the retentate was collected.

[0040] (7) The retentate obtained in step (6) was freeze-dried to obtain 54.6 g of recombinant type XVII collagen freeze-dried powder.

[0041] The purity of the recombinant type XVII collagen freeze-dried powder obtained in this example was tested to be 96.6%; the result of bacterial endotoxin test was qualified.

[0042] Example 2

[0043] (1) After the fermentation is completed, 64 L of the fermentation liquid is taken and centrifuged in a disc centrifuge (feed rate 180 L / h, slag discharge interval 700 s, slag discharge flushing water 3 s), the precipitate is discarded, and the centrifugal supernatant is collected to obtain 51 L of centrifugal supernatant;

[0044] (2) 51 L of the centrifugal supernatant collected in step (1) was filtered through a 0.22 μm hollow fiber filter membrane for clarification, and 10 L of purified water was added after the filtration to ensure the protein recovery rate, and 49 L of the filtrate was collected;

[0045] (3) The filtrate obtained in step (2) was ultrafiltered and concentrated using a 6KD spiral membrane ultrafiltration system until the permeate conductivity was 1.5 mS / cm, to obtain 20 L of ultrafiltration concentrate;

[0046] (4) The ultrafiltration concentrate obtained in step (3) was cooled to 10°C, and then 200 g of Triton X-114 was added, and the temperature of the ultrafiltration concentrate was maintained at 10°C and allowed to stand for 1 hour.

[0047] (5) The ultrafiltration concentrate obtained in step (4) is continued to be maintained at 10° C., the pH value of the ultrafiltration concentrate is adjusted to 4.3 with sodium citrate, 4500 g of ion exchange filler CM sep life FF is added, and the mixture is stirred at 200 RPM for 1 h. The ion exchange filler is collected by sieve filtration, and the filler is rinsed with 28 L of purified water. After elution, the ion exchange filler is extracted with 15 L of 16.2 g / L sodium chloride solution for 40 min, and 14.7 L of an extract of the target protein is obtained by sieve filtration. During the elution and extraction steps, the temperature of the ion exchange filler is always maintained at 10° C.

[0048] (6) The extract obtained in step (5) was ultrafiltered and concentrated through a 6KD spiral membrane ultrafiltration system until the permeate conductivity was 92uS / cm, and 2.0L of the retentate was collected.

[0049] (7) The retentate obtained in step (6) was freeze-dried to obtain 52.8 g of recombinant type XVII collagen freeze-dried powder.

[0050] The purity of the recombinant type XVII collagen freeze-dried powder obtained in this example was tested to be 96.9%; the result of bacterial endotoxin test was qualified.

[0051] Example 3

[0052] (1) After the fermentation is completed, 67 L of the fermentation liquid is taken and centrifuged in a disc centrifuge (feed rate 180 L / h, slag discharge interval 700 s, slag discharge flushing water 3 s), the precipitate is discarded, and the centrifugal supernatant is collected to obtain 53 L of centrifugal supernatant;

[0053] (2) 53 L of the centrifugal supernatant collected in step (1) was filtered through a 0.22 μm hollow fiber filter membrane for clarification, and 10 L of purified water was added after the filtration to ensure the protein recovery rate, and 51 L of the filtrate was collected;

[0054] (3) The filtrate obtained in step (2) was ultrafiltered and concentrated using a 6KD spiral membrane ultrafiltration system until the permeate conductivity was 1.2 mS / cm, obtaining 18 L of ultrafiltration concentrate;

[0055] (4) The ultrafiltration concentrate obtained in step (3) was cooled to 2°C, and then 270 g of Triton X-114 was added, the ultrafiltration concentrate was maintained at 2°C, and allowed to stand for 1 hour.

[0056] (5) The ultrafiltration concentrate obtained in step (4) was maintained at 2°C, and the pH value of the ultrafiltration concentrate was adjusted to 4.0 with sodium citrate. 3600 g of ion exchange filler SP sep life FF was added and stirred at 200 RPM for 2 h. The ion exchange filler was collected by sieve filtration and rinsed with 29 L of purified water. After rinsing, the ion exchange filler was extracted with 15 L of 20.5 g / L sodium chloride solution for 35 min. 14.8 L of extract of the target protein was obtained by sieve filtration. During the rinsing and extraction steps, the temperature of the ion exchange filler was always maintained at 10°C.

[0057] (6) The extract obtained in step (5) was ultrafiltered and concentrated through a 6KD spiral membrane ultrafiltration system until the conductivity of the permeate was 76uS / cm, and 1.8L of the retentate was collected.

[0058] (7) The retentate obtained in step (6) was freeze-dried to obtain 48.7 g of recombinant type XVII collagen freeze-dried powder.

[0059] The purity of the recombinant type XVII collagen freeze-dried powder obtained in this example was tested to be 97.4%; the result of bacterial endotoxin test was qualified.

[0060] Comparative Example 1

[0061] The difference between the preparation method of this comparative example and that of the above-mentioned Example 1 is that in step (4), the ultrafiltration concentrate is cooled to and maintained at 15°C.

[0062] By freeze-drying the obtained retentate, 48.6 g of recombinant type XVII collagen freeze-dried powder was obtained, and its purity was 96.4% after testing; its bacterial endotoxin limit exceeded 0.5 EU / mg (the prescribed limit should be lower than 0.5 EU / mg), and the test result was unqualified.

[0063] In comparative example 1, the temperature of the ultrafiltration concentrate in step (4) was too high (>10°C), resulting in insufficient encapsulation of endotoxin by the subsequent Triton X-114, and part of the endotoxin was not removed by encapsulation, resulting in the endotoxin content limit of the final pure lyophilized powder being >0.5EU / mg.

[0064] Comparative Example 2

[0065] The preparation method of this comparative example is different from that of the above-mentioned Example 1 only in that the content of Triton X-114 added in step (4) in the system is 5 g / L.

[0066] By freeze-drying the obtained retentate, 46.7 g of recombinant type XVII collagen freeze-dried powder was obtained. The purity was tested to be 96.5%, and the bacterial endotoxin limit exceeded 0.5 EU / mg (the prescribed limit should be less than 0.5 EU / mg), and the test result was unqualified.

[0067] In Comparative Example 2, the content of Triton X-114 in the system in step (4) was less than 10 g / L. Too little Triton X-114 was added, resulting in the inability to remove all endotoxin inclusions, resulting in the endotoxin content limit of the final pure lyophilized powder being greater than 0.5 EU / mg.

[0068] Comparative Example 3

[0069] The difference between the preparation method of this comparative example and that of the above-mentioned Example 1 is that in step (4), Triton X-114 is added and then the mixture is allowed to stand in a cold water bath for 30 minutes.

[0070] By freeze-drying the obtained retentate, 48.9 g of recombinant type XVII collagen freeze-dried powder was obtained. The purity was tested to be 95.7%. The bacterial endotoxin limit exceeded 0.5 EU / mg (the prescribed limit should be less than 0.5 EU / mg), and the test result was unqualified.

[0071] In comparative example 3, the cold water bath time after adding Triton X-114 in step (4) was less than 1 h. The time was too short for Triton X-114 to fully remove the endotoxin, resulting in the endotoxin content limit of the final pure lyophilized powder being greater than 0.5 EU / mg.

[0072] Comparative Example 4

[0073] The preparation method of this comparative example differs from that of the above-mentioned Example 1 only in that Triton X-114 is not added in step (4).

[0074] By freeze-drying the obtained retentate, 47.2 g of recombinant type XVII collagen freeze-dried powder was obtained. The purity was tested to be 96.7%, and the bacterial endotoxin limit exceeded 0.5 EU / mg (the prescribed limit should be lower than 0.5 EU / mg), and the test result was unqualified.

[0075] In Comparative Example 4, if Triton X-114 is not added in step (4), the endotoxin cannot be removed, resulting in the pure endotoxin content limit of the final pure lyophilized powder being > 0.5 EU / mg.

[0076] Comparative Example 5

[0077] The difference between the preparation method of this comparative example and that of the above-mentioned Example 1 is only that the content of Triton X-114 added in step (4) in the system is 20 g / L.

[0078] By freeze-drying the obtained retentate, 16.8 g of recombinant type XVII collagen freeze-dried powder was obtained, and the purity was 96.5% after testing; after bacterial endotoxin testing, the endotoxin content limit (<0.5EU / mg) was tested to be qualified.

[0079] In comparative example 5, the content of Triton X-114 in the system in step (4) was greater than 15 g / L. Excessive addition of Triton X-114 resulted in insufficient column loading of the target protein, which significantly reduced the amount of protein in the final pure lyophilized powder collected.

[0080] Comparative Example 6

[0081] The difference between the preparation method of this comparative example and that of the above-mentioned Example 1 is only that in step (5), the pH of the ultrafiltration concentrate is adjusted to 5.5.

[0082] By freeze-drying the obtained retentate, 39.5 g of recombinant type XVII collagen freeze-dried powder was obtained, and the purity of the freeze-dried powder sample was 91.5% detected by electrophoresis; the bacterial endotoxin test showed that the endotoxin content limit (<0.5EU / mg) was qualified.

[0083] In Comparative Example 6, the pH of the ultrafiltration concentrate in step (5) was adjusted too high, and some impurities combined with the ion exchange filler and were eluted along with the target protein, resulting in a significant reduction in the purity of the final pure lyophilized powder protein.

[0084] Comparative Example 7

[0085] The difference between the preparation method of this comparative example and the above-mentioned Example 1 is only that the concentration of sodium chloride in the washing liquid in step (5) is 32.1 g / L.

[0086] By freeze-drying the obtained retentate, 42.8 g of recombinant type XVII collagen freeze-dried powder was obtained, and the purity was 92.1% after testing; after bacterial endotoxin testing, the endotoxin content limit (<0.5EU / mg) was tested to be qualified.

[0087] In Comparative Example 7, the sodium chloride content in the washing solution in step (5) was too high, and some impurities were extracted together with the target protein, resulting in a significant decrease in the purity of the protein in the final pure lyophilized powder.

[0088] Comparative Example 8

[0089] The difference between the preparation method of this comparative example and that of the above-mentioned Example 1 is only that in step (5), the pH of the ultrafiltration concentrate is adjusted to 3.5.

[0090] By freeze-drying the obtained retentate, 39.5 g of recombinant type XVII collagen freeze-dried powder was obtained, and the purity was 90.4% after testing. The bacterial endotoxin test showed that the endotoxin content limit (<0.5 EU / mg) was qualified.

[0091] In Comparative Example 8, the pH of the retentate obtained in step (5) was adjusted to be too low, and some impurities combined with the ion exchange filler and were eluted along with the target protein, resulting in a significant reduction in the purity of the final pure lyophilized powder protein.

[0092] Comparative Example 9

[0093] The difference between the preparation method of this comparative example and the above-mentioned Example 1 is that the concentration of sodium chloride in the washing liquid in step (5) is 8.8 g / L.

[0094] By freeze-drying the obtained retentate, 26.8 g of recombinant type XVII collagen freeze-dried powder was obtained, and the purity was 97.1% after testing; after bacterial endotoxin testing, the endotoxin content limit (<0.5EU / mg) was tested to be qualified.

[0095] In Comparative Example 9, the sodium chloride content in the washing solution in step (5) is too low, and the target protein is not fully extracted, resulting in a significant decrease in the protein content of the final pure freeze-dried powder collected.

[0096] Comparative Example 10

[0097] The difference between the preparation method of this comparative example and that of the above-mentioned Example 1 is that the ion exchange filler is added in step (5) and then stirred at room temperature.

[0098] By freeze-drying the obtained retentate, 38.8 g of recombinant type XVII collagen freeze-dried powder was obtained, and its purity was 89.1% after testing; its bacterial endotoxin limit exceeded 0.5 EU / mg (the prescribed limit should be lower than 0.5 EU / mg), and the test result was unqualified.

[0099] In Comparative Example 10, the mixture was stirred at room temperature in step (5). Triton X-114 was turbid at room temperature, so that the subsequent filtration could not remove Triton X-114 and endotoxin substances. The pure endotoxin content limit of the pure lyophilized powder collected was >0.5EU / mg.

[0100] The results of SDS-PAGE electrophoresis of recombinant type XVII collagen in some embodiments and comparative examples are as follows Figure 1As shown; Lane 1 and Lane 2 are the electrophoresis bands of Example 1 and Example 2, both of which are normal; Lane 3 is the electrophoresis band of Comparative Example 6. Due to the high pH of the ultrafiltration concentrate, some impurities are combined with the ion exchange filler and eventually eluted together, resulting in an impurity band below the main band; Lane 4 is the electrophoresis band of Comparative Example 7. Due to the excessively high sodium chloride content in the washing solution, some impurities are extracted together with the target protein, resulting in an impurity band below the main band; Lane 5 is the electrophoresis band of Comparative Example 10. Due to the stirring operation at room temperature, Triton X-114 is turbid and cannot be removed subsequently, resulting in an impurity band below the main band.

[0101] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for purifying recombinant type XVII collagen expressed by Pichia pastoris fermentation, characterized in that: The following steps are involved: (1) centrifuging the fermentation broth of recombinant type XVII collagen expressed by Pichia pastoris engineered bacteria, collecting the centrifuge and filtering; (2) ultrafiltration and concentration of the filtered centrifuge obtained in step (1); (3) After cooling the obtained ultrafiltration concentrate to 2-10° C., Triton X-114 is added, and the feed liquid temperature is maintained at 2-10° C. and allowed to stand; the content of the Triton X-114 in the ultrafiltration concentrate is 10-15 g / L; (4) maintaining the ultrafiltration concentrate obtained after standing at 2-10° C., adjusting the pH to 4-4.5, and then adding ion exchange filler for chromatography; collecting the retained ion exchange filler by filtration and rinsing it with purified water, then leaching the ion exchange filler with the washing solution, and collecting the leaching solution; during the elution and leaching process, the temperature of the ion exchange filler is also maintained at 2-10° C.; (5) The obtained extract is concentrated by ultrafiltration to collect the retentate, and the retentate is freeze-dried to obtain recombinant type XVII collagen freeze-dried powder.

2. The method for purifying recombinant type XVII collagen expressed by Pichia pastoris fermentation according to claim 1, characterized in that: In the step (4), the ion exchange filler is a cation exchange filler; the amount of the ion exchange filler added to the ultrafiltration concentrate is not less than 200 g / L; the specific chromatography method is: maintaining the feed liquid at 2-10° C. and stirring for not less than 1 hour.

3. The method for purifying recombinant type XVII collagen expressed by Pichia pastoris fermentation according to claim 1, characterized in that: In the step (4), the washing liquid is a sodium chloride solution with a concentration of 14.6 g / L-20.5 g / L.

4. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to claim 1, characterized in that: In the step (4), the amount of purified water used in the elution process is not less than 5 times the mass of the ion exchange filler.

5. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to claim 1, characterized in that: In the step (3), the standing time is ≥ 1 h.

6. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to claim 1, characterized in that: In the step (2), ultrafiltration is performed and concentrated until the conductivity of the permeate is less than 2 mS / cm.

7. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to claim 1, characterized in that: In the step (5), ultrafiltration is performed and concentrated until the conductivity of the permeate is less than 100uS / cm.

8. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to claim 1, characterized in that: In the steps (2) and (5), 6KD roll filter membrane is used for ultrafiltration and concentration.

9. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to any one of claims 1 to 8, characterized in that: The sequence of the recombinant type XVII collagen is shown in SEQ ID NO.

1.

10. The method for purifying recombinant type XVII collagen expressed by fermentation of Pichia pastoris according to any one of claims 1 to 8, characterized in that: The purity of the recombinant type XVII collagen freeze-dried powder is ≥95%, and the endotoxin content is <0.5EU / mg.

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