Process for purifying recombinant humanized collagen easy to produce

Abstract

The invention relates to the technical field of biological materials, in particular to an easy-to-produce recombinant humanized collagen purification process which comprises the following steps: performing centrifugal separation on a fermentation broth of a pichia pastoris expression strain to obtain a fermentation supernatant, and performing ultrafiltration concentration to obtain a fermentation supernatant concentrated solution; adding an ammonium sulfate solution into the fermentation supernatant concentrated solution, stirring, carrying out centrifugal separation, and collecting a precipitate I; dissolving the precipitate I with purified water, stirring and dissolving, repeating the step S2 again, and collecting a precipitate II; and dissolving the precipitate II with purified water, and then carrying out ultrafiltration, liquid change and concentration to obtain a purified recombinant humanized collagen solution. The recombinant humanized collagen purification process provided by the invention is simple in pretreatment process, easy to produce, relatively low in cost, small in protein loss and relatively high in protein recovery rate and purity; the prepared recombinant humanized collagen can be used as a raw material in the industries of medical beauty masks or biological dressings and the like, and can also be used for a subsequent refined chromatography process.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a recombinant humanized collagen purification process that is easy to produce. Background Art

[0002] Since collagen can promote the growth of epidermal cells, maintain skin elasticity, and promote wound healing, it can be used in skin care products to moisturize, repair, and anti-aging, so collagen cosmetics are gradually occupying the market share. At the same time, collagen has a unique organizational structure and biological function, and plays an important role in maintaining the normal physiological functions and damage repair of cells, tissues, and organs. In addition, collagen has the characteristics of low immunogenicity, biodegradability, good biocompatibility and mechanical properties, and is a good wound repair material.

[0003] In addition to collagen extracted from animals, recombinant human collagen is a recombinant protein that modifies or resynthesizes a gene sequence of human collagen and then expresses the new gene sequence through a host protein expression system. Recombinant human collagen has the advantages of collagen antigenicity, biodegradability, and good biocompatibility, and does not have the disadvantages of animal collagen, such as viral risks and immune rejection, so it is widely used in the field of biomaterials.

[0004] In the prior art, the preparation of recombinant humanized collagen, especially recombinant human type III collagen, mostly adopts E. coli or yeast expression system. The collected bacteria from E. coli expression are crushed and then purified for protein, while the protein prepared by the yeast expression system is secretory expression, and the protein can be extracted directly from the fermentation broth. The commonly used recombinant humanized collagen purification process is to collect the prepared fermentation broth by centrifugation, and then use hydrophobic, gel filtration, molecular sieve, affinity chromatography, ion exchange and other multi-stage purification to obtain the target protein; this purification process is complicated, the protein loss is large, and the final protein recovery rate is low. Summary of the invention

[0005] In order to solve the problem of low protein recovery rate in the recombinant humanized collagen purification process in the prior art, the present invention provides a recombinant humanized collagen purification process that is easy to produce. The purification process is easy to produce, has a simple process, has a high protein expression amount, helps to save costs, and solves the problem of low protein recovery rate in the recombinant humanized collagen purification process in the prior art.

[0006] The technical solution adopted by the present invention to solve its technical problem is: A recombinant humanized collagen purification process that is easy to produce comprises the following steps: S1: from the fermentation broth of the Pichia pastoris expression strain, a fermentation supernatant is obtained by centrifugation, and then concentrated by ultrafiltration to obtain a fermentation supernatant concentrate; S2: adding ammonium sulfate solution to the fermentation supernatant concentrate, stirring, centrifuging, and collecting precipitate 1; S3: dissolving the precipitate 1 with purified water, stirring to dissolve, repeating step S2 again, and collecting the precipitate 2; S4: dissolving the second precipitate with purified water and then performing ultrafiltration to exchange the liquid to obtain a purified recombinant humanized collagen solution.

[0007] Optionally, the Pichia pastoris is Pichia pastoris expressing type III recombinant human collagen.

[0008] Optionally, the saturation of the ammonium sulfate solution in step S2 is 15% to 50%.

[0009] Optionally, the saturation of the ammonium sulfate solution in step S2 is 35% to 40%.

[0010] Optionally, after ultrafiltration concentration in step S1, the volume of the fermentation supernatant concentrate is 1 / 5 to 1 / 6 of the volume of the fermentation broth.

[0011] Optionally, the ultrafiltration and liquid exchange and concentration in step S4 is carried out by ultrafiltration and liquid exchange and concentration through a hollow fiber column or through a membrane package.

[0012] Optionally, the volume of the purified water in step S3 is the same as the volume of the fermentation supernatant concentrate in step S2.

[0013] Optionally, the pH range of the purified recombinant humanized collagen solution is 3.5-4.0.

[0014] Optionally, the conductivity of the purified recombinant humanized collagen solution is 200-300 μS / cm.

[0015] The beneficial effects of the present invention are: The recombinant humanized collagen purification process that is easy to produce provided by the present invention has a simple pretreatment process, is easy to produce, has low cost, and has small protein loss, and has high protein recovery rate and purity; the prepared recombinant humanized collagen can be used as raw materials in industries such as medical beauty masks or biological dressings, and can also be used in subsequent refined chromatography processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0017] Figure 1 It is the SDS-PAGE expression molecular weight identification of the recombinant humanized collagen in the present invention; Figure 2 This is the appearance of the recombinant humanized collagen solution obtained after purification in Example 1 of the present invention; Figure 3 This is the freeze-dried recombinant humanized collagen fiber sample of Example 1 of the present invention. DETAILED DESCRIPTION

[0018] The present invention is now further described in detail. The embodiments described below are exemplary and intended to be used to explain the present invention, but cannot be understood as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0019] In order to solve the problem of low protein recovery rate in the recombinant humanized collagen purification process in the prior art, the present invention provides a recombinant humanized collagen purification process that is easy to produce, and the purification comprises the following steps: S1: centrifugally separating the fermentation broth of the Pichia pastoris expression strain to obtain a fermentation supernatant containing the recombinant humanized collagen, and reducing the volume by ultrafiltration concentration to obtain a fermentation supernatant concentrate; The fermentation broth of the Pichia pastoris expression strain in this step can be obtained by fermentation using a specific fermentation method, or by fermentation using a method described in the prior art; The present invention preferably adopts the conventional methanol induction method of yeast secretory expression for fermentation, and the main process is as follows: (1) First, culture the seed solution. Calculate the required seed solution according to the inoculation amount of 5%~6%. The engineered bacteria (Pichia pastoris RX-2, constructed by our laboratory, Pichia pastoris RX-2 is deposited in Wuhan China Center for Type Culture Collection, with the deposit number of CCTCCNO. M 20242213 and the deposit date of October 15, 2024) are inoculated into a shake flask containing a certain volume of YPD medium. The temperature is controlled at 30°C and the shaker speed is set to 180~200rpm. After 18-24h of culture, inoculate it into a new secondary seed medium YPD shake flask and continue to culture until OD 600 =10-15.

[0020] (2) Tank inoculation: A 60L fermentation tank was filled with about 50L of BSM medium. After high-pressure steam sterilization, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted to 5.0 with ammonia water. First, 200mL of PTM1 was inoculated, and then the seed solution cultured at a 6% inoculation amount was inoculated into the tank, and the preliminary culture was carried out according to the set program.

[0021] (3) Feed growth stage: When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and 50% (w / v) glycerol is added (1L of 50% glycerol is added, and 12mL of PTM1 is added) until the bacterial OD reaches 600 When the concentration of glycerol is 100-150 and the wet weight is 180-220g / L, stop adding glycerol.

[0022] (4) Methanol induction stage: After the glycerol in the above tank is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12 mL of PTM1 is added for every 1 L of methanol added), and the methanol induction culture stage is entered. The rotation speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to monitor the bacterial concentration, wet weight and fermentation supernatant protein content during the fermentation process. The fermentation process can be stopped when the bacterial OD value decreases (about 70 hours) or when the protein expression level no longer changes.

[0023] (5) Collect about 60L of fermentation broth. The target protein, recombinant humanized collagen, can be obtained from the fermentation supernatant.

[0024] S2: adding ammonium sulfate solution to the fermentation supernatant concentrate obtained in step S1, stirring, preferably stirring for 0.5-4 hours, centrifuging, and collecting the precipitate, which is recorded as precipitate 1; S3: dissolving precipitate 1 with purified water, stirring to dissolve, repeating step S2 again, and collecting the precipitate, which is recorded as precipitate 2; S4: dissolving the second precipitate with purified water and then performing ultrafiltration to exchange the liquid to obtain a purified and clarified recombinant humanized collagen solution, i.e., a purified recombinant humanized collagen solution.

[0025] The recombinant humanized collagen solution after purification in step S4 is further sampled for corresponding index evaluation. The recombinant humanized collagen solution can be directly freeze-dried into recombinant humanized collagen fibers (used as raw materials in the medical beauty industry such as facial masks), or can be subsequently purified according to the intended use.

[0026] The recombinant humanized collagen purification process that is easy to produce provided by the present invention has a simple pretreatment process, is easy to produce, has low cost, and has small protein loss, and has high protein recovery rate and purity; the prepared recombinant humanized collagen can be used as raw materials in industries such as medical beauty masks or biological dressings, and can also be used in subsequent refined chromatography processes.

[0027] This method is not only simple to operate, time-saving and labor-saving, but also can reduce the loss of expensive pure fillers, extend the service life of fillers and save costs.

[0028] The preferred Pichia pastoris of the present invention is Pichia pastoris expressing type III recombinant human collagen. Specifically, the preferred Pichia pastoris species RX-2 is constructed by our laboratory and preserved in the China Center for Type Culture Collection, Wuhan, with a preservation number of CCTCC M 20242213.

[0029] In order to balance the protein recovery rate and purification cost, the saturation of the ammonium sulfate solution in step S2 of the present invention is preferably 15% to 50%, and more preferably 35% to 40%.

[0030] In order to take into account both the protein recovery rate and the purification efficiency, the volume of the fermentation supernatant concentrate obtained after ultrafiltration concentration in step S1 of the present invention is preferably 1 / 5 to 1 / 6 of the volume of the fermentation broth; preferably, the volume of the purified water in step S3 is the same as the volume of the fermentation supernatant concentrate in step S2; and preferably, the ultrafiltration liquid replacement method in step S4 is ultrafiltration liquid replacement through a hollow fiber column or ultrafiltration liquid replacement through a membrane package.

[0031] To ensure protein quality, the present invention preferably has a pH range of 3.5-4.0 for the purified recombinant humanized collagen solution; and preferably has a conductivity of 200-300 μS / cm for the purified recombinant humanized collagen solution.

[0032] The present invention provides a recombinant humanized collagen purification process that is easy to produce and has a simple process. The purification method described in the present invention achieves the purpose of increasing protein expression and saving costs, and ultimately recombinant humanized collagen freeze-dried fibers with a protein purity of 60% can be obtained.

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0034] Unless otherwise specified, the fermentation broth of the Pichia pastoris expression strain in each embodiment of the present invention and the comparative example is obtained by fermentation according to the following method: The present invention adopts the conventional methanol induction method of yeast secretory expression to carry out fermentation, and the main process is as follows: (1) First, the seed solution was cultured. The required seed solution was calculated according to the inoculation volume of 6%. The engineered bacteria (Pichia pastoris strain RX-2, constructed by our laboratory and preserved in Wuhan China Center for Type Culture Collection, with the preservation number CCTCC M20242213) were inoculated into a shake flask containing a certain volume of YPD medium. The temperature was controlled at 30°C and the shaker speed was set to 190 rpm. After culturing for 20 hours, the new secondary seed medium YPD shake flask was inoculated and cultured until OD 600 =12.

[0035] (2) Tank inoculation: A 60L fermentation tank was filled with about 50L of BSM medium. After high-pressure steam sterilization, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted to 5.0 with ammonia water. First, 200mL of PTM1 was inoculated, and then the seed solution cultured at a 6% inoculation amount was inoculated into the tank, and the preliminary culture was carried out according to the set program.

[0036] (3) Feed growth stage: When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and 50% (w / v) glycerol is added (1L of 50% glycerol is added, and 12mL of PTM1 is added) until the bacterial OD reaches 600 is 120, wet weight is 200g / L, stop adding glycerol.

[0037] (4) Methanol induction stage: After the glycerol in the above tank is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12 mL of PTM1 is added for every 1 L of methanol added), and the methanol induction culture stage is entered. The rotation speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to monitor the bacterial concentration, wet weight and fermentation supernatant protein content during the fermentation process. The fermentation process can be stopped when the bacterial OD value decreases (about 70 hours) or when the protein expression level no longer changes.

[0038] (5) Collect about 60L of fermentation broth. The target protein, recombinant humanized collagen, can be obtained from the fermentation supernatant.

[0039] The amount of ammonium sulfate added in each embodiment of the present invention and the comparative example is obtained according to Table 1.

[0040] Example 1 S1: From the 60L fermentation liquid obtained from the fermenter, a fermentation supernatant containing recombinant humanized collagen is obtained by centrifugation, and the volume is reduced to 10L by ultrafiltration concentration to obtain a fermentation supernatant concentrate; S2: Take 1L of fermentation supernatant concentrate, add 40% saturated ammonium sulfate solution according to the saturated ammonium sulfate calculation table in Table 1, slowly add 243g of ammonium sulfate while stirring, stir until completely dissolved, continue stirring for 1 hour, centrifuge and collect the precipitate, which is recorded as precipitate 1; S3: dissolving the above precipitate 1 with 1L of purified water and stirring it with a stirrer to dissolve it, repeating step S2 again, and collecting the precipitate, which is recorded as precipitate 2; S4: Dissolve the protein precipitate 2 with 1L of purified water, and ultrafilter through a hollow fiber column to an aqueous solution with a pH of about 3.5, thereby obtaining 0.5L of purified and clarified recombinant humanized collagen solution. The pH value was 3.85, and the conductivity was 243μS / cm; S5: The above protein solution is passed through a vacuum freeze dryer to obtain recombinant humanized collagen freeze-dried fibers. Figure 2 , Figure 3 As shown, the recombinant humanized collagen freeze-dried fiber has a white appearance, a smooth surface, no pigment residue, and is in the shape of a fiber sponge with certain mechanical tensile properties.

[0041] Example 2 S1: From the 60L fermentation liquid obtained from the fermenter, a fermentation supernatant containing recombinant humanized collagen is obtained by centrifugation, and the volume is reduced to 10L by ultrafiltration concentration to obtain a fermentation supernatant concentrate; S2: Take 1L of fermentation supernatant concentrate, add 35% saturated ammonium sulfate solution according to the saturated ammonium sulfate calculation table in Appendix 1, slowly add 209g of ammonium sulfate while stirring, stir until completely dissolved, continue stirring for 2 hours, centrifuge and collect the precipitate, which is recorded as precipitate 1; S3: Dissolve the above precipitate 1 with 1L of purified water and stir it with a stirrer to dissolve it. Repeat step 2b and collect the precipitate, which is recorded as precipitate 2. S4: Dissolve the protein precipitate 2 with 1L of purified water, and ultrafilter through a hollow fiber column to an aqueous solution with a pH of about 3.5, thereby obtaining 0.5L of purified and clarified recombinant humanized collagen solution. The pH value was 3.83, and the conductivity was 245μS / cm; S5: The above protein solution is passed through a vacuum freeze dryer to obtain recombinant humanized collagen freeze-dried fibers, which are white in appearance, have no obvious pigment, have a smooth surface, and have a certain mechanical tensile strength and are in the shape of a fiber sponge.

[0042] Example 3 S1: From the 60L fermentation liquid obtained from the fermenter, a fermentation supernatant containing recombinant humanized collagen is obtained by centrifugation, and the volume is reduced to 10L by ultrafiltration concentration to obtain a fermentation supernatant concentrate; S2: Take 1L of fermentation supernatant concentrate, add 40% saturated ammonium sulfate solution according to the saturated ammonium sulfate calculation table in Appendix 1, slowly add 243g of ammonium sulfate while stirring, stir until completely dissolved, continue stirring for 1 hour, centrifuge and collect the precipitate, which is recorded as precipitate 1; S3: dissolving the above precipitate 1 with 1L of purified water and stirring it with a stirrer to dissolve it, repeating step S2 twice again, and collecting the precipitate; S4: Dissolve the protein precipitate with 1L of purified water, and ultrafilter the solution through a hollow fiber column to an aqueous solution with a pH of about 3.5, thereby obtaining 0.5L of purified and clarified recombinant humanized collagen solution. The pH value was 3.77, and the conductivity was 253μS / cm; S5: The above protein solution is passed through a vacuum freeze dryer to obtain recombinant humanized collagen freeze-dried fibers. The fibers are white in appearance, pigment-free, smooth in surface, and have a certain mechanical tensile strength and are in the form of sponges.

[0043] Comparative Example 1 S1: From the 60L fermentation liquid obtained from the fermenter, a fermentation supernatant containing recombinant humanized collagen is obtained by centrifugation, and the volume is reduced to 10L by ultrafiltration concentration to obtain a fermentation supernatant concentrate; S2: Take 1L of fermentation supernatant concentrate, add 40% saturated ammonium sulfate solution according to the saturated ammonium sulfate calculation table in Appendix 1, slowly add 243g of ammonium sulfate while stirring, stir until completely dissolved, continue stirring for 1 hour, and collect the precipitate by centrifugation; S3: Dissolve the above precipitate with 1L of purified water, and ultrafilter through a hollow fiber column to an aqueous solution with a pH of about 3.5, thereby obtaining 0.5L of purified and clarified recombinant humanized collagen solution. The pH value is 3.81, and the conductivity is 248μS / cm; S4: The above protein solution is passed through a vacuum freeze dryer to obtain recombinant humanized collagen freeze-dried fibers, which are slightly green in appearance, have a smooth surface, and are in the shape of fiber sponges with certain mechanical tensile properties.

[0044] Comparative Example 2 S1: From the 60L fermentation liquid obtained from the fermenter, a fermentation supernatant containing recombinant humanized collagen is obtained by centrifugation, and the volume is reduced to 10L by ultrafiltration concentration to obtain a fermentation supernatant concentrate; S2: Take 1L of fermentation supernatant concentrate, fill the column with ion exchange filler, column volume 400mL, AKTA explorer chromatography system, purification process: water washing - alkaline washing - water washing - balance - sample loading - collection of flow-through liquid - column treatment, etc. Sample loading setting Flow: 20mL / min; finally collect the fermentation supernatant, remove most of the pigments, the treatment time takes about 5 hours; S3: The sample solution collected above was ultrafiltered through a hollow fiber column to an aqueous solution with a pH of about 3.5, and the conductivity was 500 μS / cm, thus obtaining 0.5 L of purified and clarified recombinant humanized collagen solution. The pH was tested to be 3.81 and the conductivity was 302 μS / cm; S4: The above protein solution is passed through a vacuum freeze dryer to obtain recombinant humanized collagen freeze-dried fibers, which are slightly green in appearance, have a smooth surface, and are in the shape of fiber sponges with certain mechanical tensile properties.

[0045] Comparative Example 3 S1: From the 60L fermentation liquid obtained from the fermenter, a fermentation supernatant containing recombinant humanized collagen is obtained by centrifugation, and the volume is reduced to 10L by ultrafiltration concentration to obtain a fermentation supernatant concentrate; S2: Take 1L of fermentation supernatant concentrate, add 20% saturation ammonium sulfate solution according to the saturated ammonium sulfate calculation table in Table 1, slowly add 114g of ammonium sulfate while stirring, stir until completely dissolved, continue stirring for 1 hour, centrifuge and collect the precipitate, recorded as precipitate 1; it should be noted that it is actually difficult to completely extract the protein here, there will be protein loss, a large amount of protein is still in the fermentation broth and has not been collected, and it is obviously observed here that there is less precipitation; S3: Dissolve the above precipitate 1 with 1L of purified water, and stir it with a stirrer to dissolve it. According to the saturated ammonium sulfate calculation table in Table 1, add 60% saturated ammonium sulfate solution, and slowly add 390g of ammonium sulfate while stirring. After stirring and dissolving completely, continue stirring for 1 hour, and collect the precipitate by centrifugation, which is recorded as precipitate 2; S4: Dissolve the protein precipitate 2 with 1L of purified water, and ultrafilter through a hollow fiber column to an aqueous solution with a pH of about 3.5, thereby obtaining 0.5L of purified and clarified recombinant humanized collagen solution. The pH value was 3.65, and the conductivity was 287μS / cm; S5: The above protein solution is passed through a vacuum freeze dryer to obtain recombinant humanized collagen freeze-dried fibers, which are slightly white in appearance with slightly green pigment residue, with unclear fibers and poor mechanical properties.

[0046] Table 1 Calculation table for saturated ammonium sulfate concentration at 25°C ①Note: At 25°C, when the ammonium sulfate solution is adjusted from the initial concentration to the final concentration, the number of grams of solid ammonium sulfate added per liter of solution.

[0047] The purification effects of the purification processes of the embodiments of the present invention and the comparative examples were tested. Figure 1 As shown, Figure 1 M in the middle stands for Takara Premixed Marker; No. 1 is the supernatant sample of yeast induction for 48 hours; No. 2 is the supernatant sample of yeast induction for 60 hours; No. 3 is the ultrafiltration and concentration sample of the supernatant after the fermentation is completed; No. 4 is the sample No. 3 diluted 5 times and loaded with the same volume. This figure shows the identification results of 5 μL of each sample loaded.

[0048] The products prepared in the above-mentioned embodiments and comparative examples were subjected to liquid chromatography purity detection, protein concentration (protein amount) detection, and appearance analysis after freeze-drying. The results are shown in Table 2.

[0049] Table 2 Group Purity of protein after purification Appearance after freeze drying Target protein yield Purification treatment time, (hours) Main equipment and consumables required for purification Example 1 61.76% White, smooth surface, dense fiber sponge 85% 3h+final ultrafiltration Centrifuge, ultrafiltration hollow fiber column Example 2 59.73% White, smooth and dense surface, spongy fiber 82% 3h+final ultrafiltration Centrifuge, ultrafiltration hollow fiber column Example 3 61.55% White, smooth fiber sponge 77% 4.5h+final ultrafiltration Centrifuge, ultrafiltration hollow fiber column Comparative Example 1 56.31% Light green, smooth and soft fiber sponge 92% 2h+final ultrafiltration Centrifuge, ultrafiltration hollow fiber column Comparative Example 2 60.35% Slightly green, smooth and dense fiber sponge 84% 6h+final ultrafiltration AKTA chromatography system, ion exchange filler + column, ultrafiltration hollow fiber column Comparative Example 3 40.45% Slightly green, loose surface, and no obvious fibrosis flakes 52% 3h+final ultrafiltration Centrifuge, ultrafiltration hollow fiber column Fermentation broth raw materials 46.22% / / / / By comparison, it can be seen that Example 1 has the best comprehensive effect and can be used for large-scale production.

[0050] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A recombinant humanized collagen purification process that is easy to produce, characterized in that: The steps include: S1: from the fermentation broth of the Pichia pastoris expression strain, a fermentation supernatant is obtained by centrifugation, and then concentrated by ultrafiltration to obtain a fermentation supernatant concentrate; S2: adding ammonium sulfate solution to the fermentation supernatant concentrate, stirring, centrifuging, and collecting precipitate 1; S3: dissolving the precipitate 1 with purified water, stirring to dissolve, repeating step S2 again, and collecting the precipitate 2; S4: dissolving the second precipitate with purified water and then performing ultrafiltration to exchange the liquid to obtain a purified recombinant humanized collagen solution.

2. The recombinant humanized collagen purification process that is easy to produce according to claim 1, characterized in that: The Pichia pastoris is a Pichia pastoris expressing type III recombinant human collagen.

3. The recombinant humanized collagen purification process that is easy to produce according to claim 1, characterized in that: The saturation of the ammonium sulfate solution in step S2 is 15% to 50%.

4. The recombinant humanized collagen purification process that is easy to produce as claimed in claim 3, characterized in that: The saturation of the ammonium sulfate solution in step S2 is 35% to 40%.

5. The recombinant humanized collagen purification process that is easy to produce according to any one of claims 1 to 4, characterized in that: After ultrafiltration concentration in step S1, the volume of the fermentation supernatant concentrate is 1 / 5 to 1 / 6 of the volume of the fermentation broth.

6. The recombinant humanized collagen purification process that is easy to produce according to claim 5, characterized in that: The ultrafiltration and liquid exchange and concentration in step S4 is carried out by ultrafiltration and liquid exchange and concentration through a hollow fiber column or through a membrane package.

7. The recombinant humanized collagen purification process that is easy to produce according to claim 5, characterized in that: The volume of the purified water in step S3 is the same as the volume of the fermentation supernatant concentrate in step S2.

8. The recombinant humanized collagen purification process that is easy to produce according to claim 5, characterized in that: The pH range of the purified recombinant humanized collagen solution is 3.5-4.

0.

9. The recombinant humanized collagen purification process that is easy to produce according to claim 5, characterized in that: The conductivity of the purified recombinant humanized collagen solution is 200-300 μS / cm.

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