阅读说明：本技术 利用毕赤酵母生产重组类人胶原蛋白和宿主细胞蛋白的方法 (Method for producing recombinant human-like collagen and host cell protein by using pichia pastoris ) 是由 郝东 王俊 何华斌 周浩 魏文培 宗奕珊 侯增淼 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种利用毕赤酵母生产重组类人胶原蛋白和宿主细胞蛋白的方法,利用菌株Pichia pastoris-col3-6于发酵培养过程中进行甲醇诱导,获得的发酵液经离心、过滤除菌、超滤脱盐浓缩、离子交换层析和超滤脱盐、冻干,得到重组类人胶原蛋白和目标宿主蛋白,本发明在保持发酵生产重组类人胶原蛋白的高产率、高收率及高纯度的同时,解决了工业化大规模生产毕赤酵母表达的重组类人胶原蛋白的高成本问题。(The invention discloses a method for producing recombinant human-like collagen and host cell protein by using Pichia pastoris, wherein a strain Pichia pastoris-col3-6 is used for carrying out methanol induction in the fermentation culture process, and the obtained fermentation liquor is subjected to centrifugation, filtration sterilization, ultrafiltration desalination concentration, ion exchange chromatography, ultrafiltration desalination and freeze-drying to obtain the recombinant human-like collagen and target host protein.)

1. A method for purifying protein of pichia pastoris fermentation liquor is characterized by comprising the following steps: the protein purification method comprises the following steps:

separating and removing thallus cells in the pichia pastoris fermentation liquor, then carrying out ultrafiltration, and carrying out ion exchange chromatography on the ultrafiltration product to respectively obtain recombinant human-like collagen and host cell protein produced by pichia pastoris fermentation.

2. The method for purifying protein of pichia pastoris fermentation broth according to claim 1, wherein: the protein purification method specifically comprises the following steps:

1) centrifuging the pichia pastoris fermentation liquor, filtering and sterilizing supernatant obtained by centrifuging, performing ultrafiltration desalination, and mixing concentrated solution containing recombinant human collagen obtained by ultrafiltration desalination with residual protein recovery solution of an ultrafiltration system containing host cell protein to obtain mixed feed liquid;

2) separating the recombinant human-like collagen and the host cell protein in the mixed feed liquid by ion exchange chromatography.

3. The method for purifying protein of pichia pastoris fermentation broth according to claim 1 or 2, wherein: the pichia pastoris is selected from a strain with the preservation number of CGMCC No.20458, and the protein components of the fermentation liquid of the strain comprise recombinant human collagen with 91.3KD and host cell protein with 65 KD.

4. The method for purifying protein of pichia pastoris fermentation broth according to claim 1 or 2, wherein: the preparation method of the pichia pastoris fermentation liquor comprises the following steps: culturing pichia in a fermentation culture medium and carrying out induced expression of the recombinant human-like collagen to obtain pichia fermentation liquor.

5. The method for purifying protein of pichia pastoris fermentation broth according to claim 2, wherein: the ultrafiltration desalination adopts a 6-15 KD ultrafiltration membrane, and the conductivity of the concentrated solution is lower than 1 ms/cm; the ion exchange column packing adopted by the ion exchange chromatography is selected from weak cation exchangers.

6. The method for purifying protein of pichia pastoris fermentation broth according to claim 2, wherein: the step 2) specifically comprises the following steps: adjusting the pH value of the mixed feed liquid to 6.0-6.5 by using sodium hydroxide to obtain an ion exchange chromatography stock solution, and adjusting the pH value of the ion exchange chromatography stock solution to 6.0-6.5 by using disodium hydrogen phosphate and sodium dihydrogen phosphate to carry out sample loading; after the sample loading is finished, eluting host cell protein by using a phosphate buffer solution with the pH value of 6.0-6.5 and the concentration of 8-10 mmol/L of 0.04-0.06 mol/L NaCl as an eluent, eluting by using a phosphate buffer solution with the pH value of 6.0-6.5 and the concentration of 8-10 mmol/L of 0.08-0.1 mol/L of NaCl as an impurity washing solution, and finally eluting the recombinant human-like collagen by using a phosphate buffer solution with the pH value of 6.0-6.5 and the concentration of 8-10 mmol/L of 0.3-0.4 mol/L of NaCl as an eluent.

7. The method for purifying protein of pichia pastoris fermentation broth according to claim 2, wherein: the protein purification method further comprises the following steps: after the step 2), respectively carrying out ultrafiltration and desalination on the recombinant human-like collagen solution and the host cell protein solution, and then carrying out vacuum freeze drying to obtain the recombinant human-like collagen and the host cell protein.

8. An application of pichia pastoris in fermentation production of recombinant human-like collagen is characterized in that: the protein purification method of the pichia pastoris fermentation liquor comprises the following steps: separating and removing thallus cells in the pichia pastoris fermentation liquor, then carrying out ultrafiltration, and carrying out ion exchange chromatography on an ultrafiltration product to respectively obtain recombinant human-like collagen and host cell protein generated by pichia pastoris fermentation; the pichia pastoris is selected from a bacterial strain with the preservation number of CGMCC No. 20458.

9. An application of pichia pastoris in the fermentation production of protein Marker is characterized in that: the protein purification method of the pichia pastoris fermentation liquor comprises the following steps: separating and removing thallus cells in the pichia pastoris fermentation liquor, then carrying out ultrafiltration, and carrying out ion exchange chromatography on an ultrafiltration product to respectively obtain recombinant human-like collagen and host cell protein generated by pichia pastoris fermentation; the pichia pastoris is selected from a bacterial strain with the preservation number of CGMCC No. 20458.

10. A Pichia pastoris for production of recombinant human-like collagen and host cell proteins, characterized by: the pichia pastoris is selected from a bacterial strain with the preservation number of CGMCC No. 20458.

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to a method for separating and purifying recombinant human-like collagen and host cell protein expressed by pichia pastoris.

Background

Collagen is a protein with the largest content in the body, accounts for about 25% -33% of the protein of the human body, is widely distributed in various tissues and organs of the human body, such as skin, bones, cornea, blood vessels and the like, particularly contains a large amount of collagen in the tissues of the skin and the like, and has an important role in maintaining the normal physiological functions of cells, tissues and organs as an adhesive substance of connective tissues. The recombinant collagen has the advantages of low toxicity, low antigenicity, low immunity, cell regeneration guiding performance, good biocompatibility and the like, and is widely applied to the industries of biological medicine, cosmetics, food and the like.

Protein Maker is made of a variety of purified proteins of known molecular weight and is widely used in biological experiments to monitor the migration of other proteins in gel electrophoresis and to identify the molecular weight of these proteins. Host cell protein expressed by pichia pastoris is stable in property and can be used as a potential raw material of protein marker.

The production cost of the recombinant human-like collagen expressed by pichia pastoris is higher due to the investment of equipment, personnel and production operation in the purification process of the recombinant human-like collagen at present. Meanwhile, host cell proteins expressed by pichia pastoris are not effectively utilized in the production process (the reasons include that the content of the host cell proteins in the fermentation broth is limited, and the host cell proteins are generally treated as impurities in purification, for example, chinese patent CN 108027032A).

Disclosure of Invention

The invention aims to provide a method for producing recombinant human-like collagen and host cell protein by using pichia pastoris.

In order to achieve the purpose, the invention adopts the following technical scheme:

a protein purification method of pichia pastoris fermentation liquor comprises the following steps:

separating and removing thallus cells in the pichia pastoris fermentation liquor, then carrying out ultrafiltration, and carrying out ion exchange chromatography on the ultrafiltration product to respectively obtain recombinant human-like collagen and host cell protein produced by pichia pastoris fermentation.

Preferably, the method for purifying the protein of the pichia pastoris fermentation broth specifically comprises the following steps:

1) centrifuging the fermentation liquor of the pichia pastoris, sequentially filtering and sterilizing supernatant obtained by centrifuging, ultrafiltering and desalting, and mixing concentrated solution containing the recombinant human collagen obtained by ultrafiltration and desalting with residual protein (components including host cell protein) recovery solution of an ultrafiltration system to obtain mixed feed liquid;

2) separating the recombinant human-like collagen and the host cell protein in the mixed feed liquid by ion exchange chromatography.

Preferably, the Pichia pastoris is selected from wild type or mutant Pichia pastoris (Pichia pastoris) genetically engineered bacteria.

Preferably, the Pichia pastoris is selected from a strain Pichia pastoris-col3-6 (belonging to genetically engineered bacteria), the preservation number of the strain is CGMCC No.20458, and the protein components of a fermentation liquid produced by the strain through fermentation comprise recombinant human-like collagen with the molecular weight of 91.3KD and host cell protein with the molecular weight of 65 KD.

Preferably, the preparation method of the pichia pastoris fermentation broth comprises the following steps: culturing pichia in a fermentation culture medium and carrying out induced expression of the recombinant human-like collagen to obtain pichia fermentation liquor.

Preferably, the preparation method of the pichia pastoris fermentation broth specifically comprises the following steps: culturing the pichia pastoris inoculated in the fermentation medium until a certain thallus density is reached, and performing methanol induction by replacing a carbon source of the fermentation medium with methanol in subsequent culture, wherein the culture conditions comprise: the pH value is 4.8-5.2, the temperature is 25-30 ℃, and the dissolved oxygen is controlled at 30%.

Preferably, the ultrafiltration desalination adopts a 6-15 KD ultrafiltration membrane, and the conductivity of the concentrated solution is lower than 1 ms/cm; the recovery liquid is obtained by recycling residual protein of the ultrafiltration system by using purified water.

Preferably, the ion exchange column packing used for the ion exchange chromatography is selected from weak cation exchangers taking agarose gel as a matrix.

Preferably, the step 2) specifically comprises the following steps: adjusting the pH value of the mixed feed liquid to 6.0-6.5 by using sodium hydroxide to obtain an ion exchange chromatography stock solution, and adjusting the pH value of the ion exchange chromatography stock solution to 6.0-6.5 by using disodium hydrogen phosphate and sodium dihydrogen phosphate to carry out sample loading; after the sample loading is finished, eluting host cell protein by using a phosphate buffer solution with the pH value of 6.0-6.5 and the concentration of 8-10 mmol/L of 0.04-0.06 mol/L NaCl as an eluent, eluting by using a phosphate buffer solution with the pH value of 6.0-6.5 and the concentration of 8-10 mmol/L of 0.08-0.1 mol/L of NaCl as an impurity washing solution, and finally eluting the recombinant human-like collagen by using a phosphate buffer solution with the pH value of 6.0-6.5 and the concentration of 8-10 mmol/L of 0.3-0.4 mol/L of NaCl as an eluent.

Preferably, the method for purifying the protein of the pichia pastoris fermentation broth further comprises the following steps: after the step 2), respectively carrying out ultrafiltration desalination on the recombinant human-like collagen solution and the host cell protein solution obtained by elution by adopting 6-15 KD ultrafiltration membranes, and then carrying out vacuum freeze drying to obtain the recombinant human-like collagen and the host cell protein.

The invention has the beneficial effects that:

in the process of separating and purifying the recombinant human-like collagen in the pichia pastoris fermentation liquor, other technological means are not added, so that high-purity host cell protein can be obtained simultaneously, the production cost of the recombinant human-like collagen is greatly reduced by the value generated by a byproduct (the host cell protein can be used as a protein Marker), and the method is suitable for industrial large-scale production. The invention solves the problem of high cost of industrial large-scale production of the recombinant human-like collagen expressed by pichia pastoris while maintaining high yield, high yield and high purity of the recombinant human-like collagen produced by fermentation.

Furthermore, the Pichia pastoris strain Pichia pastoris-col3-6 adopted by the invention not only can stably ferment and produce the recombinant human-like collagen, but also can produce host cell protein with higher content in fermentation compared with other known Pichia pastoris strains.

Furthermore, the invention can reduce the conductivity of the concentrated solution and improve the stability and the separation efficiency of the recombinant human-like collagen and the host cell protein by controlling the molecular interception amount of the ultrafiltration membrane.

Furthermore, the invention can simultaneously obtain two high-purity proteins by further ion exchange chromatography, wherein the recovery rates of the recombinant human collagen and the host cell protein are both up to more than 70%, and the purity is both up to more than 95%. Meanwhile, the two proteins are completely decolorized by ion exchange chromatography, and the method is suitable for industrial large-scale production.

Furthermore, the liquid reagents used in the purification process of the invention are all prepared by inorganic salt, and have simple components and low cost.

Drawings

FIG. 1 is a structural design diagram of recombinant human-like collagen: the protein contains 1011 amino acids of protein sequence full length, theoretical Molecular Weight (MW) and isoelectric Point (PI).

FIG. 2 is a SDS-PAGE electrophoresis chart of a fermentation broth purified product of the strain Pichia pastoris-col 3-6; wherein, lane 1: protein Marker, lane 2: recombinant human collagen (115KD, electrophoretic characterization error leads to higher molecular weight in the electropherogram compared to theoretical molecular weight), lane 3: target host protein (65 kD).

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

(I) fermentation strain acquisition

The invention obtains a Pichia pastoris strain (recorded as a strain Pichia pastoris-col3-6) capable of producing recombinant human-like collagen and target host protein (65KD host cell protein) by fermentation based on a previous-stage vector, genetic engineering strain construction (the Pichia pastoris host strain transformed by the vector is GS115) and a fermentation test experiment. The amino acid sequence of the recombinant human-like collagen is shown in figure 1.

The strain Pichia pastoris-col3-6 has been deposited in China general microbiological culture Collection center (CGMCC, address No. 3 Xilu No. 1 Beijing Kogyo Chengxiang) at 31.7.2020, with the deposit number of CGMCC No.20458 and is classified and named as Pichia pastoris.

(II) fermentation and protein purification

The invention carries out induction expression of recombinant human-like collagen in the fermentation culture process of strains, and obtains fermentation liquor through centrifugation, collection and centrifugation of supernatant, filtration sterilization, ultrafiltration desalination concentration, ion exchange chromatography, ultrafiltration desalination and freeze-drying to obtain the recombinant human-like collagen and target host protein, and the specific steps are as follows:

1) culturing and inducing the strain Pichia pastoris-col3-6, taking out, centrifuging the fermentation liquid with large capacity refrigerated centrifuge to remove thallus, and collecting supernatant;

wherein, the conditions of culturing and induced expression are as follows: taking an inorganic salt BSM culture medium as a bottom material, fermenting the bottom material by using a 10L fermentation tank (the pH is 5.0, the temperature is 29.0 ℃, and the dissolved oxygen is controlled to be about 30 percent), after the glycerol in the bottom material is completely consumed (generally, the wet weight of the thalli reaches 180-210 mg/mL), adding methanol for induction for 40-50 hours, and taking the bottom material out of the tank;

2) filtering the supernatant with 0.22 μm hollow fiber membrane to remove residual thallus and mechanical impurities, and collecting filtrate;

3) ultrafiltering and desalting the filtrate by a 10KD ultrafiltration membrane until the conductivity is lower than 1ms/cm, concentrating the volume to about 0.5L to obtain a concentrated solution, circularly recovering the residual protein of the ultrafiltration system by 300mL of purified water to obtain a recovered solution, fully mixing the concentrated solution and the recovered solution, and adjusting the pH value of the mixed solution to 6.4 by using a dilute sodium hydroxide solution to serve as an ion exchange chromatography stock solution for later use;

4) and (3) carrying out ion exchange chromatography purification on the stock solution (the ion exchange column is filled with CM Sepharose FF): adjusting pH of the ion exchange chromatography stock solution to 6.4 with buffer pair (disodium hydrogen phosphate and sodium dihydrogen phosphate) in 10mmol/L phosphate buffer solution with pH of 6.4 as loading solution, eluting with 10 column volumes of eluent with pH of 6.4 (specifically 10mmol/L phosphate buffer solution containing 0.06mol/L sodium chloride and pH of 6.4), collecting eluate as target host protein, eluting with 10 column volumes of eluent with pH of 6.4 (specifically 10mmol/L phosphate buffer solution containing 0.08mol/L sodium chloride and pH of 6.4), eluting with 10 column volumes of eluent with pH of 6.4 (specifically 10mmol/L phosphate buffer solution containing 0.3mol/L sodium chloride and pH of 6.4), and collecting eluate as recombinant human collagen solution;

5) respectively carrying out ultrafiltration desalination and concentration on the recombinant human-like collagen solution and the target host protein solution which are collected by ion exchange chromatography by using an ultrafiltration membrane with 10 KD;

6) respectively carrying out vacuum freeze drying on the concentrated solution obtained in the step 5) to obtain the recombinant human collagen (figure 2, 115KD) and the target host protein (figure 2, 65 KD).

Through detection, the recovery rate of the recombinant human-like collagen obtained by purification reaches more than 70%, the purity reaches more than 95%, the recovery rate of the target host protein reaches more than 70%, and the purity reaches more than 95%. The protein concentration after purification is determined by a biuret method, and the result shows that the expression quantity of the recombinant human-like collagen of the strain Pichia pastoris-col3-6 can reach 4.8g/L, and although the expression quantity of the target host protein is lower than that of the recombinant human-like collagen, the expression quantity of the target host protein is improved by 1g/L compared with the GS115 fermentation product.

In a word, compared with the existing purification method, the method has the advantages of low cost and simple process, and the obtained pure protein is completely decolorized and is suitable for industrial large-scale production.