CN112830953A - Method for extracting active protein from stem cell culture solution - Google Patents
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Abstract
The invention discloses a method for extracting active protein from stem cell culture solution, aiming at providing a method for separating and extracting stem cell active protein, which has simple operation, high protein purity and activity, high protein yield and easy industrial production, and has the technical key points that: (1) filtering with ultrafiltration membrane to remove small molecular impurities; (2) separating and purifying protein by chromatography; (3) concentrating the protein solution by ultrafiltration and removing inorganic salts; (4) freeze drying to obtain stem cell active protein product.
Description
Technical Field
The technology relates to the field of biological medicine, in particular to a method for extracting active protein from stem cell culture solution.
Background
Stem cells are a type of pluripotent cells with self-replicating ability, have potential functions of regenerating tissues and organs and human bodies, and are called "universal cells" in the medical field. Its discovery has changed the way that human beings respond to diseases. The stem cell study was first of ten scientific achievements in the world in 1999 in the journal of the United states of sciences, and was ranked prior to human genome sequencing and cloning techniques.
The stem cells have wide technical prospects, and the stem cells are known to have therapeutic effects in the research of various diseases such as diabetes, leukemia, stroke, Parkinson and spinal injuries, blood system diseases, nervous system diseases, limb ischemic diseases, cardiovascular and cerebrovascular diseases, secretory system diseases, surgical diseases, skin diseases, tumors, immune systems and the like. Stem cell technology will initiate a new medical revolution following drug and surgical treatment, bringing benefits to human health. Stem cell therapy currently carries out a number of clinical trials worldwide, covering over 140 diseases. In the global stem cell market, cell therapy occupies the semiwall of the river and has a further expanding trend. On the whole, the application prospect of the stem cell technology is wide, and the proportion of stem cell treatment is further expanded. The market size of stem cells worldwide was $ 635 billion in 2015, $ 789 billion in 2016, and $ 1745 billion in 2020.
The stem cells can secrete a large amount of active proteins into a culture medium in the culture process, the active proteins comprise cytokines such as vascular endothelial growth factor, fibroblast growth factor, stem cell growth factor, hepatocyte growth factor, vascular endothelial growth factor, nerve growth factor, keratinocyte growth factor and the like, and proteins such as biological processes, cell localization, molecular functions, KEGG channels and the like, and the active proteins have the effects of promoting cell proliferation, tissue repair, resisting oxidative stress, regulating skin pigment metabolism and the like, so that the active proteins can be used for preparing a stem cell culture solution to promote the stem cell proliferation, can also be used for clinical treatment, promote wound healing, and can also be used for resisting aging, protecting skin and beautifying.
The existing stem cell active protein extraction technology has the problems of low extraction efficiency, more foreign matters, easy pollution and the like, and is not beneficial to standardized production and preparation, so that stem cell culture solution is usually discarded as production waste liquid, is not effectively utilized, and causes great waste. The method comprehensively considers the characteristic of complex components in the stem cell culture solution, establishes a stem cell active protein separation and purification integrated technology mainly based on separation methods such as membrane separation, chromatographic separation, freeze drying and the like aiming at the research and development current situation of the stem cell extract and the urgency of the stem cell extract in application in the field of biomedicine, and realizes the large-scale separation and extraction of the stem cell active protein with high purity, high activity, high recovery rate, controllable performance and good batch repeatability.
Disclosure of Invention
The invention aims to develop a stem cell protein separation and extraction method which is simple to operate, high in protein purity and protein recovery rate and easy for industrial production.
A method for separating and purifying active protein from stem cell culture solution sequentially comprises the following steps:
(1) ultra-filtration membrane filtration
Collecting a culture solution for culturing the stem cells, adjusting the pH value to 7.0-9.0 (preferably 7.5-8.5), then performing ultrafiltration membrane filtration, wherein the cutoff molecular weight of the ultrafiltration membrane is 2,000-50,000 dalton (preferably 3,000-30,000 dalton), the operating pressure is 0.02-0.2 MPa (preferably 0.05-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-120 min (preferably 60-90 min), thereby obtaining an ultrafiltration filtrate.
Filtering and separating: and adding deionized water into the residual filtrate after the ultrafiltration is finished, wherein the addition amount of the deionized water is 1-5 times (preferably 2-4 times) of the volume of the residual filtrate, adjusting the pH to 7.0-9.0 (preferably pH to 7.5-8.5), continuously performing ultrafiltration separation, keeping the cutoff molecular weight of an ultrafiltration membrane to be 2,000-50,000 dalton (preferably 3,000-30,000 dalton), operating the pressure to be 0.02-0.2 MPa (preferably 0.05-0.15 MPa), the temperature to be 20-40 ℃ (preferably 25-35 ℃), treating the treatment time to be 30-120 min (preferably 60-90 min), repeating the filtration separation step for 1-10 times (preferably 4-8 times), and removing small molecular impurities.
(2) Chromatographic separation
Collecting the residual liquid obtained in the step (1) for chromatographic separation, selecting one or more of sephadex, sepharose, polyacrylamide gel, silica gel, macroporous adsorption resin, polyamide resin and ion exchange resin as a chromatographic medium, eluting with an eluent, selecting one or more of Phosphate Buffer (PBS) buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer and citric acid buffer as the eluent, wherein the concentration of the buffer is 0.02-0.2M (preferably 0.08-0.12M), the pH value is 4.5-9.0 (preferably 6.0-8.0), the flow rate of the eluent is 0.5-50 ml/min (preferably 10-40 ml/min), the temperature is 20-40 ℃ (preferably 28-36 ℃), the elution time is 30-240 min (preferably 120-200 min), and collecting the eluted protein solutions respectively at intervals of 5-30 min (preferably 10-20 min).
(3) Concentration of protein solution
Respectively carrying out ultrafiltration concentration on the protein solution collected by chromatographic separation to remove micromolecular inorganic salts, wherein the cutoff molecular weight of an ultrafiltration membrane is 2,000-5,000 dalton (preferably 3,000-4,000 dalton), the operating pressure is 0.05-0.2 MPa (preferably 0.08-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-90 min (preferably 45-60 min), so as to obtain a protein concentrated solution; diluting the protein concentrated solution by adding deionized water, wherein the volume of the deionized water is 5-10 times (preferably 7-9 times) of the volume of the protein concentrated solution, performing ultrafiltration again to concentrate the protein solution, wherein the cutoff molecular weight of an ultrafiltration membrane is 2,000-5,000 dalton (preferably 3,000-4,000 dalton), the operating pressure is 0.05-0.2 MPa (preferably 0.08-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-90 min (preferably 45-60 min), so that the protein concentrated solution is obtained.
(4) Freeze drying
And respectively freeze-drying the concentrated protein solution at the temperature of-20 to-50 ℃ (preferably-35 to-45 ℃), the vacuum degree of 20 to 100Pa (preferably 60 to 90Pa), and the time of 24 to 72h (preferably 48 to 64 h).
The method for extracting the active protein from the stem cell culture solution comprises the following steps of (1) filtering with an ultrafiltration membrane to remove impurities, wherein the pH of the stem cell culture solution is 7.0-9.0 (preferably the pH is 7.5-8.5), the cutoff molecular weight of the filtration membrane is 2,000-50,000 dalton (preferably 5,000-30,000 dalton), the operation pressure is 0.02-0.2 MPa (preferably 0.05-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-120 min (preferably 60-90 min); and adding deionized water into the residual filtrate after the ultrafiltration is finished, wherein the addition amount of the deionized water is 1-5 times (preferably 2-4 times) of the volume of the residual filtrate, the pH value is adjusted to 7.0-9.0 (preferably pH value is 7.5-8.5), the ultrafiltration separation is continuously carried out, the cutoff molecular weight of an ultrafiltration membrane is 2,000-50,000 dalton (preferably 5,000-30,000 dalton), the operation pressure is 0.02-0.2 MPa (preferably 0.05-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), the treatment time is 30-120 min (preferably 60-90 min), and the ultrafiltration separation is repeated for 1-10 times (preferably 4-8 times).
Further, the method for extracting active protein from the stem cell culture solution comprises the step (2) of separating and purifying protein by chromatography, wherein a chromatography medium is selected from sephadex, sepharose gel, polyacrylamide gel, silica gel, macroporous adsorption resin and polyamide resin, one or more ion exchange resins are loaded and then eluted by eluent, the eluent is selected from one or more of Phosphate Buffered Saline (PBS), phosphate buffered saline, Tris-HCl buffered saline, HEPES buffered saline and citric acid buffered saline, the concentration of the buffer is 0.02-0.2M (preferably 0.08-0.12M), the pH is 4.5-9.0 (preferably 6.0-8.0), the flow rate of the eluent is 0.5-50 ml/min (preferably 10-40 ml/min), the temperature is 20-40 ℃ (preferably 28-36 ℃), the elution time is 30-240 min (preferably 120-200 min), and the eluted protein solution is respectively collected at intervals of 5-30 min (preferably 10-20 min).
Further, the method for extracting active protein from the stem cell culture solution comprises the step (3) of concentrating the protein solution, concentrating the chromatographic separation eluent by ultrafiltration, wherein the cutoff molecular weight of an ultrafiltration membrane is 2,000-5,000 dalton (preferably 3,000-4,000 dalton), the operation pressure is 0.05-0.2 MPa (preferably 0.08-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-90 min (preferably 45-60 min).
Further, the method for extracting the active protein from the stem cell culture solution comprises the step (4) of freeze drying, wherein the freeze drying temperature is-20 to-50 ℃ (preferably-35 to-45 ℃), the vacuum degree is 20 to 100Pa (preferably 60 to 90Pa), and the time is 24 to 72 hours (preferably 48 to 64 hours).
The technical scheme provided by the invention has the following technical advantages:
(1) the invention comprehensively considers the characteristic of complex components in the stem cell culture solution, and establishes a stem cell protein extraction, separation and purification integrated technology which mainly adopts separation methods such as ultrafiltration membrane separation, chromatographic separation and purification, freeze drying and the like.
(2) The technical scheme provided by the invention adopts mild extraction conditions, so that the activity of the extracted stem cell active protein is high.
(3) The technical scheme provided by the invention comprises a chromatographic separation method, and active proteins with different molecular weights can be subjected to fractional separation to obtain active protein components with different molecular weights, so that the active protein has high purity, and the molecular weight distribution of the active protein in each component is narrow.
(4) The technical scheme provided by the invention is simple to operate and easy for process amplification and industrial production.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not to be construed as limiting the invention in any way. Any modification made by anyone within the scope of the claims of the present invention is still within the scope of the claims of the present invention.
Example 1
The invention provides a method for extracting active protein from stem cell culture solution, which sequentially comprises the following steps:
(1) ultra-filtration membrane filtration
Collecting 1L culture solution for culturing stem cells, adjusting pH to 8.0, and filtering with ultrafiltration membrane with cut-off molecular weight of 2,000dalton, operation pressure of 0.1MPa, temperature of 30 deg.C, and treatment time of 60min, wherein the volume of the filtrate is 10% of the volume of the culture solution; and adding deionized water into the residual filtrate after the ultrafiltration is finished, wherein the addition amount of the deionized water is 3 times of the volume of the residual filtrate, adjusting the pH value to 8.0, continuously performing ultrafiltration separation, performing ultrafiltration separation for 60min at the operating pressure of 0.1MPa and the temperature of 30 ℃ with the molecular weight cutoff of 2,000 daltons by using an ultrafiltration membrane, repeating the separation step for 7 times, and collecting the ultrafiltration residual filtrate.
(2) Chromatographic separation
And carrying out chromatographic separation on the residual filtrate, wherein the chromatographic medium is sephadex, eluting with an eluent after sample loading, the eluent is Phosphate Buffered Saline (PBS) buffer solution with the concentration of 0.1M, the pH value of 7.2, the flow rate of the eluent is 20ml/min, the temperature is 30 ℃, the elution time is 180min, and 12 parts of eluted protein solution is collected at the time interval of 15 min.
(3) Concentration of protein solution
Respectively carrying out ultrafiltration concentration on each protein solution component collected by chromatographic separation, wherein the cutoff molecular weight of an ultrafiltration membrane is 2,000 daltons, the operation pressure is 0.1MPa, the temperature is 30 ℃, and the treatment time is 60min to obtain a protein concentrated solution; diluting the protein concentrated solution with deionized water, wherein the volume of the deionized water is 10 times of the volume of the protein concentrated solution, ultrafiltering and concentrating the protein solution, wherein the cut-off molecular weight of the ultrafiltration membrane is 2,000 daltons, the operating pressure is 0.1MPa, the temperature is 30 ℃, and the treatment time is 60min to obtain the protein concentrated solution.
(4) Freeze drying
And freeze-drying the concentrated protein solution at-40 ℃ under the vacuum degree of 90Pa for 60 hours.
Extracting 12 parts of active protein through the extraction steps, wherein the average molecular weight of the component 1 is 127,000dalton (the molecular weight distribution range is 139,000-108,000 dalton), the amount of the active protein is 0.39g, and the protein purity is 98.4%; the average molecular weight of the component 2 is 104,000 daltons (the molecular weight distribution range is 116,000-95,000 daltons), the mass of active protein is 0.37g, and the protein purity is 97.3%; the average molecular weight of the component 3 is 93,000 daltons (the molecular weight distribution range is 107,000-82,000 daltons), the active protein amount is 0.35g, and the protein purity is 97.5%; the average molecular weight of the component 4 is 84,000dalton (the molecular weight distribution range is 91,000-73,000 dalton), the active protein amount is 0.36g, and the protein purity is 96.6%; the average molecular weight of the component 5 is 78,000dalton (the molecular weight distribution range is 89,000-68,000 dalton), the active protein amount is 0.34g, and the protein purity is 97.5%; the average molecular weight of the component 6 is 74,000dalton (the molecular weight distribution range is 83,000-65,000 dalton), the active protein amount is 0.31g, and the protein purity is 94.9%; the average molecular weight of the component 7 is 61,000dalton (the molecular weight distribution range is 67,000-55,000 dalton), the active protein amount is 0.32g, and the protein purity is 96.3%; the average molecular weight of the component 8 is 53,000dalton (the molecular weight distribution range is 64,000-48,000 dalton), the mass of active protein is 0.29g, and the protein purity is 95.7%; the average molecular weight of the component 9 is 45,000dalton (the molecular weight distribution range is 52,000-38,000 dalton), the mass of active protein is 0.28g, and the protein purity is 96.1%; the average molecular weight of the component 10 is 35,000dalton (the molecular weight distribution range is 43,000-29,000 dalton), the mass of active protein is 0.3g, and the protein purity is 97.4%; the average molecular weight of the component 11 is 26,000dalton (the molecular weight distribution range is 33,000-21,000 dalton), the mass of active protein is 0.26g, and the protein purity is 95.8%; the average molecular weight of the component 12 is 17,000 daltons (the molecular weight distribution range is 24,000-13,000 daltons), the active protein mass is 0.27g, and the protein purity is 96.1%.
Example 2
The invention provides a method for extracting active protein from stem cell culture solution, which sequentially comprises the following steps:
(1) ultra-filtration membrane filtration
Collecting 1L culture solution for culturing stem cells, adjusting pH to 8.0, and filtering with ultrafiltration membrane with cut-off molecular weight of 2,000dalton, operating pressure of 0.08MPa, temperature of 25 deg.C, and processing time of 90 min; and adding deionized water into the residual filtrate after the ultrafiltration is finished, wherein the addition amount of the deionized water is 4 times of the volume of the residual filtrate, adjusting the pH value to 8.0, continuously performing ultrafiltration separation, performing ultrafiltration separation for 90min at the operating pressure of 0.08MPa and the temperature of 25 ℃ with the ultrafiltration membrane cutoff molecular weight of 3,000dalton, repeating the separation step for 8 times, and collecting the ultrafiltration residual filtrate.
(2) Chromatographic separation
And carrying out chromatographic separation on the residual filtrate, wherein the chromatographic medium is macroporous adsorption resin, the sample is loaded and then eluted by using an eluent, the concentration of the eluent is 0.1M, the pH value is 6.8, the flow rate of the eluent is 30ml/min, the temperature is 30 ℃, the elution time is 120min, and 10 parts of the eluted protein solution is collected at 12min intervals.
(3) Concentration of protein solution
Ultrafiltering and concentrating the protein solution collected by chromatographic separation, wherein the cut-off molecular weight of the ultrafiltration membrane is 5,000dalton, the operating pressure is 0.08MPa, the temperature is 25 ℃, and the processing time is 60min to obtain a protein concentrated solution; diluting the protein concentrated solution by adding deionized water, wherein the volume of the deionized water is 15 times of that of the protein concentrated solution, and ultrafiltering and concentrating the protein solution, wherein the cut-off molecular weight of an ultrafiltration membrane is 3,000 daltons, the operating pressure is 0.08MPa, the temperature is 25 ℃, and the treatment time is 60min, so that the protein concentrated solution is obtained.
(4) Freeze drying
And freeze-drying the concentrated protein solution at-40 ℃ under the vacuum degree of 80Pa for 72 hours.
Extracting 10 parts of active protein through the extraction steps, wherein the average molecular weight of the component 1 is 133,000dalton (the molecular weight distribution range is 141,000-118,000 dalton), the amount of the active protein is 0.42g, and the protein purity is 97.7%; the average molecular weight of the component 2 is 117,000 daltons (the molecular weight distribution range is 126,000-109,000 daltons), the mass of active protein is 0.40g, and the protein purity is 97.9%; the average molecular weight of the component 3 is 102,000dalton (the molecular weight distribution range is 114,000-93,000 dalton), the active protein amount is 0.38g, and the protein purity is 97.1%; the average molecular weight of the component 4 is 89,000dalton (the molecular weight distribution range is 97,000-76,000 dalton), the active protein amount is 0.39g, and the protein purity is 96.8%; the average molecular weight of the component 5 is 77,000 daltons (the molecular weight distribution range is 86,000-64,000 daltons), the active protein content is 0.37g, and the protein purity is 96.3%; the average molecular weight of the component 6 is 66,000dalton (the molecular weight distribution range is 73,000-59,000 dalton), the active protein amount is 0.35g, and the protein purity is 95.6%; the average molecular weight of the component 7 is 57,000dalton (the molecular weight distribution range is 65,000-49,000 dalton), the active protein amount is 0.36g, and the protein purity is 96.7%; the average molecular weight of the component 8 is 45,000dalton (the molecular weight distribution range is 54,000-41,000 dalton), the mass of active protein is 0.32g, and the protein purity is 95.2%; the average molecular weight of the component 9 is 34,000dalton (the molecular weight distribution range is 41,000-31,000 dalton), the mass of active protein is 0.31g, and the protein purity is 96.9%; the component 10 has an average molecular weight of 21,000dalton (molecular weight distribution range of 23,000-17,000 dalton), an active protein mass of 0.35g, and a protein purity of 97.2%.
Example 3
The invention provides a method for extracting active protein from stem cell culture solution, which sequentially comprises the following steps:
(1) ultra-filtration membrane filtration
Collecting 1L culture solution for culturing stem cells, adjusting pH to 7.0, and filtering with ultrafiltration membrane with cut-off molecular weight of 5,000dalton, operating pressure of 0.1MPa, temperature of 20 deg.C, and treating for 45 min; and adding deionized water into the residual filtrate after the ultrafiltration is finished, wherein the addition amount of the deionized water is 4 times of the volume of the residual filtrate, adjusting the pH value to 7.0, continuously performing ultrafiltration separation, performing ultrafiltration separation for 45min at the operating pressure of 0.1MPa and the temperature of 20 ℃ with the molecular weight cutoff of 10,000dalton of an ultrafiltration membrane, repeating the separation step for 5 times, and collecting the ultrafiltration residual filtrate.
(2) Chromatographic separation
And (3) performing chromatographic separation on the residual filtrate, wherein the chromatographic medium is ion exchange resin, the sample is loaded and then eluted by using an eluent, the eluent is Tris-HCl buffer solution, the concentration of the buffer solution is 0.05M, the pH value is 7.4, the flow rate of the eluent is 20ml/min, the temperature is 20 ℃, the elution time is 180min, and 10 parts of eluted protein solution is collected at 18min intervals.
(3) Concentration of protein solution
Ultrafiltering and concentrating the protein solution collected by chromatographic separation, wherein the cut-off molecular weight of the ultrafiltration membrane is 5,000dalton, the operating pressure is 0.05MPa, the temperature is 30 ℃, and the processing time is 60min to obtain a protein concentrated solution; diluting the protein concentrated solution with deionized water, wherein the volume of the deionized water is 10 times of the volume of the protein concentrated solution, ultrafiltering and concentrating the protein solution, wherein the cut-off molecular weight of the ultrafiltration membrane is 3,000 daltons, the operating pressure is 0.05MPa, the temperature is 30 ℃, and the treatment time is 60min, so as to obtain the protein concentrated solution.
(4) Freeze drying
And freeze-drying the concentrated protein solution at-30 ℃ under the vacuum degree of 60Pa for 60 hours.
Extracting 10 parts of active protein through the extraction steps, wherein the average molecular weight of the component 1 is 128,000dalton (the molecular weight distribution range is 140,000-121,000 dalton), the amount of the active protein is 0.36g, and the protein purity is 92.1%; the average molecular weight of the component 2 is 114,000dalton (the molecular weight distribution range is 122,000-107,000 dalton), the mass of active protein is 0.33g, and the protein purity is 91.6%; the average molecular weight of the component 3 is 98,000dalton (the molecular weight distribution range is 105,000-91,000 dalton), the active protein amount is 0.32g, and the protein purity is 97.1%; the average molecular weight of the component 4 is 83,000dalton (the molecular weight distribution range is 91,000-75,000 dalton), the active protein amount is 0.31g, and the protein purity is 86.8%; the average molecular weight of the component 5 is 72,000dalton (the molecular weight distribution range is 790,000-66,000 dalton), the active protein amount is 0.33g, and the protein purity is 90.7%; the average molecular weight of the component 6 is 61,000dalton (the molecular weight distribution range is 68,000-54,000 dalton), the active protein amount is 0.31g, and the protein purity is 91.6%; the average molecular weight of the component 7 is 49,000dalton (the molecular weight distribution range is 57,000-42,000 dalton), the active protein amount is 0.32g, and the protein purity is 91.2%; the average molecular weight of the component 8 is 37,000dalton (the molecular weight distribution range is 44,000-31,000 dalton), the mass of active protein is 0.33g, and the protein purity is 89.2%; the average molecular weight of the component 9 is 28,000dalton (the molecular weight distribution range is 32,000-24,000 dalton), the mass of active protein is 0.34g, and the protein purity is 90.3%; the average molecular weight of the component 10 is 18,000 daltons (the molecular weight distribution range is 23,000-15000 daltons), the mass of active protein is 0.32g, and the protein purity is 88.7%.
Claims (3)
1. A method for extracting active protein from stem cell culture solution is characterized by sequentially comprising the following steps:
(1) ultra-filtration membrane filtration
Collecting a culture solution for culturing the stem cells, adjusting the pH value to 7.0-9.0 (preferably pH value 7.5-8.5), then performing ultrafiltration membrane filtration, wherein the cutoff molecular weight of the ultrafiltration membrane is 2,000-50,000 dalton (preferably 3,000-30,000 dalton), the operating pressure is 0.02-0.2 MPa (preferably 0.05-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-120 min (preferably 60-90 min), so as to obtain an ultrafiltration filtrate;
filtering and separating: adding deionized water into the residual filtrate after the ultrafiltration is finished, wherein the addition amount of the deionized water is 1-5 times (preferably 2-4 times) of the volume of the residual filtrate, adjusting the pH to 7.0-9.0 (preferably pH to 7.5-8.5), continuously performing ultrafiltration separation, keeping the cutoff molecular weight of an ultrafiltration membrane to be 2,000-50,000 dalton (preferably 3,000-30,000 dalton), operating the pressure to be 0.02-0.2 MPa (preferably 0.05-0.15 MPa), the temperature to be 20-40 ℃ (preferably 25-35 ℃), treating the treatment time to be 30-120 min (preferably 60-90 min), repeating the filtration separation step for 1-10 times (preferably 4-8 times), and removing small molecular impurities;
(2) chromatographic separation
Collecting the residual filtrate in the step (1) for chromatographic separation, selecting one or more of sephadex, sepharose, polyacrylamide gel, silica gel, macroporous adsorption resin, polyamide resin and ion exchange resin as a chromatographic medium, eluting with an eluent, selecting one or more of Phosphate Buffer (PBS) buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer and citric acid buffer as the eluent, wherein the concentration of the buffer is 0.02-0.2M (preferably 0.08-0.12M), the pH value is 4.5-9.0 (preferably 6.0-8.0), the flow rate of the eluent is 0.5-50 ml/min (preferably 10-40 ml/min), the temperature is 20-40 ℃ (preferably 28-36 ℃), the elution time is 30-240 min (preferably 120-200 min), and collecting the eluted protein solutions at intervals of 5-30 min (preferably 10-20 min);
(3) concentration of protein solution
Respectively carrying out ultrafiltration concentration on the protein solution collected by chromatographic separation to remove micromolecular inorganic salts, wherein the molecular weight cut-off of an ultrafiltration membrane is 2,000-2,0000 dalton (preferably 3,000-15,000 dalton), the operating pressure is 0.05-0.2 MPa (preferably 0.08-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-90 min (preferably 45-60 min), so as to obtain a protein concentrated solution; diluting the protein concentrated solution by adding deionized water, wherein the volume of the deionized water is 5-10 times (preferably 7-9 times) of the volume of the protein concentrated solution, performing ultrafiltration again to concentrate the protein solution, wherein the cutoff molecular weight of an ultrafiltration membrane is 2,000-20,000 dalton (preferably 3,000-15,000 dalton), the operating pressure is 0.05-0.2 MPa (preferably 0.08-0.15 MPa), the temperature is 20-40 ℃ (preferably 25-35 ℃), and the treatment time is 30-90 min (preferably 45-60 min), so as to obtain the protein concentrated solution;
(4) freeze drying
And respectively freeze-drying the concentrated protein solution at the temperature of-20 to-50 ℃ (preferably 35 to 45 ℃), the vacuum degree of 20 to 100Pa (preferably 60 to 90Pa), and the time of 24 to 72h (preferably 48 to 64 h).
2. The method for extracting active protein from stem cell culture solution according to claim 1, wherein: the volume of the ultrafiltration raffinate in the step (1) is 5-15% (preferably 8-12%) of the volume of a culture solution for culturing the stem cells.
3. The method for extracting active protein from stem cell culture solution according to claim 1, wherein: the total volume of the protein solution after elution in the step (2) is 10-20 times (preferably 12-16 times) of the volume of the raffinate.
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