Preparation method and purification method of human serum albumin
阅读说明：本技术 一种人血清白蛋白的制备方法及其纯化方法 (Preparation method and purification method of human serum albumin ) 是由 谢丽萍 胡又佳 朱文 吴珺艺 阮江雄 韩姝 徐磊 于 2019-03-05 设计创作，主要内容包括：本发明提供了一种人血清白蛋白的制备方法,其包括将产人血清白蛋白的巴斯德毕赤酵母菌接种于培养基中发酵,从发酵液中获得人血清白蛋白即可；所述培养基为除了甘油和毕赤酵母微量元素1两种组分外,其余组分浓度均至多降低为1/4的BSM培养基,优选降低为1/4～1/2的BSM培养基。本发明还提供了一种人血清白蛋白的纯化方法。利用本发明的制备方法制得的人血清白蛋白的产量及其占总蛋白的比例均显著高于现有技术,且该制备方法中所使用的培养基成本显著降低,且无动物源性病毒污染的风险,从而显著降低制备方法的成本。利用本发明所述的纯化方法纯化的回收率显著提升,并且最终产品的纯度也显著进一步提升。(The invention provides a preparation method of human serum albumin, which comprises inoculating pichia pastoris producing human serum albumin in a culture medium for fermentation, and obtaining human serum albumin from fermentation liquor; the culture medium is a BSM culture medium with the concentration of the rest components reduced to 1/4 at most except two components of glycerol and pichia pastoris microelement 1, and preferably is a BSM culture medium with the concentration reduced to 1/4-1/2. The invention also provides a method for purifying the human serum albumin. The yield of the human serum albumin prepared by the preparation method and the proportion of the human serum albumin in the total protein are obviously higher than those of the prior art, the cost of the culture medium used in the preparation method is obviously reduced, and the risk of animal-derived virus pollution is avoided, so that the cost of the preparation method is obviously reduced. The recovery rate of purification by the purification method is obviously improved, and the purity of the final product is also obviously further improved.)
1. A method for preparing human serum albumin is characterized in that the method comprises inoculating Pichia pastoris (Pichia pastoris) producing human serum albumin in a culture medium for fermentation, and obtaining human serum albumin from the fermentation liquid;
the culture medium is a BSM culture medium, wherein the concentration of the rest components except the glycerol and the pichia pastoris microelement 1 is reduced to 1/4 at most, and the BSM culture medium is preferably reduced to 1/4-1/2.
2. The method of claim 1, wherein the reduced-to-1/2 BSM medium comprises 4% glycerol, 1.335% phosphoric acid, 0.0465% calcium sulfate dihydrate, 0.91% potassium sulfate, 0.745% magnesium sulfate heptahydrate, 0.206% potassium hydroxide, and 0.4% Pichia pastoris microelement 1;
the BSM medium reduced to 1/4 includes 4% glycerol, 0.6675% phosphoric acid, 0.02325% calcium sulfate dihydrate, 0.455% potassium sulfate, 0.3725% magnesium sulfate heptahydrate, 0.103% potassium hydroxide, and 0.4% Pichia pastoris microelement 1;
wherein the phosphoric acid and the pichia pastoris microelement 1 are in percentage by volume of each component in the culture medium; the percentage of the other components is the mass volume percentage of each component in the culture medium.
3. The method of claim 2, wherein pichia pastoris trace element 1 includes 0.6% copper sulfate pentahydrate, 0.008% sodium iodide, 0.3% manganese sulfate monohydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate heptahydrate, 0.02% biotin, and 0.5% sulfuric acid;
wherein the percentage of the sulfuric acid is the volume percentage of the sulfuric acid in the pichia pastoris trace element 1; the percentage of the other components is the mass volume percentage of each component in the pichia pastoris microelement 1.
4. The method according to any one of claims 1 to 3, wherein the Pichia pastoris producing human serum albumin is an engineered bacterium in which an expression vector for a human serum albumin gene is integrated in Pichia pastoris, the expression vector containing a promoter, an alpha leader peptide, a human serum albumin gene and a terminator in this order; preferably:
the promoter is AOX1 promoter;
and/or, the alpha leader peptide is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae);
and/or the expression vector is provided with an geneticin resistant gene, and the genetic engineering bacteria have the performance of resisting the geneticin with the concentration of 0.25-5.0 mg/mL;
and/or the nucleotide sequence of the human serum albumin gene is shown as SEQ ID NO.1 in the sequence table;
and/or the skeleton of the expression vector is plasmid pPIC 9K.
5. An application of BSM culture medium with the concentration of the rest components reduced to 1/4 or 1/2 except two components of glycerin and pichia pastoris microelement 1 in the preparation of human serum albumin by biological fermentation;
preferably, the BSM medium reduced to 1/4 or the BSM medium reduced to 1/2 is the BSM medium reduced to 1/4 or the BSM medium reduced to 1/2 of claim 2 or 3, and/or the biofermentation is a fermentation using pichia pastoris producing human serum albumin as described in claim 4.
6. A method for purifying human serum albumin, comprising the step of subjecting human serum albumin to affinity chromatography,
the pH of the sample loading buffer solution used in the affinity chromatography is more than 7 and more than or equal to 4.3, and preferably more than or equal to 5;
the human serum albumin is obtained by fermenting Pichia pastoris which produces the human serum albumin.
7. The purification method of claim 6, further comprising the step of subjecting the human serum albumin to hydrophobic chromatography and a second affinity chromatography.
8. The purification method according to claim 6 or 7, wherein the human serum albumin is human serum albumin obtained by the production method according to any one of claims 1 to 4.
9. The purification process according to any one of claims 6 to 8, wherein the loading buffer used in the affinity chromatography comprises a potassium phosphate buffer, preferably a potassium phosphate buffer containing sodium chloride; the concentration of the potassium phosphate is preferably 20-30 mmol/L, more preferably 25mmol/L, and the concentration of the sodium chloride is preferably less than or equal to 0.1 mol/L;
and/or, in the affinity chromatography, a loading sample used before loading is mixed with a loading buffer used in the affinity chromatography, so that the concentration of sodium chloride in the loading sample is less than or equal to 0.1mol/L, and preferably 0.09 mol/L; the sample used in the affinity chromatography is preferably filtered; more preferably a sample which is filtered through a 0.45 μm microporous membrane;
and/or, the loading buffer used in the affinity chromatography is used for the column equilibrium of the chromatographic column used in the affinity chromatography;
and/or the filler of the chromatographic column used for affinity chromatography is active Blue F3GA, and the chromatographic column used for affinity chromatography is preferably Blue Sepharose6 FF;
and/or the elution solution used in the affinity chromatography is potassium phosphate buffer solution containing sodium chloride, the concentration of the potassium phosphate is preferably 20-30 mmol/L, more preferably 25mmol/L, and the concentration of the sodium chloride is preferably 1.5-2.5mol/L, more preferably 2 mol/L;
and/or the elution solution used in the affinity chromatography has a pH of 7.
10. The purification method according to any one of claims 7 to 9, wherein the solution obtained after the affinity chromatography is used as a sample for the hydrophobic chromatography, and the solution obtained by the affinity chromatography is preferably mixed with potassium phosphate buffer containing sodium chloride before the sample is loaded, so that the sodium chloride concentration of the solution obtained by the affinity chromatography is 1.5 to 2.5mol/L, preferably 2 mol/L; wherein the concentration of potassium phosphate in the potassium phosphate buffer solution containing sodium chloride is preferably 20-30 mmol/L, and more preferably 25 mmol/L;
and/or, the solution obtained by the hydrophobic chromatography is used as a sample for loading during the second affinity chromatography, and the solution obtained by the hydrophobic chromatography is preferably mixed with an elution solution used during the hydrophobic chromatography before loading, so that the sodium chloride concentration of the solution obtained by the hydrophobic chromatography is less than or equal to 0.1 mol/L;
and/or the filler of the chromatographic column used in the second affinity chromatography is active Blue F3GA, and the chromatographic column used in the second affinity chromatography is preferably Blue Sepharose6 FF;
and/or, the chromatographic column used in the hydrophobic chromatography is Phenyl Sepharose6 FF;
and/or the buffer solution used for column equilibrium of the chromatographic column in hydrophobic chromatography or the elution solution used in the second affinity chromatography is potassium phosphate buffer solution containing sodium chloride, the concentration of the potassium phosphate is preferably 20-30 mmol/L, more preferably 25mmol/L, and the concentration of the sodium chloride is preferably 1.5-2.5mol/L, more preferably 2 mol/L;
and/or, the buffer used for the column equilibrium of the chromatographic column in the second affinity chromatography is the same as the loading buffer in the affinity chromatography;
and/or the elution solution used in the hydrophobic chromatography is potassium phosphate buffer solution, and the concentration of potassium phosphate is preferably 20-30 mmol/L, and more preferably 25 mmol/L;
and/or the pH of the buffer solution used in the column equilibration of the chromatography column used in the hydrophobic chromatography and the elution solution used in the hydrophobic chromatography or the second affinity chromatography is 7.
Technical Field
The invention relates to a preparation method and a purification method of human serum albumin.
Background
Human Serum Albumin (HSA) is the most abundant protein in human plasma, accounts for 60% of total serum protein, and is about 35-40g/L, and has the main functions of maintaining plasma osmotic pressure and the transportation of various substances. HSA is widely used in shock and burn treatment, postoperative recovery, drug carriers, cell culture media and the like, and the worldwide demand of HSA reaches about 600 tons/year, and the demand of HSA accounts for about one third of that of China. At present, human serum albumin is mainly extracted from human blood, a corresponding purification process is a low-temperature ethanol precipitation method, the source of the human serum albumin is severely limited by blood supply, the risk of blood virus transmission and diffusion exists, and the low efficiency of the human serum albumin is caused by the requirements of a large amount of organic reagents and low-temperature control in the purification process.
The construction of engineering bacteria and the production of foreign proteins by transgenic technology originated in the last 70 th century, and since then, a series of foreign protein expression systems including escherichia coli, saccharomyces cerevisiae, pichia pastoris, insects, animal and plant cells and the like were established and rapidly developed, providing a great promotion for the industrial application of precious protein drugs and more efficient and more environmentally friendly industrial enzymes. At present, exogenous expression and related purification work of human serum albumin have been realized in various expression systems by using transgenic technology, including escherichia coli, saccharomyces cerevisiae, pichia pastoris, animal and plant cells, and animals and plants.
Escherichia coli (Escherichia coli) is a prokaryote and is widely used for expression of foreign proteins due to its clear genetic background, simple nutritional requirements and safety. As early as 1981, Richard et al successfully expressed HSA using E.coli, but only qualitatively detected its expression, and the yield was unknown; SLEEP equals 1990, HSA is successfully expressed by Saccharomyces cerevisiae (Saccharomyces cerevisiae), but the yield of a Saccharomyces cerevisiae expression system is low, and the system can highly glycosylate HSA and easily causes immunogenicity; wuhan university researchers Yang He and the like apply transgenic technology to traditional crop rice, and the wedding achievement of obtaining 2.75g of high-purity HSA per kilogram of rice is achieved. However, the culture period of the transgenic plants is long, the transgenic plants are greatly influenced by environmental factors, and the resistance genes of the transgenic crops are easy to diffuse; taiwan scholars Yu-Kuo Liu et al realized 75mg/L HSA expression using a transgenic plant in vitro cell expression system. However, in general, the yield of the transgenic animal and plant cell expression system is not optimistic and the culture cost is high, so that the transgenic animal and plant cell expression system is generally not the first choice for the industrial production of HSA.
Pichia pastoris (Pichia pastoris) is a eukaryotic expression system, which was discovered by Koichi et al in 1969 to have the property of growing on methanol as the sole carbon source and was further first reported to be successful for foreign protein expression in 1987. Compared with the traditional prokaryotic expression system, the pichia pastoris has the remarkable advantages of high expression quantity, strong post-processing capacity, high exogenous gene stability and the like, and compared with transgenic animal and plant cells and animal and plant cells, the pichia pastoris has the characteristics of simple genetic operation, large thallus growth quantity, low culture cost, small industrialization difficulty and the like. Currently, pichia expression systems have achieved the expression of over 500 foreign proteins. The Studies of Merck company, Sehoon Kim et al, obtained 10g/L of HSA expression in the Pichia system by optimizing the amount of the initial inducing bacteria (Kim S, Meehl M, d' Anjou M. Maximizationrecombinant μmangser μmangese expression in a mutsppichia pastoris expression [ J ]. Biotechnology expression, 2014,00(00):1-9.), Kobayashi et al, explored the feed process and also achieved 11g/L of HSA expression (Ohya T, Ohyama M, Koyashi K. optimization of μmangeser μmangese expression in a thiotrophphic procedure [ J ]. Biotechnology, 8790, and 887). According to the literature contents of the two paragraphs that show high expression of HSA, the methanol induction time is 450 hours and 250 hours, respectively, and the expression efficiency of the corresponding target protein is only 22 mg/(L × h). The patent published by Gaojian et al shows that it also achieves HSA expression of about 10g/L in Pichia pastoris, but the methanol induction time is at least 200 hours (patent application CN1854301A, Gaojian, Jiamada, plum oil, Dengjian Hui. a carrier for expressing human serum albumin and engineering bacteria [ P ]. 2005-04-29). Such long induction time obviously prolongs the production cycle, increases the time cost, and simultaneously, the long-time storage of a large amount of methanol also becomes a safety hazard which cannot be ignored. In order to overcome the defects that the expression quantity of the human serum albumin is low, the fermentation time is long and the like are not beneficial to industrialization, the inventor constructs and obtains a pichia pastoris engineering bacterium GS115-pPIC9K-hsa-5-4 capable of efficiently producing the human serum albumin through multiple rounds of screening, and applies for a related patent on 12/25/2017 (patent application No. 201711421153.3). When the genetic engineering bacteria are used for on-tank production of human serum albumin, the yield of the target protein can reach 8.86g/L after the methanol is induced for 96 hours, the production efficiency can reach 92.29mg/(L × h), and sterile air is used in the whole fermentation process without pure oxygen. The fermentation liquor can realize the recovery rate of 58.1 percent and the purity of target protein of 96.47 percent through simple separation and purification steps of centrifugation, micro-membrane filtration and one-step affinity chromatography. Although the production efficiency of human serum albumin is considerable at 92.29mg/(L × h), the yield of target protein of 8.86g/L, the recovery rate of 58.1% and the purity of target protein of 96.47% still have room for further increase, the BMGY medium used in the production process is expensive (about 50 yuan/liter), and the tryptone contained in the BMGY medium brings the risk of contamination with viruses of animal origin.
Disclosure of Invention
In order to overcome the defects that the yield of human serum albumin, the proportion of the human serum albumin in the total protein, the recovery rate of downstream separation and purification and the purity of a final product are not ideal while ensuring the production efficiency in the process of producing the human serum albumin by pichia pastoris engineering bacteria (Pichiapastoris) for expressing the human serum albumin in the prior art, and a culture medium used in the production process is expensive and is easy to introduce animal-derived viruses, a preparation method of the human serum albumin and a purification method of the human serum albumin are provided. The yield of the human serum albumin prepared by the preparation method and the proportion of the human serum albumin in the total protein are both obviously higher than the prior art, the cost of the culture medium used in the preparation method is obviously reduced, the risk of animal-derived viral pollution is avoided, and the cost of the preparation method is obviously reduced. The recovery rate of purification by the purification method is obviously improved, the purity of the final product is also obviously further improved, and the method is suitable for industrial production.
Because the expression level of the exogenous protein is related to a plurality of factors, including the specificity of a strain, the nature of the exogenous protein, the components of a culture medium, a culture environment, a fermentation process and the like, and a strategy for improving the expression level of the exogenous protein correspondingly has a plurality of points of entry, the inventor unexpectedly discovers that the yield of the original Pichia pastoris engineering bacteria with high human serum albumin expression is cultured by fermenting the BSM culture medium with reduced salt concentration, finally discovers that the yield of the obtained human serum albumin is further obviously improved by reducing the osmotic pressure of the culture medium, and discovers that the direct relationship between the osmotic pressure of the culture medium and the yield of the human serum albumin is improved for the first time. The art has used BSM with reduced salt concentrations to culture fermenting yeast strains to increase their production of expressed foreign proteins, all starting from lower initial concentrations. However, it is very rare in the art that the present inventors start from a strain expressing a target protein with a high initial concentration (8.86g/L) through optimization of a technical scheme, and the strain expresses the target protein with a yield of up to 17.47g/L after final optimization.
In the process of protein purification, because the purification system is relatively fixed, the technicians in the field do not think of optimizing the pH value of affinity chromatography, but the inventor surprisingly finds that the purity of the final product is more than 99.9% and the recovery rate is greatly improved by changing the pH value of the loading buffer solution of the affinity chromatography and creatively applying the experimental scheme of the affinity chromatography-hydrophobic chromatography-secondary affinity chromatography to the process of purifying the human serum albumin.
In order to solve the technical problem, the invention provides a preparation method of human serum albumin, which comprises the steps of inoculating pichia pastoris for producing human serum albumin in a culture medium for fermentation, and obtaining the human serum albumin from fermentation liquor;
the culture medium is a BSM culture medium, wherein the concentration of the rest components except the glycerol and the
Preferably, the BSM medium reduced to 1/2 (i.e., 1/2BSM medium of the prior art) includes 4% glycerol, 1.335% phosphoric acid, 0.0465% calcium sulfate dihydrate, 0.91% potassium sulfate, 0.745% magnesium sulfate heptahydrate, 0.206% potassium hydroxide, and 0.4% pichia pastoris trace element 1(PTM 1).
Preferably, the reduced-to-1/4 BSM medium (i.e., 1/4BSM medium in the prior art) includes 4% glycerol, 0.6675% phosphoric acid, 0.02325% calcium sulfate dihydrate, 0.455% potassium sulfate, 0.3725% magnesium sulfate heptahydrate, 0.103% potassium hydroxide, and 0.4%
Wherein, the percentage of the phosphoric acid and the
In the present invention, the mass volume percentage may be conventional in the art, such as g/ml, unless otherwise specified.
Preferably, the pichia
wherein the percentage of the sulfuric acid is the volume percentage of the sulfuric acid in the pichia
Preferably, the Pichia pastoris for producing human serum albumin is an engineering bacterium of an expression vector integrating human serum albumin gene in Pichia pastoris (Pichia pastoris), and the expression vector sequentially contains a promoter, alpha guide peptide, a human serum albumin gene and a terminator.
The human serum albumin producing pichia pastoris strain can be the strain used in example 5 of the background-mentioned patent application No. 201711421153.3, the preparation of which is incorporated herein by reference in its entirety.
More preferably, the promoter is AOX1 promoter.
More preferably, the alpha leader peptide is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae).
More preferably, the expression vector has an geneticin-resistant gene, and the genetically engineered bacterium has the performance of resisting geneticin with the concentration of 0.25-5.0 mg/mL.
More preferably, the nucleotide sequence of the human serum albumin gene is shown as SEQ ID NO.1 in the sequence table.
More preferably, the skeleton of the expression vector is plasmid pPIC 9K.
In order to solve the technical problems, the invention provides an application of a BSM culture medium with the concentration of the rest components reduced to 1/4 or 1/2 in preparation of human serum albumin by biological fermentation, except two components of glycerol and
Preferably, the BSM reduced to 1/4 medium or the BSM reduced to 1/2 medium is the above-mentioned BSM reduced to 1/4 medium or the BSM reduced to 1/2 medium.
Preferably, the biological fermentation is fermentation by using the pichia pastoris producing human serum albumin.
In order to solve the above technical problems, the present invention provides a method for purifying human serum albumin, comprising the step of subjecting human serum albumin to affinity chromatography,
the pH of the sample loading buffer solution used in the affinity chromatography is more than 7 and more than or equal to 4.3, and preferably more than or equal to 5;
the human serum albumin is obtained by fermenting Pichia pastoris which produces the human serum albumin.
The fermentation according to the invention, which may be conventional in the art, may be carried out in a fermenter, for example usually in a 3L, 5L or 10L fermenter.
Preferably, the applicant finds that the charge properties of the target protein and the hybrid protein are very close in further research, the separation effect is poor by adopting the ion exchange in the prior art, and the effect of changing the last step of ion exchange into the second affinity chromatography is good in an unexpected way; therefore, the purification method of the invention also comprises the steps of carrying out hydrophobic chromatography and second affinity chromatography on the human serum albumin.
4.3 is the isoelectric point of human serum albumin, and when the pH of the loading buffer used in the affinity chromatography is less than 4.3, the albumin is likely to precipitate or be unstable, so peracid conditions are not adopted.
The loading buffer used in the affinity chromatography according to the present invention may be a loading buffer for affinity chromatography as conventionally defined in the art, i.e., a buffer used in diluting a sample to be loaded or a raw material solution to be purified when performing affinity chromatography.
Preferably, the human serum albumin is obtained by the preparation method.
Preferably, the loading buffer used in the affinity chromatography comprises potassium phosphate buffer, preferably potassium phosphate buffer containing sodium chloride; the concentration of the potassium phosphate is preferably 20-30 mmol/L, more preferably 25mmol/L, and the concentration of the sodium chloride is preferably less than or equal to 0.1 mol/L.
Preferably, in the affinity chromatography, a loading sample used before loading is mixed with a loading buffer used in the affinity chromatography, so that the concentration of sodium chloride in the loading sample is less than or equal to 0.1mol/L, and preferably 0.09 mol/L; the sample used in the affinity chromatography is preferably filtered; more preferably a sample which has been filtered through a 0.45 μm microfiltration membrane.
Preferably, the loading buffer used in the affinity chromatography is used for the column equilibration of the chromatography column used in the affinity chromatography.
Preferably, the filler of the chromatographic column used for affinity chromatography is active Blue F3GA (Cibacron Blue F3GA, Blue gel for short), and the chromatographic column used for affinity chromatography is preferably Blue Sepharose6 FF.
Preferably, the elution solution used in the affinity chromatography is potassium phosphate buffer containing sodium chloride, the concentration of the potassium phosphate is preferably 20-30 mmol/L, more preferably 25mmol/L, and the concentration of the sodium chloride is preferably 1.5-2.5mol/L, more preferably 2 mol/L.
Preferably, the elution solution used in the affinity chromatography has a pH of 7.
Preferably, the solution obtained after the affinity chromatography is used as a sample for loading in the hydrophobic chromatography, and the solution obtained by the affinity chromatography is preferably mixed with potassium phosphate buffer solution containing sodium chloride before loading, so that the sodium chloride concentration of the solution obtained by the affinity chromatography is 1.5-2.5mol/L, preferably 2 mol/L; wherein the concentration of potassium phosphate in the potassium phosphate buffer solution containing sodium chloride is preferably 20 to 30mmol/L, and more preferably 25 mmol/L.
Preferably, the solution obtained by the hydrophobic chromatography is used as a sample for loading in the second affinity chromatography, and before loading, the solution obtained by the hydrophobic chromatography is preferably mixed with an elution solution used in the hydrophobic chromatography, so that the sodium chloride concentration of the solution obtained by the hydrophobic chromatography is less than or equal to 0.1 mol/L.
In the present invention, the elution solution is a solution used for eluting the target protein (for example, it may be referred to as a chromatography B solution in the prior art), and is not an elution solution obtained after elution.
Preferably, the packing of the column used in the second affinity chromatography is active Blue F3GA, and the column used in the second affinity chromatography is preferably Blue Sepharose6 FF.
Preferably, the chromatographic column used in the hydrophobic chromatography is Phenyl Sepharose6 FF.
Preferably, the buffer used for column equilibration of the chromatography column in hydrophobic chromatography or the elution solution used for the second affinity chromatography is potassium phosphate buffer containing sodium chloride, the concentration of potassium phosphate is preferably 20-30 mmol/L, more preferably 25mmol/L, and the concentration of sodium chloride is preferably 1.5-2.5mol/L, more preferably 2 mol/L.
Preferably, the column equilibration of the column for the second affinity chromatography is performed using the same buffer as the loading buffer used for the affinity chromatography.
Preferably, the elution solution used in the hydrophobic chromatography is potassium phosphate buffer, and the concentration of potassium phosphate is preferably 20-30 mmol/L, and more preferably 25 mmol/L.
Preferably, the buffer used in column equilibration of the column used in the hydrophobic chromatography, and the elution solution used in the hydrophobic chromatography or the second affinity chromatography have a pH of 7.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the yield of the human serum albumin prepared by the preparation method and the proportion of the human serum albumin in the total protein are obviously higher than those of the prior art
In a preferred embodiment of the invention, the 1/2BSM medium is used for fermentation culture of related strains, and the yield of human serum albumin of 17.47g/L can be achieved after 192 hours of induction (the yield of human serum albumin in the background-mentioned patent 201711421153.3 is 8.86g/L), and compared with the related documents and patents of human serum albumin reported at present, the advantages of high expression amount and high expression efficiency are obvious. And the proportion of the target protein in the induction end fermentation broth supernatant was as high as 76.86% of total protein (human serum albumin in the background-mentioned patent 201711421153.3 accounts for about 50.19% of total protein). Is more beneficial to the post-treatment of separation and purification.
(2) The cost of the culture medium used in the preparation method is obviously reduced, and the risk of animal-derived virus pollution is avoided, so that the cost of the preparation method is obviously reduced
The BSM media with reduced salt concentration (e.g., 1/2BSM and 1/4BSM) described in the present invention are fully synthetic media with all components and amounts known, good batch-to-batch stability and low cost (about 3-4 yuan/liter). Meanwhile, the culture medium does not contain any animal-derived components, so that the risk of animal-derived virus pollution is eliminated.
(3) The recovery rate of the purification by using the purification method provided by the invention is obviously improved, and the purity of the final product is also obviously further improved
The purification method provided by the invention adopts a separation and purification scheme of affinity chromatography-hydrophobic chromatography-secondary affinity chromatography, and can simultaneously realize a very high recovery rate (the recovery rate is up to 67.53% in a certain preferred embodiment of the invention) on the basis of obtaining a target protein with higher purity (the purity is more than 99% in a certain preferred embodiment of the invention), thereby indicating that the downstream separation and purification scheme established by the invention has a better industrialization prospect.
Drawings
FIG. 1 is a schematic diagram of electrophoresis of supernatant proteins from fermentation broth in BMGY medium; m is Marker; 1-14 are electrophorograms of fermentation supernatants at 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144 and 156 hours post-induction, respectively.
FIG. 2 is a schematic diagram of electrophoresis of supernatant proteins of fermentation broth in BSM medium; m is Marker; 1-9 are electrophoresis patterns of fermentation broth supernatants at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours after induction, respectively.
FIG. 3 is a schematic diagram of electrophoresis of supernatant proteins of fermentation broth in BSM medium; m is Marker; 1-9 are electrophoresis images of fermentation broth supernatants at 108, 120, 132, 144, 156, 168, 180, 192, and 204 hours after induction, respectively.
FIG. 4 is a graph showing comparison of wet weight of cells in BMGY and BSM medium.
FIG. 5 is a graph showing comparison between BMGY and BSM in terms of the yield of target protein.
FIG. 6 is a schematic representation of the electrophoresis of the supernatant proteins of a fermentation broth in 1/2BSM medium; m is Marker; 1-9 are electrophoresis patterns of fermentation broth supernatants at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours after induction, respectively.
FIG. 7 is a schematic representation of the electrophoresis of the supernatant proteins of a fermentation broth in 1/2BSM medium; m is Marker; 1-9 are electrophoresis images of fermentation broth supernatants at 108, 120, 132, 144, 156, 168, 180, 192, and 204 hours after induction, respectively.
FIG. 8 is a schematic representation of the electrophoresis of the supernatant proteins of a fermentation broth in 1/4BSM medium; m is Marker; 1-9 are electrophoresis patterns of fermentation broth supernatants at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours after induction, respectively.
FIG. 9 is a schematic representation of the electrophoresis of the supernatant proteins of a fermentation broth in 1/4BSM medium; m is Marker; 1-9 are electrophoresis of fermentation broth supernatants at 108, 120, 132, 144, 156, 168, 180, 192, and 204 hours after induction, respectively.
FIG. 10 is a comparison of wet weight of cells in BSM medium with three different salt concentrations.
FIG. 11 is a schematic diagram of electrophoresis of supernatant proteins of fermentation broth in BSM medium with three different salt concentrations.
FIG. 12 is a graph showing the comparison of the osmotic pressure of fermentation broth in BSM medium with three different salt concentrations.
FIG. 13 is a comparison of BMGY and 1/2BSM medium with the target protein.
FIG. 14 is a graph showing the effect of loading pH on Blue Sepharose6FF affinity chromatography.
FIG. 15 is a Blue Sepharose6FF affinity chromatography elution profile.
FIG. 16 is a Blue Sepharose6FF affinity chromatography protein electrophoresis analysis; m is Marker; 1-4 are 0, 333, 666 and 1000mg/L HSA standards, respectively; 5, separating and purifying raw material liquid; 6, diluting the raw material liquid; 7 is affinity chromatography sample liquid; 8 is flow-through liquid; 9 is eluent; 10 is regeneration liquid.
FIG. 17 shows the elution profile of Phenyl Sepharose6FF (HS) hydrophobic chromatography.
FIG. 18 shows the electrophoretic analysis of Phenyl Sepharose6FF (HS) hydrophobic protein chromatography. M is Marker; 1-4 are 0, 333, 666 and 1000mg/L HSA standards, respectively; 5 is hydrophobic chromatography sample liquid; 6 is flow-through liquid; 7 is eluent.
FIG. 19 is a Blue Sepharose6FF secondary affinity chromatography elution profile.
FIG. 20 is a Blue Sepharose6FF secondary affinity chromatography protein electrophoresis analysis; m is Marker; 1-4 are 0, 333, 666 and 1000mg/L HSA standards, respectively; 5 is sample liquid of secondary affinity chromatography; 6 is flow-through liquid; 7 is eluent; 8 is regeneration liquid.
FIG. 21 shows a comparison of protein electrophoresis of three-step chromatography eluates.
FIG. 22 shows HPLC purity identification of the eluate after concentration by secondary affinity chromatography.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Tris (Tris hydroxymethyl aminomethane), acrylamide, ammonium persulfate (for preparing protein electrophoresis gel), TEMED (tetramethylethylenediamine), SDS (sodium dodecyl sulfate), DTT (dithiothreitol), biotin, YNB (yeast basic nitrogen group), bromophenol blue, EDTA (ethylene diamine tetraacetic acid), glycine, modified BCA (bisquinolinecarboxylic acid) protein concentration detection kit and 0.45 μm microporous filter membrane were purchased from Shanghai Biotech Limited; blue Sepharose6FF affinity chromatography pre-column and phenyl Sepharose6FF (HS) hydrophobic chromatography pre-column were purchased from Shanghai Booglong Biotechnology Ltd; tryptone and yeast extract were purchased from Oxiod, uk; human serum albumin standards were purchased from Sigma-Aldrich (shanghai) trade ltd; the other common reagents such as methanol, sodium chloride, ammonia water and the like are purchased from chemical reagents of national drug group, ltd.