WO2020119609A1 - 一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法 - Google Patents

一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法 Download PDF

Info

Publication number
WO2020119609A1
WO2020119609A1 PCT/CN2019/123762 CN2019123762W WO2020119609A1 WO 2020119609 A1 WO2020119609 A1 WO 2020119609A1 CN 2019123762 W CN2019123762 W CN 2019123762W WO 2020119609 A1 WO2020119609 A1 WO 2020119609A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant human
human fibronectin
buffer
exchange chromatography
chromatography
Prior art date
Application number
PCT/CN2019/123762
Other languages
English (en)
French (fr)
Inventor
杨代常
占全
Original Assignee
武汉禾元生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉禾元生物科技股份有限公司 filed Critical 武汉禾元生物科技股份有限公司
Priority to EP19894910.9A priority Critical patent/EP3896163A4/en
Priority to KR1020217019636A priority patent/KR102689084B1/ko
Priority to JP2021533137A priority patent/JP7312830B2/ja
Priority to US17/312,875 priority patent/US20220056071A1/en
Publication of WO2020119609A1 publication Critical patent/WO2020119609A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/203—Equilibration or regeneration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for separating and purifying recombinant human fibronectin.
  • Fibronectin (Fibronectin, Fn), also known as fibronectin, is a highly distributed high molecular weight glycoprotein. It is present in plasma, intracellular material of cells and different cell surfaces. It usually exists in the form of a dimer with a molecular weight of about 450kD. The monomer has a molecular weight of 220-250kD and is connected by a disulfide bond at the carboxyl end of the protein. Fibronectin is mostly present in body fluids such as plasma in soluble form, and in extracellular matrix in insoluble form. It has a combination with extracellular matrix proteins such as collagen, circulating blood proteins such as fibrin, glycosaminoglycans such as heparin, etc. Therefore, it is involved in many important physiological processes such as embryonic development, wound healing, hemostasis and coagulation Plays an important role.
  • Fn and some contaminated proteins will precipitate out in the form of precipitates when standing below 4°C, and then be further purified by precipitation and ion exchange chromatography; from cultured fibrin
  • the surface separation of Fn protein uses the extraction method of low-concentration urea; Anti-Fn medium can be widely used in the purification of Fn in plasma and cell culture. But these methods are gradually replaced by affinity chromatography, the principle is based on its high specific affinity with denatured collagen (usually gelatin), and then washed with 1mol L KBr, 1-8mol Urea or amine salt Off.
  • fibronectin in plasma is extremely rich, about 300mg/L, so plasma is a major source of Fn.
  • the Fn products produced abroad are all extracted from human plasma.
  • cosmetic additives and can be used in medicine to treat wounds, burns, shock, etc. it has significant social benefits and economic value, but the plasma source is limited, and the production process is complicated, not Conducive to scale-up.
  • the object of the present invention is to provide a chromatography method for separating and purifying recombinant human fibronectin from genetically engineered rice seeds expressing recombinant fibronectin.
  • the present invention provides the following technical solutions:
  • a method for separating and purifying recombinant human fibronectin from genetically engineered rice seeds includes the following steps in sequence:
  • step 1) the recombinant human fibronectin genetically engineered rice seeds are used as raw materials, the rice is hulled and polished into semi-polished rice, and ground into 80-100 mesh rice flour; the rice flour and the extraction buffer are weight/volume 1: Mix at a ratio of 5-1:10 and extract for 0.5-2 hours at room temperature to obtain crude protein extract;
  • the extraction buffer is: 0-50mM Tris, 0-50mM PB, 0-110mM NaCl, pH 5.9-8.0; the preferred extraction component can be added 0.8-1mM PMSF or 5-10mM GSH or 0.05-0.1% Tween One or more of 80.
  • Step 2 The packing material for cation exchange chromatography is selected from NanoGel 30/50 SP, UniGel 30/80 SP, SP Bestarose FF, SP Bestarose HP, Bestarose Diamond MMC, Uniphere S, MacroPrep S, POROS XS, SP-6FF, SP -6HP, SP SepharoseTM Fast Flow; NanoGel 50 SP or SP Bestarose HP is preferred.
  • pH gradient elution or sodium chloride concentration gradient elution can be selected, preferably sodium chloride gradient elution.
  • a combination of pH and sodium chloride gradient is selected for elution.
  • the washing buffer includes phosphate buffer, 0.13M sodium chloride, 0.3M sodium chloride, pH 5.9, and elution buffer. Including phosphate buffer, 0.1M sodium chloride, 0.3M sodium chloride, pH 7.0.
  • sodium chloride gradient elution is selected, and the washing and elution buffers of the chromatography process include phosphate buffer, 0.15M sodium chloride, 0.3M sodium chloride, and pH 7.0.
  • Step 3 The packing material for anion exchange chromatography is selected from Q Bestarose Fast, Q Bestarose HP, Bestarose DEAE, Q SepharoseTM HP, Q SepharoseTM Fast Flow, DEAE SepharoseTM Fast Flow, UniGel 30/80Q, NanoGel 30/50Q, UNO Sphere Q. It is preferable to use Borgron QFF or Huiyan QHP.
  • Anion exchange chromatography can choose sodium chloride concentration gradient elution.
  • the washing and elution buffers of the chromatography process include phosphate buffer, 0.2M sodium chloride, 0.3M sodium chloride, and pH are both 7.0.
  • the target elution component obtained by anion exchange chromatography can be concentrated, freeze-dried, etc. into a finished product by a known method.
  • the loading buffer used includes phosphate buffer with a pH of less than 7.5 and a salt concentration of less than 0.12M.
  • the elution buffer of the target substance (recombinant human fibronectin) in cation exchange chromatography includes phosphate buffer and sodium chloride; the pH is 6.8-7.1, the preferred sodium chloride concentration is 0.3M, and the pH of the buffer is 7.0.
  • the buffer used includes phosphate buffer, 0.09-0.13M sodium chloride, and the pH is 5.8-7.1.
  • the buffer used includes phosphate buffer, 0.12-0.3M sodium chloride, and the pH is 6.8-7.1.
  • the buffer used when the filler of anion exchange chromatography is Huiyan QHP, the buffer used includes phosphate, 0.1-0.3M sodium chloride, and the pH is 6.8-7.1.
  • the buffer used when the filler for anion exchange chromatography is Borgron QFF, the buffer used includes phosphate, 0.1-0.3M sodium chloride, and the pH is 6.8-7.1.
  • the invention also provides a plant expression vector for preparing the genetically engineered rice seed.
  • the expression vector is constructed by introducing a gene expressing human fibronectin and a rice-specific promoter Gt13a and its signal peptide into a plasmid vector.
  • the nucleotide sequence of the gene expressing human fibronectin is shown in SEQ ID NO. 1, and the plasmid vector is pOsPMP626.
  • the raw materials used in the present invention are derived from genetically engineered rice seeds expressing recombinant fibronectin, using promoters and signal peptides specifically expressed in rice endosperm to mediate Fn into the inner membrane system of rice endosperm cells and store the protein in rice endosperm In the body, so that Fn can accumulate in rice seeds in large quantities, and eventually reach a higher expression level. Since the rice-specific expression system does not have plasma-specific impurities such as Fg and vWF, the separation and purification of Fn with rice seeds has a great advantage, and it has a different purification method from other sources.
  • the invention utilizes cation and anion exchange two-step chromatography to extract and purify Fn from genetically engineered rice seeds expressing recombinant fibronectin, and explores and optimizes the process conditions, providing separation and purification of recombinant human fibronectin from rice seed species.
  • a purification process that can be scaled up.
  • Figure 1 is a schematic diagram of the structure of plasmid pOsPMP626.
  • Figure 2 is a schematic diagram of the structure of plasmid pOsPMP627.
  • Figure 3 is a schematic diagram of the structure of plasmid pOsPMP628.
  • Figure 4 is the positive detection of the target gene in T1 generation genetic engineering materials, where M is the DNA standard molecular weight marker; PMP628-51, 628-62, 628-63, 628-64, 628-65, 628-68, 628-69 And 628-71 are T1 transgenic materials; NC, negative control recipient varieties; P is positive control plasmid.
  • Figure 5 is the positive detection of marker genes in T1 generation genetic engineering materials, where M is the DNA standard molecular weight marker; PMP628-51, 628-62, 628-63, 628-64, 628-65, 628-68, 628-69 And 628-71 are T1 generation genetic engineering materials; NC, the negative control recipient variety; P is the positive control plasmid.
  • Figure 6 is the detection of the expression level of FN in the genetic engineering seeds of T1 generation identified by SDS-PAGE, where M is the standard molecular weight Marker; PC is 220 kD Human Plasma Fibronectin (FN) pure protein (Merck); PMP628-51, 628- 62, 628-63, 628-64, 628-65, 628-68, 628-69 and 628-71 are genetically modified materials.
  • M is the standard molecular weight Marker
  • PC 220 kD Human Plasma Fibronectin (FN) pure protein (Merck)
  • PMP628-51, 628- 62, 628-63, 628-64, 628-65, 628-68, 628-69 and 628-71 are genetically modified materials.
  • Figure 7 is the SDS-PAGE detection of different Heparin affinity chromatography eluted samples, in which nano-micro, Qianchun, Boglong, GE, Huiyan correspond to NW, QC, BGL, GE, HY respectively;
  • Figure 8 is the SDS-PAGE detection of samples obtained under the optimized washing conditions of Heparin chromatography, where M is the standard molecular weight Maker, Load is the protein extraction solution, FT is the penetrating solution, 10% B is 100 mM NaCl, 18% B, 21%B, 29%B, 36%B correspond to 180mM, 210mM, 290mM, 360mM NaCl respectively.
  • Figure 9 is the SDS-PAGE detection of the samples obtained under the optimized washing conditions of Heparin chromatography, where Load is the protein extract and FT is the penetrant, corresponding to 12%B, 14%B, 16%B, and 29%B, respectively 120mM, 140mM, 160mM, 290mM NaCl, CIP is the regeneration solution.
  • Fig. 10 shows the results of the prediction of the DoE experiment on the pH, sample conductance, and flow rate of Huiyan QFF chromatogram.
  • Figure 11 shows the predicted results of the DoE experiment on the conductivity, elution pH, and elution conductance of QFFF chromatogram.
  • Figure 12 is the SDS-PAGE test results of the comparative experimental samples of GE QHP and Huiyan QFF chromatographic packing materials, where M is the standard molecular weight marker, Load is the protein extraction solution, FT is the penetrating solution, Wash is the washing solution, Elu It is the eluent.
  • Figure 13 shows the results of SDS-PAGE test of the comparative experimental samples of GE QHP and Huiyan Q HP chromatography packing, where M is the standard molecular weight marker, Load is the protein extraction solution, FT is the penetrating solution, Wash is the washing solution, Elu For the eluent, CIP is the regeneration liquid.
  • Fig. 14 is the SDS-PAGE detection of the pH stability study sample of the extract, in which M is the standard molecular weight marker, the supernatant is the supernatant after the sample is centrifuged after pH adjustment, and the precipitate is the reconstituted precipitate after centrifugation after the pH adjustment.
  • Figure 15 is the SDS-PAGE detection of Bogelon SP HP chromatography samples, where M is the standard molecular weight marker, Load is the protein extract, FT is the penetrant, Wash is the washing solution, and Elu (10X) is the eluent Concentrate 10 times.
  • Fig. 16 is the SDS-PAGE detection of chromatographic samples of Nano-Gel Nano 50 Gel Pack, where M is the standard molecular weight marker, Load is the protein extraction solution, FT is the penetrating solution, Wash is the washing solution, and Elu is the eluent.
  • Figure 17 is the SDS-PAGE detection results of chromatographic samples of Huiyan Q HP, Nano Micro UniGel 80 Q, Boglong Q FF packing, where M is the standard molecular weight Marker, Load is NanoGel 50 SP SP elution collection solution, FT For the penetrating fluid, Wash is the washing solution, Elu is the eluent, and CIP is the regeneration solution.
  • Figure 18 is the SDS-PAGE detection result of the two-step chromatography purification sample of Bogelong SP-HP-HuiQ HP, where M is the standard molecular weight Marker, and it is reduced to Huiyan Q HP chromatographic eluent added with reduced sample loading solution
  • the non-reduced sample is the sample processed by Huiyan Q HP chromatography eluent and non-reducing sample loading solution.
  • Figure 19 is the SDS-PAGE detection results of the two-step chromatography purification sample of Nano-Gel Nano SP 50-SP-Borgron Q, FF, where M is the standard molecular weight marker, Load is the sample pH after protein extraction is adjusted to the loading pH, and FT is the wear Permeabilization, Wash is the washing solution and Elu is the eluent.
  • Fig. 20 is the chromatogram of Nano 50 Gel SP.
  • Figure 21 is Boglong Q FF chromatogram.
  • Figure 22 shows the SDS-PAGE test results of the two-step chromatography purification process of Nano-Gel Nano SP 50-SP-Borgron Q FF, where M is the standard molecular weight marker, P is the Fn positive reference, and 0927, 0928, and 0929 are different Batch samples are reduced to reductive electrophoresis, and non-reduced to non-reduced electrophoresis.
  • Fig. 23 is the SEC-HPLC chart of the verification sample of Nano-Gel Nano 50 Gel SP-Borgron Q FF two-step chromatography purification process.
  • Figure 24 shows the detection results of the cell viability of the target protein obtained by the Nano-Gel 50 SP-Borgron Q FF two-step chromatography purification process.
  • Example 1 Preparation of recombinant human fibronectin genetically engineered rice
  • rice-specific promoter Gt13a and its signal peptide are used to mediate the expression of recombinant human fibronectin gene in rice endosperm cells.
  • construct the rice-specific expression recombinant of the present invention Human fibronectin carrier and genetically engineered rice plants were screened, and the recombinant human serum albumin described therein was exchanged for the recombinant human fibronectin of the invention.
  • the plasmid pOsPMP626 shown in Figure 1 was used to construct a rice endosperm-specific expression cassette.
  • the synthesized codon-optimized human FN gene (SEQ ID NO.1) was digested with MylI and XhoI and cloned into pOsPMP02 to construct plasmid pOsPMP627, as shown in Figure 2; then pOsPMP627 was digested with HindIII and EcoRI , Inserting the entire expression cassette with a length of 7152bp containing the Gt13a promoter and its signal peptide sequence and the codon-optimized FN gene and Nos terminator into the binary expression vector 1300 to construct an Agrobacterium-mediated bacterial plasmid, named pOsPMP628, as shown in Figure 3.
  • the pOsPMP628 plasmid was transformed into Agrobacterium tumefaciens EHA105 (Invitrogen Corporation, USA), and pOsPMP628 was transformed into the callus regeneration tissue of rice variety TP309 by Agrobacterium tumefaciens-mediated co-transformation, and formed a complete after culture, screening and induction Plants; Then, identify positive transformed plants by PCR amplification, using the forward primer FN-F1 (SEQ ID NO.
  • the expression level of OsrhFn in the above four genetically engineered rice was also determined by SDS-PAGE method. The results showed that the expression of FN was highest in the PMP628-71 family. As shown in Figure 6. Through the above various detection methods, the final selection of genetically engineered rice plants.
  • Example 2 Preparation of recombinant human fibronectin (hFn) crude extract
  • the recombinant human fibronectin genetically engineered rice is hulled and polished into semi-polished rice, and ground into 80-100 mesh rice flour.
  • the rice flour and the extraction buffer were mixed at a ratio of 1:5 (weight/volume, kg/L), and extracted at room temperature for 1 hour.
  • the composition of the extraction buffer is: 20 mM Phosphate Sodium (PB) pH 8.0, and 5 mM glutathione, 1 mM PMSF, and 0.1% Tween 80 are added to the extract in advance.
  • the mixture obtained above was filtered through a filter cloth plate and frame filter press to obtain a clear crude hFn protein extract.
  • the addition of glutathione can increase the extraction amount of hFn and reduce dimers and multimers.
  • the addition of PMSF is a broad-spectrum protease inhibitor that can reduce the degradation during hFn extraction.
  • the addition of Tween 80 can maintain the stability of hFn extraction. .
  • Heparin affinity chromatography is mainly characterized by ion exchange, combining the results of Heparin affinity chromatography and anion exchange chromatography, it can be replaced by anion exchange chromatography.
  • anionic fillers Q Bestarose Fast Flow, Q Bestarose HP, Bestarose DEAE, Q Sepharose TM HP, Q Sepharose TM Fast Flow, DEAE Sepharose TM Fast Flow, UniGel 30/80Q, NanoGel 30/50Q, UNO Sphere Q, all Effectively enrich the target protein, and can remove part of the miscellaneous protein.
  • the model shows that when the sample conductance is 9.6mS/cm, the elution conductance is 30mS/cm, and the pH is 7.0, the maximum recovery of Fn is 58.5% and the purity is 13.2%.
  • the predicted results of DoE experiment are shown in Fig. 11.
  • Huiyan QFF has penetrated, resulting in low recovery rate.
  • Huiyan Q and HP can be used as replacement fillers. The results are shown in Figure 12 and Figure 13.
  • the composition of the washing buffer is 50 mM Tris-HCl, 194 mM NaCl, pH 7.6, and the composition of the elution buffer is 50 mM Tris-HCl, 268 mM NaCl, pH 7.6.
  • the Tris buffer system was changed to the PB buffer system. After extraction under the same conditions, the clarity of the sample was greatly improved.
  • the stability of the sample was studied by adjusting the pH of the extraction solution. The results are shown in Figure 14.
  • the pH is less than 6.5, the extract begins to become turbid, so the pH of the Borglon SP SP chromatography is 7.0.
  • the optimized washing conditions are 20 mM PB, 130 mM NaCl, pH 5.9 is the washing buffer, and the elution buffer is 20 mM PB, 100 mM NaCl, pH 6.95.
  • the test results of the chromatographic samples are shown in Figure 15.
  • Nano Micro Gel 50 SP is a highly cross-linked porous polystyrene microsphere with high flow rate, high load capacity, high salt resistance, low back pressure and other characteristics; the experiment found that Nano 50 Gel SP at high flow rate
  • the download volume is up to 22.7g rice noodles/mL filler.
  • the safety load is still 12.5g rice noodles/mL filler, which is more suitable as a capture step.
  • the optimized washing buffer composition is 20 mM PB, 150 mM NaCl, pH 7.0; the elution buffer composition is 20 mM PB, 300 mM NaCl, pH 7.0.
  • the test results of the chromatographic samples are shown in Figure 16.
  • the two-step chromatography of Boglon SP and Huiyan Q HP is one of the preferred processes for the separation and purification of OsrhFn.
  • Cation exchange chromatography as the primary purification: 235ml SP Bestarose HP column with 20mM PB, pH 7.0 as equilibration buffer, 20mM PB with pH 5.9, 130mM NaCl as washing buffer, 20mM PB with pH 7.0, 100mM NaCl is used as the elution buffer for chromatography.
  • Nano-Gel NanoGel 50 SP-Borgron QFF two-step chromatography is one of the preferred processes for the separation and purification of OsrhFn.
  • Cation exchange chromatography as the primary purification: 90ml Nano Gel 50 SP column with 20mM PB, pH 7.0 as equilibrium buffer, 20mM PB, 145mM NaCl, pH 7.0 as washing buffer, 20mM PB, 300mM NaCl, pH 7.0
  • the elution buffer was purified by chromatography.
  • Cation exchange chromatography is used as the primary purification: the column is equilibrated with 20mM PB, pH 7.0, 20mM PB, 140mM NaCl, pH 7.0 as washing buffer, 20mM PB, 300mM NaCl, pH 7.0 as elution buffer. Chromatography, the chromatogram is shown in Figure 20.
  • FIG. 22 is the SEC-HPLC chart of the verification sample of Nano-Gel Nano 50 Gel SP-Borgron Q FF two-step chromatography purification process.
  • Figure 24 shows the detection results of the cell viability of the target protein obtained by the Nano-Gel 50 SP-Borgron Q FF two-step chromatography purification process. The purity and activity of the recombinant human fibronectin obtained by the extraction and purification method of the present invention are satisfactory.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physiology (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Water Supply & Treatment (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种表达重组纤维连接蛋白的基因工程水稻种子中分离纯化重组人纤维连接蛋白的层析方法。将基因工程稻谷粉碎后与提取缓冲液混合提取后过滤，得到含重组人纤维连接蛋白的粗提液；将含重组人纤维连接蛋白的粗提液经过阳离子交换层析，进行初级分离纯化，获得含重组人纤维连接蛋白的初级产物；将初级产物经过阴离子交换层析，进行末级分离纯化，获得重组人纤维连接蛋白目标物。该方法成本低，易于工业化放大。所获得的OsrhFn目标物SEC-HPLC纯度大于95％。生物活性较好。

Description

一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法 技术领域
本发明属于生物技术领域，具体涉及一种分离纯化重组人纤维连接蛋白的方法。
背景技术
纤维连接蛋白(Fibronectin，Fn)，也做纤维结合蛋白，是一种分布极为广泛的高分子量糖蛋白。存在于血浆、细胞胞内物质及不同细胞表面中，它通常以分子量为450kD左右的二聚体形式存在，单体分子量为220-250kD，由位于蛋白质羧基端的二硫键相连。纤维连接蛋白大多以可溶的形式存在于血浆等体液中，以不可溶的形式存在于细胞外基质中。它具有与细胞外基质蛋白如胶原蛋白、循环血液蛋白如血纤蛋白、糖胺聚糖如肝素等物质的结合，因此，在很多重要的生理过程如胚胎发育、伤口愈合、止血和凝血中都发挥着重要的作用。
早期从组织中纯化Fn的方法多为冷沉淀法，Fn与一些杂蛋白在4℃以下静置时会以沉淀形式析出，之后再以沉淀和离子交换层析等方法进一步纯化；从培养纤维原表面分离Fn蛋白则采用低浓尿素的提取方法；而Anti-Fn介质则可广泛地应用于血浆及细胞培养物中Fn的纯化。但这些方法都逐渐以亲和层析所取代，原理是基于它与变性胶原蛋白(通常为明胶)的高特异亲和性的结合，然后以1mol L KBr、1-8mol L尿素或胺盐洗脱。
纤维连接蛋白在血浆中的含量极为丰富，约为300mg/L，因此血浆是获得Fn的一个主要来源。国外生产的Fn产品都是从人血浆中提取的,作为化妆品添加剂并可用于药物中治疗创伤、烧伤、休克等，具有重大的社会效益及经济价值，但血浆来源有限，且生产工艺复杂，不利于规模化放大。
发明内容
本发明的目的在于提供一种从表达重组纤维连接蛋白的基因工程水稻种子中分离纯化重组人纤维连接蛋白的层析方法。
为实现上述目的，本发明提供以下技术方案：
一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法，依次包括以下步骤：
1)从重组人纤维连接蛋白的基因工程水稻种子中提取含有重组人纤维连接蛋白的粗提取液；
2)将含有重组人纤维连接蛋白的粗提取液经阳离子交换层析，得到初级产物；
3)将初级产物经阴离子交换层析，得到纯化的重组人纤维连接蛋白。
步骤1)中以重组人纤维连接蛋白基因工程水稻种子为原料，将稻谷脱壳抛光成半精米，并研磨成80-100目的米粉；将所述米粉与提取缓冲液以重量/体积为1:5-1:10的比例混合，常温下提取0.5-2小时，获得粗蛋白提取物；
所述提取缓冲液为：0-50mM Tris、0-50mM PB，0-110mM NaCl，pH 5.9-8.0；优选的提取组分中可添加0.8-1mM PMSF或5-10mM GSH或0.05-0.1％Tween 80中的一种或数种。
步骤2)中阳离子交换层析的填料选自NanoGel 30/50 SP、UniGel 30/80 SP、SP Bestarose FF、SP Bestarose HP、Bestarose Diomond MMC、Uniphere S、MacroPrep S、POROS XS、SP-6FF、SP-6HP、SP SepharoseTM Fast Flow；优选使用NanoGel 50 SP或SP Bestarose HP。阳离子交换层析可选择pH梯度洗脱或者氯化钠浓度梯度洗脱，优选氯化钠梯度洗脱。
在一种实施方式中，选择pH和氯化钠梯度结合的方式洗脱，洗杂缓冲液包括磷酸盐缓冲液，0.13M氯化钠，0.3M氯化钠，pH为5.9，洗脱缓冲液包括磷酸盐缓冲液，0.1M氯化钠，0.3M氯化钠，pH为7.0。
在另一种实施方式中，选择氯化钠梯度洗脱，层析过程的洗杂和洗脱缓冲液包括磷酸盐缓冲液，0.15M氯化钠，0.3M氯化钠，pH为7.0。
步骤3)中阴离子交换层析的填料选自Q Bestarose Fast Flow、Q Bestarose HP、Bestarose DEAE、Q SepharoseTM HP、Q SepharoseTM Fast Flow、DEAE SepharoseTM Fast Flow、UniGel 30/80Q、NanoGel 30/50Q、UNO Sphere Q。优选使用博格隆Q FF或汇研Q HP。
阴离子交换层析可选择氯化钠浓度梯度洗脱。在一种实施方式中，层析过程的洗杂和洗脱缓冲液包括磷酸盐缓冲液，0.2M氯化钠，0.3M氯化钠，pH均为7.0。
经阴离子交换层析获得的目标洗脱组分可用已知的方法进行浓缩、冷冻干燥法等制成成品。
在阳离子交换层析中，使用的上样缓冲液包括磷酸盐缓冲液，其pH小于7.5，盐浓度小于0.12M。
阳离子交换层析中目标物(重组人纤维连接蛋白)的洗脱缓冲液包括磷酸盐缓冲液和氯化钠；pH为6.8-7.1，优选的氯化钠浓度为0.3M，缓冲液的pH为7.0。
在一种实施方式中，当阳离子交换层析的填料为SP Bestarose HP时，使用的缓冲液包括磷酸盐缓冲液、0.09-0.13M氯化钠，pH为5.8-7.1。
在另一种实施方式中，当阳离子交换层析的填料为Nano Gel 50 SP时，使用的缓冲液包括磷酸盐缓冲液、0.12-0.3M氯化钠，pH为6.8-7.1。
在另一种实施方式中，当阴离子交换层析的填料为汇研Q HP时，使用的缓冲液包括磷酸盐、0.1-0.3M氯化钠，pH为6.8-7.1。
在另一种实施方式中，当阴离子交换层析的填料为博格隆Q FF时，使用的缓冲液包括磷酸盐、0.1-0.3M氯化钠，pH为6.8-7.1。
本发明还提供一种用于制备所述的基因工程水稻种子的植物表达载体，所述表达载体是将表达人纤维连接蛋白的基因与水稻特异性启动子Gt13a及其信号肽导入质粒载体构建。优选的，所述表达人纤维连接蛋白的基因的核苷酸序列如SEQ ID NO.1所示，所述质粒载体为pOsPMP626。
本发明所用的原料来源于表达重组纤维连接蛋白的基因工程水稻种子，利用水稻胚乳特异性表达的启动子和信号肽，介导Fn进入水稻胚乳细胞的内膜系统，并储存到水稻胚乳的蛋白体中，从而使Fn能在水稻种子内大量积累，最终达到较高的表达水平。由于水稻表达体系中不存在Fg和vWF等血浆特有的杂质，因此以水稻种子进行Fn的分离纯化具有非常大的优势，具有与其他来源不同的纯化方法。
本发明利用阳离子和阴离子交换两步层析对表达重组纤维连接蛋白的基因工程水稻种子中提取Fn并进行纯化，并对工艺条件进行摸索和优化，为水稻种子种分离纯化重组人纤维连接蛋白提供了一种可规模化放大的纯化工艺。
附图说明
图1为质粒pOsPMP626结构示意图。
图2为质粒pOsPMP627结构示意图。
图3为质粒pOsPMP628结构示意图。
图4为T1代基因工程材料中目的基因的阳性检测，其中M为DNA标准分子量Marker；PMP628-51、628-62、628-63、628-64、628-65、628-68、628-69和628-71为T1代转基因材料；NC，阴性对照受体品种；P为阳性对照质粒。
图5为T1代基因工程材料中标记基因的阳性检测，其中M为DNA标准分子量Marker；PMP628-51、628-62、628-63、628-64、628-65、628-68、628-69和628-71为T1代基因工程材料；NC，阴性对照受体品种；P为阳性对照质粒。
图6为SDS-PAGE鉴定T1代基因工程种子中FN的表达量检测，其中M为标准分子量Marker；PC为220 kD的Human Plasma Fibronectin(FN)纯蛋白(Merck公司)；PMP628-51、628-62、628-63、628-64、628-65、628-68、628-69和628-71为转基因材料。
图7为不同Heparin亲和层析洗脱样品的SDS-PAGE检测，其中纳微、千纯、博格隆、GE、汇研分别对应NW、QC、BGL、GE、HY；
图8为Heparin层析优化的洗杂条件所获样品的SDS-PAGE检测，其中M 为标准分子量Maker，Load为蛋白提取液，FT为穿透液，10％B为100mM NaCl，18％B、21％B、29％B、36％B分别对应180mM、210mM、290mM、360mM NaCl。
图9为Heparin层析优化的洗杂条件所获样品的SDS-PAGE检测，其中Load为蛋白提取液，FT为穿透液，12％B、14％B、16％B、29％B分别对应120mM、140mM、160mM、290mM NaCl，CIP为再生液。
图10为汇研Q FF层析pH、上样电导、流速DoE实验预测刻画结果。
图11为汇研Q FF层析上样电导、洗脱pH、洗脱电导DoE实验预测刻画结果。
图12为GE Q HP和汇研Q FF层析填料的对比实验样品SDS-PAGE检测结果，其中M为标准分子量Marker，Load为蛋白提取液，FT为穿透液，Wash为洗杂液，Elu为洗脱液。
图13为GE Q HP和汇研Q HP层析填料的对比实验样品SDS-PAGE检测结果，其中M为标准分子量Marker，Load为蛋白提取液，FT为穿透液，Wash为洗杂液，Elu为洗脱液，CIP为再生液。
图14为提取液pH稳定性研究样品SDS-PAGE检测，其中M为标准分子量Marker，上清为调pH后样品离心后的上清，沉淀为调pH后样品离心后的沉淀再次复溶。
图15为博格隆SP HP层析样品的SDS-PAGE检测，其中M为标准分子量Marker，Load为蛋白提取液，FT为穿透液，Wash为洗杂液，Elu(10X)为洗脱液浓缩10倍。
图16为纳微Nano Gel 50 SP填料的层析样品SDS-PAGE检测，其中M为标准分子量Marker，Load为蛋白提取液，FT为穿透液，Wash为洗杂液，Elu为洗脱液。
图17为汇研Q HP、纳微UniGel 80 Q、博格隆Q FF填料的层析样品SDS-PAGE检测结果，其中M为标准分子量Marker，Load为NanoGel 50 SP层析洗脱收集液，FT为穿透液，Wash为洗杂液，Elu为洗脱液，CIP为再生液。
图18为博格隆SP HP-汇研Q HP两步层析纯化样品的SDS-PAGE检测结果，其中M为标准分子量Marker，还原为汇研Q HP层析洗脱液加入还原型上样液处理的样品，非还原为汇研Q HP层析洗脱液加入非还原型上样液处理的样品。
图19为纳微Nano Gel 50 SP-博格隆Q FF两步层析纯化样品的SDS-PAGE检测结果，其中M为标准分子量Marker，Load为蛋白提取后样品调pH至上样pH，FT为穿透液，Wash为洗杂液，Elu为洗脱液。
图20为纳微Nano Gel 50 SP层析图谱。
图21为博格隆Q FF层析图谱。
图22为纳微Nano Gel 50 SP-博格隆Q FF两步层析纯化工艺验证样品的 SDS-PAGE检测结果，其中M为标准分子量Marker，P为Fn阳性参照，0927、0928、0929为不同批次样品，还原为还原性电泳，非还原为非还原型电泳。
图23为纳微Nano Gel 50 SP-博格隆Q FF两步层析纯化工艺验证样品的SEC-HPLC图谱。
图24为纳微Nano Gel 50 SP-博格隆Q FF两步层析纯化工艺得到的目标蛋白细胞活性检测结果。
具体实施方式
下文将通过实施例和附图以详细说明本发明的技术方案，从而更好地阐述本发明的特点和优势。所提供的实施例应被解释为对本发明方法的举例说明，而不以任何方式限制本发明揭示的技术方案。
以下实施例中使用的试剂和仪器，除特别说明以外，均为普通市售。
实施例1：重组人纤维连接蛋白基因工程稻谷的制备
本实施例选用水稻特异性启动子Gt13a及其信号肽来介导重组人纤维连接蛋白基因在水稻胚乳细胞中的表达，具体参考公开号为CN100540667中的方法来构建本发明的水稻特异性表达重组人纤维连接蛋白载体以及筛选基因工程水稻植株，将其中所述的重组人血清白蛋白换成本发明的重组人纤维连接蛋白。用如图1所示的质粒pOsPMP626来构建水稻胚乳特异性表达盒。将所述合成的经密码子优化的人FN基因(SEQ ID NO.1)用MylI和XhoI酶切后克隆到pOsPMP02中构建成质粒pOsPMP627，如图2所示；然后用HindIII和EcoRI酶切pOsPMP627，将长度为7152bp的含Gt13a启动子及其信号肽序列还有经密码子优化的FN基因以及Nos终止子的整个表达盒插入到双元表达载体1300，构建农杆介导菌质粒，命名为pOsPMP628，具体如图3所示。将所述pOsPMP628质粒转化根癌农杆菌EHA105(美国Invitrogen公司)，通过根癌农杆菌介导共转化将pOsPMP628转化到水稻品种TP309的愈伤再生组织中，经培养、筛选和诱导后形成完整的植株；然后，通过PCR扩增来鉴别阳性转化植株，用目的基因的正向引物FN-F1(SEQ ID NO.2:5’-ATCAACTACCGCACCGAGAT-3’)和反向引物FN-R1(SEQ ID NO.3:5’-TCTTCTCCTTCGGGGTCAC-3’)进行PCR扩增，产物大小为679bp。鉴别结果表明，通过根癌农杆菌介导转化获得72株独立的重组人纤维连接蛋白转基因水稻和4株独立的高产重组人纤维连接蛋白基因工程水稻。鉴定结果如图4和图5所示。
本实施例还通过SDS-PAGE方法测定上述4株基因工程水稻中OsrhFn的表达水平，结果显示PMP628-71家系中FN的表达最高。如图6所示。通过以 上多种检测方法，最终筛选获得基因工程水稻植株。
Figure PCTCN2019123762-appb-000001
Figure PCTCN2019123762-appb-000002
Figure PCTCN2019123762-appb-000003
实施例2：重组人纤维连接蛋白(hFn)粗提液的制备；
将重组人纤维连接蛋白基因工程稻谷脱壳抛光成半精米，研磨成80-100目的米粉。将米粉与提取缓冲液以1：5(重量/体积，kg/L)的比例混合，于常温提取1小时。提取缓冲液的成分为：20mM磷钠盐(PB)pH 8.0，提取液中预先加入5mM谷胱甘肽、1mM PMSF、0.1％Tween80。将上述得到的混合物经滤布式板框压滤机压滤，得到澄清的hFn蛋白粗提液。加入谷胱甘肽可提高hFn的提取量，减少二聚体和多聚体，加入PMSF是广谱蛋白酶抑制剂，可以减少hFn提取过程中的降解，加入Tween 80可保持hFn提取过程中稳定性。
实施例3：OsrhFn小试纯化工艺优化
1. OsrhFn粗纯条件优化
1.1作为粗纯的亲和层析介质筛选及层析条件优化
本发明选取纳微、千纯、博格隆、GE、汇研五家公司生产的不同类型的Heparin亲和填料在同一条件下进行对比实验。实验最终步骤的样品用SDS-PAGE进行检测，结果见图7。
经过比较发现，纳微、博格隆、GE三家的Heparin填料纯化效果较好，考虑到纳微填料基架为聚苯乙烯，稳定性和重复性更适合作为捕获步骤层析介质。通过实验条件摸索，Heparin在高盐条件下未能减少非特异性吸附且有大量目的蛋白损失，因此选择低盐条件进行上样。在此基础上进行了洗杂条件优化，洗杂的盐浓度范围为100-180mM，pH为8.0，洗脱盐浓度为290mM，pH为8.0。最终确定Heparin最佳层析条件为120mM NaCl，洗脱条件为290mM NaCl。优化结果见图8和图9。
1.2作为粗纯的阴离子层析介质筛选及层析条件优化
由于Heparin亲和层析特征主要表现为离子交换作用，结合Heparin亲和层析和阴离子交换层析结果，可用阴离子交换层析进行替换。从37种阴离子填料Q Bestarose Fast Flow、Q Bestarose HP、Bestarose DEAE、Q Sepharose TM HP、Q Sepharose TM Fast Flow、DEAE Sepharose TM Fast Flow、UniGel 30/80Q、NanoGel 30/50Q、UNO Sphere Q，均能有效富集目的蛋白，且能去除部分杂蛋白。
以汇研Q FF用于层析条件摸索，选取pH值、电导、流速三个因素，每个因素选择三个水平，进行DoE实验。最终确定电导9.6mS/cm，pH为7.6、流速为0.58mL/min作为层析的最佳上样条件，此条件下Fn回收率最高为58.13％，DoE实验预测结果见图10。为进一步提高Q FF的回收率，固定上样pH，选取上样电导、洗脱电导、洗脱pH三个因素，每个因素选择三个水平进行DoE实验。模型显示当上样电导为9.6mS/cm，洗脱电导为30mS/cm，pH为7.0时，Fn回收率最高为58.5％，纯度为13.2％，DoE实验预测结果见图11。
无论在低电导还是在高电导条件下，汇研Q FF均有穿透，导致回收率偏低，根据阴离子填料筛选复筛结果，可尝试用汇研Q HP和GE Q HP作为替换填料，替换结果见图12和图13。优化之后的洗杂缓冲液成份为50mM Tris-HCl，194mM NaCl，pH 7.6，洗脱缓冲液成份为50mM Tris-HCl，268mM NaCl，pH 7.6。
1.3作为粗纯的阳离子层析介质筛选及层析条件优化
1.3.1博格隆SP HP层析条件优化
为更好衔接阳离子交换层析，将Tris缓冲体系改变为PB缓冲体系，按照同样条件进行提取后，上样样品的澄清度有较大改善，通过调节提取液的pH对样 品的稳定性进行研究，结果见图14，当pH＜6.5时，提取液开始变浑浊，因此选择博格隆SP HP层析上样pH为7.0。在此条件下，优化的洗杂条件为20mMPB，130mM NaCl，pH 5.9为洗杂缓冲液，洗脱缓冲液为20mM PB，100mM NaCl，pH 6.95。层析样品检测结果见图15。
1.3.2纳微Nano Gel 50 SP层析条件优化
纳微Nano Gel 50 SP层析介质是一种高交联的多孔聚苯乙烯微球，具有高流速、高载量、耐高盐、低反压等特性；实验发现Nano Gel 50 SP在高流速下载量高达22.7g米粉/mL填料，经过放大确认后安全载量依然有12.5g米粉/mL填料，作为捕获步骤比较合适。优化的洗杂缓冲液成分为20mM PB，150mM NaCl，pH为7.0；洗脱缓冲液成分为20mM PB，300mM NaCl，pH 7.0。层析样品检测结果见图16。
2. OsrhFn精纯条件优化
为了获得简单的工艺，根据前期研究结果，选择汇研Q HP、博格隆Q FF、纳微UniGel 80 Q进行实验。发现汇研Q HP和博格隆Q FF均有较好的分离效果，在同样条件下UniGel 80 Q目的蛋白的洗脱盐浓度降低，有利于后期工艺。综合考虑，选择粒径较大的Q FF作为精纯介质，Q HP作为备选方案。检测结果见图17。
实施例4：博格隆SP HP-汇研Q HP两步纯化工艺
根据实施例3粗纯和精纯层析条件筛选结果，博格隆SP HP和汇研Q HP两步层析作为优选的工艺之一进行OsrhFn的分离纯化。
提取：称取1000g基因工程水稻米粉，按照实施例1进行提取。
阳离子交换层析作为初级纯化：235ml SP Bestarose HP层析柱以20mM PB，pH 7.0为平衡缓冲液，采用pH为5.9的20mM PB，130mM NaCl为洗杂缓冲液，采用pH 7.0的20mM PB，100mM NaCl为洗脱缓冲液进行层析。
阴离子交换层析作为末级纯化：28ml汇研Q HP层析柱以pH为7.0的20mM PB为平衡缓冲液，采用pH为7.0的20mM PB，200mM NaCl为洗杂缓冲液，采用pH为7.0的20mM PB，300mM NaCl为洗脱液进行层析。层析样品SDS-PAGE检测结果见图18。
实施例5：纳微NanoGel 50 SP–博格隆Q FF两步纯化工艺
根据实施例3粗纯和精纯层析条件筛选结果，纳微NanoGel 50 SP–博格隆Q FF两步层析作为优选的工艺之一进行OsrhFn的分离纯化。
提取：称取1000g基因工程水稻米粉，按照实施例1进行提取。
阳离子交换层析作为初级纯化：90ml Nano Gel 50 SP层析柱以20mM PB， pH 7.0为平衡缓冲液，20mM PB，145mM NaCl，pH 7.0为洗杂缓冲液，20mM PB，300mM NaCl，pH 7.0为洗脱缓冲液进行层析纯化。
阴离子交换层析作为末级纯化：16ml博格隆Q FF层析柱以pH为7.0的20mM PB为平衡缓冲液，采用pH为7.0的20mM PB，200mM NaCl为洗杂缓冲液，采用pH 7.0的20mM PB，300mM NaCl为洗脱缓冲液进行层析。层析样品SDS-PAGE检测结果见图19。
实施例6：OsrhFn纯化工艺验证
为验证小试工艺的重复性，将优选的纳微Nano Gel 50 SP–博格隆Q FF两步层析工艺进行了三次验证，验证程序具体实施步骤如下：
提取：向5000mL 20mM PB，pH 8.0提取液中加入7.7g GSH(100mL超纯水溶解)，用2M NaOH复调pH至8.0；加入0.87g PMSF(435mL异丙醇溶解)，5ml Tween 80，搅拌混匀；称取1kg Fn米粉，加入提取液中室温搅拌提取1h，加入250g助滤剂，正压过滤，压滤液用0.45μm滤膜过滤获得含有OsrhFn的粗提液。
阳离子交换层析作为初级纯化：层析柱以20mM PB，pH 7.0为平衡缓冲液，20mM PB，140mM NaCl，pH 7.0为洗杂缓冲液，20mM PB，300mM NaCl，pH 7.0为洗脱缓冲液进行层析，层析图谱见图20。
阴离子交换层析作为末级纯化：层析柱以20mM PB，pH 7.0为平衡缓冲液，20mM PB，200mM NaCl，pH 7.0为洗杂缓冲液，20mM PB，300mM NaCl，pH 7.0为洗脱缓冲液进行层析，层析图谱见图21。
三批验证批次纯度、蛋白浓度和产率结果见下表。
Figure PCTCN2019123762-appb-000004
三批验证批次的SDS-PAGE检测结果见图22。根据实验数据，所获得的目标蛋白的纯度大于95％。图23为纳微Nano Gel 50 SP-博格隆Q FF两步层析纯化工艺验证样品的SEC-HPLC图谱。图24为纳微Nano Gel 50 SP-博格隆Q FF两步层析纯化工艺得到的目标蛋白细胞活性检测结果。本发明提取纯化方法所获得的重组人纤维连接蛋白的纯度和活性均令人满意。

Claims (10)

  1. 一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的层析方法，依次包括以下步骤：
    1)从重组人纤维连接蛋白的基因工程水稻种子中提取含有重组人纤维连接蛋白的粗提取液；
    2)将含有重组人纤维连接蛋白的粗提取液经阳离子交换层析，得到初级产物；
    3)将所述初级产物经阴离子交换层析，得到纯化的重组人纤维连接蛋白目标物；
    其中阳离子交换层析的填料选自Nano Gel 30/50 SP、Uni Gel 30/80 SP、SP Bestarose FF、SP Bestarose HP、Bestarose Diomond MMC、Uniphere S、MacroPrep S、POROS XS、SP-6FF、SP-6HP和SP Sepharose TM Fast Flow；
    阴离子交换层析的填料选自Q Bestarose Fast Flow、Q Bestarose HP、Bestarose DEAE、Q Sepharose TM HP、Q Sepharose TM Fast Flow、DEAE Sepharose TMFast Flow、UniGel 30/80Q、NanoGel 30/50Q和UNO Sphere Q。
  2. 根据权利要求1所述的方法，其中阳离子交换层析的填料为Nano Gel 50 SP。
  3. 根据权利要求1所述的方法，其中阴离子交换层析的填料为Q Bestarose FF。
  4. 根据权利要求1所述的方法，其中步骤1)所述重组人纤维连接蛋白粗提取液是通过下述方法制备，包括步骤：
    4a)以表达重组人纤维连接蛋白的基因工程水稻种子为原料，将稻谷脱壳抛光成半精米并研磨成80-100目的米粉；
    4b)将所述米粉与提取缓冲液以重量/体积为1:5-1:10的比例混合，常温下提取0.5-2小时，获得蛋白粗提取物；所述提取缓冲液为：10-50mM Tris、10-50mM PB，0-110mM NaCl，pH 5.9-8.0；
    4c)将粗蛋白提取物过滤，得到含重组人纤维连接蛋白的粗提取液。
  5. 根据权利要求4所述的方法，其特征在于步骤4b)中的提取缓冲液中添加0.8-1mM PMSF、5-10mM GSH和0.05-0.1％Tween 80中的一种或数种。
  6. 根据权利要求1所述的方法，将由步骤1)获得的含重组人纤维连接蛋白的粗蛋白提取液经过阳离子交换层析，进行初级分离纯化，获得含重组人纤维连接蛋白的初级产物；其中：所述阳离子交换层析介质为纳微Nano Gel 50 SP阳离子交换填料，层析步骤为：
    6a)采用5-15倍柱体积的组分为10-50mM PB，0-120mM NaCl，pH为6.8-7.1的平衡缓冲液，以50-200cm/小时的流速平衡层析柱；
    6b)以步骤1)的蛋白粗提取液作为上样样品，其中样品电导为2.5-13.5ms/cm，pH为6.8-7.1；
    6c)采用组分为10-50mM PB，130-200mM NaCl，pH为6.8-7.1的洗杂缓冲液，以50-200cm/小时的流速洗脱杂蛋白，洗杂缓冲液体积为20-40倍柱体积；
    6d)采用组分为10-50mM PB，250-300mM NaCl，pH为6.8-7.1的洗脱缓冲液，以50-200cm/小时的流速洗脱样品，所得洗脱液为初级产物。
  7. 根据权利要求1所述的方法，将由步骤1)获得的含重组人纤维连接蛋白的蛋白粗提取液经过阳离子交换层析，进行初级分离纯化，获得含重组人纤维连接蛋白的初级产物；其中所述阳离子交换层析介质为博格隆SP Bestarose HP阳离子交换填料，层析步骤为：
    7a)采用5-15倍柱体积组分为10-50mM PB，pH为6.8-7.1的平衡缓冲液，以50-200cm/小时的流速平衡层析柱；
    7b)以步骤1)的蛋白粗提取液作为上样样品，其中样品电导为2.5ms/cm，pH为6.8-7.1；
    7c)用7a)中的平衡缓冲液20-40倍柱体积进行再平衡，采用组分为10-50mM PB，100-130mM NaCl，pH为5.8-6.1的洗杂缓冲液，以50-200cm/小时的流速洗脱杂蛋白；
    7d)用组分为10-50mM PB，90-110mM NaCl，pH为6.8-7.1的洗脱缓冲液，以50-200cm/小时的流速洗脱样品，所得洗脱液为初级产物。
  8. 根据权利要求1所述的方法，将由步骤2)获得的含重组人纤维连接蛋白的初级产物经过阴离子交换层析，获得纯化的重组人纤维连接蛋白目标物；其中：所述阴离子交换层析介质为博格隆Q Bestarose FF或汇研Q HP阴离子交换填料；具体步骤如下：
    8a)采用5-15倍柱体积的平衡缓冲液，以50-200cm/小时的流速平衡层析柱，平衡缓冲液的组分为10-50mM PB，0-150mM NaCl，pH为6.8-7.1；
    8b)以步骤2)的初级产物作为上样样品，其中样品电导为12.5-17.6ms/cm，pH为6.8-7.1；
    8c)采用组分为10-50mM PB，200-220mM NaCl，pH为6.8-7.1的洗杂缓冲液，以50-200cm/小时的流速洗脱杂蛋白；
    8d)采用组分为10-50mM PB，250-300mM NaCl，pH为6.8-7.1的洗脱缓冲液，以50-200cm/小时的流速洗脱样品，所得洗脱液为终级产物。
  9. 一种用于制备权利要求1所述的基因工程水稻种子的植物表达载体，其特征在于，所述表达载体是将表达人纤维连接蛋白的基因与水稻特异性启动子Gt13a及其信号肽导入质粒载体构建。
  10. 根据权利要求9所述的植物表达载体，其特征在于所述表达人纤维连 接蛋白的基因的核苷酸序列如SEQ ID NO.1所示，所示质粒载体为pOsPMP626。
PCT/CN2019/123762 2018-12-10 2019-12-06 一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法 WO2020119609A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP19894910.9A EP3896163A4 (en) 2018-12-10 2019-12-06 METHOD FOR THE SEPARATION AND PURIFICATION OF RECOMBINANT HUMAN FIBRONECTIN FROM GENETICALLY MODIFIED RICE SEEDS
KR1020217019636A KR102689084B1 (ko) 2018-12-10 2019-12-06 유전자 조작 벼 종자로부터 재조합 인체 피브로넥틴의 분리 및 정제 방법
JP2021533137A JP7312830B2 (ja) 2018-12-10 2019-12-06 遺伝子改良水稲の子実から組換えヒトフィブロネクチンを分離精製する方法
US17/312,875 US20220056071A1 (en) 2018-12-10 2019-12-06 Method for separating and purifying recombinant human fibronectin from genetically engineered rice seed

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811505129.2 2018-12-10
CN201811505129.2A CN111285932B (zh) 2018-12-10 2018-12-10 一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法

Publications (1)

Publication Number Publication Date
WO2020119609A1 true WO2020119609A1 (zh) 2020-06-18

Family

ID=71021300

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/123762 WO2020119609A1 (zh) 2018-12-10 2019-12-06 一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法

Country Status (6)

Country Link
US (1) US20220056071A1 (zh)
EP (1) EP3896163A4 (zh)
JP (1) JP7312830B2 (zh)
KR (1) KR102689084B1 (zh)
CN (1) CN111285932B (zh)
WO (1) WO2020119609A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274091A1 (zh) * 2021-06-30 2023-01-05 武汉禾元生物科技股份有限公司 一种以基因工程水稻表达和制备重组瑞替普酶的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117645664A (zh) * 2024-01-29 2024-03-05 英特菲尔(成都)生物制品有限责任公司 重组人纤连蛋白标准品及其制备方法、鉴定方法与应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100540667C (zh) 2005-07-13 2009-09-16 杨代常 利用水稻胚乳细胞作为生物反应器生产重组人血清白蛋白
CN102532254A (zh) * 2010-12-24 2012-07-04 武汉禾元生物科技有限公司 一种从水稻种子中分离纯化重组人血清白蛋白的方法
CN102994514A (zh) * 2012-11-07 2013-03-27 武汉禾元生物科技有限公司 一种从水稻种子生产和分离纯化重组人抗胰蛋白酶（OsrAAT)的方法
CN103589706A (zh) * 2013-04-25 2014-02-19 珠海市御品堂生物科技有限公司 纳豆激酶的提纯方法及制备方法
CN103880947A (zh) * 2012-12-21 2014-06-25 武汉禾元生物科技有限公司 一种分离纯化高纯度重组人血清白蛋白的层析方法
CN104109204A (zh) * 2013-04-16 2014-10-22 武汉禾元生物科技有限公司 一种从水稻种子中分离纯化重组人乳铁蛋白的方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5460955A (en) * 1991-01-03 1995-10-24 Wisconsin Alumni Research Foundation Fibronectin purification vector
US6531577B1 (en) * 1997-12-15 2003-03-11 Hemasure Denmark A/S von Willebrand factor (vWF)-containing preparation, process for preparing vWF-containing preparations, and use of such preparations
MXPA05001034A (es) * 2002-07-29 2005-06-08 Univ Laval Metodo para mejorar el rendimiento de produccion de proteina recombinante de plantas.
JP4960701B2 (ja) * 2003-08-27 2012-06-27 オーアールエフ・リフタエクニ・エイチエフ 組換えタンパク質のタンパク質分解性切断および精製のためのプロセス
EP1711048A4 (en) * 2003-12-23 2008-05-14 Ventria Bioscience METHODS FOR EXPRESSING A HETEROLOGOUS PROTEIN IN VEGETABLE SEEDS USING MONOCOTYLEDONE SEED PROTEIN PROMOTERS OTHER THAN RESIST PROTEINS
CN102812121A (zh) * 2010-01-25 2012-12-05 文特里亚生物科学公司 用于改善蛋白质生产的方法和组合物
CA2876135A1 (en) * 2012-06-07 2013-12-12 National Cheng Kung University Modified fibronectin fragments or variants and uses thereof
CN103865932A (zh) 2012-12-11 2014-06-18 武汉禾元生物科技有限公司 一种从水稻种子生产重组人碱性成纤维细胞生长因子的方法
CN107827974B (zh) * 2017-11-10 2021-01-01 中国科学院过程工程研究所 一种人纤维蛋白原的制备方法
CN108794639B (zh) * 2018-07-03 2020-11-20 广州澳特朗生物技术有限公司 一种重组纤连蛋白及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100540667C (zh) 2005-07-13 2009-09-16 杨代常 利用水稻胚乳细胞作为生物反应器生产重组人血清白蛋白
CN102532254A (zh) * 2010-12-24 2012-07-04 武汉禾元生物科技有限公司 一种从水稻种子中分离纯化重组人血清白蛋白的方法
CN102994514A (zh) * 2012-11-07 2013-03-27 武汉禾元生物科技有限公司 一种从水稻种子生产和分离纯化重组人抗胰蛋白酶（OsrAAT)的方法
CN103880947A (zh) * 2012-12-21 2014-06-25 武汉禾元生物科技有限公司 一种分离纯化高纯度重组人血清白蛋白的层析方法
CN104109204A (zh) * 2013-04-16 2014-10-22 武汉禾元生物科技有限公司 一种从水稻种子中分离纯化重组人乳铁蛋白的方法
CN103589706A (zh) * 2013-04-25 2014-02-19 珠海市御品堂生物科技有限公司 纳豆激酶的提纯方法及制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Wuhan Healthgen Launches Rice Recombinant Fibronectin: Fibronectin OsrFn", WUHAN HEALTHGEN LAUNCHES RICE RECOMBINANT FIBRONECTIN: FIBRONECTIN OSRFN-BIO-EQUIP, 5 December 2018 (2018-12-05), pages 1 - 5, XP055811301, Retrieved from the Internet <URL:https://www.bio-equip.com/news.asp?id=453079117> *
See also references of EP3896163A4
SHOJI, Y. ET AL.: "A Fibronectin-binding Protein from Rice Bran with Cell Adhesion Activity for Animal Tumor Cells", BIOSCIENCE, BIOTECHNOLOGY , AND BIOCHEMISTRY, vol. 65, no. 5, 31 December 2001 (2001-12-31), pages 1181 - 1186, XP009024726, DOI: 10.1271/bbb.65.1181 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274091A1 (zh) * 2021-06-30 2023-01-05 武汉禾元生物科技股份有限公司 一种以基因工程水稻表达和制备重组瑞替普酶的方法

Also Published As

Publication number Publication date
EP3896163A4 (en) 2022-08-10
JP7312830B2 (ja) 2023-07-21
EP3896163A1 (en) 2021-10-20
JP2022511925A (ja) 2022-02-01
CN111285932A (zh) 2020-06-16
KR20210099030A (ko) 2021-08-11
CN111285932B (zh) 2023-07-11
US20220056071A1 (en) 2022-02-24
KR102689084B1 (ko) 2024-07-26

Similar Documents

Publication Publication Date Title
Hansson et al. Single–Step Recovery of a Secreted Recombinant Protein by Expanded Bed Adsorption
CA2132533C (en) Protein purification
KR101868858B1 (ko) 형질전환 쌀 알곡으로부터 인간 혈청 알부민을 정제하는 방법
WO2020119609A1 (zh) 一种从基因工程水稻种子中分离纯化重组人纤维连接蛋白的方法
JP5868999B2 (ja) 植物において組換えタンパク質を発現および精製するための費用効果が高い方法
CN107286249A (zh) 一种寡聚赖氨酸修饰的重组去铁蛋白纳米笼及其制备
EP2102335B1 (en) Purification of factor xi
EP4143331A2 (en) Fusion polypeptides for target peptide production
US11325953B2 (en) Protein of ‘dangshan suli’ having function of promoting growth of pollen tube, encoding gene PBRTTS1 and use thereof
CN111484557B (zh) 一种从基因工程水稻种子中分离纯化重组人血清白蛋白-表皮生长因子融合蛋白的方法
CA2890659C (en) Method for producing, isolating and purifying recombinant human antitryptase (osraat) from rice seeds
CA2537102A1 (en) A process for proteolytic cleavage and purification of recombinant proteins produced in plants
CN111808881A (zh) 一种高效表达afp3-pten融合蛋白的方法
CN109942700A (zh) 一种重组型荞麦胰蛋白酶抑制剂突变体以及胰蛋白酶亲和材料
CN109776654B (zh) 一种亲和肽及其应用
CN109609539B (zh) 一种从重组毕赤酵母中制备elabela多肽的方法
CN111285929B (zh) 一种从基因工程水稻种子中分离纯化重组人表皮细胞生长因子的方法
CN112142848A (zh) 一种重组人胰岛素及其纯化制备方法
KR101906319B1 (ko) 재조합 3형 아데노바이러스 섬유 폴리펩타이드의 정제방법
CN108484738B (zh) 辐抗肽突变蛋白及其制备与纯化方法
CN115873833B (zh) 生产免疫球蛋白g降解酶的工程菌株和工艺
CN106566819B (zh) 一种低温嗜盐α—淀粉酶的基因克隆、表达及分离纯化方法
CN118879716A (zh) 一种核酸、重组质粒、重组人fgf-9蛋白的制备方法
CN116063571A (zh) 重组ssb抗原的制备方法和应用
CN114854782A (zh) 一种高效表达具有高活性的重组多肽连接酶原的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19894910

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021533137

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20217019636

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019894910

Country of ref document: EP

Effective date: 20210712