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WO2020088207A1 - Procédé de séparation et de purification de lactoferrine humaine de recombinaison à faible saturation en fer à partir de graines de riz génétiquement modifiées - Google Patents

Procédé de séparation et de purification de lactoferrine humaine de recombinaison à faible saturation en fer à partir de graines de riz génétiquement modifiées Download PDF

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Publication number
WO2020088207A1
WO2020088207A1 PCT/CN2019/110384 CN2019110384W WO2020088207A1 WO 2020088207 A1 WO2020088207 A1 WO 2020088207A1 CN 2019110384 W CN2019110384 W CN 2019110384W WO 2020088207 A1 WO2020088207 A1 WO 2020088207A1
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WIPO (PCT)
Prior art keywords
recombinant human
human lactoferrin
volume
genetically engineered
buffer
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PCT/CN2019/110384
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English (en)
Chinese (zh)
Inventor
杨代常
董亮亮
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Wuhan Healthgen Biotechnology Co Ltd
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Wuhan Healthgen Biotechnology Co Ltd
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Publication of WO2020088207A1 publication Critical patent/WO2020088207A1/fr
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    • 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
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • 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
    • 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 lactoferrin with low iron saturation from genetically engineered rice seeds.
  • Lactoferrin is a non-heme iron-binding glycoprotein that is widely present in the exudates of mammals and is the first line of defense against infection in the human body. It has a broad-spectrum antibacterial effect and can act on bacteria, fungi, Protozoa, viruses. Lactoferrin mainly has four kinds of antibacterial mechanisms: "iron deprivation", membrane penetration, enzyme inhibition and auxiliary antibacterial. (1) Iron deprivation mechanism. Almost all bacteria need iron for growth. Iron ions are necessary for oxidase in bacteria.
  • Lactoferrin as an iron-binding protein, can competitively bind iron ions with microorganisms, so that the microorganisms lose the iron ions on which they depend, which in turn leads to the death of the microorganisms.
  • the activity of lactoferrin depends on the degree of saturation of iron ions. In general, the lower the saturation, the higher the antibacterial activity.
  • Membrane penetration mechanism Lactoferrin is positively charged and has a strong affinity for negatively charged phospholipids, nucleic acids, and lipopolysaccharides, which leads to the release of lipopolysaccharides, which in turn destroys the bacterial plasma membrane.
  • Enzyme inhibition mechanism is bind iron ions with microorganisms, so that the microorganisms lose the iron ions on which they depend, which in turn leads to the death of the microorganisms.
  • the activity of lactoferrin depends on the degree of saturation of iron ions. In general, the lower the saturation, the higher the antibacterial
  • lactoferrin has a strong inhibitory effect on cysteine proteases, and cysteine protease inhibitors can effectively inhibit the growth of S. aureus. (4) Auxiliary antibacterial mechanism. Lactoferrin can indirectly achieve antibacterial effect by changing the relationship between bacteria and host.
  • Patent CN102459328A discloses a method for preparing lactoferrin with low iron saturation. The method includes using water-miscible solvent and acid to deprive iron in lactoferrin, and then using ultrafiltration or diafiltration to remove iron, solvent and acid The iron saturation of the obtained lactoferrin is less than 10%.
  • Some other research results on reducing iron saturation of lactoferrin involve the use of chelating agents and low pH environment to remove iron in lactoferrin, but the resulting iron saturation of lactoferrin is usually greater than 10% and is not suitable for commercial production .
  • the object of the present invention is to provide a method for separating and purifying recombinant human lactoferrin with low iron saturation from genetically engineered rice seeds.
  • the separation and purification method of the present invention includes the following steps:
  • the extraction buffer contains 50-100 mM citric acid-trisodium citrate, 100-200 mM NaCl, 1-10 mM EDTA- 2Na, pH is 3.5 ⁇ 5.0;
  • the further above method includes the following steps:
  • Hulling genetically engineered rice into semi-refined rice and grinding into 80-100 mesh rice flour The rice flour and the extraction buffer are mixed at a ratio of 1: 5 to 1:10 (weight / volume, kg / L), and extracted at 15 to 35 ° C for 1 to 24 hours.
  • the components of the extraction buffer are: 50-100 mM citric acid-trisodium citrate, 100-200 mM NaCl, 1-10 mM EDTA-2Na, pH 3.8-5.0.
  • the above-obtained mixture is added to 2 to 5% of perlite for pressure filtration. After the pressure filtration is completed, the filtrate is adjusted to pH 4.0 to 5.0 with 0.5 to 2M NaOH or dilute hydrochloric acid.
  • the 0.22 ⁇ m filter membrane is recombinant human lactoferrite
  • the crude protein extract is the loading solution for cation chromatography.
  • step (1) The crude extract of step (1) is used as the chromatographic loading liquid, wherein the pH of the loading liquid is 4.0 to 5.0, the conductivity is 20 to 30 mS / cm, and the loading volume is 20 to 40 CV;
  • the buffer protein containing 5-50 mM PB and 300-500 mM NaCl with a pH of 7.0-8.0 is used to elute the mixed protein at a linear flow rate of 280-315 cm / h, and the volume of the washing solution is 5-7 CV;
  • Another object of the present invention is to provide a method for extracting recombinant human lactoferrin with low iron saturation from genetically engineered rice seeds, which specifically includes:
  • the composition of the extraction buffer is 50 to 100mM citric acid-trisodium citrate, 100 ⁇ 200mMNaCl, 1 ⁇ 10mM EDTA-2Na, pH 3.8 ⁇ 5.0;
  • step (3) The mixture obtained in the above step (2) is added to 2 to 5% perlite for pressure filtration, and the filtrate is adjusted to pH 4.0 to 5.0 with 0.5 to 2M NaOH. After filtration, an extract containing recombinant human lactoferrin is obtained. Used as sample loading solution for chromatography;
  • the more preferred method of the present invention includes:
  • (2a) Mix rice flour with extraction buffer at a ratio of 1: 5 (weight / volume, kg / L), and extract at 25 ° C ⁇ 2.5 ° C for 5 to 17 hours.
  • the composition of the extraction buffer is 100 mM citric acid-citric acid Trisodium, 150mMNaCl, 10mM EDTA-2Na, pH 4.0;
  • the method of the invention can produce low-iron saturation lactoferrin on a large scale, at low cost and low cost.
  • Figure 1 SP BB load measurement chromatography UV absorption spectrum, where the ordinate indicates: UV 280 detection value, the abscissa indicates: chromatography volume (mL).
  • Figure 2 SDS-PAGE detection chart of chromatographic samples of SP load measurement.
  • FIG. 3 SDS-PAGE detection chart of chromatographic loading solution (A) and eluent (B) at different extraction pH.
  • FIG. 4 SDS-PAGE detection charts of chromatographic loading solution (A) and eluent (B) at different extraction temperatures.
  • Figure 5 SDS-PAGE detection chart of chromatographic loading solution (A) and eluent (B) at different extraction times.
  • SP Bestarose BigBeads (SP) BB packing used in the following examples, the manufacturer is Borglon (Shanghai) Biotechnology Co., Ltd .; B1030 chromatography column, purchased from Borglon (Shanghai) Biotechnology Co., Ltd .; other materials or reagents If there is no special instructions, they are all conventional commercial products.
  • the recombinant human lactoferrin genetically engineered rice (origin reference patent CN104109204B) is dehulled 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 25 ⁇ 2.5 ° C for 7 ⁇ 1h hours.
  • the components of the extraction buffer are: 100 mM citric acid-trisodium citrate, 150 mM NaCl, 10 mM EDTA-2Na, pH 4.0.
  • the above-obtained mixture is added to 2 to 5% perlite for pressure filtration.
  • the filtrate is adjusted to pH 5.0 with 0.5 to 2M NaOH and filtered through a 0.22 ⁇ m filter membrane to obtain a crude extract of recombinant human lactoferrin. It is the loading solution of SP BB chromatography.
  • Figure 1 shows that as the sample volume continues to increase, the penetration peak begins to rise significantly (as indicated by the red arrow).
  • the maximum capacity of SPBB is set to 37CV (less than 10% L, if calculated according to 10% penetration, the maximum dynamic binding capacity is 19 tubes or 41CV).
  • the maximum load of 37CV according to 80% of the sample load, the maximum sample load is 30CV, that is, the sample load does not exceed 30CV.
  • the target protein extracted with pH 3.5 is the least, and its output is only 0.77g / kg, which is far lower than other pH extraction conditions.
  • the pH of the extraction solution gradually increased from 3.8 to 5.0, the SDS-PAGE electrophoretic purity of the product decreased, and the yield and iron saturation gradually increased.
  • the output of pH 5.0 was the highest, up to 5.64g / kg , Iron saturation 15.35%.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Water Supply & Treatment (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de séparation et de purification de lactoferrine humaine de recombinaison à faible saturation en fer à partir de graines de riz génétiquement modifiées, le procédé consiste à : extraire d'abord un extrait brut contenant une lactoferrine de recombinaison à partir d'une lactoferrine humaine de recombinaison de graines de riz génétiquement modifiée, une solution tampon d'extraction contenant de 50 à 100 mM d'acide citrique-citrate trisodique, de 100 à 200 mM de NaCl, de 1 à 10 mM d'EDTA-2Na, le pH de celle-ci étant de 3,8 à 5,0 ; réaliser ensuite une chromatographie par cations sur l'extrait brut obtenu contenant la lactoferrine humaine de recombinaison pour obtenir un produit de lactoferrine à faible saturation en fer ayant une pureté > 95 %. La présente invention concerne en outre un procédé d'extraction de lactoferrine humaine de recombinaison à faible saturation en fer à partir de graines de riz génétiquement modifiées, la solution d'extraction utilisée contenant de l'acide citrique-citrate trisodique et de l'EDTA-2Na, qui peut réduire de manière significative la saturation en fer de la lactoferrine humaine de recombinaison.
PCT/CN2019/110384 2018-11-02 2019-10-10 Procédé de séparation et de purification de lactoferrine humaine de recombinaison à faible saturation en fer à partir de graines de riz génétiquement modifiées Ceased WO2020088207A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811299207.8 2018-11-02
CN201811299207.8A CN111138524B (zh) 2018-11-02 2018-11-02 一种从基因工程水稻种子中分离纯化低铁饱和度的重组人乳铁蛋白的方法

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WO2020088207A1 true WO2020088207A1 (fr) 2020-05-07

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Cited By (1)

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WO2024109532A1 (fr) 2022-11-24 2024-05-30 武汉禾元生物科技股份有限公司 Conjugué médicament-albumine sérique humaine recombinante

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EP4623934A1 (fr) 2022-11-24 2025-10-01 Wuhan Healthgen Biotechnology Corp Conjugué médicament-albumine sérique humaine recombinante

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