WO2020108390A1 - 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 - Google Patents
参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 Download PDFInfo
- Publication number
- WO2020108390A1 WO2020108390A1 PCT/CN2019/120118 CN2019120118W WO2020108390A1 WO 2020108390 A1 WO2020108390 A1 WO 2020108390A1 CN 2019120118 W CN2019120118 W CN 2019120118W WO 2020108390 A1 WO2020108390 A1 WO 2020108390A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- particles
- protein
- hydrogel
- liquid sample
- detergent
- Prior art date
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28047—Gels
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- 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
-
- 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
- B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D36/00—Filter circuits or combinations of filters with other separating devices
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D36/00—Filter circuits or combinations of filters with other separating devices
- B01D36/04—Combinations of filters with settling tanks
- B01D36/045—Combination of filters with centrifugal separation devices
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
- B01D63/16—Rotary, reciprocated or vibrated modules
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
- B01J20/267—Cross-linked polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
-
- 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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03001—Alkaline phosphatase (3.1.3.1)
Definitions
- the invention relates to a dry hydrogel particle mixed with a detergent, which realizes the mass concentration of a high multiple and a high recovery macromolecule and the improvement of protein specific activity.
- the macromolecules referred to in the present invention include polysaccharides, nucleic acids, proteins and aggregates formed by their single or complex components such as ribosomes, exosomes, inclusion bodies, chromosomes, organelles and viruses and other natural biological macromolecules or aggregates It also includes water-soluble synthetic polymers or colloids such as polyacrylamide, polyacrylic acid, polyacrylate, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, latex particles, colloidal gold, etc. When preparing, modifying or analyzing these macromolecular liquid samples, they often need to be concentrated because their concentration is too low.
- the hydrogel concentration is a good solution to the slowness of ultrafiltration, membrane clogging, and partial concentration of protein membranes Problems such as aggregation caused by polarization and pressure-induced degeneration.
- high-power concentration because the volume ratio of the swollen hydrogel particles is relatively large, the macromolecular concentrated solution trapped on its surface is relatively large, which seriously reduces the recovery rate.
- the thickness of the hydrogel intercepted by the filter membrane in the centrifugal force direction during filtration centrifugation cannot be too high, thus severely limiting the volume of the macromolecular liquid sample that can be concentrated.
- the mixture of hydrogel and detergent has been used to make a fixed or controlled release detergent or perfume, but prior to the present invention, there has been no report of the prior art that the mixture is used for the concentration of macromolecules .
- the invention provides a hydrogel particle mixed with a detergent, and the particles dried according to the gel provide a method for concentrating a macromolecular liquid sample with high mass and high recovery rate, and also provide a companion The method of improving the specific activity of protein liquid sample in the concentration process.
- Figure 1 Filtration centrifugation of a mixture of swelling hydrogel and macromolecular concentrate.
- Figure 4 Macromolecule concentration horizontal rotary head filter centrifugation.
- the protein formed by the alternate polymerization of more than 20 amino acid monomers has a diverse primary sequence structure and corresponding secondary spatial conformation. These conformations are often the structural basis required to perform various specific active functions of proteins, but they are often unstable and easily change under the influence of time, physical or chemical factors and lead to the loss of corresponding active functions, which is the phenomenon of protein denaturation .
- the hydrogel contains a three-dimensional polymer network with hydrophilic groups that are cross-linked by chemical covalent or physical non-covalent interactions.
- the hydrophilic groups can contain an aqueous solvent or an aqueous solution in the network.
- Common natural or artificial hydrogels are, but not limited to, polyacrylamide, polyacrylic acid, polyacrylic acid salt, polydextran, cellulose, polyvinyl alcohol, polyethylene glycol, agarose, polyhyaluronic acid, polyji Modified hydrogel or a plurality of the above hydrogel monomers obtained by polymerizing and cross-linking butylin, polyalginic acid, sodium polyalginate, polyvinylpyrrolidone, polypeptide, polyprotein, derivatives of the above hydrogel monomers Or a hybrid hydrogel formed by polymer mixing and crosslinking.
- Hydrogels can be prepared as single-phase agglomerates by polymerization and cross-linking of monomer solutions or cross-linking reactions of high-molecular polymer solutions, and then prepared as pellets through procedures such as dicing, washing, drying, and grinding; if The hydrogel formed by the reaction of these solutions is not enough to absorb all the liquid, the hydrogel will separate from the solution, and then be prepared into particles through the procedures of washing, drying and grinding if necessary; the hydrogel particles are also It can be directly prepared by the polymerization of monomers in the dispersed suspended droplets and the cross-linking reaction or the cross-linking reaction of high molecular polymers, followed by washing and drying to complete the preparation.
- the above hydrogel particles can all be fully hydrated, swelled and suspended in a detergent solution with a certain doping concentration, and then a preferred filtration method is used to remove excess decontamination. Agent solution, and drying the obtained wet granules to obtain the hydrogel dried granules mixed with the detergent according to the present invention.
- detergents used in common biological sciences include but are not limited to Tween-20, Tween-40, Tween-60, Tween-80, Brij-35, Brij -58, NP-40, Triton X-100, Triton X-114, octyl glucoside, octyl thioglucoside, 3-[3-(cholamidopropyl) dimethylamino] propanesulfonate Acid internal salt, 3-[(3-cholaminepropyl) dimethylamino]-2-hydroxy-1-propanesulfonic acid internal salt, sodium cholate, sodium deoxycholate, cetyl trimethyl bromide Ammonium chloride or sodium lauryl sulfate.
- the detergent can be distributed in the form of monomers and micelles within or outside the hydrogel network. After removing excess liquid and drying the hydrogel, the detergent should be distributed in the inner space and outer surface of the net.
- the detergents distributed locally on the surface of the particles have a sufficiently high concentration, which can effectively reduce the adsorption of macromolecular solutes on the surface of the particles.
- the doping of the detergent enhances the recovery rate of the solute mass and solution volume of the macromolecular concentrated solution, that is, the recovery rate of the total mass of the solute. It should be noted that if the detergent outside the hydrogel network is worn out due to interaction with the target macromolecule or diffusion into the concentrated solution, the original detergent inside the network can also diffuse out to supplement the decontaminated consumption Agent to maintain its function of improving the recovery rate of target macromolecules.
- the high-volume large-molecule liquid sample is concentrated at a multiple, it is necessary to recover the concentrated liquid with a small volume ratio in the direction of the centrifugal force in the following filtration centrifugal separation method for the large thickness of the swelling hydrogel particle layer.
- the effect of detergent doping on the dried hydrogel particles on the efficient recovery of the total mass of the macromolecular solute in the macromolecular concentrate is particularly obvious and important.
- a protein liquid sample usually contains a tightly folded conformation, a reversible loose intermediate conformation and an irreversible loose conformation, respectively corresponding to an active functional conformation, a reversible functionally inactive intermediate conformation and an irreversible functionally inactive conformation. If a protein liquid sample undergoes the above concentration process of dry hydrogel particles mixed with active functional detergent, it is conceivable that the water molecules hydrated on the surface of the protein molecules are quickly sucked away on the surface of the hydrogel particles.
- the conformation of the reversible loose intermediate will shrink and return to the tightly folded conformation due to dehydration, which means that the non-functionally active intermediate conformation component is converted into an active functional conformation component, which improves the total activity of the protein liquid sample or Specific activity is also equal to the purified and enriched active functional conformation of protein samples.
- the active function of the protein liquid sample in the concentration process of the dried hydrogel particles is not reduced but increased. It is a gaining sample processing, and the improvement of the conformational purity of the protein helps the crystallization process that requires the coordinated arrangement of homogeneous components and conformational molecules and Various structural analysis.
- Filtration centrifugation is a mature method for efficiently separating solid-liquid mixtures and efficiently recovering the liquids. It is especially suitable for the separation of a large number of hydrogel particles and a small amount of concentrated liquid volume produced when the above-mentioned hydrogels concentrate large molecules at high multiples. demand.
- Both conventional laboratory or industrial filter centrifuges can be used in the present invention.
- the mixture of the above-mentioned swelling hydrogel particles 1 and the macromolecular concentrated solution 2 moves toward the membrane wall 3 with a suitable pore diameter under the action of centrifugal force in the direction indicated by the arrow. Under the appropriate centrifugal strength and time, the dehydrated hydrogel particles 4 are intercepted on the filter membrane, and the concentrated liquid 5 is efficiently thrown out and collected from the filter membrane pores.
- a technically mature continuous filtration and centrifugal system for solid-liquid separation can be used to continuously remove the swelling hydrogel particles accumulated on the filter membrane and collect the macromolecular concentrate at the same time In order to ensure that the hydrogel particles are not accumulated on the filter membrane, the concentrated liquid is trapped and the target macromolecules are lost.
- a preferred simple continuity operating system is shown in Figure 2.
- the main component is a conical drum with filter holes.
- the mixture 1 of swollen hydrogel particles and macromolecular concentrate is continuously thrown in from the bottom of the small diameter; under the action of centrifugal force in the direction indicated by the arrow, the surface dehydrated water intercepted on the filter membrane
- the gel particles 2 continuously move along the upper side of the large diameter and are thrown out; and the concentrated liquid 3 is continuously thrown out of the pores of the filter membrane 4 and collected as the concentrated liquid 5.
- the above process constitutes a continuous operation of feeding a concentrated large-molecular liquid sample, removing the hydrogel particles dehydrated on the surface, and collecting the concentrated liquid.
- the above-mentioned macromolecule concentration factor is controlled by the ratio of the mass of the dried hydrogel particles and the volume of the liquid to be concentrated, that is, the more the input of the hydrogel relative to the liquid sample, the greater the concentration factor.
- different volumes of macromolecules to be concentrated need to weigh different dry hydrogel particles. If there is a small volume of large molecular liquid sample that needs to be concentrated, accurate weighing of a small amount of dried hydrogel particles is an inconvenient operation, that is, if the weighing is too low, the concentration factor is not enough, or if the weighing is too large, the liquid sample is all Blots dry and cannot be recycled.
- the present invention constructs a zoned contact and pipetting system for excess dried hydrogel particles as shown in FIG. 3 and a macromolecular liquid sample to be concentrated.
- One side of the system is the area of excess dry hydrogel particles containing unhumidified dry hydrogel particles 1, swelling hydrogel particles 2 and macromolecular concentrate 3; the other side is the inlet of macromolecular liquid sample 4
- the sample area; the bottom of the system contains the filter membrane 5 that intercepts the swollen and unswollen hydrogel particles; the macromolecular liquid sample and the excessively dried hydrogel particles that were originally isolated from each other come into contact through the contact membrane 6 at the bottom of the system.
- Capillary action drives liquid absorption from left to right.
- the pore size and density of the membrane 6 should be set appropriately to prevent too much hydrogel particles from entering the sample injection area, but the macromolecular liquid sample 4 can penetrate into the excessively dry hydrogel particle area without being absorbed too quickly. Unable to recycle effectively.
- the entire concentration system is centrifuged immediately. Under the action of the centrifugal force in the direction indicated by the arrow, the hydrogel particles 7 that swell but are dehydrated on the surface are filtered Retentively, the concentrated liquid 8 is collected through the filter membrane.
- part of the concentrated solution 3 may return to the sampling area through the contact membrane 6 to be mixed with the macromolecular liquid sample 4, but this mixture will also be collected in the concentrated solution 8 during the final centrifugation, ensuring a large High efficiency recovery of the entire molecule.
- the substance of the present invention includes but is not limited to the following embodiments. Without detailed description, the raw materials, equipment, and methods involved in the embodiments can be obtained from normal commercial channels, public literature, or prior art in the art.
- Type of hydrogel particles Select the type of hydrogel particles depending on the ease of preparation, cost of preparation, and the need for interaction with the target macromolecule.
- Types and concentrations of ginseng detergents according to the characteristics of the interaction between the target macromolecules and hydrogel particles, the types of ginseng detergents and the ginseng liquids with the highest quality and active functional recovery are selected The concentration range is 0.001 to 10%.
- mild non-ionic hybrid detergents such as Tween series, Brij series, NP-40, Qu Laton series; Tween series is particularly preferred, because they have no negative effect on the active function of most biological macromolecules, and can often prevent the aggregation of these macromolecular active functions.
- Hydrogel particle cross-linked network pore size This pore size is usually positively related to the concentration of monomer or polymer in the hydrogel preparation and the degree of cross-linking, but it must be smaller than the size of the target macromolecule; the size of the target macromolecule is mainly It is positively related to its molecular weight, the looseness of the conformation of the group arrangement and the thickness of the hydration layer.
- Hydrogel particle size If the particle size is too small, it will cause the particle size to be too large, it is easy to adsorb the target macromolecule and intercept the macromolecular concentrate, and the pore size of the filtration centrifugal filter membrane will be reduced accordingly, which will reduce the filtration speed and efficiency; If the diameter is too large, the inflation process is too slow.
- the particle size of the dried or swollen hydrogel particles preferably ranges from 1 to 5000 microns, and a further preferred range is from 5 to 1000 microns.
- Filtration pore size and material of the centrifugal filter membrane The membrane pore size must be small enough to intercept dry or swollen hydrogel particles. However, if the pore size is too small, the filtration speed is too low; if the pore size is too large, it is not conducive to carrying the mixture of the macromolecular concentrated solution and the swelling hydrogel before centrifugation, because it is necessary to prevent the concentrated liquid from leaking from the excessively large pore size of the filter pores . Therefore, the pore size preferably ranges from 0.5 to 1000 microns.
- the filter membrane should not adsorb large molecules, and hydrophilic or hydrophobic substrates can be used.
- the small pore size filter membrane is preferably a hydrophilic substrate to facilitate the penetration of the liquid driven by the centrifugal force
- the large pore size filter membrane is preferably a hydrophobic substrate with a water-repellent function in order to carry the liquid sample before filtration and centrifugal separation without leakage .
- Example 2 Preparation of detergent-doped and dried polyacrylamide hydrogel particles by single-phase polymerization method
- Example 3 Preparation of detergent doped polyacrylamide particles by inverse suspension polymerization
- Water phase configuration Mix 29 grams of acrylamide, 1 gram of N,N-methylenebisacrylamide, 100 milliliters of deionized water, and 0.2 grams of ammonium persulfate.
- Oil phase configuration Mix 200 ml cyclohexane and 3 ml Span-80.
- Steps Configure the oil phase in a three-necked flask equipped with mechanical agitation and a spherical condenser, heat to 57°C, and stir at 240 rpm; configure the water phase in the beaker, add to the flask and mix with the oil phase, During stirring, the water phase is dispersed into the oil phase, and the temperature is maintained at 57°C for 30 minutes; the temperature is raised to 67°C, and a large amount of reflux is generated when the system exotherms.
- the temperature is maintained for 30 minutes; the temperature is increased to 70°C and the temperature is maintained for 30 minutes;
- the cyclohexane was removed by vacuum filtration with a Büchner funnel; washed three times with 250 ml of 50% ethanol aqueous solution, and then washed three times with 250 ml of deionized water; with 250 ml, the preferred concentration was 0.01 to 1%, particularly preferred It was washed twice with 0.2% Tween-20 in deionized water; the resulting wet particles were dried at 90°C and sealed for storage.
- the first step is to weigh 2g of the dried polyacrylamide hydrogel particles mixed with the detergent prepared in Example 2 or 3, placed on a filter membrane with a pore size of 20 microns and open at the upper end
- a plastic tube with a protruding bayonet add about 12 ml of pH7.4 phosphate buffer solution with a concentration of 10 to 500 ⁇ g of bovine serum albumin (black dots in the figure) at a concentration of 10 to 500 ⁇ g per ml;
- Step 3 Put the above plastic tube into a 50 ml centrifuge tube, and follow the thin arrow in the circumferential direction on the horizontal rotating head Centrifuge at 3000xg for 3 minutes;
- Step 4 is to collect about 2 ml of bovine serum albumin concentrate in the direction of the centrifugal force indicated by the thick arrow of the centrifuge tube. It has been determined that the protein recovery rate of the concentrated liquid is above 90%,
- Example 4 Add 10 ml of deionized water to the bovine serum albumin concentrate obtained in Example 4, and then put in the dried polyacrylamide hydrogel particles with 2 g of the detergent prepared in Example 2 or 3 In the plastic membrane filter tube; repeat the static hydration and centrifugation operation described in Example 4 to obtain 2 ml of concentrated liquid. Compared with the concentrated solution obtained in Example 4, the concentrated solution reduced salt and phosphate buffer strength by nearly 83%. Repeat the above desalting and buffer replacement operations once, the desalting and buffer replacement rate will be further increased to about 97%.
- Example 6 Improve the specific activity of alkaline phosphatase
- the concentration operation of the sample volume of 12 ml was carried out according to Example 4, except that the bovine serum albumin sample was replaced with a pH7.5 trishydroxymethylaminomethane buffer solution of bovine small intestine alkaline phosphatase at a concentration of 20 to 200 ⁇ g per ml.
- the mass recovery rate of the quantitative determination of the resulting concentrated protein was 90%, and the activity recovery rate of the alkaline phosphatase activity measurement was 100%, so the specific activity of the alkaline phosphatase increased by about 11%.
- Concentration of trace protein contained in human urine can increase its concentration and detectability.
- Normal human urine is mixed with 2 ⁇ g of horseradish peroxidase per ml as a model protein urine sample.
- the concentration operation of the sample volume of 12 ml was performed as in Example 4, but the bovine serum albumin sample was replaced with the model protein urine sample.
- the concentration of the resulting concentrated solution was measured by horseradish peroxidase activity, and it was found that the enzyme protein was concentrated about 6 times, and the recovery rate was more than 95%.
- Example 8 Concentration of M13 phage secreted into the medium
- the target phage needs to be screened, and the M13 phage secreted into the extracellular medium after assembly in E. coli must be concentrated.
- the existing M13 concentration method is based on polyethylene glycol precipitation, and the recovery rate is low at a large medium volume or low titer, and the operation requires low temperature and a long time.
- a concentration operation of a sample volume of 12 ml was performed according to Example 4 of the present invention, but the bovine serum albumin sample was replaced with culture medium to secrete M13 phage sample. Finally, after titer determination, M13 phage was concentrated about 6 times, and the recovery rate was about 95%.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Dispersion Chemistry (AREA)
- Water Supply & Treatment (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Detergent Compositions (AREA)
- Peptides Or Proteins (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
一种参杂去污剂的干燥水凝胶颗粒以及据此颗粒而实现的一种大分子液样质量浓缩和蛋白质液样比活性提升的方法,克服了现有技术的低效率和活性功能丧失等缺点,适合处理大分子液样的各种大小体积。水凝胶颗粒充分悬浮并水化于去污剂溶液中,沥去多余去污剂溶液,干燥此湿颗粒即得参杂去污剂的干燥水凝胶颗粒;将交联网络孔径小于目标大分子的此干燥颗粒按比例与该大分子液样接触,水凝胶颗粒吸胀后做过滤离心或锥形滤膜转鼓离心,去除表层脱水的水凝胶颗粒,得到大分子质量浓缩滤液或蛋白质比活性提升的浓缩滤液。
Description
本发明涉及一种参杂去污剂的干燥水凝胶颗粒,实现了高倍数及高回收率的大分子的质量浓缩和蛋白质比活性的提升。
本发明所指的大分子包括多糖、核酸、蛋白质以及它们单个或复合组分所形成的聚集体如核糖体、外泌体、内含体、染色体、细胞器和病毒等天然生物大分子或聚集体,亦包括诸如聚丙烯酰胺、聚丙烯酸、聚丙烯酸盐、聚乙烯醇、聚乙二醇、聚乙烯吡咯烷酮、乳胶颗粒、胶体金等水溶性合成高分子或胶体。在制备、修饰或分析这些大分子液体样品时,经常因为它们的浓度过低而需要浓缩。操纵有机溶剂、酸碱度、离子浓度、水化竞争剂或温度等条件所造成的沉淀是浓缩大分子液样的常用方法,但存在用户不友好、低浓度样沉淀效率差和生物大分子活性功能丧失等一系列问题。相对来说,基于压力和纳米级孔径膜的超滤方法来浓缩大分子具有温和及浓缩效率与浓度无关等优点,但也经常遇到过程缓慢和膜孔堵塞的难题,对蛋白质样品来说还有膜局部浓度极化诱导的聚集以及压力诱导的构象改变等变性风险。
我们注意到,干燥的水凝胶颗粒在大分子液样中水化膨胀时,它的交联网状结构可以让水溶剂和小分子溶质自发而迅速地进入网内空间而同时把大分子排阻在外,即实现了大分子在网外空间里的浓缩,并经过简单的过滤离心的固液分离可以很方便地得到该浓缩样(Analytical Biochemistry,1984,138,451)。由于均匀分布在大分子液样全体空间的水凝胶网状表面积远远高于单层超滤膜面积,水凝胶浓缩很好地解决了超滤法的缓慢、膜堵塞、蛋白质膜局部浓缩极化导致的聚集和压力诱导的变性等问题。但是,在进行高倍数浓缩时,因为吸胀的水凝胶颗粒体积占比比较大,其表面截留的大分子浓缩液就相对较多,严重降低了回收率。因此,在执行诸如7倍以上浓缩时,在过滤离心分离时离心力方向上被滤膜拦截的水凝胶厚度就不能太高,因而严重限制了可以浓缩的大分子液样的体积。
水凝胶和去污剂的混合体已经被用来制作固定成型或可控释放的洗涤剂或香水剂,但在本发明之前,未见该混合体用于大分子浓缩的现有技术的报道。
发明内容
本发明提供一种参杂去污剂的水凝胶颗粒,并据此胶干燥而成的颗粒提供一种大分子液样的质量高倍数和高回收率的浓缩方法,还提供了一种伴随该浓缩过程的蛋白质液样比活性提高的方法。计量取交联网络孔径小于目标大分子尺寸的参杂有去污剂的干燥水凝胶颗粒,使它与待浓缩的各种大小体积的大分子水溶液接触,让水凝胶颗粒在大分子液中吸走液体及小分子溶质、膨胀并将大分子排阻在交联网络之外而形成浓缩液;再经过过滤离心或锥形滤膜转鼓离心分离该水凝胶颗粒与大分子液样的混合物;去除表层脱水但内孔吸胀的水凝胶颗粒,滤出液即为排阻在水凝胶外的目标大分子浓缩液。如果该大分子是蛋白质,上述的浓缩过程还提高了该蛋白质液样的比活性,因此可以成为一种蛋白质比活性提升的方法。本发明克服了现有大分子浓缩技术的用户不友好、缓慢、堵塞、局部浓度低效、极化或活性功能丧失等缺点。
图1.吸胀水凝胶与大分子浓缩液混合物的过滤离心分离。
图2.浓缩大体积大分子液样的连续过滤离心分离。
图3.干燥水凝胶颗粒与大分子液样的分区接触和吸液以控制液样浓缩速度。
图4.大分子浓缩的水平转头过滤离心法。
由20多种氨基酸单体交替聚合形成的蛋白质具有多样化的一级序列结构及相应的次级空间构象。这些构象往往是执行蛋白质各种特定活性功能所需的结构基础,但它们常常不稳定,易在时间、物理或化学等因素的作用下而改变并导致相应活性功能的丧失,此为蛋白质变性现象。
水凝胶含化学共价或物理非共价作用下而相互交联的带有亲水基团的三维聚合物网络,其亲水基团可以在该网络内包纳水溶剂或水溶液。常见的天然或人造水凝胶有但不限于聚丙烯酰胺、聚丙烯酸、聚丙烯酸盐、聚葡聚糖、纤维素、聚乙烯醇、聚乙二醇、琼脂糖、聚透明质酸、聚几丁质、聚海藻酸、聚海藻酸钠、聚乙烯吡咯烷酮、聚多肽、聚蛋白质、上述水凝胶单体的衍生物聚合交联而成的修饰水凝胶或多个上述水凝胶单体或聚合体混合聚合交联而成的杂交水凝胶。
水凝胶可以经过单体溶液的聚合和交联反应或高分子聚合物溶液的交联反应而单相成块制备,再经过切块、清洗、干燥和研磨等程序而被制备为颗粒;如果这些溶液反应所形成的水凝胶不足以吸收全部液体,该水凝胶就会从溶液中分相出来,再经过清洗、干燥和必要时的研磨等程序被制备为颗粒;水凝胶颗粒亦可通过分散悬浮液滴内的单体聚合和交联反应或高分子聚合物交联反应而直接制成,再加以清洗和干燥等工序完成制备。作为一种优选的去污剂参杂方法,上述水凝胶颗粒最终都可以充分水化、膨胀和悬浮于一定参杂浓度的去污剂水溶液中,再优选的过滤方法沥去多余的去污剂溶液,对所得湿颗粒做干燥处理,即得本发明所指的去污剂参杂的水凝胶干燥颗粒。按对蛋白质活性功能友好度大体顺序排列,常见生物科学领域所应用的去污剂包括但不限于吐温-20、吐温-40、吐温-60、吐温-80、Brij-35、Brij-58、NP-40、曲拉通X-100、曲拉通X-114、辛基葡糖苷、辛基硫代葡糖苷、3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐、3-[(3-胆胺丙基)二甲基氨基]-2-羟基-1-丙磺酸内盐、胆酸钠、脱氧胆酸钠、十六烷基三甲基溴化铵或十二烷基硫酸钠。
当上述干燥水凝胶颗粒按比例定量投入大分子水溶液时,可以想象,这些颗粒巨大表面所包裹的网状结构的亲水基团将导引并吸入水溶液,而该网状结构在干燥塌陷时产生的扭曲张力可以因为被吸入水溶液的空间填充而释放,因而水凝胶干燥颗粒的立体网状空间对水溶液的吸纳是无需额外能量支持的自发而迅速的过程。如果水凝胶交联网状的孔径比目标大分子尺寸小,该大分子被排阻在网外而与网外的水溶液一起形成了浓缩液。因为去污剂对水凝胶的参杂起始于这两种组分在液体中的混合体,去污剂可以以单体和微团的形式分布于水凝胶网内或网外,在去除多余液体并干燥水凝胶后去污剂应该分布在网内空间和网外表面。在参杂去污剂的干燥水凝胶颗粒吸收水溶液而浓缩大分子时,局部分布于颗粒网状表面的去污剂具有足够高的浓度,可以有效减少大分子溶质的在颗粒表面的吸附,也有效降低了网外大分子浓缩液在颗粒表面的张力而有利于它与颗粒在诸如下述的过滤离心驱动下发生有效的相向相分离。因此,去污剂的参杂增强了大分子浓缩液的溶质质量和溶液体积的回收率,也就是提高了溶质总质量的回收率。应该注意的是,如果水凝胶网外的去污剂因为与目标大分子相互作用或扩散到浓缩液里而耗损,原来在网内的去污 剂还可以扩散出来,补充被消耗的去污剂以保持它提高目标大分子回收率的功能。如果对大体积大分子液样做高倍数浓缩,就是要在下述的过滤离心分离方法中的离心力方向上对大厚度的吸胀水凝胶颗粒层回收体积占比很小的浓缩液,这时在干燥水凝胶颗粒上的去污剂参杂对大分子浓缩液的大分子溶质总质量的高效率回收所起的作用尤为明显和重要。
可以想象,一个蛋白质液样通常含有紧密折叠构象、可逆松散中间体构象和不可逆松散构象,分别对应着活性功能构象、可逆功能失活中间体构象和不可逆功能失活构象。如果一个蛋白质液样经历上述的参杂有活性功能友好的去污剂的干燥水凝胶颗粒的浓缩过程,可以想象,蛋白质分子表面水化的水分子在水凝胶颗粒表面被快速吸走,可逆松散中间体构象会因为去水化而收缩回归到紧密折叠构象,也就是说没有功能活性的中间体构象成分转变为一种有活性功能的构象成分,提高了该蛋白质液样的总活性或比活性,也等于纯化、富集了蛋白质样品的活性功能构象。蛋白质液样在干燥水凝胶颗粒浓缩过程的活性功能不降反升是增益性的样品处理,而蛋白质构象纯度的提高有助于需要同质的成分和构象的分子间协同排列的结晶过程以及各种结构分析。
过滤离心是高效率分离固液混合体并高效率回收该液体的成熟方法,尤其适用于上述的水凝胶高倍数浓缩大分子时产生的大量水凝胶颗粒和少量浓缩液体积混合体的分离需求。常规实验室或工业过滤离心机均可以用在本发明中。如图1所示,在一个旋转容器里,上述的吸胀水凝胶颗粒1与大分子浓缩液2混合体在箭头所指方向的离心力作用下均向含有合适孔径的滤膜壁3运动,在合适离心强度和时间作用下表层脱水的水凝胶颗粒4被拦截在滤膜上,浓缩液5则高效率地从滤膜孔中被甩出和收集。
对于诸如工业化规模大体积液样的大分子浓缩,可使用技术上成熟的连续性固液分离的过滤离心系统,不断去除堆积在滤膜上的吸胀水凝胶颗粒并同时收集大分子浓缩液,保证水凝胶颗粒不在滤膜上不断堆积而造成的浓缩液被截留和目标大分子的损失。一个优选的简单连续性操作系统如图2所示,主要部件是一个带有滤孔的锥形转鼓。转鼓在旋转时,吸胀的水凝胶颗粒和大分子浓缩液的混合物1不断地从小口径的底部投入;在箭头所指方向的离心力作用下,被拦截在滤膜上的表层脱水的水凝胶颗粒2不断地顺着大口径的上方移动并被甩出;而浓缩液3则不断从滤膜4的孔中被甩出和被收集为浓缩液体5。上述过程构成了浓缩大体积的大分子液样的投料、表层脱水的水凝胶颗粒的刨除和浓缩液的收集的连续性操作。
上述的大分子浓缩倍数依靠干燥水凝胶颗粒质量和待浓缩液体积的比例来控制,即水凝胶相对液样的投入越多浓缩倍数就越大。为了一定的浓缩倍数和浓缩效果,不同体积的大分子待浓缩液样就需要称量不同的干燥水凝胶颗粒。如果有小体积的大分子液样需要浓缩,小量的干燥水凝胶颗粒的准确称量是不方便的操作,即称量过低则浓缩倍数不够,或称量过多则液样全部被吸干而无法回收。为此,本发明构建了一种图3所示的过量干燥水凝胶颗粒与待浓缩大分子液样的分区接触和吸液系统。系统的一侧是含有未湿化的干燥水凝胶颗粒1、吸胀水凝胶颗粒2和大分子浓缩液3的过量干燥水凝胶颗粒区;另一侧是大分子液样4的进样区;系统底部含拦截吸胀和未吸胀水凝胶颗粒的滤膜5;而原本相互隔离的大分子液样和过量干燥水凝胶颗粒通过系统底部的触膜6而发生接触,在毛细作用驱动下发生液体从左到右迁移的吸液。触膜6的孔径大小和密度应适当设置,防止过多的水凝胶颗粒进入进样区,但大分子液样4可以顺利渗入过量干燥水凝胶颗粒区又不会过快被吸干而无法有效回收。在操作应用中,大分子液样4被吸液收缩到合适程度时,即刻将整个浓缩系统离心,在箭头所指方向的离心力作用下,吸胀但表层脱水的水凝胶颗粒7被滤膜截留,浓缩液8则通过滤膜而被收集。需要注意的是,可能会有部分浓缩液3通过触膜6回流到进样区与大分子液样4混合,但这个混合体也会在最终离心时被收集到浓缩液8里,保证了大分子整体的高效率回收。
本发明的实质性内容包括但不限于以下实施例。若无细述,实施例所涉及的原材料、设备和方法均可从本领域的正常的商业渠道、公开文献或现有技术获取。
实施例1:优选条件
水凝胶颗粒种类:视制备难易、制备成本以及与目标大分子的相互作用的需要而选择水凝胶颗粒种类。优选聚丙烯酰胺和聚葡聚糖这两种水凝胶颗粒,因为它们具有制备相对简便和经济以及不与大分子相互作用的惰性的优点;优选聚丙烯酸或聚丙烯酸盐水凝胶颗粒则是因为除了它制备的简便经济性外,它还具有巨大的单位胶质量的吸水量,虽然需要仔细考量它所携带的负电荷与目标大分子相互作用对浓缩工艺和回收率的影响。
参杂去污剂的种类和浓度:视目标大分子及水凝胶颗粒相互作用的特性而选定能带来最高质量及活性功能回收率的参杂去污剂种类和去污剂参杂液浓度范围为0.001~10%。对于除多糖和核酸外的敏感生物大分子如蛋白质,尤其是活性功能对构象特别敏感的酶,优选使用温和的非离子型参杂去污剂如吐温系列、Brij系列、NP-40、曲拉通系列;特别优选吐温系列,因为它们对绝大多数生物大分子活性功能没有负性影响,还经常可以防止这些大分子活性功能丧失的聚集。
水凝胶颗粒交联网络孔径:该孔径通常与水凝胶制备中单体或聚合物的浓度以及交联度正相关,但必须比目标大分子的尺寸更小;而目标大分子的尺寸主要与它的分子量、基团排列的构象的松散度和水化层厚度等因素正相关。
水凝胶颗粒粒径:粒径过小会导致颗粒比表面过大,容易吸附目标大分子并截留大分子浓缩液,也使过滤离心滤膜的孔径相应下降而降低了过滤速度和效率;粒径过大则吸胀过程太慢。干燥或吸胀水凝胶颗粒的粒径优选范围为1~5000微米,更进一步的优选范围为5~1000微米。
过滤离心滤膜孔径和材料:滤膜孔径必须足够小,能够拦截干燥或吸胀的水凝胶颗粒。但孔径过小则导致过滤速度过低;孔径过大则不利于在离心前承载大分子浓缩液和吸胀水凝胶的混合物,因为这时需要防止浓缩液从过大的滤孔孔径中泄漏。因此,孔径优选范围为0.5~1000微米。滤膜应该不吸附大分子,可以使用亲水或疏水基材。一般而言,小孔径滤膜优选亲水基材以利于离心力驱动下的液体通透,大孔径滤膜优选具有斥水功能的疏水基材以便在过滤离心分离前承载液样而不发生渗漏。
实施例2:单相聚合法制备去污剂参杂干燥聚丙烯酰胺水凝胶颗粒
组分:丙烯酰胺29克,N,N-亚甲基双丙烯酰胺1克,去离子水100毫升,过硫酸铵0.2克,四甲基乙二胺40微升。
步骤:在烧杯中将上述丙烯酰胺、N,N-亚甲基双丙烯酰胺和过硫酸铵固体溶解、混匀于去离子水中,加入四甲基乙二胺启动聚合反应,立即倒入底面积较大的具有惰性内表面的容器中,静置约1小时完成聚合;将聚合而成的凝胶切碎,90℃烘干;烘干的块状聚合物在粉碎机中粉碎,筛选出60~150目标颗粒,倒入到去离子水中进行充分溶胀;用250毫升去离子水清洗两次,再用250毫升优选浓度为0.01~1%,特别优选浓度为0.2%的吐温-20去离子水溶液清洗两次;所得湿颗粒90℃烘干后密封保存。
实施例3:反相悬浮聚合法制备去污剂参杂干燥聚丙烯酰胺颗粒
水相配置:混合29克丙烯酰胺,1克N,N-亚甲基双丙烯酰胺,100毫升去离子水和0.2克过硫酸铵。油相配置:混匀200毫升环己烷和3毫升司盘-80。
步骤:在配置有机械搅拌和球形冷凝管的三口烧瓶中配置好油相,升温至57℃,搅拌转速至每分钟240转;在烧杯中配置好水相,加入到烧瓶中与油相混合,在搅拌作中水相分散到油相中,57℃保温30分钟;升温至67℃,待体系放热产生大量回流,当放热结束后继续保温30分钟;升温至70℃,保温30 分钟;反应结束后,用布氏漏斗真空抽滤去除环己烷;用250毫升50%乙醇水溶液清洗三次,再用250毫升去离子水清洗三次;用250毫升优选浓度为0.01~1%,特别优选浓度为0.2%的吐温-20去离子水溶液清洗两次;所得湿颗粒90℃烘干后密封保存。
实施例4:牛血清白蛋白浓缩
如图4所示意,第1步为称量2克实施例2或3所制备的去污剂参杂的干燥聚丙烯酰胺水凝胶颗粒,置于带有20微米孔径滤膜和在上端开口处有凸出卡口的塑料管中,再往该塑料管投入约12毫升浓度为每毫升10~500微克牛血清白蛋白(图中的黑点)的pH7.4磷酸盐缓冲液;第2步让水凝胶和蛋白液混合体静置5分钟以充分水化吸胀;第3步将上述塑料管卡套在50毫升的离心管中,在水平转头按细箭头所示的圆周方向以3000xg离心3分钟;第4步即在离心套管粗箭头所示的离心力方向上收集到约2毫升牛血清白蛋白浓缩液。经测定,浓缩液蛋白质的质量回收率在90%以上,浓度比初始值提高约6倍。
实施例5:牛血清白蛋白溶液脱盐和缓冲液置换
在实施例4所获得的牛血清白蛋白浓缩液中加入10毫升去离子水,再放入带有2克实施例2或3所制备的去污剂参杂的干燥聚丙烯酰胺水凝胶颗粒的滤膜塑料管中;重复实施例4所述的静置水化和离心分离操作,得到2毫升浓缩液。该浓缩液比起实施例4所得浓缩液减少了近83%的盐及磷酸缓冲液强度。重复上述脱盐和缓冲液置换操作一次,脱盐及缓冲液置换率将进一步提升到97%左右。
实施例6:提升碱性磷酸酶比活性
按实施例4做12毫升样品体积的浓缩操作,只是牛血清白蛋白样换成浓度每毫升20~200微克的牛小肠碱性磷酸酶的pH7.5三羟甲基氨基甲烷缓冲溶液。所得浓缩液蛋白定量测定的质量回收率为90%,而碱性磷酸酶活性测定的活性回收率为100%,因而碱性磷酸酶的比活性提升了约11%。
实施例7:尿液蛋白质浓缩
人尿液所含微量蛋白质经过浓缩可以提升它的浓度和相应的可检测性。正常人尿液混入每毫升2微克的辣根过氧化物酶作为模式蛋白尿液样。按实施例4做12毫升样品体积的浓缩操作,但牛血清白蛋白样换成该模式蛋白尿液样。最终得到的浓缩液经辣根过氧化物酶活性测定,得知该酶蛋白被浓缩6倍左右,而回收率在95%以上。
实施例8:分泌到培养基中的M13噬菌体浓缩
无论是为了序列测定还是进行噬菌体展示,需要筛选目标噬菌体,也就需要浓缩大肠杆菌内组装后分泌到细胞外培养基中的M13噬菌体。现有的M13浓缩方法基于聚乙二醇沉淀,在大培养基体积或低滴度时回收率偏低,操作也需要低温和较长的时间。作为一种改进的方法,按本发明实施例4做12毫升样品体积的浓缩操作,但牛血清白蛋白样换成培养基分泌M13噬菌体样。最终经滴度测定,M13噬菌体被浓缩6倍左右,回收率约为95%。
Claims (9)
- 一种用于大分子质量浓缩或蛋白质比活性提升的参杂去污剂的干燥水凝胶颗粒,其特征为所述的颗粒的交联网络孔径小于所述的大分子或蛋白质的尺寸。
- 根据权利要求1所述的颗粒,其特征为所述的颗粒选自聚丙烯酰胺、聚丙烯酸、聚丙烯酸盐、聚葡聚糖、纤维素、聚乙烯醇、聚乙二醇、琼脂糖、聚透明质酸、聚几丁质、聚海藻酸、聚海藻酸钠、聚乙烯吡咯烷酮、聚多肽、聚蛋白质、上述水凝胶单体的衍生物聚合交联而成的修饰水凝胶、多个上述水凝胶单体或聚合体混合聚合交联而成的杂交水凝胶。
- 根据权利要求1所述的颗粒,其特征为所述的去污剂选自吐温-20、吐温-40、吐温-60、吐温-80、Brij-35、Brij-58、NP-40、曲拉通X-100、曲拉通X-114、辛基葡糖苷、辛基硫代葡糖苷、3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐、3-[(3-胆胺丙基)二甲基氨基]-2-羟基-1-丙磺酸内盐、胆酸钠、脱氧胆酸钠、十六烷基三甲基溴化铵、十二烷基硫酸钠。
- 根据权利要求1所述的颗粒,其特征为所述的去污剂的参杂浓度为0.001~10%。
- 一种大分子质量浓缩的方法,其特征为:所述的大分子液样的体积和权利要求1~4所述的颗粒的质量按一定比例进行接触,让所述的颗粒吸胀并将所述的大分子排阻在外;将所述的大分子液样和所述的颗粒的混合体进行过滤离心;去除表层脱水但内孔吸胀的所述的颗粒,收集被所述的颗粒排阻在外的所述的大分子的浓缩滤液。
- 根据权利要求5所述的方法,其特征在于所述的过滤离心在一个锥形滤膜转鼓中执行,其中所述的大分子液样和所述的颗粒的混合体连续不断地投入到小口径底部,表层脱水但内孔吸胀的所述的颗粒连续不断地在离心力作用下向大口径方向向上移动并被甩出,所述的大分子的浓缩滤液连续不断地从所述的滤膜中被甩出而被收集。
- 根据权利要求5所述的方法,其特征在于所述的大分子液样被触膜隔离的过量的所述的颗粒定量可控性地吸走液体。
- 一种蛋白质比活性提升的方法,其特征为:所述的蛋白质液样的体积和权利要求1~4所述的颗粒的质量按一定比例进行接触,让所述的颗粒吸胀并将所述的蛋白质排阻在外;将所述的蛋白质液样和所述的颗粒的混合体进行过滤离心;去除表层脱水但内孔吸胀的所述的颗粒,收集被所述的颗粒排阻在外的所述的蛋白质的浓缩滤液。
- 一种处理用于结晶或构象分析的蛋白质液样的方法,其特征为:所述的蛋白质液样的体积和权利要求1~4所述的颗粒的质量按一定比例进行接触,让所述的颗粒吸胀并将所述的蛋白质排阻在外;将所述的蛋白质液样和所述的颗粒的混合体进行过滤离心;去除表层脱水但内孔吸胀的所述的颗粒,收集被所述的颗粒排阻在外的所述的蛋白质的浓缩滤液。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP19889316.6A EP3885739B1 (en) | 2018-11-29 | 2019-11-22 | Method to mass concentrate a macromolecule with detergent-mixed dry hydrogel particles |
US17/298,007 US20220097025A1 (en) | 2018-11-29 | 2019-11-22 | Detergent-mixed dry hydrogel particle and concentration and specific activity enhancement of macromolecule |
JP2021530303A JP2022509979A (ja) | 2018-11-29 | 2019-11-22 | 洗剤を混入した乾燥ハイドロゲル粒子および高分子の濃縮と比活性の向上 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811485330.9A CN109569023B (zh) | 2018-11-29 | 2018-11-29 | 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 |
CN201811485330.9 | 2018-11-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020108390A1 true WO2020108390A1 (zh) | 2020-06-04 |
Family
ID=65926469
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2019/120118 WO2020108390A1 (zh) | 2018-11-29 | 2019-11-22 | 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20220097025A1 (zh) |
EP (1) | EP3885739B1 (zh) |
JP (1) | JP2022509979A (zh) |
CN (1) | CN109569023B (zh) |
WO (1) | WO2020108390A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023212067A1 (en) | 2022-04-28 | 2023-11-02 | Sorrento Therapeutics, Inc. | Sample collection and concentration device and methods of its use |
EP4212873A4 (en) * | 2020-09-11 | 2024-06-05 | FUJIFILM Corporation | CONCENTRATION DEVICE, METHOD FOR CONCENTRATING A SAMPLE SOLUTION, METHOD FOR TESTING A SAMPLE SOLUTION AND TEST KIT |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109569023B (zh) * | 2018-11-29 | 2021-03-02 | 杭州立昂科技有限公司 | 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 |
CN112295263A (zh) * | 2019-07-26 | 2021-02-02 | 苏州星烁纳米科技有限公司 | 去除纳米线水溶液中水组分的方法 |
WO2021065300A1 (ja) * | 2019-09-30 | 2021-04-08 | 富士フイルム株式会社 | 免疫検査方法及び濃縮用治具 |
EP4099017A4 (en) * | 2020-01-31 | 2023-02-15 | FUJIFILM Corporation | IMMUNOLOGICAL TEST METHOD |
CN111398008A (zh) * | 2020-03-11 | 2020-07-10 | 易春 | 一种快速定量细胞外囊泡样本浓缩方法 |
CN111707816A (zh) * | 2020-06-12 | 2020-09-25 | 郑州方欣生物科技有限责任公司 | 通过浓缩介质提升尿液检测试剂盒检测灵敏度的方法 |
CN111562366A (zh) * | 2020-06-15 | 2020-08-21 | 郑州方欣生物科技有限责任公司 | 尿液hiv抗体快速检测试剂盒、制备方法及使用方法 |
CN111562367A (zh) * | 2020-06-15 | 2020-08-21 | 郑州方欣生物科技有限责任公司 | 尿液新型冠状病毒快速检测试剂盒、制备方法及使用方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4555344A (en) * | 1983-08-25 | 1985-11-26 | Regents Of The University Of Minnesota | Method of size-selective extraction from solutions |
CN1153298A (zh) * | 1995-12-26 | 1997-07-02 | 黄潮 | 在凝胶过滤介质上连接进行重复固相析出的分离纯化方法 |
US20050115890A1 (en) * | 2003-09-26 | 2005-06-02 | Sartorius Ag | Adsorption membranes, method of producing same and equipment, including the adsorption membranes |
CN102072836A (zh) * | 2010-11-12 | 2011-05-25 | 深圳华大基因科技有限公司 | 生物大分子浓缩除盐方法及试剂盒 |
CN109569023A (zh) * | 2018-11-29 | 2019-04-05 | 杭州立昂科技有限公司 | 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2794592B2 (ja) * | 1989-05-29 | 1998-09-10 | ハイモ株式会社 | 生体液中の高分子物質の濃縮方法 |
JP4080565B2 (ja) * | 1996-04-26 | 2008-04-23 | 大日本インキ化学工業株式会社 | 多孔質体の製造方法および多孔質体 |
US20050059024A1 (en) * | 2003-07-25 | 2005-03-17 | Ambion, Inc. | Methods and compositions for isolating small RNA molecules |
WO2005058453A1 (en) * | 2003-12-15 | 2005-06-30 | Preentec Ag | Method for the concentration and purification of biological compounds |
CN100562358C (zh) * | 2007-05-17 | 2009-11-25 | 浙江大学 | 以水凝胶微球为模板制备中空微球的方法 |
BRPI0913733A2 (pt) * | 2008-09-30 | 2015-08-18 | 3M Innovative Properties Co | Artigos para detecção de células, dispositivo de aquisição de amostra, kit e métodos para detecção de células |
EP2536786B1 (en) * | 2010-02-18 | 2015-12-30 | Dow Corning Corporation | Surface-modified hydrogels and hydrogel microparticles |
EP2614148A1 (en) * | 2010-09-08 | 2013-07-17 | Qiagen GmbH | Method and device for concentrating target compounds |
KR101539838B1 (ko) * | 2013-03-22 | 2015-07-28 | 성균관대학교산학협력단 | 도파 함유 고흡수성 하이드로젤 |
-
2018
- 2018-11-29 CN CN201811485330.9A patent/CN109569023B/zh active Active
-
2019
- 2019-11-22 WO PCT/CN2019/120118 patent/WO2020108390A1/zh unknown
- 2019-11-22 US US17/298,007 patent/US20220097025A1/en active Pending
- 2019-11-22 JP JP2021530303A patent/JP2022509979A/ja active Pending
- 2019-11-22 EP EP19889316.6A patent/EP3885739B1/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4555344A (en) * | 1983-08-25 | 1985-11-26 | Regents Of The University Of Minnesota | Method of size-selective extraction from solutions |
CN1153298A (zh) * | 1995-12-26 | 1997-07-02 | 黄潮 | 在凝胶过滤介质上连接进行重复固相析出的分离纯化方法 |
US20050115890A1 (en) * | 2003-09-26 | 2005-06-02 | Sartorius Ag | Adsorption membranes, method of producing same and equipment, including the adsorption membranes |
CN102072836A (zh) * | 2010-11-12 | 2011-05-25 | 深圳华大基因科技有限公司 | 生物大分子浓缩除盐方法及试剂盒 |
CN109569023A (zh) * | 2018-11-29 | 2019-04-05 | 杭州立昂科技有限公司 | 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 |
Non-Patent Citations (3)
Title |
---|
ANALYTICAL BIOCHEMISTRY, vol. 138, 1984, pages 451 |
DU, CUIHONG: "Enzyme Engineering", 31 October 2014, HUAZHONG UNIVERSITY OF SCIENCE & TECHNOLOGY PRESS, CN, ISBN: 978-7-5609-9695-0, article DU CUIHONG : "Passage; Enzyme Engineering", pages: 55 - 70, XP009528648 * |
LIN, AIGUO: "New Functional Membrane Technology and Application Thereof", 30 November 2013, CHINA UNIVERSITY OF PETROLEUM PRESS, CN, ISBN: 978-7-5636-3902-1, article LIN AIGUO: "Passage", pages: 36 - 38, XP009528635 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4212873A4 (en) * | 2020-09-11 | 2024-06-05 | FUJIFILM Corporation | CONCENTRATION DEVICE, METHOD FOR CONCENTRATING A SAMPLE SOLUTION, METHOD FOR TESTING A SAMPLE SOLUTION AND TEST KIT |
WO2023212067A1 (en) | 2022-04-28 | 2023-11-02 | Sorrento Therapeutics, Inc. | Sample collection and concentration device and methods of its use |
Also Published As
Publication number | Publication date |
---|---|
EP3885739A4 (en) | 2022-01-19 |
EP3885739A1 (en) | 2021-09-29 |
JP2022509979A (ja) | 2022-01-25 |
CN109569023B (zh) | 2021-03-02 |
EP3885739C0 (en) | 2023-08-30 |
EP3885739B1 (en) | 2023-08-30 |
US20220097025A1 (en) | 2022-03-31 |
CN109569023A (zh) | 2019-04-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2020108390A1 (zh) | 参杂去污剂的干燥水凝胶颗粒及大分子浓缩和比活性提升 | |
RU2698822C1 (ru) | Упорядоченный фильтрующий материал для очистки биоматериала | |
JP5032324B2 (ja) | 複合濾過物品 | |
CN101903296B (zh) | 复合聚合物过滤介质 | |
EA013385B1 (ru) | Устройство для удаления целевых агентов из образца и способ | |
CN113195095A (zh) | 大孔琼脂糖 | |
US11364480B2 (en) | Chromatography medium with bound microglobules and method for the preparation thereof | |
JP6159737B2 (ja) | 分離方法における、フィブリルセルロースを含む固定相の使用 | |
US9606032B2 (en) | Preparation of samples for analysis and sampling device therefor | |
US4555344A (en) | Method of size-selective extraction from solutions | |
CN108855003B (zh) | 一种用于清除血液中炎性因子的免疫吸附剂及其制备方法 | |
CN105658313A (zh) | 硫酸化纤维素水合物膜、其制备方法和该膜作为病毒纯化用吸附膜的用途 | |
JP2022184990A (ja) | バイオセパレーションのための複合材料 | |
CN103173934B (zh) | 一种半乳糖乙烯酯/异丙基丙烯酰胺共聚物纳米纤维膜的制备方法 | |
CN107406824B (zh) | 纯化生物组合物的方法及用于其的制品 | |
CN111701581A (zh) | 低密度脂蛋白、胆固醇及甘油三酯吸附剂及其制备方法和应用 | |
CN105693922A (zh) | 一种两性蛋白质冰胶印迹聚合物及其制备方法和应用 | |
BR102020004927A2 (pt) | Adsorvente monolítico macroporoso polimérico funcionalizado com anilina, seu processo de obtenção e aplicação. | |
Mattheus et al. | Chitosan-alginate affinity microcapsules for isolation of bovine serum albumin | |
JP7344232B2 (ja) | バイオセパレーションのための複合材料 | |
EP4385618A1 (en) | Composite material for bioseparations | |
Baydemir et al. | A novel chromatographic media: histidine-containing composite cryogels for HIgG separation from human serum | |
BR102020005020A2 (pt) | Adsorvente monolítico macroporoso polimérico funcionalizado com butilamina, seu processo de obtenção e aplicação. | |
Josić et al. | The application of immobilized affinity ligands in high performance liquid chromatography | |
JP2022097368A (ja) | 回収用キットおよび核酸含有溶液の調製方法 |
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: 19889316 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2021530303 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: 2019889316 Country of ref document: EP Effective date: 20210624 |