Method for regulating and controlling density of grafted polymer on surface of microsphere

Abstract

The invention provides a method for regulating and controlling the density of a polymer on the surface of a microsphere, which is characterized by mainly comprising the following steps of: treating a microsphere body with TBAF (Tetrabutylammonium Fluoride), and reacting with a silane coupling agent and a monomer in sequence to obtain a modified microsphere. The method disclosed by the invention is beneficial to control of reaction sites on the surfaces of the microspheres, reduces the density of a polymer grafted on the surfaces of the microspheres, is more beneficial to grabbing of target products (such as protein, nucleic acid and the like), and improves the adsorption capacity of the microspheres.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to a method for regulating the density of grafted polymers on the surface of microspheres. Background Art

[0002] By modifying the surface of silica microspheres with polymers, the physicochemical properties of the silica microsphere surface can be significantly improved, making it widely used in biomedical-related fields such as drug release, drug synthesis and purification, and medical device manufacturing. There are mainly two modification strategies for the surface of silica microspheres: one is the polymer chain post-grafting strategy (Grafting to), that is, the synthesized polymer chain is linked to the surface of the silica microsphere through a chemical reaction (such as click reaction) or other special interactions; the other is the polymerization strategy on the surface of silica microspheres (Grafting from), that is, after special treatment of the silica microsphere surface, the surface has active sites that can initiate polymerization, and then an in-situ polymerization reaction is initiated to prepare polymer chains linked on the surface. In the actual R & D and large-scale production processes, radical polymerization is used as a common technical means for the Grafting from modification strategy because of its good reaction tolerance, ability to polymerize more types of monomers, and high monomer conversion rate.

[0003] In the fields of pharmaceutical purification and protein synthesis, the performance of silica microspheres used for protein molecule purification depends entirely on the performance of the polymer chains grafted on their surfaces. The regulation of the polymer chains on the surface of these silica microspheres mainly focuses on the polymer chain length (i.e., the size of the molecular weight) and the grafting density of the polymer chains. The regulation of the polymer chain length is mainly achieved by changing the ratio of the polymer monomer to the initiator (sometimes including the ratio to the chain transfer agent). However, the related research on the regulation of the polymer chain density on the surface of silica microspheres is still limited to small-scale laboratory research, which greatly restricts the application of such silica microspheres in the field of biomedical purification. Therefore, developing a method for regulating the grafting density of polymer chains on the surface of silica microspheres that is easy to operate and scale up is of great significance for the development of biomedical purification in the industrial scale-up production process. Summary of the Invention

[0004] In the present invention, the inventor uses a fluorinating agent as a reagent for controlling the density of polymerization initiation sites or the density of polymer chains grafted on the surface of microspheres. The fluorinating agent provides fluoride ions (F — ) to cleave the link between the silane coupling agent and the microsphere, resulting in a reduction in the initiation sites or a direct reduction in the grafted polymer chains, and then the density of the grafted polymer chains is adjusted by the fluorinating agent: during the reaction, F —It tends to attack the Si atom in the Si-O bond. Compared with hydrofluoric acid (HF) reported in the literature, the fluorinating agent of the present invention has the advantages of convenient use and lower toxicity, and thus has the potential for industrial production scale-up.

[0005] In the first aspect of the present invention, a method for regulating the polymer density on the surface of microspheres is provided, which is characterized by mainly including the following steps: treating the microsphere body with a silane coupling agent, then reacting with a fluorinating agent in sequence, and finally reacting with a monomer in the presence of an initiator to obtain modified microspheres.

[0006] Further preferably, the fluorinating agent is selected from one or more of alkyl fluorides, aryl fluorides, arylalkyl fluorides, fluorinated salts or fluorocyanates.

[0007] Further preferably, the number of alkyl groups of the alkyl fluoride is 1-30, preferably 4-20, and further preferably 4-16.

[0008] Further preferably, the alkyl fluoride is an alkyl fluorinated salt, such as alkyl ammonium fluoride, sodium alkyl fluoride, potassium alkyl fluoride, cesium alkyl fluoride, etc.

[0009] Further preferably, the alkyl fluoride is selected from tetramethylammonium fluoride (TMAF), tetraethylammonium fluoride (TEAF), tetrapropylammonium fluoride (TPAF), tetrabutylammonium fluoride (TBAF), sodium tetrabutyl fluoride.

[0010] Further preferably, the number of alkyl groups of the arylalkyl fluoride is 1-30, preferably 3-20, and further preferably 3-15.

[0011] Further preferably, the arylalkyl fluoride is an arylalkyl fluorinated salt, such as arylalkyl ammonium fluoride, arylalkyl sodium fluoride, arylalkyl potassium fluoride, arylalkyl cesium fluoride, etc.

[0012] Further preferably, the aryl is a phenyl group.

[0013] Further preferably, the arylalkyl fluoride is phenyltrimethylammonium fluoride, phenyldiethylammonium fluoride, phenyltripropylammonium fluoride or phenyltributylammonium fluoride.

[0014] Further preferably, the fluorinated salt is selected from ammonium fluoride, sodium fluoride, potassium fluoride or cesium fluoride.

[0015] Further preferably, the fluorocyanate is selected from sodium fluorocyanate or potassium fluorocyanate.

[0016] Further preferably, before the microsphere body is treated with the silane coupling agent, it is ultrasonically cleaned and treated with an alkaline solution.

[0017] Further preferably, the alkaline solution is selected from one or more of hydroxide solutions, carbonate solutions, bicarbonate solutions, etc.

[0018] Further preferably, the alkaline solution is selected from sodium hydroxide solution, potassium hydroxide solution or a combination thereof.

[0019] Further preferably, the silane coupling agent contains an unsaturated bond in its structure.

[0020] Further preferably, the silane coupling agent is selected from one or more of γ-methacryloxypropyltrimethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, vinyltris(β-methoxyethoxy)silane, dimethylethenyethoxysilane, methylethenyldiethoxysilane or vinyltrimethoxysilane oligomer.

[0021] Further preferably, the monomer is an acrylic monomer.

[0022] Further preferably, the monomer is selected from one or more of substituted or unsubstituted acrylic acid, acrylate, and acrylate ester.

[0023] Further preferably, the substituent is selected from alkyl, cycloalkyl, aryl, alkoxy, cycloalkoxy, aryloxy, etc.

[0024] Further preferably, the monomer is selected from one or more of alkyl acrylic acid, alkyl acrylate, or alkyl acrylate ester.

[0025] Further preferably, the acrylic monomer is selected from one of acrylic acid, methacrylic acid, methacrylate, methacrylate ester, methyl methacrylate, butyl acrylate, isooctyl acrylate or a combination thereof.

[0026] Further preferably, the surface of the microsphere body contains silicon, silica or hydroxyl groups; preferably, the material of the microsphere body is selected from glass, ceramic or magnetic material. When the microsphere body is a magnetic microsphere, its surface is coated with silica or hydroxyl groups.

[0027] Further preferably, the microsphere body is a silica microsphere.

[0028] Further preferably, the size of the microsphere body is selected from any one of the following particle size scales or the range between any two particle size scales: 0.1μm, 0.15μm, 0.2μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm, 0.45μm, 0.5μm, 0.55μm, 0.6μm, 0.65μm, 0.7μm, 0.75μm, 0.8μm, 0.85μm, 0.9μm, 0.95μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm, 3.5μm, 4μm, 4.5μm, 5μm, 5.5μm, 6μm, 6.5μm, 7μm, 7.5μm, 8μm, 8.5μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter size is the average value;

[0029] One of the preferred ways, the diameter of the microsphere body is selected from 0.1 - 10μm;

[0030] One of the preferred ways, the diameter of the microsphere body is selected from 0.2 - 6μm;

[0031] One of the preferred ways, the diameter of the microsphere body is selected from 0.4 - 5μm;

[0032] One of the preferred ways, the diameter of the microsphere body is selected from 0.5 - 3μm;

[0033] One of the preferred ways, the diameter of the microsphere body is selected from 0.2 - 1μm;

[0034] One of the preferred ways, the diameter of the microsphere body is selected from 0.5 - 1μm;

[0035] One of the preferred ways, the diameter of the microsphere body is selected from 1μm to 1mm;

[0036] One of the preferred ways, the average diameter of the microsphere body is 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, with a deviation of ±20%, more preferably ±10%.

[0037] Further preferably, the method further comprises the following steps:

[0038] (1) Ultrasonically wash the microspheres to obtain microspheres A;

[0039] (2) React microspheres A with an alkaline solution and wash to obtain microspheres B;

[0040] (3) React microspheres B with a silane coupling agent to obtain microspheres C;

[0041] (4) React microspheres C with a fluorinating agent to obtain microspheres D;

[0042] (5) React microspheres D with a monomer under initiator conditions to obtain microspheres E;

[0043] (6) Treat microspheres E with an alkaline solution to obtain microspheres F, i.e., the target modified microspheres.

[0044] Further preferably, the feeding ratio (L: mol) of the silane coupling agent to the fluorinating agent is 1:0.1 - 20; more preferably 1:0.1 - 15; even more preferably any value or the range between any two values among 1:1, 1:1.5, 1:2, 1:15 or 10:1.

[0045] Further preferably, the feeding ratio (L: g) of the monomer to the initiator is (10 - 70):(10 - 50); more preferably 0.5 - 5:1; further preferably 0.5 - 2:1, and even more preferably any value or the range between any two values among 0.5:1, 0.6:1, 1:1, 1.5:1 or 2:1.

[0046] Further preferably, step (1) further comprises putting the microspheres into deionized water and stirring for 10 - 60 minutes, and then ultrasonically cleaning for 10 - 60 minutes.

[0047] Further preferably, repeat step (1) 2 - 3 times.

[0048] Further preferably, step (2) further comprises adding 5 - 50 g of sodium hydroxide to the microspheres A system in batches and stirring for 0.1 - 8 h under the condition that the reaction temperature is 15 - 80°C.

[0049] Further preferably, step (2) further comprises washing the reacted microspheres with water or an alcohol solvent until the pH of the solution is 6 - 9.5 to obtain microspheres B.

[0050] Further preferably, in the step (3), the reaction temperature is 0 to 150 °C, more preferably 10 to 100 °C, and even more preferably 50 to 100 °C; the reaction time is 1 to 48 h, preferably 5 to 24 h.

[0051] Further preferably, the step (4) further includes reacting the microsphere C with a fluorinating agent solution, and the solution is preferably THF, and the reaction temperature is preferably 0 to 150 °C, more preferably 10 to 100 °C, and even more preferably 50 to 70 °C; the reaction time is 1 to 48 h, preferably 12 to 36 h, and more preferably 24 to 26 h.

[0052] Further preferably, the step (4) further includes, after the reaction is completed, washing the microsphere D, and the washing is carried out with a solvent selected from water or alcohol solvents, and further preferably deionized water or absolute ethanol.

[0053] Further preferably, the step (4) further includes storing the washed microsphere D in a sealed manner.

[0054] Further preferably, the step (5) further includes: after mixing the microsphere D with the monomer and the initiator, sealing the reaction system, bubbling, and then heating for reaction.

[0055] Further preferably, the bubbling time in the step (5) is 0.5 to 5 h, more preferably 1 to 2 h.

[0056] Further preferably, the heating temperature in the step (5) is 50 to 150 °C, more preferably 60 to 100 °C.

[0057] Further preferably, the initiator is selected from azo initiators, peroxide initiators or redox initiators.

[0058] Further preferably, the initiator is selected from one or more of 4,4'-azobis(4-cyanovaleric acid), azobisisobutyronitrile, azobisisoheptonitrile, dimethyl azobisisobutyrate, azobisisobutamidine hydrochloride, azobisisobutimidazoline hydrochloride, and azoisobutyronitrile formamide.

[0059] Further preferably, the feeding ratio (mL:mg) of the monomer to the initiator is 1:01 to 10; further preferably 1:0.5 to 5, and more preferably 1:1 to 2.

[0060] Further preferably, the basic solution in the step (6) is selected from one or more of hydroxide solutions, carbonate solutions, bicarbonate solutions, etc.

[0061] Further preferably, the step (6) further includes, after the microsphere E is treated with the basic solution, standing, washing, and storing in a sealed manner.

[0062] Further preferably, the standing is carried out in water or an alcohol solvent, more preferably deionized water or absolute ethanol.

[0063] Further preferably, the washing is carried out using a solvent selected from water or an alcohol solvent, more preferably deionized water or absolute ethanol.

[0064] Further preferably, the temperature for airtight storage is 0 - 10°C, preferably 4°C.

[0065] The second aspect of the present invention provides a modified microsphere, which is prepared by the method described in the first aspect of the present invention.

[0066] Further preferably, the density of the polymer chains grafted on the modified microsphere is reduced.

[0067] The third aspect of the present invention provides the use of the modified microsphere prepared by the method of the first aspect of the present invention or the modified microsphere provided by the second aspect of the present invention in cell-free protein synthesis, protein separation and purification, target antibody drug enrichment, targeted drug delivery, nucleic acid separation and extraction, cell sorting, enzyme immobilization, and kits.

[0068] The main advantages and positive effects of the present invention include:

[0069] (1) For the first time, a fluorinating agent is used to regulate the density of the polymer grafted on the microsphere surface, which has pioneering significance.

[0070] (2) Using a fluorinating agent, especially an alkyl fluoride, to treat the microsphere surface is beneficial to controlling the reaction sites on the microsphere surface, reducing the density of the polymer grafted on the microsphere surface, being more conducive to capturing target products (such as proteins, nucleic acids, etc.), and increasing the adsorption capacity of the microsphere. Detailed implementation manners

[0071] The present invention will be further described below in conjunction with specific embodiments and examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the conditions guided by the specific embodiments described above shall be preferentially followed and referred to, and then the conventional conditions can be followed, such as the experimental conditions described in "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-Free Protein Synthesis Experimental Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008" and other documents, or the conditions recommended by the manufacturer.

[0072] Unless otherwise specified, the percentages and parts mentioned in the present invention are weight percentages and weight parts.

[0073] Unless otherwise specified, the materials and reagents used in the embodiments of the present invention are all commercially available products.

[0074] Unless otherwise specified, the temperature unit in this application is degrees Celsius (°C).

[0075] Nouns and Terms

[0076] The following are the explanations or descriptions of the meanings of some relevant "nouns" and "terms" adopted in the present invention, so as to better understand the present invention. The corresponding explanations or descriptions apply to the whole text of the present invention, both to the following text and to the above text. When the present invention involves the citation of documents, the definitions of relevant terms, nouns, and phrases in the cited documents are also cited. However, when there is a conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When there is a conflict between the definitions in the cited documents and the definitions in the present invention, it does not affect the components, substances, compositions, materials, systems, formulations, species, methods, equipment, etc. determined in the cited documents to prevail.

[0077] Polymer, as used in the present invention, broadly includes oligomers and polymers, having at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be characterized by suitable methods, such as number-average molecular weight, weight-average molecular weight, viscosity-average molecular weight, etc.).

[0078] Acrylic monomers: Monomer molecules that can be used to synthesize acrylic polymers, having a basic structure of C(COO-)=C. Examples include CH(COOH)=CH2, CH(COONa)=CH2, CH3C(COOH)=CH2, CH3C(COONa)=CH2, CH(COOCH3)=CH2, CH(COOCH2CH2OH)=CH2, CH3C(COOCH3)=CH2, CH3C(COOCH2CH2OH)=CH2, etc.

[0079] "Fixation", such as "fixed to", "fixed with", "fixed on", etc., refers to a covalent binding method.

[0080] "Connection" / "Binding" methods, such as "carrying", "connected with", "connected to", "connected at", "binding", "capturing", "captured to", etc., are not particularly limited and include, but are not limited to, covalent and non-covalent methods.

[0081] Purification substrate, also known as the target substance, is the substance to be separated from the mixed system. The purification substrate in the present invention is not particularly limited. For example, it is a protein substance (also called the target protein at this time).

[0082] "Modified" products include, but are not limited to, derivatives of the present invention, modified products, genetically modified products, fusion products, etc., which can maintain the original functions or properties, or can optimize or change their functions or properties.

[0083] Eluent (taking the target protein as an example): Elute the target protein; after elution, the target protein exists in the eluent.

[0084] Washing solution (taking the target protein as an example): Elute impurities such as miscellaneous proteins; after elution, the miscellaneous proteins are carried away by the washing solution.

[0085] Binding force: Binding ability, such as the binding ability of a biological microsphere to a certain protein.

[0086] Affinity: Using substrate solutions with different concentration gradients, it is the substrate concentration when the biological microsphere binds only 50% of the substrate.

[0087] IVTT: In vitro transcription and translation, an in vitro transcription and translation system, i.e., a cell-free protein synthesis system. A cell-free protein synthesis system uses exogenous target mRNA or DNA as a template for protein synthesis, and by artificially controlling the addition of substrates required for protein synthesis, as well as transcription- and translation-related protein factors and other substances, can achieve the synthesis of the target protein. The cell-free protein synthesis system of the present invention is not particularly limited and can be any one or any combination of cell-free protein synthesis systems based on yeast cell extracts, Escherichia coli cell extracts, mammalian cell extracts, plant cell extracts, or insect cell extracts.

[0088] In the present invention, "translation-related enzymes (TRENs)" refer to enzyme substances required during the process of synthesizing protein products from nucleic acid templates, not limited to enzymes required during the translation process.

[0089] Nucleic acid template: Also known as a genetic template, it refers to the nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates, and their combinations.

[0090] Flow-through: The supernatant collected after incubating the biological microspheres with the system containing the target protein, which contains the residual target protein not captured by the biological microspheres.

[0091] RFU, Relative Fluorescence Unit.

[0092] eGFP: Enhanced green fluorescence protein. In the present invention, the eGFP broadly includes the wild type and its variants, including but not limited to the wild type and its mutants.

[0093] mEGFP: An A206K mutant of eGFP.

[0094] "Optionally" means that it can be present or absent, with the criterion of being able to implement the technical solution of the present invention as the selection standard.

[0095] In the present invention, "optional manner" means that as long as it is applicable to the technical solution of the present invention, it can be used to implement the present invention.

[0096] In the present invention, preferred embodiments such as "preferred", "more preferred", "most preferred", etc. do not constitute any limitation on the scope of coverage and protection scope of the invention, and are not used to limit the scope and implementation manner of the present invention, but are only used to provide some embodiments as examples.

[0097] In the description of the present invention, for the preferred modes such as "one of the preferences", "one of the preferred modes", "one of the preferred embodiments", "one of the preferred examples", "preferred examples", "in a preferred embodiment", "in some preferred examples", "in some preferred modes", "preferably", "preferred", "preferably", "more preferably", "more preferably", "further preferably", "most preferably", etc., and the illustrative listing modes such as "one of the embodiments", "one of the modes", "examples", "specific examples", "for example", "as an example", "for example", "such as", "like", etc., they do not constitute any limitation to the scope of coverage and protection scope of the invention in any sense, and the specific features described by each mode are included in at least one specific embodiment of the present invention. In the present invention, each mode

[0098] The specific features described by each mode can be combined in a suitable manner in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred mode can also be combined by any suitable means.

[0099] In the present invention, "any combination thereof" means "greater than 1" in terms of quantity, and in terms of the scope of coverage, it means the group composed of the following situations: "optionally one of them, or the group composed of optionally at least two of them".

[0100] In the present invention, the descriptions of "one or more", "one or more kinds", etc. in the form of "one or more" have the same meaning as "at least one", "at least one kind", "its combination", "or its combination", "and its combination", "or any combination thereof", "and any combination thereof", etc., and can be used interchangeably, indicating that the quantity is equal to "1" or "greater than 1".

[0101] In the present invention, the use of "or / and", "and / or" means "optionally one of them or optionally their combination", and also means at least one of them.

[0102] The prior art means described in the present invention in the ways such as "usually", "conventionally", "generally", "often", "tend to", etc. are also cited as references for the content of the present invention. If there is no special description, they can be regarded as one of the preferred modes of some technical features of the present invention, and it should be noted that they do not constitute any limitation to the scope of coverage and protection scope of the invention in any sense.

[0103] All the documents mentioned in the present invention and the documents directly or indirectly cited by these documents are cited as references in this application, just as if each document is cited separately as a reference.

[0104] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (including but not limited to the embodiments) can be combined with each other to form new or preferred technical solutions, as long as they can be used to implement the present invention. Due to space limitations, they will not be elaborated one by one.

[0105] 1. Purified substrate (preferably a proteinaceous substance)

[0106] The purified substrate of the present invention refers to the substance that the magnetic microspheres of the present invention are used to capture and separate, and there is no particular limitation as long as the purified substrate can specifically bind to the purification medium of the biological microspheres of the present invention (such as the metal chelate part at the end of the modified biological microspheres of the present invention).

[0107] When the purified substrate is a proteinaceous substance, the purified substrate is also referred to as the target protein.

[0108] 2. Purification tag in the target protein

[0109] The target protein may not carry a purification tag. In this case, the target protein itself should be able to be captured by the purification medium in the biological microspheres.

[0110] In some preferred embodiments, the target protein carries a purification tag, and the purification tag can specifically bind to the purification medium. In a target protein molecule, the number of purification tags is one, two or more; when there are two or more purification tags, the types of purification tags are one, two or more. It should be noted that as long as the amino acid sequences of the tags are different, they are regarded as different types of tags.

[0111] The purification tag in the target protein can be selected from the group consisting of but not limited to the following tags: histidine tag, avidin, avidin analog, Streg tag, FLAG tag or its variants, C tag and its variants, Spot tag and its variants, GST tag and its variants, MBP tag and its variants, SUMO tag and its variants, CBP tag and its variants, HA tag and its variants, Avi tag and its variants, affibody protein, antibody-like tag, antigen-like tag, and their combinations. The purification tag can be fused at the N-terminus or C-terminus.

[0112] The histidine tag generally contains at least 5 histidine residues, such as 5×His tag, 6×His tag, 8×His tag, etc.

[0113] The C tag contains the EPEA sequence.

[0114] The GST tag refers to the glutathione S-transferase tag.

[0115] The MBP tag refers to the maltose-binding protein tag.

[0116] The SUMO tag is a known small ubiquitin-like modifier protein and is one of the important members of the ubiquitin polypeptide chain superfamily. In terms of primary structure, SUMO has only 18% homology with ubiquitin. However, their tertiary structures and biological functions are very similar.

[0117] The Avi tag is a known small tag composed of 15 amino acid residues, which can be specifically recognized by the desthiobiotin ligase BirA.

[0118] Antibody-based tags include, but are not limited to, the complete structure of an antibody (whole antibody), domain, subunit, fragment, heavy chain, light chain, single-chain fragment (such as nanobody, heavy chain lacking the light chain, heavy chain variable region, complementarity-determining region, etc.), and the like.

[0119] Antigen-based tags include, but are not limited to, the complete structure of an antigen (whole antigen), domain, subunit, fragment, heavy chain, light chain, single-chain fragment (such as antigenic determinant, etc.), and the like.

[0120] In some preferred embodiments, a purification tag is linked to the N-terminus or C-terminus of the target protein, or purification tags are linked to both ends.

[0121] All kinds of purification tags described in this part can be candidates for the purification medium in the biospheres of the present invention.

[0122] 3. Type of Target Protein

[0123] The target protein can be a natural protein or its modified product, or an artificially synthesized sequence. The source of the natural protein is not particularly limited and includes, but is not limited to: eukaryotic cells, prokaryotic cells, pathogens; wherein the eukaryotic cell sources include, but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell sources can include, but are not limited to, murine (including rats, mice, guinea pigs, golden hamsters, hamsters, etc.), rabbit-derived, monkey-derived, human-derived, porcine-derived, ovine-derived, bovine-derived, canine-derived, equine-derived, etc. The pathogens include viruses, chlamydia, mycoplasma, etc. The viruses include HPV, HBV, TMV, coronaviruses, rotaviruses, etc.

[0124] The types of the target protein include, but are not limited to, polypeptides (in the present invention, the "target protein" broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies, antigens, immunoglobulins, hormones, collagens, polyamino acids, vaccines, etc., partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins, and variants of any of the foregoing proteins. The "subunits or fragments of any of the foregoing proteins" include the subunits or fragments of "partial domains of any of the foregoing proteins". The "variants of any of the foregoing proteins" include the variants of "partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins". The "variants of any of the foregoing proteins" include, but are not limited to, mutants of any of the foregoing proteins. In the present invention, in the case of two or more consecutive "foregoing" at other positions, the meaning is interpreted similarly.

[0125] The structure of the target protein can be either a complete structure or selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, etc. Examples of incomplete antibody structures include, but are not limited to, nanobodies (heavy-chain antibodies lacking the light chain, VHH, which retain the complete antigen-binding ability of the heavy-chain antibody), heavy-chain variable regions, complementarity-determining regions (CDRs), etc.

[0126] For example, the target protein that can be synthesized by the in vitro protein synthesis system of the present invention can be selected from any one protein, fusion proteins in any combination, and compositions in any combination, including but not limited to: luciferase (such as firefly luciferase), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase (e.g., murine catalase), actin, antibody, variable region of antibody (such as single-chain variable region of antibody, scFV), single chain and fragments of antibody (such as heavy chain of antibody, nanobody, light chain of antibody), α-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin and its precursors, glucagon-like peptide (GLP-1), interferon (including but not limited to interferon α, such as interferon αA, interferon β, interferon γ, etc.), interleukin (such as interleukin-1β, interleukin 2, interleukin 12, etc.), lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), transthyretin, tyrosinase, xylanase, β-galactosidase (LacZ, e.g., Escherichia coli β-galactosidase), etc., partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins, or variants of any of the foregoing (as defined above, the variants include mutants, e.g., luciferase mutants, mutants of eGFP, and the variants can also be homologs). The aminoacyl-tRNA synthetase, for example, human lysine-tRNA synthetase, human leucine-tRNA synthetase, etc. The glyceraldehyde-3-phosphate dehydrogenase, for example, Arabidopsis thaliana glyceraldehyde 3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase. Patent document CN109423496A can also be referred to. The composition in any combination can include any of the foregoing proteins or can also include fusion proteins in any combination of the foregoing.

[0127] In some preferred embodiments, the protein synthesis ability of the in vitro protein synthesis system is evaluated using a target protein with fluorescence properties such as GFP, eGFP, mScarlet, etc., or a similar substance or a mutant thereof.

[0128] The application fields of the target protein include, but are not limited to, biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, cosmetics, food, food additives, nutritional agents, agriculture, feed, daily necessities, washing, environment, chemical staining, fluorescent labeling and other fields.

[0129] 4. Mixed system containing the target protein

[0130] The biological microspheres of the present invention can be used to separate the target protein from its mixed system. The target protein is not limited to one substance and allows for a combination of multiple substances, as long as the purpose of purification is to obtain this composition, or the form of this composition can meet the purification requirements.

[0131] The mixed system containing the target protein is not particularly limited, as long as the purification medium of the biological microspheres of the present invention can specifically bind to the target protein; generally, it is also required that the purification medium has no specific binding or non-specific binding effect with other substances in the mixed system other than the target protein.

[0132] In the examples of the present invention, the mixed system containing the target protein can be of natural origin or an artificially constructed or obtained mixed system.

[0133] For example, a specific protein can be isolated and purified from commercially available serum.

[0134] For example, the target protein can be separated from the system after the reaction of an in vitro protein synthesis system.

[0135] In vitro protein synthesis reaction refers to the reaction of synthesizing proteins in a cell-free in vitro synthesis system, which at least includes the translation process. It includes, but is not limited to, IVT reaction (in vitro translation reaction), IVTT reaction (in vitro transcription and translation reaction), IVDTT reaction (in vitro replication, transcription and translation reaction). In the present invention, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as the D2P system, D-to-P system, D_to_P system, DNA-to-Protein system; the corresponding in vitro protein synthesis method is also referred to as the D2P method, D-to-P method, D_to_P method, DNA-to-Protein method.

[0136] "Cell-free system" refers to a method of in vitro protein synthesis that does not rely on the secretion and expression of intact cells. It should be noted that in the cell-free protein synthesis system of the present invention, cell components are allowed to be added to facilitate the reaction, but the added cells do not primarily aim to secrete and express exogenous target proteins. In addition, in the D2P system without intact cells constructed under the guidance of the present invention, a small amount of intact cells are deliberately added (for example, the protein content provided by them does not exceed 30 wt% compared to the protein content provided by cell extracts). Such an "evasion" method is also within the scope of protection of the present invention.

[0137] One of the specific embodiments of the in vitro protein synthesis system further includes, but is not limited to, for example, the cell-free protein synthesis system based on Escherichia coli described in WO2016005982A1. The in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, etc., which are described in other cited documents of the present invention and their directly and indirectly cited documents, are also incorporated into the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, the in vitro cell-free protein synthesis system (In vitro cell-free protein synthesis system) described in the cited documents on pages 27-28 of the section "2.1 Systems and Advantages" in the literature "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" can be used as the in vitro protein synthesis system for implementing the present invention.For example (unless it conflicts with the present invention, the following documents and their cited documents are cited in their entirety and for all purposes), the cell-free in vitro protein synthesis systems, DNA template construction and amplification methods described in documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622, CN110938649A, CN110964736A, CN111378706A, CN111378707A, CN111378708A, CN111718419A, CN111748569A, CN2019107298813, CN2019112066163, CN2018112862093, CN2019114181518, CN2020100693833, CN2020101796894, CN202010269333X, CN2020102693382, CN2020113574616 and their cited documents can all be used as the in vitro protein synthesis system of the present invention and the DNA template construction and amplification method of the present invention.

[0138] There is no particular limitation on the source cells of the cell extract of the in vitro protein synthesis system as long as they can express the target protein in vitro. Exogenous proteins of in vitro protein synthesis systems derived from prokaryotic cell extracts and eukaryotic cell extracts (yeast cell extracts can be preferably used, and Kluyveromyces lactis can be more preferably used) disclosed in the prior art, or endogenous proteins of prokaryotic cell systems and eukaryotic cell systems (yeast cell systems can be preferably used, and Kluyveromyces lactis systems can be more preferably used) applicable to intracellular synthesis can also be synthesized using the in vitro protein synthesis system of the present invention, or attempts can be made to synthesize them using the in vitro protein synthesis system provided by the present invention.

[0139] One of the preferred modes of the in vitro protein synthesis system is the IVTT system. The liquid after the IVTT reaction (denoted as the IVTT reaction solution) contains, in addition to the target protein expressed, the residual reaction raw materials in the IVTT system, and particularly contains various factors from the cell extract (such as ribosomes, tRNAs, translation-related enzymes, initiation factors, elongation factors, termination factors, etc.). The IVTT reaction solution can, on the one hand, provide the target protein for binding to the biological microspheres, and on the other hand, can also provide a mixed system for testing the separation effect of the target protein.

[0140] "The expression system of the present invention", "the in vitro expression system of the present invention", "the cell-free in vitro expression system", and "the cell-free in vitro expression system" can be used interchangeably and all refer to the in vitro protein expression system of the present invention. Other description methods can also be used, such as: in vitro protein synthesis system, in vitro protein synthesis system, cell-free system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. According to the reaction mechanism, it can include an in vitro translation system (which can be abbreviated as the IVT system, a type of mR2P system), an in vitro transcription and translation system (which can be abbreviated as the IVTT system, a type of D2P system), an in vitro replication, transcription and translation system (which can be abbreviated as the IVDTT system, a type of D2P system), etc. In the present invention, the IVTT system is preferred. We also refer to the in vitro protein synthesis system as the "Protein Factory" ("Protein Factory" or "proteinfactory" or "Proteinfactory"). The in vitro protein synthesis system provided by the present invention describes its components in an open manner. The cell-free protein synthesis system of the present invention uses exogenous DNA, mRNA, or a combination thereof as the nucleic acid template for protein synthesis, and realizes the in vitro synthesis of the target protein by artificially controlling the addition of substrates required for protein synthesis and transcription, translation-related protein factors, etc.

[0141] In the present invention, "protein" and "protein" have the same meaning and are both translated as protein and can be used interchangeably.

[0142] In the present invention, both "system" and "system" are translated as system and can be used interchangeably.

[0143] In the present invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

[0144] In the present invention, cell extract, cell extract solution, cell lysate, cell disrupted product, and cell dissolved product have the same meaning and can be used interchangeably. In English, they can be described as cell extract, cell lysate, etc.

[0145] In the present invention, energy system, energy supply system, and energy supply system have the same meaning and can be used interchangeably. Energy regeneration system and energy regeneration system have the same meaning and can be used interchangeably. The energy regeneration system is a preferred embodiment or a component of the energy system.

[0146] The in vitro synthesis system of the present invention further comprises one or more components selected from the following group: substrates for protein synthesis, substrates for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, buffer, energy regeneration system, polyethylene glycol (PEG) or its analog, dithiothreitol (DTT), and an optional solvent, wherein the solvent is water or an aqueous solvent. Further, the cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.

[0147] Further, the substrate for synthesizing RNA comprises: one of nucleoside monophosphates, nucleoside triphosphates, or a combination thereof.

[0148] Further, the substrate for synthesizing protein comprises: 20 natural amino acids and non-natural amino acids.

[0149] Further, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.

[0150] Further, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.

[0151] Further, the energy regeneration system is selected from the following group: phosphocreatine / phosphocreatinase system, one of the energy systems of glycolytic pathway intermediates, sucrose, or a combination thereof.

[0152] Further, the buffer is selected from the following group: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0153] Further, the protein synthesis system contains polyethylene glycol (PEG) or its analog. The concentration of polyethylene glycol or its analog is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analog is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the following group: PEG3000, PEG3350, PEG6000, PEG8000, or a combination thereof.

[0154] Further, the polyethylene glycol includes polyethylene glycols with molecular weights (Da) ranging from 200 to 10,000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc. Preferably, the polyethylene glycol has a molecular weight of 3000 - 10,000.

[0155] An alternative embodiment is that the protein synthesis system provided by the present invention includes: yeast cell extract, 4 - (2 - hydroxyethyl)-1 - piperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphokinase, RNA polymerase, polyethylene glycol, and sucrose.

[0156] In the present invention, the cell extract does not contain intact cells. Typical cell extracts include ribosomes for protein translation, transfer RNAs, aminoacyl - tRNA synthetases, initiation factors and elongation factors required for protein synthesis, as well as termination release factors. In addition, the cell extract also contains some other proteins derived from the cytoplasm of the cells, especially soluble proteins.

[0157] In the present invention, the protein content in the cell extract is 20 - 100 mg / ml, preferably 50 - 100 mg / ml. The method for measuring the protein content is the Coomassie Brilliant Blue assay method.

[0158] In the present invention, the method for preparing the cell extract or cell lysate is not limited. A preferred preparation method includes the following steps:

[0159] (i) Provide cells;

[0160] (ii) Wash the cells to obtain washed cells;

[0161] (iii) Disrupt the washed cells to obtain a crude cell extract;

[0162] (iv) Perform solid - liquid separation on the crude cell extract to obtain the liquid part, which is the cell extract.

[0163] In the present invention, the method of solid - liquid separation is not particularly limited. A preferred method is centrifugation.

[0164] In the present invention, the centrifugation conditions are not particularly limited. A preferred centrifugation condition is 5000 - 100,000×g, preferably 8000 - 30,000×g.

[0165] In the present invention, the centrifugation time is not particularly limited. A preferred centrifugation time is 0.5 min - 2 h, and preferably, 20 min - 50 min.

[0166] In the present invention, the temperature of centrifugation is not particularly limited. Preferably, the centrifugation is carried out at 1 - 10 °C, and preferably, at 2 - 6 °C.

[0167] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to treat with a washing solution at a pH of 7 - 8 (preferably, 7.4). The washing solution is not particularly limited. Typically, the washing solution is selected from the group consisting of: potassium 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0168] In the present invention, the method of cell disruption treatment is not particularly limited. A preferred cell disruption treatment includes high - pressure disruption, freeze - thaw (such as liquid nitrogen low - temperature) disruption.

[0169] The nucleoside triphosphate mixture in the protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate. In the present invention, the concentration of each mononucleotide is not particularly limited. Generally, the concentration of each mononucleotide is 0.5 - 5 mM, and preferably, 1.0 - 2.0 mM.

[0170] The amino acid mixture in the protein synthesis system may include natural or non - natural amino acids, and may include D - type or L - type amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is generally 0.01 - 0.5 mM, and preferably, 0.02 - 0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.

[0171] In a preferred embodiment, the cell - free protein synthesis system in vitro further contains sucrose. The concentration of the sucrose is 0.03 - 40 wt%, preferably, 0.08 - 10 wt%, more preferably, 0.1 - 5 wt%, based on the total weight of the protein synthesis system.

[0172] A particularly preferred cell-free protein synthesis system in vitro contains the following components in addition to yeast cell extract: 22 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid with a pH of 7.4, 30 - 150 mM potassium acetate, 1.0 - 5.0 mM magnesium acetate, 1.5 - 4 mM nucleoside triphosphate mixture, 0.08 - 0.24 mM amino acid mixture, 25 mM phosphocreatine, 1.7 mM dithiothreitol, 0.27 mg / mL phosphocreatine kinase, 1% - 4% polyethylene glycol, 0.5% - 2% sucrose, 0.027 - 0.054 mg / mL T7 RNA polymerase.

[0173] In the present invention, the cell extract and the cell lysate have the same meaning, both referring to the substances obtained after cell disruption. Specific examples

[0175] Example 1:

[0176] Take 500 mL of silica microspheres and place them in a 10 L beaker. Add deionized water and stir for 10 - 60 minutes, then ultrasonically clean for 10 - 60 minutes, repeating 3 times to obtain the silica microspheres for standby;

[0177] Transfer the above silica microspheres to a reaction vessel, add 5 - 50 g of sodium hydroxide in batches, and stir for 0 - 8 h under the condition that the reaction temperature is 15 - 80 °C;

[0178] Use deionized water to repeatedly rinse the silica microspheres in (2) until the pH value of the cleaning solution is about 6 - 9.5.

[0179] Example 2:

[0180] (1) Take 50 mL of the silica microspheres (average particle size 100 μm) treated in Example 1, wash them 2 times with absolute ethanol (100 mL in total), and then transfer them to a reaction vessel;

[0181] (2) Gradually add 100 mL of absolute ethanol under mechanical stirring conditions. While maintaining the rotation speed not exceeding 240 revolutions per minute, dropwise add 10 mL of the silane coupling agent γ-methacryloxypropyltrimethoxysilane, and then raise the temperature to 50 °C and react for 24 hours;

[0182] (3) Maintain the reaction temperature at 50 °C, add 20 mL of TBAF-THF solution (1 M, 0.02 mol) to the reaction system, continue to react for 24 hours, and raise the temperature to 70 °C and continue to react for 2 hours;

[0183] (4) Wash with absolute ethanol three times, with a dosage of 100 mL each time; wash with deionized water 3 times, with a dosage of 200 mL each time;

[0184] (5) After the washing is completed, store the silica microspheres in deionized water, place them in a sealed container, and store them at 4 °C.

[0185] (6) Take 20 mL of the silica microspheres prepared in (5), add 80 mL of deionized water, and transfer them to a reaction vessel.

[0186] (7) Add acrylic acid monomer (15 mL) and initiator 4,4'-azobis(4-cyanovaleric acid) (30 mg) to the reaction system. After sealing the reaction system, keep stirring and bubbling with nitrogen for 1 hour.

[0187] (8) Stir at 60 °C for 8 hours. After the reaction is completed, open the sealed reaction system. After the reaction solution cools to room temperature, add 200 mL of sodium hydroxide solution (0.75 M) and stir for 3 hours.

[0188] (9) Transfer the reaction solution in (8) to 2 L of deionized water and let it stand for 24 hours. Then precipitate with sodium chloride, remove the supernatant, and wash with deionized water. Repeat this step three times, and then store the silica microspheres in deionized water, place them in a sealed container, and store them at 4 °C.

[0189] Example 3

[0190] (1) Take 50 mL of the silica microspheres (average particle size 100 μm) treated in Example 1, wash them twice with anhydrous ethanol (100 mL in total), and transfer them to a reaction vessel.

[0191] (2) Gradually add 110 mL of anhydrous ethanol under mechanical stirring. While keeping the rotation speed not exceeding 240 revolutions per minute, add dropwise 10 mL of silane coupling agent γ-methacryloxypropyltrimethoxysilane, and then raise the temperature to 50 °C and react for 24 hours.

[0192] (3) Keep the reaction temperature at 50 °C, add 10 mL of TBAF-THF solution (1 M, 0.01 mol) to the reaction system, continue to react for 24 hours, and then raise the temperature to 70 °C and continue to react for 2 hours.

[0193] (4) Wash three times with anhydrous ethanol, 100 mL each time; wash three times with deionized water, 200 mL each time.

[0194] (5) After the washing is completed, store the silica microspheres in deionized water, place them in a sealed container, and store them at 4 °C.

[0195] (6) Take 10 mL of the silica microspheres prepared in (5), add 80 mL of deionized water, and transfer them to a reaction vessel.

[0196] (7) Add acrylic acid monomer (15 mL) and initiator 4,4'-azobis(4-cyanovaleric acid) (50 mg) into the reaction system. After sealing the reaction system, bubble nitrogen while stirring for 1 hour.

[0197] (8) Stir for 8 hours under the heating condition of 60 °C. After the reaction ends, open the sealed reaction system. After the reaction solution cools to room temperature, add 200 mL of sodium hydroxide solution (0.75 M) and stir for 3 hours.

[0198] (9) Transfer the reaction solution in (8) to 2 L of deionized water and let it stand for 24 hours. Then precipitate with sodium chloride, remove the supernatant, and wash with deionized water. Repeat this step three times, and store the silica microspheres in deionized water. Place them in a sealed container and store at 4 °C.

[0199] Example 4

[0200] (1) Take 50 mL of the silica microspheres (average particle size 100 μm) treated in Example 1, wash them twice with absolute ethanol (100 mL in total), and transfer them to a reaction vessel.

[0201] (2) Gradually add 100 mL of absolute ethanol under mechanical stirring. While maintaining the rotation speed not exceeding 240 revolutions per minute, dropwise add 10 mL of silane coupling agent γ-methacryloxypropyltrimethoxysilane, and then raise the temperature to 50 °C and react for 24 hours.

[0202] (3) Maintain the reaction temperature at 50 °C, add 20 mL of TBAF-THF solution (1 M) to the reaction system, continue to react for 24 hours, and then raise the temperature to 70 °C and continue to react for 2 hours.

[0203] (4) Wash three times with absolute ethanol, 100 mL each time; wash three times with deionized water, 200 mL each time.

[0204] (5) After washing, store the silica microspheres in deionized water. Place them in a sealed container and store at 4 °C.

[0205] (6) Take 20 mL of the silica microspheres prepared in (5), add 80 mL of absolute ethanol, and transfer them to a reaction vessel.

[0206] (7) Add n-butyl acrylate monomer (15 mL) and initiator azobisisobutyronitrile (15 mg) into the reaction system. After sealing the reaction system, bubble nitrogen while stirring for 1 hour.

[0207] (8) Stir for 8 hours under heating conditions at 60 °C. After the reaction is completed, open the sealed reaction system. After the reaction solution is cooled to room temperature, add 200 mL of sodium hydroxide solution (0.75 M) and stir for 3 hours;

[0208] (9) Transfer the reaction solution in (8) to 500 mL of absolute ethanol and let it stand for 24 hours, then wash it three times with absolute ethanol. Subsequently, store the silica microspheres in deionized water and keep them in a sealed container at 4 °C.

[0209] Example 5 (control example)

[0210] (1) Take 50 mL of the silica microspheres ((average particle size 100 μm)) treated in Example 1, wash them 2 times with absolute ethanol (100 mL in total), and then transfer them to a reaction vessel;

[0211] (2) Gradually add 110 mL of absolute ethanol under mechanical stirring conditions. While keeping the rotation speed not exceeding 240 revolutions per minute, dropwise add 10 mL of the silane coupling agent γ-methacryloxypropyltrimethoxysilane, and then raise the temperature to 50 °C and react for 24 hours;

[0212] (3) Wash three times with absolute ethanol, with 100 mL used each time; wash 3 times with deionized water, with 200 mL used each time;

[0213] (4) After washing, store the silica microspheres in deionized water and keep them in a sealed container at 4 °C.

[0214] (5) Take 10 mL of the silica microspheres prepared in (4), add 80 mL of deionized water, and then transfer them to a reaction vessel;

[0215] (6) Add acrylic monomer (15 mL) and initiator 4,4'-azobis(4-cyanovaleric acid) (50 mg) to the reaction system. After sealing the reaction system, keep stirring and pass nitrogen gas to bubble for 1 hour;

[0216] (7) Stir for 8 hours under heating conditions at 60 °C. After the reaction is completed, open the sealed reaction system. After the reaction solution is cooled to room temperature, add 200 mL of sodium hydroxide solution (0.75 M) and stir for 3 hours;

[0217] (8) Transfer the reaction solution in (7) to 2 L of deionized water and let it stand for 24 hours, then precipitate with sodium chloride to remove the supernatant and wash with deionized water. Repeat this step three times, and then store the silica microspheres in deionized water and keep them in a sealed container at 4 °C.

[0218] Example 6 verification experiment

[0219] (1) The test material (taking the microspheres obtained in Example 3 as an example) was filled into a purification column, and the column volume was 10 mL. The D2P system was used to express the EGFP protein for testing the loading capacity of the purification column.

[0220] (2) The volume of the reaction solution containing the EGFP protein was 1 L. After centrifugation at 3792 g for 20 min, the precipitate was discarded and the supernatant was taken, and the fluorescence value of the supernatant was measured and recorded as Total.

[0221] (3) The purification column was equilibrated with 2 L of equilibration buffer (equilibration buffer: 50 mM Tris-Cl pH 8.0, 500 mM NaCl)

[0222] (4) The sample was loaded, the flow-through was collected, and the fluorescence value of its EGFP was measured and recorded as FT.

[0223] (5) After the sample loading was completed, the column was washed with 1 L of equilibration buffer, the washing solution was collected, and the fluorescence value of its EGFP was measured and recorded as W0.

[0224] (6) The column was washed with 200 mL of washing solution A, the washing solution was collected, and the fluorescence value of its EGFP was measured and recorded as W10 (washing solution A: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 10 mM imidazole).

[0225] (7) The column was washed with 200 mL of washing solution B, the washing solution was collected, and the fluorescence value of its EGFP was measured and recorded as W20-0.1 (washing solution B: 50 mM Tris-Cl pH 8.0, 100 mM NaCl, 20 mM imidazole).

[0226] (8) The column was washed with 200 mL of washing solution C, the washing solution was collected, and the fluorescence value of its EGFP was measured and recorded as W20-0.3 (washing solution C: 50 mM Tris-Cl pH 8.0, 300 mM NaCl, 20 mM imidazole).

[0227] (9) The column was washed with 200 mL of washing solution D, the washing solution was collected, and the fluorescence value of its EGFP was measured and recorded as W20-0.5 (washing solution D: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 20 mM imidazole).

[0228] (10) The column was washed with 200 mL of washing solution E, the washing solution was collected, and the fluorescence value of its EGFP was measured and recorded as W40 (washing solution E: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 40 mM imidazole).

[0229] (11) Finally, elute with 200 mL of eluent F, collect the obtained eluate, and measure the fluorescence value of EGFP, denoted as E250 (eluent F: 50 mM Tris-Cl pH 8.0, 1 M NaCl, 250 mM imidazole).

[0230] (12) According to the RFU value, convert it to the corresponding protein concentration, and calculate relevant data such as protein amount and loading capacity.

[0231] Based on the purified EGFP, a standard curve was measured. The formula for converting the RFU value of EGFP to mass concentration is: X = -1 / 0.0005 * ln(1 - (Y - 38.089) / 31692). Where X is the mass concentration (μg / mL) and Y is the EGFP RFU fluorescence reading. Calculate the mass concentration of EGFP through the above formula. As shown in Table 1 below:

[0232] Table 1

[0233]

[0234]

[0235] Note: Binding capacity (mg / ml) = total mass of the target protein washed (g) × 1000 / column volume (ml) (where the total mass of the target protein washed is 0.001 g and the column volume is 10 ml)

[0236] Example 7 Verification Experiment (Control)

[0237] (1) Pack the purification column with the test material (the microspheres prepared in Example 5), and the packed column volume is 10 mL. Use the D2P system to express EGFP protein for testing the loading capacity of the purification column.

[0238] (2) The volume of the reaction solution containing EGFP protein is 0.7 L. After centrifuging at 3792 g for 20 min, discard the precipitate and take the supernatant, and measure the fluorescence value of the supernatant, denoted as Total.

[0239] (3) Equilibrate the purification column with 2 L of equilibration buffer (equilibration buffer: 50 mM Tris-Cl pH 8.0, 500 mM NaCl)

[0240] (4) Load the sample, collect the flow-through fraction, and measure the fluorescence value of EGFP, denoted as FT.

[0241] (5) After the loading is completed, wash the column with 1 L of equilibration buffer, collect the washing solution, and measure the fluorescence value of EGFP, denoted as W0.

[0242] (6) Wash the column with 1.0 L of Wash Buffer A, collect the wash buffer, and measure the fluorescence value of EGFP in it, denoted as W20 (Wash Buffer A: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 20 mM imidazole).

[0243] (7) Wash the column with 1.0 L of Wash Buffer B, collect the wash buffer, and measure the fluorescence value of EGFP in it, denoted as W40 (Wash Buffer B: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 40 mM imidazole).

[0244] (8) Wash the column with 0.5 L of Elution Buffer C, collect the eluate, and measure the fluorescence value of EGFP in it, denoted as E250 (Elution Buffer C: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 250 mM imidazole).

[0245] (9) Finally, wash the column with 0.5 L of Elution Buffer D, collect the eluate, and measure the fluorescence value of EGFP in it, denoted as W20-0.5 (Elution Buffer D: 50 mM Tris-Cl pH 8.0, 500 mM NaCl, 500 mM imidazole).

[0246] (10) According to the RFU value, convert it to the corresponding protein concentration, and calculate relevant data such as protein amount and loading capacity.

[0247] Based on the purified EGFP, a standard curve was determined. The formula for converting the RFU value of EGFP to mass concentration is: X = -1 / 0.0005 * ln(1 - (Y - 38.089) / 31692). Where X is the mass concentration (μg / mL) and Y is the EGFP RFU fluorescence reading. Calculate the mass concentration of EGFP through the above formula. As shown in Table 2 below:

[0248] Table 2

[0249]

[0250] Note: Binding capacity (mg / ml) = total mass of the target protein washed (g) × 1000 / column volume (ml) (where the total mass of the target protein washed is 0 g and the column volume is 10 ml)

[0251] By comparing Example 6 and Example 7, it can be seen that the protein loading of the microspheres without TBAF treatment is finally 0, while the final loading of the microspheres prepared by the method of the present application can reach 0.1 g / L. The reason is that TBAF can cleave the link between the silane coupling agent and the silica microspheres, resulting in a reduction in the initiation sites on the surface of the microspheres, and ultimately leading to a direct reduction in the polymer chains grafted on the surface of the microspheres, causing the polymer chains to stretch, which can more favorably adsorb proteins. In the comparative example, there are too many sites on the microspheres, which may ultimately cause the polymer chains to aggregate into a ball, seriously affecting the activity of the polymer chains, severely affecting the adsorption of proteins, and ultimately resulting in the microspheres being unable to adsorb proteins (such as Example 7 of the present application) or adsorbing very little protein. Thus, it can be seen that the modified microspheres prepared by the technical solution of the present application have made remarkable progress.

[0252] Taking the above ideal embodiments based on the present application as an inspiration, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of this application. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A method for regulating the polymer density on the surface of microspheres, characterized in that, It mainly includes: The microsphere body is treated with a silane coupling agent, then reacted with a fluorinating agent, and finally reacted with a monomer in the presence of an initiator to obtain a modified microsphere.

2. The method for regulating the surface polymer density of microspheres according to claim 1, characterized in that, The fluorinating agent is selected from one or more of alkyl fluorides, aryl fluorides, arylalkyl fluorides, fluorinated salts or fluorocyanates.

3. A method for regulating the polymer density on the surface of microspheres according to claim 1 or 2, characterized in that, The fluorinating agent is selected from one or more of TBAF, tetramethylammonium fluoride, tetraethylammonium fluoride, tetrapropylammonium fluoride, sodium tetrabutylfluoride, phenyltrimethylammonium fluoride, phenyltriethylammonium fluoride, phenyltripropylammonium fluoride, phenyltributylammonium fluoride, ammonium fluoride, sodium fluoride, potassium fluoride, cesium fluoride, sodium fluorocyanate or potassium fluorocyanate; more preferably TBAF.

4. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-3, characterized in that, The structure of the silane coupling agent contains an unsaturated bond; preferably, the silane coupling agent is selected from one or more of γ-methacryloxypropyltrimethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, vinyltris(β-methoxyethoxy)silane, dimethylethenyl ethoxysilane, methylethenyldiethoxysilane or vinyltrimethoxysilane oligomer.

5. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-4, characterized in that, The monomer is an acrylic monomer; preferably, the acrylic monomer is selected from one or more of substituted or unsubstituted acrylic acid, acrylate, acrylic acid ester; further preferably, the monomer is selected from one of acrylic acid, methacrylic acid, methacrylate, methacrylic acid ester, methyl methacrylate, butyl acrylate, isooctyl acrylate or a combination thereof.

6. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-5, characterized in that, The surface of the microsphere body contains silicon, silica or hydroxyl groups.

7. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-6, characterized in that, The size of the microsphere body is selected from any one of the following particle size scales or the range between any two particle size scales: 0.1μm, 0.15μm, 0.2μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm, 0.45μm, 0.5μm, 0.55μm, 0.6μm, 0.65μm, 0.7μm, 0.75μm, 0.8μm, 0.85μm, 0.9μm, 0.95μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm, 3.5μm, 4μm, 4.5μm, 5μm, 5.5μm, 6μm, 6.5μm, 7μm, 7.5μm, 8μm, 8.5μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter size is the average value.

8. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-7, characterized in that, The feeding ratio (L: mol) of the silane coupling agent to the fluorinating agent is 1:0.1 to 20; preferably 1:0.1 to 15; more preferably any value or the range between any two values among 1:1, 1:1.5, 1:2, 1:15 or 10:

1.

9. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-8, characterized in that, The feeding ratio (L: g) of the monomer to the initiator is (10 - 70):(10 - 50); preferably 0.5 - 5:1; more preferably 0.5 - 2:1, and further preferably any value or the range between any two values among 0.5:1, 0.6:1, 1:1, 1.5:1 or 2:

1.

10. A method for regulating the polymer density on the surface of microspheres according to any one of claims 1-9, characterized in that, Specifically, it includes the following steps: (1) Ultrasonically wash the microspheres to obtain microspheres A; (2) React the microspheres A with an alkaline solution and wash to obtain microspheres B; (3) React the microspheres B with a silane coupling agent to obtain microspheres C; (4) React the microspheres C with a fluorinating agent to obtain microspheres D; (5) React the microspheres D with a monomer under the condition of an initiator to obtain microspheres E; (6) Treat the microspheres E with an alkaline solution to obtain microspheres F, which are the target modified microspheres.

11. A method for regulating the polymer density on the surface of microspheres according to claim 10, characterized in that, The initiator is selected from azo initiators, peroxide initiators or redox initiators; preferably one or more of 4,4'-azobis(4-cyanovaleric acid), azobisisobutyronitrile, azodiisooctanenitrile, dimethyl 2,2'-azobis(2-methylpropionate), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methyl-N-(2-hydroxyethyl)propionamide) dihydrochloride, azoisobutyronitrile carboxamide.

12. A modified microsphere, characterized in that: Prepared by the method according to any one of claims 1 - 11.

13. Use of the modified microspheres according to claim 12 in cell-free protein synthesis, protein separation and purification, enrichment of target antibody drugs, nucleic acid separation and extraction, cell sorting, enzyme immobilization and kits.

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