CN113564214A

CN113564214A - Integrated preparation method for in-vitro synthesis and purification of protein, kit and application thereof

Info

Publication number: CN113564214A
Application number: CN202011511566.2A
Authority: CN
Inventors: 郭敏; 吴亮; 徐丽琼; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2020-04-28
Filing date: 2020-12-18
Publication date: 2021-10-29
Also published as: CN113564213A; WO2022111661A1

Abstract

The invention discloses an integrated preparation method for in vitro synthesis and purification of protein, a kit and application thereof, and belongs to the technical field of protein synthesis. The preparation method comprises the steps of forming a reaction system by an in vitro protein synthesis system and a nucleic acid template for coding a target protein, adding a protein fixing system capable of combining the target protein into the reaction system, and specifically combining a target protein product on the protein fixing system in the in vitro protein synthesis reaction process. The invention breaks through the traditional strategy of synthesis and purification, adopts a brand-new coupling method integrating synthesis and purification, combines the traditional protein synthesis and purification steps into one, greatly saves the time cost for purifying the protein product, greatly improves the purification efficiency, obviously shortens the preparation period, has simple flow and convenient operation, and can be widely applied to the aspects of in vitro protein synthesis and detection; and the preparation period is obviously shortened, and the practicability is greatly improved.

Description

Integrated preparation method for in-vitro synthesis and purification of protein, kit and application thereof

Technical Field

The invention relates to the technical field of protein synthesis, in particular to the technical field of in-vitro protein synthesis, and specifically relates to an integrated preparation method, a kit and application of in-vitro protein synthesis and purification.

Background

Proteins are important molecules in cells, and are involved in performing almost all functions of cells. Protein synthesis mainly includes conventional intracellular synthesis techniques and a new generation of in vitro synthesis techniques. The conventional protein expression system refers to a molecular biological technique for expressing foreign genes by model organisms such as bacteria, fungi, plant cells or animal cells. In vitro protein synthesis systems, also known as cell-free expression systems, which have been developed in the 1960 s, have been used for synthesizing foreign proteins by artificially controlling and adding substances such as substrates, energy, transcription and/or translation-related protein factors, and the like, which are required for protein synthesis, to exogenous mRNA or DNA as a protein synthesis template. The in vitro protein synthesis system is generally characterized in that components such as a nucleic acid template (an mRNA template or a DNA template), RNA polymerase, amino acids, ATP and the like are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of target proteins. The in vitro protein synthesis system is a relatively rapid, time-saving, and convenient means of protein expression without the need for plasmid construction, transformation, cell culture, cell collection, and disruption, and is an important tool in the protein field ("Garcia RA, Riley MR. applied biology and biotechnology. Humana Press.1981, 263-264"; "Fromm HJ, Hargrove M. essences of biochemistry.2012"; CN 109988801A; "Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in biological gene research. Current in Structural biology.2013, 23; (3): 402. uk" Zene media, Lenaissance, culture and culture of cell culture and culture of 31. cell culture of 14. Purchase. Biochemical engineering of cell culture of 32. 393. A. In vitro protein synthesis system May also express non-natural amino acids (e.g. having a deleterious effect on the cell)_DAmino acids) capable of synthesizing a plurality of proteins simultaneously in parallel, facilitating the development of high-throughput drug screening and proteomics research (Spirin AS, Swartz JR. Chapter 1.Cell-Free Protein Synthesis Systems: Historical Landmarks, Classification, and General methods. Wiley-VCH Verlag GmbH)&KGaA,2008: 1-34.). The protein product produced by the in vitro synthesis system can be widely applied to various fields such as medicine, food, nutriment, dietary supplement, cosmetics and the like, including but not limited to Proteinn of applicant^TM、PROTN^TMProlondon, Prolondon^TMGeneral, general^TMAnd the like.

For protein synthesis systems, especially for the fields of in vitro detection, experimental research and the like, important requirements on the aspects of high efficiency, high throughput, simple operation and the like are met, and two important links of protein synthesis and protein product purification are often involved. Both synthesis efficiency and purification efficiency are important aspects of protein production efficiency. The purification process of the protein product generally includes separating the protein product from the synthesis system to obtain a primary purified product (or a solution of the primary purified product), and performing one or more subsequent purification operations according to specific purity requirements, such as ultrafiltration membrane, column chromatography, ion exchange column, hydrophobic column, salting out, recrystallization, vacuum drying, freeze drying, and the like. At present, both the traditional intracellular expression system and the new-generation in vitro synthesis system adopt a two-step operation mode of synthesis and purification; after the protein synthesis reaction is finished, an additional purification device is required to be introduced, and after long-time incubation, the protein product can be separated and purified from the reaction mixed system. Although researchers have made a series of efforts to improve the efficiency of protein synthesis, many times the time saved in the synthesis step is a premium relative to the time spent in subsequent purification. The control of the incubation time becomes a key for restricting the overall efficiency of protein production.

At present, agarose gel and other materials are commonly used as purification columns or purification microsphere carriers. The three-dimensional porous structure of the gel material is beneficial to improving the specific surface area of the material, thereby increasing the sites capable of being combined with a purification medium and improving the specific binding capacity to a protein product. Although the three-dimensional porous structure of the carrier material can greatly increase the number of protein binding sites, the porous structure inside the carrier can also increase the retention time of the protein during protein elution, and discontinuous spaces or dead spaces inside the carrier can also prevent the protein from being eluted from the material, thereby increasing the retention ratio. If the site for binding with the protein is fixed only on the outer surface of the carrier, although the anti-protein product can be prevented from entering the interior of the material, the retention time and the retention ratio of the protein during elution can be greatly reduced; however, if only the outer surface of the carrier is used, the specific surface area of the carrier is greatly reduced, and the number of binding sites of the protein is greatly reduced, thereby reducing the purification efficiency.

In the prior art, a purification column (such as a protein A column for purifying antibodies) or a purification microsphere for protein separation and purification mainly adopts a covalent coupling mode to fix a purification medium. Although the covalent coupling mode can ensure that the purification medium is firmly fixed on the carrier, after the purification column is used for many times, the binding performance of the purification medium is reduced, and the purification effect is reduced. Therefore, in order to guarantee higher purification efficiency and quality, operating personnel need in time with the whole renewal or change of filler in the affinity chromatography column, and this process not only consumptive material quantity is big, consumes a large amount of manual works and time moreover, leads to the purification with high costs.

Disclosure of Invention

Aiming at the technical problem of restricting the overall efficiency of protein production, the invention discloses a simple, convenient, more efficient and higher-throughput integrated preparation method for in vitro synthesis and purification of protein, a kit and application thereof. The in vitro synthesis and purification integrated preparation method of the protein is also called a coupled purification method (also called a coPure method) and an integrated purification method (simply called an intePure method), breaks through the traditional strategy of synthesis and purification, adopts a brand new coupling method integrating synthesis and purification, greatly saves the time cost of purifying a protein product, greatly improves the purification efficiency, obviously improves the protein production efficiency, and has simple flow and convenient operation.

1. The invention provides an integrated preparation method for in vitro synthesis and purification of protein. The integrated preparation method for in vitro synthesis and purification of the protein at least comprises the following steps:

step i: at least providing an in vitro protein synthesis system, a nucleic acid template for coding target protein and a protein fixing system, and placing the in vitro protein synthesis system, the nucleic acid template for coding the target protein and the protein fixing system in the same reaction container to form a reaction purification mixed system;

Wherein the in vitro protein synthesis system is capable of providing translation-related elements required for synthesis of the protein of interest in combination with the nucleic acid template encoding the protein of interest; the target protein is capable of specifically binding to the Protein Immobilization System (PIS);

step ii: carrying out incubation reaction under a proper condition to obtain a target protein product; during the incubation reaction, i.e. during the in vitro protein synthesis reaction, the target protein product can specifically bind to the protein immobilization system to obtain loaded-PIS (protein immobilization system bound to the target protein product);

step iii: separating the loaded-PIS obtained in the step ii from the reaction purification mixed system;

step iv: eluting the target protein from the loaded-PIS separated in the step iii to obtain an eluent containing the target protein.

Optionally further comprising the step v: separating the target protein from the eluent to obtain a purified product of the target protein.

Further optionally comprising step vi: and further purifying the target protein to obtain a repurified product of the target protein.

Optionally also comprising step vii: detecting any product of the target protein.

The implementation time of step vii is not particularly limited, and the sample to be tested may be performed in any step of the preparation method, and may be a solution, a liquid mixture, or a solid-liquid mixture containing a protein product, a wet powder or a dry powder, a liquid, wet powder, or dry powder sample containing no protein product as a reference, or a sample at the reaction zero point.

The reaction purification mixed system is also called an intePure system or a coPure system.

In a preferred embodiment, the protein immobilization system comprises a purification medium; the protein of interest is capable of specifically binding to the purification medium.

In one preferred embodiment, the protein immobilization system comprises a solid matrix and a purification medium (purification element) attached to an outer surface of the Solid Matrix (SM).

In a preferred form, the solid substrate has a diameter size of any one of the following particle size scales (the deviation may be ± 25%, ± 20%, ± 15%, ± 10%) or a range between any two of the particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 5 μm, 1 μm, 5 μm, and a, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm. Unless otherwise specified, the diameter size refers to an average size.

In one preferred form, the protein immobilization system comprises a solid substrate, a purification medium, and a polymer connecting the solid substrate and the purification medium.

In one preferred embodiment, the polymer is hydrophilic, does not precipitate out of an aqueous solution, and can be sufficiently stretched in an in vitro protein synthesis system (typically an aqueous system) to provide a larger mobility zone for the purification medium.

In one preferred embodiment, a branched polymer is attached to the outer surface of the solid substrate, one end of the branched polymer is covalently fixed to the outer surface of the solid substrate, and branches having independent ends are distributed outside the solid substrate; the purification medium is attached to the branched end of the branched polymer.

The branched polymer is a nonlinear polymer having a branched structure and at least 2 branches. The branched structure refers to a structure having a branch point.

The branched chain refers to a chain having a branch point and an independent end in a branched polymer.

In one preferred embodiment, the branched polymer is covalently coupled to the outer surface of the solid substrate directly or indirectly via a linking member.

The branched structure of the branched polymer includes, but is not limited to, a branched structure, a comb structure, a tree structure, a hyperbranched structure, a cyclic branched structure, and the like, and a combination thereof.

In a preferred embodiment, the branched polymer has a comb-like structure. In this case, the comb structure has a linear main chain and at least 3 side branches; one end of the linear main chain is covalently fixed on the outer surface of the solid matrix, and the other end of the branched chain type polymer is distributed outside the solid matrix.

In a preferred embodiment, the branched polymer has a cyclic branched structure. The cyclic branched structure has a cyclic skeleton, is directly or indirectly fixed on the outer surface of the solid matrix through one site of the cyclic skeleton, and is connected with a large amount of purification media (at least three purification media) through a large amount of branch points on the cyclic skeleton.

In one preferred embodiment, at least 3 of the purification media are linked to one molecule of the branched polymer.

In a preferred embodiment, the protein immobilization system is a magnetic microsphere system. The magnetic microsphere system has a magnetic microsphere body.

In one preferred embodiment, the magnetic microsphere body constitutes a solid matrix of the protein immobilization system, the outer surface of the magnetic microsphere body is connected with a branched-chain polymer, one end of the branched-chain polymer is covalently immobilized on the outer surface of the magnetic microsphere body, and branched chains with independent ends are distributed outside the magnetic microsphere body; the purification medium is attached to the branched end of the branched polymer.

In one preferred embodiment, the magnetic microsphere body is SiO₂Coated magnetic material particles.

In a preferred embodiment, the magnetic material particles have a chemical composition selected from the group consisting of: iron oxides, iron compounds, iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides, chromium oxides, and combinations thereof.

In a further preferred mode, the magnetic material particles have a chemical composition selected from the group consisting of: fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、AlNiCo、FeCrCo、FeCrMo、FeAlC、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo、FeSi、FeAl、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃、PbO·6Fe₂O₃GdO, and combinations thereof.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm.

In one preferred mode, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.5 to 3 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.5 to 1 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In a preferred embodiment, the magnetic microsphere body has an average diameter of 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, or 1000nm, with a deviation of ± 20%, more preferably ± 10%.

In one preferred form, the polymer has a linear backbone that is either a polyolefin backbone or an acrylic polymer backbone.

More preferably, the linear backbone of the polymer is a polyolefin backbone and is provided by the backbone of an acrylic polymer.

In a more preferred embodiment, the monomer unit of the acrylic polymer is acrylic acid, acrylate, methacrylic acid, methacrylate ester, or a combination thereof.

In a preferred embodiment, the purification medium is attached to the end of the branched chain of the branched polymer in such a manner that: covalent bonding, dynamic covalent bonding, supramolecular interactions, or combinations thereof.

In a preferred embodiment, the dynamic covalent bond includes a reversible linking group such as an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In a preferred form, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, combinations thereof.

In one of the preferred modes, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

Preferably, the purification medium comprises a metal ion, a biotin-type tag, an avidin-type tag, a polypeptide-type tag, a protein-type tag, an immunological-type tag, or a combination thereof. In one of the preferred modes, the purification medium is selected from: a metal ion, avidin, an avidin analog that can bind biotin or an analog thereof, biotin, a biotin analog that can bind avidin or an analog thereof, an affinity protein, an antibody, an antigen, DNA, or a combination thereof.

In a preferred embodiment, the metal ion is Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof.

In one preferred embodiment, the biotin-type tag is biotin, a biotin analogue capable of binding avidin-analogue, or a combination thereof.

In one preferred embodiment, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

In a more preferred embodiment, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

In a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof.

In a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, said affinity protein is selected from the group consisting of: protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

In a preferred embodiment, the immunological label is either an antibody-type label or an antigen-type label.

In a preferred embodiment, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one.

In a preferred embodiment, the antibody type tag is an anti-protein antibody.

In a preferred embodiment, the antibody-type tag is an antibody against a fluorescent protein.

In a preferred embodiment, the antibody type tag is an antibody against green fluorescent protein or a mutant thereof.

In a preferred embodiment, the antibody-type tag is a nanobody.

In one preferred embodiment, the antibody-type tag is a nanobody against a protein.

In a preferred embodiment, the antibody type tag is a single domain antibody against a protein.

In a preferred embodiment, the antibody-type tag is a single domain antibody against a protein.

In a preferred embodiment, the antibody type tag is an antibody VHH fragment of an anti-protein.

In a preferred embodiment, the antibody type tag is an anti-protein antibody scFV fragment.

In a preferred embodiment, the antibody-type tag is a nanobody against a fluorescent protein.

In one preferred embodiment, the antibody type tag is a nanobody against green fluorescent protein or a mutant thereof.

In a preferred embodiment, the antibody type tag is an antibody Fab fragment.

In a preferred embodiment, the antibody-type tag is an antibody F (ab') 2 fragment.

In a preferred embodiment, the antibody-type tag is an antibody Fc fragment.

In a preferred embodiment, the target protein product is bound to the protein immobilization system by the following force: biotin-avidin binding, Streg tag-avidin binding, avidin-avidin binding, histidine tag-metal ion affinity, antibody-antigen binding, or a combination thereof. The Streg tag, which refers to a peptide tag developed by IBA for specific binding to avidin or its analogs, typically contains the WSHPQFEK sequence or its variant sequences.

In one preferred form, the protein of interest carries a purification tag capable of specifically binding to the purification medium. One, two or more purification tags per target protein molecule; when two or more purification tags are contained, the kinds of the purification tags are one, two or more. It should be noted that, as long as the amino acid sequences of the tags are different, the tags are regarded as different types of tags.

In a preferred embodiment, the purification tag in the target protein is selected from the following classes: a histidine tag, avidin, a Streg tag (a tag comprising a WSHPQFEK sequence or a variant thereof), a tag comprising a WRHPQFGG sequence or a variant thereof, a tag comprising a RKAAVSHW sequence or a variant thereof, a FLAG tag, a Spot tag, a C tag, a GST tag, an MBP tag, a SUMO tag, a CBP tag, an HA tag, an Avi tag, an affinity protein, an antibody, an antigen, a combination thereof.

In one preferred embodiment, the target protein is linked to a purification tag at the N-terminus or C-terminus, or to both termini.

In the integrated preparation method for in vitro protein synthesis and purification, after the in vitro protein synthesis reaction is started, the step iv needs to be controlled at a proper time, so that a higher protein synthesis amount and a higher protein purification yield are realized under the condition of saving an independent incubation step.

In a preferred embodiment, the end point of the in vitro protein synthesis reaction is monitored as the amount of protein synthesis or the reaction time, and the timing of performing step iv is controlled.

In a preferred embodiment, the translation-related elements in the in vitro protein synthesis system are provided by a cell extract, exogenously added translation-related elements, or a combination thereof.

In a preferred embodiment, the translation-related elements of the in vitro protein synthesis system include, but are not limited to: tRNA, ribosome, translation-related enzyme, initiation factor, elongation factor, termination factor.

In one preferred embodiment, the translation-related enzymes include, but are not limited to: the aminoacyl tRNA synthetase, RNA polymerase, peptidyl transferase, or a combination thereof, and further optionally comprises a transcriptase, a DNA polymerase, or a combination thereof.

In a preferred embodiment, the in vitro protein synthesis system comprises a cell extract (i.e., the translation-related elements in the in vitro protein synthesis system are provided in a manner comprising a cell extract), optionally comprising exogenously added translation-related elements.

In a preferred embodiment, the cell source of the cell extract is Escherichia coli.

In another preferred mode, the cell source of the cell extract is eukaryotic cells.

In a further preferred embodiment, the cell extract is derived from a yeast cell, a mammalian cell, a plant cell, an insect cell, a nematode cell, a pathogen, or a combination thereof.

In a further preferred embodiment, the cellular source of the cellular extract is Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof.

In a further preferred embodiment, the cellular source of the cellular extract is Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybuvinii, Kluyveromyces hainanensis, Kluyveromyces williamsii, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensis, Kluyveromyces lactis, or a combination thereof.

In another preferred embodiment, the translation-related elements of the in vitro protein synthesis system are provided by exogenous addition, i.e., do not include the provision of a cell extract.

In a preferred embodiment, the exogenously added translation-related element is a purified translation-related element or a combination thereof.

In a more preferred embodiment, the exogenously added translation-related element is derived from Escherichia coli.

In another more preferred mode, the exogenously added translation-related element is derived from a eukaryotic cell.

In another more preferred mode, the exogenously added translation-related element is derived from a combination of E.coli and a eukaryotic cell.

In a further more preferred form, the exogenously added translation-related element is derived from a yeast cell, a mammalian cell, a plant cell, an insect cell, a nematode cell, a pathogen, or a combination thereof.

In a further more preferred manner, the exogenously added translation-related element is derived from kluyveromyces, saccharomyces cerevisiae, saccharomyces pichia pastoris, or a combination thereof.

In a further more preferred form, the exogenously added translation-related element is derived from kluyveromyces lactis, kluyveromyces marxianus, kluyveromyces polybuhitensis, kluyveromyces hainanensis, kluyveromyces wilcoxielli, kluyveromyces fragilis, kluyveromyces hubeiensis, kluyveromyces polyspora, kluyveromyces siamensis, kluyveromyces aureoides, or a combination thereof.

In one preferred embodiment, the in vitro protein synthesis system is capable of recognizing a promoter in the nucleic acid template encoding the protein of interest.

In one preferred embodiment, the in vitro protein synthesis system comprises an RNA polymerase.

In a preferred embodiment, the source of the RNA polymerase is selected from any one of the following: a cell extract comprising an endogenously expressed RNA polymerase, an exogenous RNA polymerase, a translation product of an exogenous nucleic acid template encoding an RNA polymerase, or a combination thereof.

In a more preferred embodiment, the RNA polymerase is T7 RNA polymerase.

In one preferred embodiment, the nucleic acid template encoding the target protein comprises a T7 promoter capable of initiating a gene transcription process for the target protein, and the in vitro protein synthesis system comprises T7 RNA polymerase, an exogenous nucleic acid template encoding T7 RNA polymerase, or a combination thereof.

In a more preferred form, the in vitro cell-free protein synthesis system comprises a cellular extract comprising endogenously expressed T7 RNA polymerase.

In a preferred embodiment, the in vitro protein synthesis system further comprises an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

In one preferred embodiment, the in vitro protein synthesis system comprises a cell extract (optionally containing an endogenously expressed RNA polymerase), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, and an exogenously added RNA polymerase.

In a preferred embodiment, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis; the cell extract comprises an endogenously expressed RNA polymerase.

In a preferred embodiment, the in vitro protein synthesis system comprises purified translation-related elements, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis; the purified translation-related elements include: tRNA, ribosome, aminoacyl tRNA synthetase, RNA polymerase, initiation factor, elongation factor, termination factor.

The in vitro protein synthesis system optionally includes a DNA polymerase.

In a preferred embodiment, the source of the DNA polymerase is selected from any one of the following: a cell extract comprising an endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding a DNA polymerase, or a combination thereof.

In one preferred embodiment, the DNA polymerase is phi29 DNA polymerase.

In one preferred embodiment, the in vitro protein synthesis system comprises a cell extract (optionally containing endogenously expressed RNA polymerase and/or DNA polymerase), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, an exogenously added RNA polymerase, an exogenously added DNA polymerase, a substrate for DNA synthesis.

In a preferred embodiment, the in vitro protein synthesis system optionally comprises any one or a combination of the following exogenously added components: DNA amplification related elements, RNA amplification related elements, RNase inhibitors, crowding agents, magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, antifreeze agents, trehalose, reaction promoters, antifoam agents, alkanes, buffers, aqueous solvents.

In a preferred embodiment, the in vitro protein synthesis system optionally comprises any one or a combination of the following exogenously added components: rnase inhibitors, crowding agents, magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoaming agents, alkanes, buffers, aqueous solvents, exogenous nucleic acid templates encoding RNA polymerase, DNA polymerase, exogenous nucleic acid templates encoding DNA polymerase, other DNA amplification related elements, substrates for synthesizing DNA, RNA amplification related elements. When the in vitro protein synthesis system contains a DNA polymerase or an exogenous nucleic acid template encoding a DNA polymerase, it preferably also contains a substrate for synthesizing DNA.

The nucleic acid template encoding the protein of interest may be a DNA template, an mRNA template, or a combination thereof.

In a preferred embodiment, the target protein is selected from any one of the following proteins, fusion proteins in any combination, and mixtures in any combination: luciferase, green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody, variable region of antibody, single-chain and single-chain fragments of antibody, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and its precursor, glucagon-like peptide, interferon, interleukin, lysozyme, serum albumin, transthyretin, tyrosinase, xylanase, beta-galactosidase, a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, a variant of any of the foregoing. In a preferred form, the variant is a mutant. Preferably, the variant is a homologue.

In one preferred embodiment, the protein immobilization system is a magnetic microsphere system; the step i of providing the magnetic microsphere system comprises preparing the magnetic microsphere system, and optionally comprises regenerating the magnetic microsphere system or/and replacing the purification medium.

Regeneration of the magnetic microsphere system refers to renewal of the purification media, wherein the purification media before and after renewal are of the same type.

And the types of the purification media before and after the replacement are different, including not completely the same or completely different.

2. The second aspect of the present invention also provides an in vitro protein synthesis purification kit, comprising:

(k1) any of the in vitro protein synthesis systems of the invention;

(k2) optionally including a nucleic acid template encoding a protein of interest, a nucleic acid vector having a multiple cloning site, or a combination thereof;

(k3) any one of the protein immobilization systems of the invention, or a component thereof; the constituent elements of the protein fixing system comprise any one of a solid matrix, a solid matrix wrapped by a branched chain type polymer, a reaction raw material of the branched chain type polymer, a purification medium or a combination thereof.

(k4) Optionally, a position control member of the protein fixation system for manipulating the position of the protein fixation system.

(k5) Optionally, washing solution A, for washing the protein immobilization system bound to the target protein.

(k6) Optionally, eluent B, can elute the target protein from the protein immobilization system.

(k7) Optionally, a regeneration reagent for the protein immobilization system, to effect regeneration of the protein immobilization system.

(k8) Optionally, the purification media is replaced with a reagent to effect replacement of the purification media.

The (k1) can constitute an in vitro protein synthesis kit.

The above-mentioned (k2), (k4), (k5), (k6), (k7) and (k8) may be present or absent independently of each other.

The in vitro protein synthesis system is capable of providing translation-related elements required for synthesis of a protein of interest in conjunction with the nucleic acid template encoding the protein of interest.

The protein immobilization system is provided with a purification medium.

The protein of interest is capable of specifically binding to the purification medium.

Preferably, the protein immobilization systems described in (k3), (k4), (k5), (k6) and (k7) are all magnetic microsphere systems.

Preferably, said (k3) comprises a magnetic microsphere system or a component thereof; the magnetic microsphere system comprises a magnetic microsphere body, magnetic microspheres with the outer surfaces connected with branched chain polymers, reaction raw materials of the branched chain polymers and a purification medium or a combination of the reaction raw materials and the purification medium.

Preferably, the components of the kit are placed in one or more containers as a solid, semi-solid, liquid, emulsion, suspension, or combination thereof.

Preferably, said (k1) and said (k2) are packaged separately.

Preferably, (k1) comprises the cell extract and is separately packaged in a container.

Preferably, the translation-related components in (k1) are independently packaged together in a container.

Preferably, (k1) comprises purified translation-related elements or a combination thereof (i.e. comprises a purified translation-related element or a combination of purified translation-related elements), and are packaged together in a single container.

Preferably, said nucleic acid template encoding a protein of interest comprises a promoter element capable of being recognized by said in vitro protein synthesis system of (k 1).

Preferably, the nucleic acid template encoding the target protein contains a T7 promoter capable of initiating a gene transcription process for the target protein, and the in vitro protein synthesis system comprises T7 RNA polymerase, an exogenous nucleic acid template encoding T7 RNA polymerase, or a combination thereof.

Preferably, the nucleic acid template encoding the target protein contains a T7 promoter, and the in vitro protein synthesis system comprises a cell extract including endogenously expressed T7 RNA polymerase.

3. The third aspect of the invention also provides the use of a protein immobilisation system according to any of the invention in the in vitro synthesis of a protein,

Preferably, the protein fixing system is coupled with any one of the in vitro protein synthesis systems (or the in vitro protein synthesis kit) of the invention for use in constructing a reaction and purification mixed system integrating protein synthesis and purification.

Preferably, the protein immobilization system is applied to the kit for in vitro protein synthesis and purification according to the second aspect, and can be used for implementing the method for preparing the inteure.

Preferably one, for use in protein manufacture, or for use in assays based on protein synthesis.

Preferably, the protein fixing system is the magnetic microsphere system of the invention; more preferably, the magnetic microsphere system carries a purification medium through a branched chain polymer coated on the outer surface of a solid matrix, and the amount of the purification medium can realize multiple times, more than ten times, more than one hundred times and even more than one thousand times of amplification.

Preferably, the application in the in vitro protein synthesis of the antibody substances or the application in the in vitro synthesis and purification of the antibody substances.

Has the advantages that:

the in vitro synthesis and purification integrated preparation method (the intePure preparation method or the coPure preparation method) breaks through the traditional strategy of synthesis and purification, adopts a brand-new synthesis and purification integrated coupling method, combines the traditional incubation and purification steps into a whole, omits the traditional independent purification incubation step, greatly saves the time cost of purifying a protein product, greatly improves the purification efficiency, obviously shortens the preparation period, has simple flow and convenient operation, and can be widely applied to the aspects of in vitro protein synthesis and detection. Especially for in vitro detection, the preparation period is obviously shortened, and the practicability is greatly improved.

The method for preparing the intePure can adopt a magnetic microsphere system, not only can combine protein products at high flux, but also can effectively reduce the retention time and the retention proportion of the protein during elution, can conveniently update and replace a purification medium (for example, affinity protein), has the characteristics of rapidness, high flux, reusability and renewable use, and can greatly reduce the cost; the magnetic microsphere system can be matched with the in-vitro protein synthesis system for use, so that the integrated preparation of protein synthesis and purification can be smoothly implemented, the preparation period is shortened, and meanwhile, higher purification yield can be realized.

Taking a magnetic microsphere system as a protein fixing system as an example:

firstly, the surface of the magnetic microsphere can be coated by a branched chain type polymer carrying a large number of branched chains, the limitation of specific surface area is overcome, a large number of purification medium binding sites are provided, the number of purification media which can be bound on the surface of the magnetic microsphere is amplified by multiple times, more than ten times, more than one hundred times and even more than one thousand times, and then the high-flux binding of a target protein product is realized.

Secondly, the flexibility of the polymer chain can be utilized, the polymer chain can flexibly swing in a reaction and purification mixed system, the activity space of a purification medium is enlarged, the capture rate and the combination amount of the protein are increased, the rapid and sufficient combination of a target protein product is promoted, and the high efficiency and the high flux are realized.

Moreover, the purification medium can be connected to the tail end of the branched chain of the polymer, on one hand, the branched chain structure can not form a net structure, so that the branched chain is not accumulated, discontinuous space and dead angle can be avoided, and high detention time and high detention proportion caused by the traditional net structure are avoided; on the other hand, the branched chain of the polymer further plays a space separation role, so that a purification medium can be fully distributed in a reaction and purification mixed system and is far away from the surface of the magnetic microsphere and the internal skeleton of the polymer, the efficiency of capturing protein is increased, the retention time and the retention proportion of a purified substrate can be effectively reduced in the subsequent elution step, and the separation with high flux, high efficiency and high proportion is realized. Particularly, the branched chain type polymer can adopt a special comb-shaped structure (or brush-shaped structure), a large number of branched chains are suspended at the side end of the linear main chain of the polymer, and a large number of purification media are combined on the outer surface of the magnetic microsphere, so that the high flexibility of the linear main chain can be utilized, the advantage of high magnification of the number of the branched chains is also achieved, and the separation with high speed, high flux, high efficiency and high proportion (high yield) is better realized.

In a fourth aspect, the purification medium can be attached to the ends of the polymeric arms on the outer surface of the magnetic microspheres by means of an affinity complex with a strong non-covalent binding force; when the purification medium needs to be updated and replaced, the purification medium can be conveniently and quickly eluted from the magnetic microspheres, and then the purification performance of the magnetic microspheres can be quickly recovered by combining with the new purification medium, so that the magnetic microsphere system can be repeatedly recycled, and the cost of a separation and purification link is reduced.

And in the fifth aspect, when the target protein product is separated from the system, the operation is convenient, the aggregation state and the position of the magnetic microspheres can be efficiently controlled by only using a small magnet, the rapid dispersion or rapid sedimentation of the magnetic microspheres in liquid is realized, the separation and purification of the protein product become simple and rapid, large-scale experimental equipment such as a high-speed centrifuge and the like are not needed, and the separation and purification cost is greatly reduced.

In a sixth aspect, the purification medium is selective. According to the specific type of the purification substrate, a corresponding purification medium can be flexibly carried in the magnetic microsphere system, so that the capture of specific target molecules (particularly biochemical molecules) is realized. For example, affinity proteins can be selected for targeting, and are generally applied to separation and purification of antibody substances on a large scale.

The magnetic microsphere system provided by the invention can be stably suspended in aqueous liquid and can not settle within 2 days or even longer. Moreover, the magnetic microsphere system can be stably suspended in an aqueous liquid system without continuous stirring, which is an excellent property brought by the unique structural design; on one hand, the size of the magnetic microsphere body can be controlled below 10 micrometers, even below 1 micrometer, on the other hand, a hydrophilic polymer can be wrapped on the outer surface of the magnetic microsphere body, the grafting density of the polymer on the outer surface of the magnetic microsphere body can be adjusted, the characteristics of the hydrophilicity, the structure type, the hydrodynamic radius, the chain length, the number of branched chains, the length of the branched chains and the like of the polymer can be adjusted, and the suspension state of the magnetic microsphere system in a system can be better controlled.

Drawings

FIG. 1 is a schematic view of a protein fixation system. The protein immobilization system is a biotin magnetic microsphere and biotin is used as a purification medium. Wherein, the magnetic microsphere body used as the solid matrix is made of SiO₂Encapsulated Fe₃O₄For example. In the figure, the number of polymer molecules (4) is only a simple illustration, and does not mean that the number of polymer molecules on the outer surface of the magnetic microsphere is limited to 4, but can be controlled and adjusted according to the content of each raw material in the preparation process. Similarly, the number of branches pendant from the side ends of the linear backbone is also illustrative and is not intended to limit the number of side branches of the polymer molecules of the present invention.

FIG. 2 is a schematic view of a protein fixation system. The protein fixing system is affinity protein magnetic microspheres, protein A is used as a purification medium, and the protein A is combined at the tail end of a branched chain of the brush-shaped structure in a biotin-avidin-protein A mode. Wherein, the magnetic microsphere body used as the solid matrix is made of SiO₂Encapsulated Fe₃O₄For example. In the figure, the number of polymer molecules and the number of branches at the side end of the main chain are only illustrative, and the wrapping density of the polymer and the number of branches at the side end of the molecule are not limited in the present invention.

FIG. 3 shows the results of RFU assay before and after binding of biomagnetic microspheres D to protein A-eGFP-avidin. Protein A-eGFP-avidin (SPA-eGFP-avidin) is prepared through an in-vitro protein synthesis system, supernatant (abbreviated as IVTT supernatant) after IVTT reaction is obtained, and the change of solution RFU values before and after combination with the biomagnetic microspheres D is compared. Of the two proteins, protein A-eGFP-avidin, avidin with the number 1 was Streptavidin, and avidin with the number 2 was Tamavidin 2. "Total" corresponds to the RFU value of IVTT Supernatant before binding (before biomagnetic microsphere treatment), and "Supernatant" corresponds to the RFU value of IVTT Supernatant after binding (after biomagnetic microsphere treatment).

FIG. 4 shows the RFU test results of preparing biomagnetic microspheres F; protein a-eGFP-avidin saturation binding. And repeatedly reacting the biomagnetic microspheres D with the solution (abbreviated as IVTT supernatant) obtained after the IVTT expressing the protein A-eGFP-avidin to obtain the biomagnetic microspheres F which are saturated and combined by the protein A-eGFP-avidin. Wherein, the avidin corresponding to 1 is Streptavidin, the avidin corresponding to 2 is Tamavidin2, and the super corresponds to IVTT supernatant which is not treated by the biomagnetic microspheres; flow-through1 (Flow through liquid 1), Flow-through2 (Flow through liquid 2), and Flow-through3 (Flow through liquid 3) are respectively corresponding to the magnetic microspheres to be incubated with avidin-protein A continuously (capture binding) and eluted (unbind release) for three times, and IVTT supernatant with the same source and the same dosage is used each time to obtain Flow through

liquids

1, 2 and 3 in sequence.

FIG. 5 is a plasmid map of plasmid vector pD2P _1.06e-8His-eGFP vector, also referred to as pD2P _1.06e vector, of 5914bp, including functional elements such as T7 promoter, 5 'UTR, leader peptide coding sequence (leading peptide), 8 XHis (histidine tag), MCS sequence (MCS, multiple cloning site), mEGFP coding sequence, 3' UTR, LAC4 terminator (not shown), replication initiation site (f1 ori), AmpR promoter, ampicillin resistance gene (AmpR gene), high copy number replication initiation site (ori), lacI promoter, and lacI coding gene. In the figure, D2P _1.06e _ F and D2P _1.06e _ R indicate the binding sites of the forward primer and the reverse primer, respectively.

FIG. 6 shows the results of fluorescence measurement of PRTa-GFP prepared by the integrated method of in vitro protein synthesis and purification (IVTT-co-purification method), example 5. Wherein Flow-through corresponds to the supernatant collected after the IVTT reaction is completed. Wash 1, Wash 2, and Wash 3 correspond to washes for washing the magnetic beads to which the protein products are bound three times, respectively. Elution corresponds to the Elution of protein-containing product.

FIG. 7 fluorescent assay results for PRTa-GFP prepared by a stepwise procedure of IVTT followed by purification, example 5. Wherein, Total corresponds to the reaction solution after the IVTT reaction is finished, Supernatant corresponds to the collected Supernatant, Flow-through corresponds to the Supernatant collected after the incubation with the magnetic beads, Wash 1, Wash 2 and Wash 3 correspond to the cleaning solution for washing the magnetic beads combined with the protein product for three times respectively, and Elution corresponds to the eluent containing the protein product.

FIG. 8 shows an SDS-PAGE electropherogram of the eluate containing the protein product, example 5. Wherein, intePure corresponds to an integrated method of protein in vitro synthesis and purification (IVTT-co-purification method), and control corresponds to a stepwise method of IVTT followed by purification.

FIG. 9 shows the results of RFU fluorescence detection, example 6. Wherein, intePure corresponds to an integrated method of protein in vitro synthesis and purification (IVTT-co-purification method), and control corresponds to a stepwise method of IVTT followed by purification. Total, Flow-through, Wash1, Wash 2, Wash 3 and Elution correspond to reaction liquid (Total) after IVTT reaction finishes reaction, a penetrating liquid (Flow-through) separated from loaded-BIS magnetic beads, three groups of cleaning liquids (Wash 1, Wash 2 and Wash 3) obtained by cleaning the magnetic beads after penetrating liquid separation and an eluent (Elution) containing protein products in sequence.

FIG. 10 shows an SDS-PAGE electropherogram of a sample containing a protein product, example 6. Wherein, the inteure-E1 and the inteure-E2 respectively correspond to an integrated method for in vitro synthesis and purification of protein (IVTT-co-purification method), 10 mu L and 30 mu L of eluent of nickel magnetic beads are added, the control corresponds to the eluent of a step-by-step method of IVTT purification, and the Wash1 corresponds to the Wash1 when the IVTT-co-purification method is adopted and 10 mu L of nickel magnetic beads are used, namely the corresponding first washing in the inteure-E1 group.

FIG. 11 shows the results of RFU fluorescence detection, example 7. An integrated method of in vitro protein synthesis and purification (IVTT-co-purification method) is adopted. PRTb-1 used 0.2mM protease inhibitor (PMSF) and 10. mu.L of nickel magnetic beads, PRTb-2 used 1.0mM protease inhibitor (PMSF) and 30. mu.L of nickel magnetic beads, and the control group did not have protease inhibitor and nickel magnetic beads added. Total, FT, W1, W2 and E correspond to a reaction solution (Total) after reaction is finished, a penetrating solution (FT) obtained by separating loaded-BIS magnetic beads, two groups of cleaning solutions (W1 and W2) obtained by cleaning the magnetic beads after penetrating solution separation and an eluent (E) containing protein products in sequence.

FIG. 12 is a microphotograph of nickel magnetic beads having different particle diameters. (A) Diameter 1 μm, static state; (B) diameter 1 μm, flow state; (C) diameter of 10 μm; (D) the diameter is 100 μm. Wherein the scales of (A) and (B) are 10 μm; (C) and (D) is 100 μm.

FIG. 13 is a comparison of the purification effect of nickel magnetic beads prepared by using 1 μm and 10 μm magnetic microsphere bodies. Wherein, PC corresponds to a stock solution to be purified (IVTT reaction solution, 1mL reaction system); ni1 beads and Ni10 beads adopt magnetic microsphere bodies with the particle size of 1 mu m and 10 mu m respectively; the volume of the nickel magnetic beads used was 10. mu.L, 5. mu.L, and 1. mu.L, respectively.

FIG. 14 is a comparison of the purification effect of nickel magnetic beads prepared by using 1 μm and 100 μm magnetic microsphere bodies. Wherein, PC corresponds to a stock solution to be purified (IVTT reaction solution, 1mL reaction system); ni1 beads and Ni100 beads adopt magnetic microsphere bodies with the particle size of 1 micron and 100 microns respectively; the volume of the nickel magnetic beads used was 10. mu.L, 5. mu.L, and 1. mu.L, respectively.

FIG. 15. map of plasmid DNA encoding 8 His-eGFP. In contrast to fig. 5, no MCS site is set.

FIG. 16. results of electrophoretic testing of different samples of the 8His-eGFP prepared by IVTT-co-purification reaction.

FIG. 17 shows the results of electrophoresis of different samples of IVTT-co-purification reactions and stepwise preparation of 8 His-eGFP.

Nucleotide and/or amino acid sequence listing

SEQ ID No. 1, nucleotide sequence of protein A (protein A), length 873 bases.

SEQ ID No. 2, nucleotide sequence of tamavidin2, 423 bases in length.

3, nucleotide sequence of mEGFP, 714 bases in length.

SEQ ID No. 4, amino acid sequence of mEGFP, 238 amino acids in length.

SEQ ID No. 5, nucleotide sequence of protein G antibody binding region, 585 bases in length.

SEQ ID No. 6, amino acid sequence of the nano antibody anti-eGFP, length 117 amino acids.

SEQ ID No. 7, the nucleotide sequence of mScarlet, 693 bases in length.

Detailed Description

The meaning of the terms, nouns, phrases of the present invention.

The meaning of this section is to be interpreted as applying to the invention in its entirety, both as follows and as above. In the present invention, when a cited document is referred to, the definitions of the related terms, nouns and phrases in the cited document are also incorporated, but in case of conflict with the definitions in the present invention, the definitions in the present invention shall control. In the event that a definition in a cited reference conflicts with a definition in the present disclosure, the cited components, materials, compositions, materials, systems, formulations, species, methods, devices, etc. are not to be construed as limiting.

In vitro protein synthesis reaction refers to a reaction for synthesizing a protein in an in vitro cell-free synthesis system, and at least comprises a translation process. Including but not limited to IVT response (in vitro translation reaction), IVTT response (in vitro transcription translation reaction), IVDTT response (in vitro replication transcription translation reaction). In the present invention, IVTT reaction is preferred. Since the IVTT reaction, corresponding to the IVTT system, is a process of in vitro transcription and translation of DNA into Protein (Protein), we also refer to such in vitro Protein synthesis systems as the D2P system, the D-to-P system, the D _ to _ P system, and the DNA-to-Protein system; the corresponding in vitro Protein synthesis methods are also called D2P method, D-to-P method, D _ to _ P method, DNA-to-Protein method.

"cell-free system" refers to a system in which protein synthesis is performed in vitro, but not by secretory expression from intact cells. In the in vitro cell-free protein synthesis system of the present invention, it is also permissible to add a cell component to promote the reaction, but the added cells do not mainly aim at secretory expression of the foreign target protein. In addition, in the D2P system without intact cells constructed under the guidance of the present invention, a small amount of intact cells (for example, the protein content provided by the system is not more than 30 wt% compared with the protein content provided by the cell extract) is intentionally added, and such a "evasion" mode is also included in the scope of the present invention.

The target protein: the target expression product of the in vitro protein synthesis system of the present invention is not secreted and synthesized by host cells, but synthesized in vitro based on an exogenous nucleic acid template, and thus may be referred to as an exogenous protein. The target protein can be a protein, a fusion protein, a protein-containing molecule or a mixture of fusion protein molecules; also broadly included are polypeptides. The product obtained after the in vitro protein synthesis reaction based on the nucleic acid template encoding the target protein may be a single substance or a combination of two or more substances. The terms "target protein", "target protein" and "target translation product" have the same meaning, and can be translated into the forms of "objective protein", "interested protein", "objective translated product", "interested protein product" and the like, and can be used interchangeably in the present invention.

The target protein product refers to a protein product synthesized by the in vitro protein synthesis reaction.

D2P, DNA-to-Protein, from DNA template to Protein product. For example, D2P technology, D2P system, D2P method, D2P kit, and the like.

mR2P, mRNA-to-Protein, from mRNA template to Protein product. For example, mR2P technology, mR2P system, mR2P method, mR2P kit, and the like.

IVTT, in vitro transcription translation.

IVDTT, in vitro replication transcription translation, replication transcription translation in vitro.

CFPS system: cell-free protein synthesis system, cell-free protein synthesis system.

The terms "expression system of the invention", "in vitro cell-free expression system" and "in vitro cell-free expression system" are used interchangeably and refer to in vitro protein expression systems of the invention, and can also be used in other descriptive ways, such as: protein in vitro synthesis system, in vitro protein synthesis system, cell-free protein synthesis system, cell-free in vitro 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, an in vitro translation system (abbreviated to IVT system, an mR2P system), an in vitro transcription translation system (abbreviated to IVTT system, a D2P system), an in vitro replication transcription translation system (abbreviated to IVDTT system, a D2P system) and the like can be included. In the present invention, the IVTT system is preferred. We also refer to the in vitro Protein synthesis system as a "Protein Factory" ("Protein Factory" or "Protein Factory"). The components of the in vitro protein synthesis system provided by the invention are described in an open mode. The cell-free protein synthesis system of the invention takes exogenous DNA, mRNA or the combination thereof as a nucleic acid template for protein synthesis, and realizes the in vitro synthesis of target protein by artificially controlling and supplementing substrates required by protein synthesis and substances such as transcription and translation related protein factors.

In the present invention, "protein" and "protein" have the same meaning, and are each translated into protein, and they can be used interchangeably.

In the present invention, both "system" and "system" are translated into system and used interchangeably.

In the present invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and are used interchangeably.

In the present invention, the cell extract, the cell lysate, the cell disruptant, and the cell lysate have the same meaning and can be used interchangeably, and english can adopt the descriptions of cell extract, cell lysate, and the like.

In the present invention, the energy system, and the energy supply system have equivalent meanings and can be used interchangeably. The energy regeneration system and the energy regeneration system have equivalent meanings and can be used interchangeably. The energy regeneration system is a preferred embodiment or component of the energy system.

The invention relates to an inteure preparation method, which is characterized in that an in-vitro protein synthesis reaction and a purification process are carried out together in a system, and is different from the traditional stepwise preparation method of synthesis before purification, wherein the inteure preparation method is a coupling type synthesis and purification integrated preparation method and can also be described as an inteure method, a coPure method, a coupling synthesis-purification method (synthesis coupled with purification method), "synthesis-co-purification" (synthesis-co-purification) method, "reaction-co-purification" method, "expression-co-purification" method, "translation-co-purification" method and the like; wherein the purification process performed together with the protein synthesis reaction is a local separation process for capturing the protein product in a free state in the mixed system to the protein immobilization system, and therefore, the intePure method can also be described as a "synthesis-co-separation" method, a "reaction-co-separation" method, an "expression-co-separation" method, a "translation-co-separation" method, and the like. Depending on the type of in vitro protein synthesis reaction, the inteure may also correspond to the descriptions "IVT-co-purification", "mR 2P-co-purification", "IVTT-co-purification", "D2P-co-purification", "mR 2P-co-purification", "IVDTT-co-purification", "CFPS-co-purification", "CFS-co-purification", etc.

An in vitro protein synthesis reaction mixed system, also described as an in vitro protein synthesis reaction mixture, a reaction mixed system, a reaction mixture, refers to a mixed system comprising an in vitro protein synthesis system, a nucleic acid template encoding a target protein; the system may be homogeneous or heterogeneous, and may be a liquid system such as a solution, an emulsion, or a suspension.

The intePure system, also called as reaction purification mixed system, intePure mixed system, coPure system, refers to a mixed system of an in vitro protein synthesis system and a protein product purification system, and is a mixed system which can carry out the in vitro protein synthesis process and the product purification process together.

The inteure kit, also known as the coPure kit, refers to the implementation of the method for preparation of inteure kit, also known as in vitro protein synthesis purification kit.

Purification of the protein product: at least comprises a process of separating the protein product from the mixed system in which the reaction is carried out, and optionally comprises a process of refining the protein product. The purification process of a protein product typically comprises the following four steps: (1) partially separating from a reaction mixed system, and combining the reaction mixed system with a Protein Immobilization System (PIS) to obtain a target protein-combined immobilization system (loaded-PIS), which is a partial separation and purification process in the intePure process; (2) a process of separating the loaded-PIS (protein immobilization system combined with protein product) from the intePure system, namely a process of separating the loaded-PIS from the reaction mixed system; (3) eluting the target protein from the loaded-PIS to obtain an eluent (a solution of a primary purified product) containing the target protein; (4) optionally, the eluate is dried to obtain a primary purified product of the target protein. The refining process is a process of further purifying on the basis of a primary purified product of the target protein or a solution thereof to obtain a repurified product of the target protein.

PIS, protein immobilization system, capable of capturing protein products from a reaction mixture and capable of being separated from a reaction purification mixture, thereby releasing the captured proteins.

loaded-PIS, refers to a protein immobilization system that binds a protein product of interest.

SM, Solid Matrix (SM), is an essential supporting part of protein immobilization systems, insoluble in the in vitro protein synthesis system, and capable of achieving physical separation from the in vitro protein synthesis system. For example, magnetic microspheres are used as a solid matrix, and physical separation of a protein immobilization system can be achieved by using the action of a magnet.

PE, purification element, is a functional element in protein immobilization systems for specifically capturing a protein of interest.

Immobilization, immobilized, immobilization, and the like "immobilization" means a covalent bonding means.

The "linkage"/"binding" means of carrying, linking to, binding, capturing, etc. is not particularly limited and includes, but is not limited to, covalent means, dynamic covalent means, non-covalent means, etc.

Linking element, refers to an element used to link two or more non-adjacent groups. The linking means between the linking member and the adjacent group is not particularly limited, and includes, but is not limited to, covalent means, dynamic covalent means, non-covalent means, and the like. The internal connection mode of the linking unit (linking means) is not particularly limited, and includes, but is not limited to, covalent mode, dynamic covalent mode, non-covalent mode, and the like. The non-covalent means includes, but is not limited to, coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interaction, and other supramolecular interaction means.

The outer surface of the solid substrate refers to the surface that is in contact with the reactive mixing system, as opposed to the area that is not in contact with the reactive mixing system.

Polymers, broadly included in the present invention are oligomers and polymers having at least three structural units or a molecular weight of at least 500Da (which molecular weight may be characterized in a suitable manner, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.).

The branched polymer in the present invention means a nonlinear polymer having a branched structure and at least two branches. The structure of the branched polymer is also referred to as a branched structure. The branched structure is preferably not a crosslinked structure, such as preferably a degree of crosslinking of not more than 40%, more preferably not more than 30%, more preferably not more than 20%, more preferably not more than 10%, more preferably not more than 5%, more preferably no crosslinked network. The branched structure refers to a structure having a branch point, including but not limited to a branched structure, a comb structure, a tree structure, a hyperbranched structure, a cyclic branched structure, and the like, and a combination structure thereof. The branched polymer comprises at least two branches. The branched polymers optionally have a linear backbone, e.g., there may be no linear backbone in some branched structures, such as dendrimers, hyperbranched structures, and the like. In the present invention, one end of the branched polymer is covalently immobilized on the outer surface of the solid matrix of the PIS. The branched polymer has at least one branch point, each branch point is connected with at least one branch chain, each branch chain has an independent terminal, and all the branch chains are distributed outside the solid matrix of the PIS and distributed in the liquid part of the reaction purification mixed system.

The linear main chain, linear main chain have the same meaning in the present invention, and may be used interchangeably.

Branched chain: in the present invention, the term "branched polymer" refers to a chain having a branch point and an independent end. In some structures, the side chain is also referred to as a side chain, for example, when the branched polymer is a comb-like structure, the polymer has both a linear main chain and a plurality of side chains, and the side chains are suspended at the side ends of the linear main chain. The length and size of the branched chain are not particularly limited, and the branched chain may be a short branched chain such as a carboxyl group, a hydroxyl group, or an amino group, or may be a long branched chain containing a large number of atoms. The structure of the branched chain is not particularly limited, and the branched chain may be linear or branched with a branched structure. The branches may also contain additional side chains or side groups. The number, length, size, degree of re-branching, and other structural features of the branched chains are preferably such that the net structure is not formed as much as possible, and the retention ratio is preferably not increased by accumulation of the branched chains.

The end of the branch includes the end of all branches. For branched polymers having a linear backbone, the other end of the linear backbone must be attached to a branch point in addition to being anchored to one end of the solid matrix of the PIS, and is therefore also broadly encompassed within the scope of the present invention as "branch end". Thus, the branched polymer has at least 1 branch point.

Tree structure: mainly refers to a nonlinear structure formed by divergently and repeatedly arranging structural units in a regular and exponential manner, has the symmetry of a chemical topological structure, and is called tree-shaped in a plurality of fields because the arrangement manner is similar to tree-shaped. In the present invention, unless otherwise specified, the "tree structure" also includes incomplete tree structures that are slightly imperfect in symmetry, including incomplete tree structures resulting from deliberate circumvention of design, and incompleteness of tree arrangement resulting from "unintentional" omission of structural units during synthesis. The term "tree" is broadly included in the present invention when the number of branches in the incomplete tree is at least 50% of the number of branches in the complete tree. In some preferred embodiments, the number of branches in the incomplete tree is at least 60% of the number of branches in the complete tree. In some preferred embodiments, the number of branches in the incomplete tree is at least 70% of the number of branches in the complete tree. In some preferred embodiments, the number of branches in the incomplete tree is at least 80% of the number of branches in the complete tree. In some preferred embodiments, the number of branches in the incomplete tree is at least 90% of the number of branches in the complete tree. In the preparation method, the tree structure is usually prepared by different generations in a stepwise manner.

The comb structure, also referred to as a brush structure in the present invention, mainly refers to a nonlinear structure formed by arranging structural units in series. The comb structure has a linear main chain and at least three side branched chains hanging from the side ends of the linear main chain. The spatial orientation of the side branched chains is not particularly limited, and the side branched chains may be arranged unilaterally, bilaterally, or stereoscopically around the circumference with the linear main chain as an axis. The structural units can adopt a repeated arrangement mode, such as an alpha-polylysine structure (a linear main chain formed by head-adjacent alpha-amino and alpha-carboxyl of lysine and regular epsilon-amino suspended on a side chain). The structural units may also be arranged in a random manner, such as a comb structure formed by randomly copolymerizing a structural unit with a branch chain with another structural unit, specifically an ethylene-acrylic acid copolymer. Comb structures in the present invention also broadly include those having comb-like topologies, but not necessarily non-linear structures having repeating units. That is, even if the structural units providing the side branches are different and even all the side branches are from the same molecule (e.g., some open chain monosaccharide structures having side hydroxyl groups), as long as the topology of "linear backbone + side branches" is formed, it is within the scope of the comb structure of the present invention.

The cyclic branched structure has both cyclic skeleton and nonlinear structure of branched chain, and the invention mainly refers to the nonlinear structure of branched chain with branched chain distributed in the cyclic skeleton, allowing to include nested cyclic structure. Examples thereof include cyclic monosaccharide structures, cyclodextrin structures and the like.

Hyperbranched structure mainly refers to a nonlinear structure formed by irregularly arranging structural units. The preparation method is generally a one-pot method, and the structural control is realized by controlling the structure, the dosage and the like of raw materials.

Different linear structures may be obtained from the same starting material by different reaction modes. For example, for trifunctional small molecular raw materials such as lysine, ornithine, aspartic acid and glutamic acid, a dendrimer can be prepared by repeatedly performing condensation and deprotection steps step by using selective protection raw materials, a comb-shaped polymer can be prepared by selectively protecting the raw materials in a one-pot manner, two ends of a linear main chain of the comb-shaped structure can be connected to form an annular branched structure, and a hyperbranched polymer can be prepared by using raw materials without a protective group in a one-pot manner.

And purifying the substrate, namely the substance to be separated from the reaction and purification mixed system, wherein the purified substrate is the target protein product.

The purification medium, purification element, can be specifically combined with the purification substrate in the protein immobilization system, thereby capturing the purification substrate, and further separating the purification substrate from the reaction purification mixed system.

Magnetic microspheres: the ferromagnetic or magnetizable microspheres, which can also be described as magnetic beads, have a fine particle size, preferably in the range from 0.1 μm to 1000. mu.m in diameter.

The "particle diameter" and "diameter" in the present invention mean an average particle diameter and an average diameter unless otherwise specified. The deviation is preferably. + -. 20%, more preferably. + -. 10%.

A magnetic microsphere body: magnetic microspheres with modified sites (magnetic microspheres with bindable sites). For example silica coated magnetic material particles, more specifically as aminated silica coated magnetic material particles.

Polyolefin chain: refers to a polymer chain free of heteroatoms covalently linked only by carbon atoms. The invention mainly relates to a polyolefin main chain in a comb structure; such as the linear backbone of an acrylic polymer.

Acrylic polymer: refers to a homopolymer or copolymer having a structure of-C (COO-) -C-unit, the copolymerization form of said copolymer being not particularly limited, preferably capable of providing a linear main chain and an appropriate amount or amount of pendant group COO-; the acrylic polymer is allowed to contain a hetero atom in the linear main chain. Wherein further substituents on the carbon-carbon double bond are allowed, as long as the progress of the polymerization reaction is not impaired, e.g. methyl substituents (corresponding to-CH) ₃C (COO-) -C-). Wherein storage of COO-In the form of-COOH, or else in the form of a salt (e.g. sodium salt), or else in the form of a formate (preferably an alkyl formate, for example methyl formate-COOCH)₃Ethyl formate-COOCH₂CH₃(ii) a May also be hydroxyethyl formate-COOCH₂CH₂OH), and the like. Specific structural forms of the-C (COO-) -C-unit structure include, but are not limited to, -CH (COOH) -CH₂-、-CH(COONa)-CH₂-、-MeC(COOH)-CH₂-、-MeC(COONa)-CH₂-、-CH(COOCH₃)-CH₂-、-CH(COOCH₂CH₂OH)-CH₂-、-MeC(COOCH₃)-CH₂-、-MeC(COOCH₂CH₂OH)-CH₂-any one of the like or any combination thereof. Wherein Me is methyl. The linear main chain of one polymer molecule may have only one kind of the above-mentioned unit structure (corresponding to a homopolymer), or may have two or more kinds of unit structures (corresponding to a copolymer). After the side carboxyl group of the acrylic polymer is functionalized, the-C (COO-) -C-unit structure forms a covalent bond with an adjacent group, such as an amide bond, an ester bond and the like, generally in the form of-C (CO-) -C-, preferably an amide bond.

Acrylic monomer molecule: the monomer molecule which can be used for synthesizing the acrylic polymer has a basic structure of C (COO ═ C, and examples thereof include CH (cooh) ═ CH₂、CH(COONa)＝CH₂、CH₃C(COOH)＝CH₂、CH₃C(COONa)＝CH₂、CH(COOCH₃)＝CH₂、CH(COOCH₂CH₂OH)＝CH₂、CH₃C(COOCH₃)＝CH₂、CH₃C(COOCH₂CH₂OH)＝CH₂And the like.

Specific binding site: in the present invention, the specific binding site refers to a group or a structural site having a binding function on a polymer branch chain, the group or the structural site having a specific recognition and binding function for a specific target, and the specific binding can be achieved by a binding action such as coordination, complexation, electrostatic force, van der waals force, hydrogen bond, covalent bond, or other interaction.

Affinity complex: non-covalently linked complexes formed by two or more molecules by means of at least one specific binding interaction, relying on very strong affinity, such as: the complex formed by the interaction of biotin and avidin. The manner of binding of biotin to an affinity complex of avidin is well known to those skilled in the art.

Biotin: the biotin can be combined with avidin, and has strong binding force and good specificity.

Avidin: avidin, which can bind with biotin, has strong binding force and good specificity, such as Streptavidin (SA), including its protein subunit, its modified product, its mutant, etc.

Biotin analogues, meaning non-biotin molecules capable of forming a specific binding with avidin similar to "avidin-biotin", preferably one of them being a polypeptide or protein, such as those developed by IBA

Polypeptides comprising the WSHPQFEK sequence used in the series (e.g.

Etc.), and similar polypeptides containing the WNHPQFEK sequence. WNHPQFEK can be regarded as WSMutated sequences of HPQFEK.

Avidin analogs, which refer to non-avidin molecules capable of forming specific binding with biotin similar to "avidin-biotin", preferably one of which is a polypeptide or protein. The avidin analogs include, but are not limited to, derivatives of avidin, homologous species of avidin (homologues), variants of avidin, and the like. Such avidin analogs are, for example, Tamavidin1, Tamavidin2, etc. (ref.: FEBS Journal,2009,276, 1383-.

The term "a-or" an "means a chemical linkage, which may be a chemical bond, a linking element, a covalent linkage or a non-covalent linkage, such as a conjugate or a complex referred to in the present invention, including a" - "symbol. For example, "-" in the biotin-avidin complex is non-covalent; for example, "-" in an avidin-purification medium covalent conjugate is covalent; for example, biotin-avidin proteins are either covalent or noncovalent. Specifically, it is determined by the specific molecular design and the nature of the linkage of two adjacent components.

Biotin-type label: the biotin type label comprises the following units: biotin, an avidin analog capable of binding avidin analogs, and combinations thereof. The biotin-type tag is capable of specifically binding avidin, an avidin analog, or a combination thereof. Therefore, the method can be used for separating and purifying protein substances including but not limited to protein substances marked by avidin type labels.

Avidin-type tag: the avidin type tag comprises the following units: avidin, avidin analogs that bind biotin analogs, and combinations thereof. The avidin-type tag is capable of specifically binding biotin, a biotin analog, or a combination thereof. Therefore, the method can be used for separating and purifying protein substances including but not limited to protein substances labeled by biotin type labels.

Polypeptide type tag: the polypeptide-type tag of the present invention refers to a tag comprising a polypeptide tag or a derivative of a polypeptide tag. The polypeptide tag refers to a tag of a polypeptide structure consisting of amino acid units, wherein the amino acid can be a natural amino acid or an unnatural amino acid.

Protein type tag: the protein-type tag of the present invention includes a tag comprising a protein tag or a derivative of a protein tag. The protein tag refers to a tag of a protein structure consisting of amino acid units, wherein the amino acid can be natural amino acid or unnatural amino acid.

Antibody type tag: the antibody-type tag of the present invention refers to a tag containing an antibody-type substance, which is capable of specifically binding to a corresponding target, such as an antigen. Examples of the antibody type tag also include an anti eGFP nanobody that can specifically bind to eGFP protein.

Antigenic tag: the antigenic tag of the present invention refers to a tag containing an antigenic substance, which is capable of specifically binding to an antibody substance.

A peptide is a compound in which two or more amino acids are linked by peptide bonds. In the present invention, the peptide and the peptide fragment have the same meaning and may be used interchangeably.

Polypeptide, peptide composed of 10-50 amino acids.

Protein, peptide composed of more than 50 amino acids. The fusion protein is also a protein.

Derivatives of polypeptides, derivatives of proteins: any polypeptide or protein to which the present invention relates, unless otherwise specified (e.g., specifying a particular sequence), is understood to also include derivatives thereof. The derivatives of the polypeptide and the derivatives of the protein at least comprise C-terminal tags, N-terminal tags, C-terminal tags and N-terminal tags. Wherein, C terminal refers to COOH terminal, N terminal refers to NH₂The meaning of which is understood by those skilled in the art. The label can be a polypeptide label or a protein label. Some examples of tags include, but are not limited to, histidine tags (typically containing at least 5 histidine residues; such as 6 XHis, HHHHHHHHHHHH; such as 8 XHis tags, HHHHHHHHHHHHHHHH), Glu-Glu, c-myc epitopes (EQKLISEEDL),

A Tag (DYKDDDDK), a protein C (EDQVDPRLIDGK), Tag-100(EETARFQPGYRS), a V5 epitope Tag (V5 epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYDVPDYA), beta-galactosidase (beta-galactosidase), thioredoxin (thioredoxin), histidine-site thioredoxin (His-notch thioredoxin), IgG-binding domain (IgG-binding domain), intein-chitin binding domain (intein-chitin binding domain), T7 gene 10(T7 gene 10), glutathione S-transferase (glutathione-S-transferase, GST), green fluorescent protein (GST), maltose binding protein (maltose binding protein, MBP), and the like.

Protein-based substances, in the present invention, broadly refer to substances containing polypeptides or protein fragments. For example, polypeptide derivatives, protein derivatives, glycoproteins, and the like are also included in the category of protein substances.

Antibody, antigen: the present invention relates to antibodies, antigens, and, unless otherwise specified, domains, subunits, fragments, single chains, single chain fragments, variants thereof are also understood to be encompassed. For example, reference to an "antibody" includes, unless otherwise specified, fragments thereof, heavy chains lacking light chains (e.g., nanobodies), Complementarity Determining Regions (CDRs), and the like. For example, reference to "antigen" includes, unless otherwise specified, epitopes (epitopes), epitope peptides. Examples of the fragment of the antibody include an Fc fragment.

The antibody-like substance, including but not limited to antibodies, fragments of antibodies, single chains of antibodies, single chain fragments, antibody fusion proteins, fusion proteins of antibody fragments, and the like, and derivatives and variants thereof, of the present invention may be used as long as the antibody-antigen specific binding can be generated.

The antigenic substances, as used herein, include, but are not limited to, antigens known to those skilled in the art and substances capable of performing an antigenic function and specifically binding to the antibody substances.

Anti-protein antibodies: refers to an antibody that specifically binds to a protein.

Nanobody against fluorescent protein: refers to a nanobody capable of specific binding to a fluorescent protein.

Nanobody (nanobody): also known as single domain antibodies (sdabs), or single chain antibodies, or single domain antibodies, have only one heavy chain variable domain (VHH).

scFV: a single chain antibody variable fragment is a small molecule consisting of the variable region of an antibody heavy chain and the variable region of a light chain linked by a peptide chain, and is the smallest functional structural unit with antibody activity.

Fab: is the antigen-binding region of an antibody, which consists of a constant and a variable domain of each of the heavy and light chains, which domains form a paratope at the amino terminus of the monomer, the antigen-binding site, and which variable domains bind to epitopes on their particular antigen.

F (ab') 2: is the product of antibody formation by pepsin which catalyzes antibody cleavage below the hinge region to form an F (ab ') 2 fragment and a pf' fragment. After mild reduction, the F (ab ') 2 fragment can be split into two Fab' fragments.

Homology (homology), unless otherwise specified, means at least 50% homology; preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology, more preferably at least 80% homology, more preferably at least 85% homology, more preferably at least 90% homology; also such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. The description object is exemplified by homologous sequences such as the omega sequences mentioned in the present description. Homology here refers to similarity in sequence, and may be equal in numerical similarity (identity).

Homologs, which refer to substances having homologous sequences, may also be referred to as homologues.

"variant," or "variant," refers to a substance that has a different structure (including, but not limited to, minor variations) but retains or substantially retains its original function or property. Such variants include, but are not limited to, nucleic acid variants, polypeptide variants, protein variants. Means for obtaining related variants include, but are not limited to, recombination, deletion or deletion, insertion, displacement, substitution, etc. of the building blocks. Such variants include, but are not limited to, modified products, genetically engineered products, fusion products, and the like. To obtain the gene modification product, the gene modification can be performed by, but not limited to, gene recombination (corresponding to the gene recombination product), gene deletion or deletion, insertion, frame shift, base substitution, and the like. Gene mutation products, also called gene mutants, belong to one type of gene modification products. One of the preferred modes of such variants is a homologue.

Modified product: including but not limited to chemically modified products, amino acid modifications, polypeptide modifications, protein modifications, and the like. The chemical modification product refers to a product modified by chemical synthesis methods such as organic chemistry, inorganic chemistry, polymer chemistry and the like. Examples of the modification method include ionization, salinization, desalinization, complexation, decomplexing, chelation, decomplexing, addition reaction, substitution reaction, elimination reaction, insertion reaction, oxidation reaction, reduction reaction, and post-translational modification, and specific examples thereof include oxidation, reduction, methylation, demethylation, amination, carboxylation, and vulcanization.

"mutant", mutant, as used herein, unless otherwise specified, refers to a mutant product that retains or substantially retains its original function or property, and the number of mutation sites is not particularly limited. Such mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. Mutants are one type of variant. Means for obtaining relevant mutants include, but are not limited to, recombination, deletion or deletion of structural units, insertion, displacement, substitution, and the like. The structural unit of the gene is basic group, and the structural units of the polypeptide and the protein are amino acid. Types of gene mutations include, but are not limited to, gene deletions or deletions, insertions, frameshifts, base substitutions, and the like.

"modified" products, including but not limited to derivatives, modified products, genetically engineered products, fusion products, etc., of the present invention, can retain their original function or property, and can optimize, alter their function or property.

Amino acid mixture refers to a mixture containing at least two amino acids.

Amino acids: in the present invention, the amino acid may be a natural amino acid, an unnatural amino acid, or a mixture thereof, unless otherwise specified_L-an amino acid,_DAmino acids or combinations thereof, and may also be radiolabeled amino acids, modified amino acids, and the like. The modified amino acid refers to an amino acid to which a chemical modification group is attached, and the structure thereof is not particularly limited, including but not limited to modification by amino acid side groups. The above definition of amino acid encompasses any substance of the invention that includes an amino acid unit, including but not limited to: polypeptide and its derivative, protein and its derivative, polypeptide tag, protein tag, polypeptide sequence, protein sequence, amino acid modifier, polypeptide modifier, protein modifier, partial domain of any of the above, subunit or fragment of any of the above (including any of the above Domain), variants of any of the foregoing (including variants of domains, subunits, fragments of any of the foregoing). The "variant of any of the foregoing" includes, but is not limited to "a mutant of any of the foregoing. In the present invention, for compounds representing chiral types "_L-”、“_D- ", subscript form has the same meaning as non-subscript form.

In the present invention, "translation-related elements" (TRELs) refer to functional elements required for the synthesis of protein products from a nucleic acid template, and are not limited to functional elements required for the translation process; when the nucleic acid template is DNA, functional elements required in the transcription process are also included in a broad sense. The translation-related elements can be provided by cell extracts (various endogenous factors), other exogenously added components of the in vitro protein synthesis system (e.g., translation-related elements such as exogenous RNA polymerase, tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, or combinations thereof), functional elements on the nucleic acid template (e.g., functional elements that control transcription/translation of a protein of interest, resistance gene translation systems, lac repressor translation systems, translation systems that control plasmid copy number, etc.), and the like. The functional elements for controlling transcription/translation of a target protein are exemplified by a promoter, a terminator, an enhancer, an IRES element, a kozak sequence, other elements for regulating the level of translation, a signal sequence, a leader sequence, a functional tag (e.g., a selection marker tag, a tag for enhancing the level of translation), and the like.

Purified translation-related elements, wherein "purified" is provided in relation to integration of a cell extract. A purified translation-related element refers to a single kind of translation-related element or a combination of different kinds of translation-related elements obtained by artificial synthesis or extraction. For the integrated provision mode, the kinds and ratios of the respective translation-related elements depend on the inherent properties of the source strain, and the adjustability of the kinds and ratios of the translation-related elements provided by the cell extract is extremely low except by endogenous genetic modification of the strain. The 'purified' supply mode can flexibly combine different kinds of translation-related elements, and can accurately adjust and control the kinds and the proportion of the translation-related elements. The "purified" does not necessarily limit the purity to 100%, and the corresponding purity depends mainly on the preparation method and the purification method, preferably the purity is higher than 80%, more preferably higher than 85%, more preferably higher than 90%, such as the purity is higher than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, such as the purity is 100%.

In the present invention, "translation-related enzymes" (TRENs) refer to enzyme substances required in the synthesis process from a nucleic acid template to a protein product, and are not limited to enzymes required in the translation process; when the nucleic acid template is DNA, it also broadly includes enzymes required for the transcription process. Such translation-related enzymes include, but are not limited to: aminoacyl tRNA synthetases, RNA polymerases, peptidyl transferases, and the like, or combinations thereof, and may also include transcriptases, DNA polymerases, the like, or combinations thereof.

Post-translational modification: also known as post-translational processing, post-translational modification, PTM. The PTM system plays a significant role in the normal folding, activity and stability of proteins.

And the amplification related elements comprise DNA amplification related elements and RNA amplification related elements.

DNA amplification related elements including at least a DNA polymerase. Other factors such as helicases (HDA amplification), recombinases and single stranded DNA binding proteins (RPA amplification), etc. may also be included, depending on the amplification mechanism.

Gene: including coding and non-coding regions.

The nucleotide sequence is as follows: a sequence consisting of nucleotide units.

Nucleic acid sequence: the sequence of the nucleic acid substance includes DNA sequence and RNA sequence.

A coding sequence: coding sequence, abbreviated CDS. A nucleotide sequence corresponding exactly to a codon of a protein, which sequence does not contain other sequences corresponding to the protein in between (irrespective of sequence changes during mRNA processing etc.).

The coding gene is as follows: the useful gene segments encoding the protein may be contiguous or non-contiguous. The coding gene necessarily includes a coding sequence.

Nucleic acid template: also referred to as genetic template, refers to a nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates, and combinations thereof. In any embodiment of the invention, the nucleic acid templates may each independently be DNA templates, mRNA templates, or a combination thereof. In any embodiment of the invention, the nucleic acid templates may each independently preferably be DNA templates. In the present invention, the nucleic acid template encoding the target protein is preferably a DNA template unless otherwise specified.

"nucleic acid template encoding a protein X" refers to a nucleic acid template that contains a coding sequence for the protein X, on the basis of which the protein X can be synthesized by at least translation (e.g., by transcription and translation), and that allows the nucleic acid template to contain non-coding regions and also allows the nucleic acid template to contain coding sequences for polypeptides or proteins other than the protein X. For example, a "nucleic acid template encoding RNA polymerase" includes at least the coding sequence of RNA polymerase, and further allows the inclusion of other nucleic acid sequences such as non-coding regions, fusion tags, and the like; accordingly, the expression product contains at least an RNA polymerase structure, and may be an RNA polymerase molecule or a fusion protein thereof, or may be a mixed component including an RNA polymerase molecule or/and a fusion protein molecule thereof.

Nucleic acid vectors, including cloning vectors and expression vectors. The expression vector comprises a DNA expression vector and an RNA expression vector. The cloning vector has sites into which foreign nucleic acid fragments can be inserted, for example, foreign DNA fragments can be inserted into multiple cloning sites of the DNA cloning vector.

A reinforcing element: unless otherwise specified, the present invention refers to a sequence which functions to promote transcription or/and translation processes in a nucleic acid sequence located between a promoter and a coding sequence of a target protein, such as an omega sequence, a kozak sequence, an IRES sequence, and the like. Including transcription enhancing elements, translation enhancing elements.

Endogenous/endogenous: depending on the activity of the active cell metabolism. Endogenously expressed proteins are endogenously secreted by the cells as they grow and can be processed to be present in the cell extracts of the invention.

Exogenous/exogenous: independent of active cellular metabolic activity. The exogenous components are added directly to the in vitro protein synthesis system, rather than by way of adding cells or cell extracts. Such as: exogenous RNA polymerase can be added to the reaction system by exogenous means by adding a precursor (e.g., an inactive precursor that can be enzymatically or otherwise activated to produce RNA polymerase), a nucleic acid template (which can be translated into a protein by the system), a fusion protein, a pure substance, or a mixture. For another example: exogenous DNA polymerase can also be added to the reaction system by exogenous means as described above.

Exogenous RNA polymerase: has the same meaning as that of exogenous RNA polymerase.

Exogenous DNA polymerase: has the same meaning as that of an exogenous DNA polymerase.

"nucleic acid template encoding RNA polymerase (or nucleic acid template encoding DNA polymerase)" includes at least the coding sequence of RNA polymerase (or DNA polymerase), and further allows the inclusion of non-coding regions, fusion tags, and other nucleic acid sequences; accordingly, the expression product contains at least an RNA polymerase structure (or a DNA polymerase structure). Taking RNA polymerase as an example, the RNA polymerase can be an RNA polymerase molecule or a fusion protein thereof, and can also be a mixed component comprising the RNA polymerase molecule or/and a fusion protein molecule thereof.

Crowding agents, agents used to mimic the macromolecular environment of intracellular crowding in vitro. References "X Ge, D Luo and J xu. cell-free protein expression under macromolecular growth conditions [ J ]. PLoS One,2011,6(12): e 28707" and citations thereof, among others.

Sucrose polymer: refers to a polymer containing at least 2 sucrose units. Including but not limited to polysucrose.

Ficoll sucrose polymer: unless otherwise specified, refer in particular to

The reagent, a non-ionic synthetic sucrose polymer, is a highly branched polymer obtained by copolymerizing sucrose and epichlorohydrin, and can be selected from commercially available products. For example, Ficoll-400 (Polysucrose 40)0, CAS:26873-85-8), Ficoll-70 (Polysucrose 70, CAS: 72146-89-5). Wherein the content of the first and second substances,

PM 400(Sigma Aldrich) is a highly branched polymer copolymerized from sucrose and epichlorohydrin, with an average molecular weight of 400 kg/mol; ficoll PM 70(Sigma Aldrich) has an average molecular weight of 70 kg/mol.

The phosphoric acid compound comprises organic matters and inorganic matters.

The phosphate refers to an inorganic phosphate unless otherwise specified.

PNA: peptide nucleic acids, a class of DNA analogs with polypeptide backbones substituted for sugar phosphate backbones, are novel nucleic acid sequence specific reagents. The third-generation antisense reagent is constructed by computer design on the basis of first-generation and second-generation antisense reagents and finally synthesized artificially, is a brand-new DNA analogue, namely a pentose phosphodiester bond framework in DNA is replaced by a neutral peptide chain amide 2-aminoethylglycine bond, the rest is the same as the DNA, and PNA can recognize and combine with DNA or RNA sequence in a Watson-Crick base pairing mode to form a stable double-helix structure. Because PNA has no negative charge and has no electrostatic repulsion with DNA and RNA, the stability and specificity of combination are greatly improved; unlike the hybridization between DNA or DNA and RNA, the hybridization between PNA and DNA or RNA is hardly affected by the salt concentration of the hybridization system, and the hybridization ability with DNA or RNA molecules is far superior to that of DNA/DNA or DNA/RNA, which is characterized by high hybridization stability, excellent specific sequence recognition ability, no hydrolysis by nuclease and protease, and capability of linking with ligand for cotransfection into cells. These are advantages not possessed by other oligonucleotides.

Solution X: the final concentrations are 0.01-1 mol/L2-morpholine ethanesulfonic acid (CAS: 4432-31-9) and 0.1-2 mol/L NaCl respectively.

Solution Y: PBS buffer solution pH 7.2-7.5, such as: the final concentrations are 0.0684mol/L disodium hydrogen phosphate, 0.0316mol/L sodium dihydrogen phosphate and 0.15mol/L sodium chloride aqueous solution respectively.

In the present invention, the "normal temperature" is preferably room temperature to 37 ℃, specifically, preferably 20 to 37 ℃, and more preferably 25 to 37 ℃.

In the present invention, preferred embodiments such as "preferred" (e.g., preferred, preferable, preferably, preferred, etc.), "preferred", "more preferred", "better", "most preferred", etc. do not limit the scope and protection of the invention in any sense, do not limit the scope and embodiments of the invention, and are provided as examples only.

In the description of the invention, references to "one of the preferred", "in a preferred embodiment", "some preferred", "preferably", "preferred", "more preferred", "further preferred", "most preferred", etc. preferred, and references to "one of the embodiments", "one of the modes", "an example", "a specific example", "an example", "by way of example", "for example", "such as", "such", etc. do not constitute any limitation in any sense to the scope of coverage and protection of the invention, and the particular features described in each mode are included in at least one embodiment of the invention. The particular features described in connection with the various modes can be combined in any suitable manner in any one or more of the particular embodiments of the invention. In the invention, the technical schemes corresponding to the preferred modes can also be combined in any suitable mode; for example, an exogenous RNA polymerase and an exogenous DNA polymerase can be added simultaneously, see patent publication CN 108642076A.

In the present invention, "optionally" means either the presence or absence thereof. Whether the technical scheme of the invention is suitable or not is taken as a selection basis. In the present invention, the term "optional" means that the present invention can be implemented as long as it is applied to the technical means of the present invention.

In the present invention, "any combination thereof" means "more than 1" in number, and means a group consisting of the following cases in an inclusive range: "optionally one of them, or optionally a group of at least two of them".

In the present invention, the description of "one or more", etc. "has the same meaning as" at least one "," a combination thereof "," or a combination thereof "," and a combination thereof "," or any combination thereof "," any combination thereof ", etc., and may be used interchangeably to mean" 1 "or" greater than 1 "in number.

In the present invention, "and/or" means "either one of them or any combination thereof, and also means at least one of them. By way of example, "comprising a substrate for a synthetic RNA and/or a substrate for a synthetic protein", it is meant that the substrate for a synthetic RNA alone may be included, the substrate for a synthetic protein alone may be included, and the substrate for a synthetic RNA and the substrate for a synthetic protein may be included at the same time.

The prior art means described in the modes of "usually", "conventionally", "generally", "often", etc. are also referred to as the content of the present invention, and if not specifically stated, they may be regarded as one of the preferred modes of the partial technical features of the present invention, and it should be noted that they do not constitute any limitation to the scope of the invention and the protection scope.

mol%: mole percent, represents the amount of a substance as a percentage.

wt% or% (wt): are mass concentration units and all represent mass percent.

(v/v)% or% (v/v): all represent volume percent.

% (w/v): mass volume concentration units, corresponding to g/100 mL.

RFU, Relative Fluorescence Unit value (Relative Fluorescence Unit).

eGFP: enhanced green fluorescence protein (enhanced green fluorescence protein). In the present invention, the eGFP broadly includes wild-type and variants thereof, including but not limited to wild-type and mutants thereof.

mEGFP: a206K mutant of eGFP.

KH 570: 3- (methacryloyloxy) propyltrimethoxysilane,also known as gamma-methacryloxypropyltrimethoxysilane, CAS:2530-85-0, an acryloyl-functional silane coupling agent. Structural formula is

KH 550: 3-aminopropyltriethoxysilane, CAS:919-30-2, an aminated silane coupling agent. Molecular formula is NH₂-(CH₂)₃-Si(OCH₂CH₃)₃。

NTA: nitrilotriacetic acid, also known as nitrilotriacetic acid, nitrilotriacetic acid.

All documents cited herein, and documents cited directly or indirectly by such documents, are hereby incorporated by reference into this application as if each were individually incorporated by reference.

It is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to the examples) can be combined with each other to constitute a new or preferred technical solution, as long as the target protein can be synthesized in vitro or, preferably, efficiently. Not described in detail.

1. The invention provides an integrated preparation method for in vitro synthesis and purification of protein, wherein a protein-based synthesis process and a protein product purification process can be jointly carried out in the same system, and the integrated preparation method of coupled synthesis-co-purification is also called an inteure method or a coPure method.

The integrated preparation method for in vitro synthesis and purification of the protein at least comprises the following steps:

Step i: at least providing an in-vitro protein synthesis system, a nucleic acid template for coding a target protein and a protein fixing system, and placing the in-vitro protein synthesis system, the nucleic acid template for coding the target protein and the protein fixing system in the same reaction container to form a reaction purification mixed system (an intePure system or a coPure system);

step ii: carrying out incubation reaction under a proper condition to obtain a target protein product; during the course of the incubation reaction, i.e., during the course of an in vitro protein synthesis reaction, the protein product of interest is capable of specifically binding to the protein immobilization system;

step iii: separating the protein immobilization system bound to the target protein product from the reaction purification mixed system;

step iv: eluting the target protein from the protein immobilization system combined with the target protein product separated in the step iii to obtain an eluate containing the target protein;

optionally further comprising the step v: separating the target protein from the eluate; the purified product of the target protein can be obtained after separation.

Further optionally comprising step vi: further purifying the target protein; can further improve the purity of the protein product and obtain the repurified product of the target protein.

By limiting the technical features that enable "obtaining a protein product of interest", the present invention covers only those combinations of technical features that enable the above-described functions, and combinations of technical features that do not enable the above-described functions are, of course, excluded from the scope of the present invention. That is, the intePure system, also called coPure system, includes the in vitro protein synthesis system and the in vitro protein synthesis reaction mixed system, and firstly, the system is a workable system and is a system capable of expressing the target protein.

1.1. In vitro protein Synthesis System (in vitro cell-free protein Synthesis System, CFPS System)

The in vitro protein synthesis system can provide various factors required by the in vitro protein synthesis process. The extract can be provided in an integrated form by means of a cell extract, can be provided by means of an external source (e.g., the Japanese PURE system, such as the PURExpress kit), and can be provided by a combination of the two.

The species and content of each component of the in vitro protein synthesis system are not particularly limited as long as the system is constructed to be capable of reacting with a nucleic acid template encoding a target protein to synthesize the target protein. Among some preferred modes, those combinations capable of expressing the target protein with high efficiency are preferred. Combinations that do not express the protein of interest because of concentrations of certain components that are too low or too high are, of course, excluded from the scope of the invention.

The order of addition of the components of the in vitro protein synthesis system is not particularly limited.

The concentrations of the components of the in vitro protein synthesis system refer to the initial concentrations in the in vitro protein synthesis reaction mixture system, unless otherwise specified. The in vitro protein synthesis reaction mixed system comprises the in vitro protein synthesis system and a nucleic acid template for coding a target protein.

The translation-related elements in the in vitro protein synthesis system may be provided by cell extracts, exogenously added translation-related elements, or combinations thereof.

In some preferred embodiments, the in vitro protein synthesis system comprises at least a cell extract; optionally including exogenously added translation-related elements.

In some preferred embodiments, the in vitro protein synthesis system comprises exogenously added translation-related elements.

In some preferred embodiments, the in vitro protein synthesis system is capable of supplying an RNA polymerase, which may be from a source selected from the group consisting of: endogenously expressed RNA polymerase (provided via cell extract), exogenously added RNA polymerase, translation products of an exogenous nucleic acid template encoding RNA polymerase, and combinations thereof.

In some preferred embodiments, the in vitro protein synthesis system is capable of recognizing a promoter in the nucleic acid template encoding the protein of interest.

In some preferred embodiments, the in vitro protein synthesis system comprises an RNA polymerase provided by cell extraction, exogenous addition, or a combination thereof.

In some preferred embodiments, the RNA polymerase that can be supplied by the in vitro protein synthesis system is T7 RNA polymerase.

In some preferred embodiments, the nucleic acid template encoding the target protein comprises a T7 promoter capable of initiating a gene transcription process for the target protein, and the in vitro protein synthesis system comprises T7 RNA polymerase, an exogenous nucleic acid template encoding T7 RNA polymerase, or a combination thereof.

In some preferred embodiments, the in vitro cell-free protein synthesis system comprises a cellular extract comprising endogenously expressed T7 RNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises an energy system.

In some preferred embodiments, the in vitro protein synthesis system comprises a substrate for RNA synthesis.

In some preferred embodiments, the in vitro protein synthesis system comprises a substrate for a synthetic protein.

In some preferred embodiments, the in vitro protein synthesis system is capable of supplying a DNA polymerase, which may be from a source selected from the group consisting of: endogenously expressed DNA polymerase (provided via cell extract), exogenously added DNA polymerase, translation products of an exogenous nucleic acid template encoding the DNA polymerase, and combinations thereof.

In some preferred embodiments, the DNA polymerase that the in vitro protein synthesis system can supply is phi29 DNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises a DNA polymerase, a substrate for synthesizing DNA.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for protein synthesis, an RNA polymerase (contained in the cell extract or/and independently added exogenously), and a substrate for RNA synthesis. Said "comprised in a cellular extract" means that said cellular extract comprises an endogenously expressed RNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract (optionally containing endogenously expressed RNA polymerase and/or DNA polymerase), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, and exogenously added RNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for synthesizing a protein, an RNA polymerase (contained in the cell extract or/and independently added exogenously), a substrate for synthesizing RNA, a DNA polymerase (contained in the cell extract or/and independently added exogenously), and a substrate for synthesizing DNA.

In some preferred embodiments, the in vitro protein synthesis system comprises a kluyveromyces lactis cell extract (containing endogenously expressed T7 RNA polymerase), an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

In some preferred embodiments, the in vitro protein synthesis system comprises a kluyveromyces lactis cell extract (the host cell does not endogenously integrate the coding gene of RNA polymerase), an energy system, exogenously added RNA polymerase, a substrate for RNA synthesis, and a substrate for protein synthesis.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract (optionally containing endogenously expressed RNA polymerase and/or DNA polymerase), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, an exogenously added RNA polymerase, an exogenously added DNA polymerase, a substrate for DNA synthesis.

In some preferred embodiments, the in vitro protein synthesis system comprises purified translation-related elements, an energy system, a substrate for synthetic RNA, a substrate for synthetic protein; the purified translation-related elements include: tRNA, ribosome, aminoacyl tRNA synthetase, RNA polymerase, initiation factor, elongation factor, termination factor. May further comprise a peptidyl transferase, etc.

The in vitro protein synthesis system optionally comprises any one or combination of the following exogenous additional components: DNA amplification related elements, RNA amplification related elements, RNase inhibitors, crowding agents, magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, antifreeze agents, trehalose, reaction promoters, antifoam agents, alkanes, buffers, aqueous solvents.

In some preferred embodiments, the in vitro protein synthesis system optionally comprises any one or a combination of the following exogenously added components: rnase inhibitors, crowding agents, magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoaming agents, alkanes, buffers, aqueous solvents, exogenous nucleic acid templates encoding RNA polymerase, DNA polymerase, exogenous nucleic acid templates encoding DNA polymerase, other DNA amplification related elements, substrates for synthesizing DNA, RNA amplification related elements. When the in vitro protein synthesis system comprises a DNA polymerase or an exogenous nucleic acid template encoding a DNA polymerase, some preferred embodiments also comprise a substrate for synthesizing DNA.

1.1.1. Translation-related elements of in vitro protein synthesis systems

In some preferred embodiments, the translation-related elements of the in vitro protein synthesis system include, but are not limited to: tRNA, ribosome, translation-related enzyme, initiation factor, elongation factor, termination factor.

In some preferred embodiments, the translation-related enzymes include, but are not limited to: aminoacyl tRNA synthetases, RNA polymerases, peptidyl transferases, and the like, or combinations thereof, and further optionally includes transcriptases, DNA polymerases, and the like, or combinations thereof.

The translation-related elements in the in vitro protein synthesis system are provided in a manner that includes cell extracts and also allows supplementation by exogenous addition of translation-related elements.

The cell extract is intended to provide a structure or biological factor for the transcription and translation of proteins. The selection criteria of the cell extract are as follows: the target protein can be synthesized by in vitro protein synthesis reaction based on the nucleic acid template for encoding the target protein. The cell extract of the present invention may be derived from a wild type or a non-wild type. Non-wild-type modifications include, but are not limited to, gene modifications. The cellular extracts of the present invention are derived in some preferred ways from eukaryotic cells, in some preferred ways from yeast cells, and in some preferred ways from kluyveromyces lactis cells.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract comprising an endogenously expressed RNA polymerase corresponding to a promoter element on a nucleic acid template. Specifically, for example, the kluyveromyces lactis cell extract contains endogenously expressed T7 RNA polymerase, which can recognize the T7 promoter on the nucleic acid template.

In some preferred embodiments, the translation-related elements of the in vitro protein synthesis system are provided by exogenous addition, i.e., do not include the provision of a cell extract.

In some preferred embodiments, the exogenously added translation-related element is a purified translation-related element or a combination thereof.

In some preferred embodiments, the in vitro protein synthesis system comprises a system component capable of recognizing a promoter element on a nucleic acid template, e.g., an RNA polymerase that corresponds to the promoter element.

The components of the system (e.g., the corresponding RNA polymerase) that are capable of recognizing the promoter element in the nucleic acid template may be provided by a cell extract in the system, may be provided by exogenous addition, or may be provided by a combination of the two.

1.1.1.1. Cell extract

The cell extract should be capable of expressing the nucleic acid template encoding the protein of interest, i.e., capable of synthesizing the protein of interest encoded thereby, using the nucleic acid template encoding the protein of interest as a template.

The cell extract is intended to provide structural factors or/and biological factors for protein expression (such as transcription, translation).

Cell extracts can provide most of the key translation-related elements required for synthesis of the protein of interest; this is the endogenous way of supply.

The cell extract, including yeast cell extracts, is typically used to provide ribosomes, transfer RNA (tRNA), aminoacyl tRNA synthetases, initiation and elongation factors for protein synthesis, and stop release factors, and may also be engineered to provide other enzymatic materials, such as polymerases (RNA polymerases and/or DNA polymerases), endogenously.

The cell extract preferably does not contain intact cells because the method for producing the cell extract comprises a step of disrupting cells (also referred to as a cell disruption treatment, a lysis step, etc.). In contrast to the traditional synthetic approach of secreting expressed proteins from intact cells, the in vitro protein synthesis system thus constructed is referred to as a cell-free system.

The cell extract may also contain some other proteins, especially soluble proteins, originating from the cytoplasm of the cell.

In some preferred embodiments, the cell extract contains various factors required for protein synthesis.

The related coding gene can be naturally present in the genome of the cell, or can be integrated into the genome of the cell (integrated into a chromosome), or can be inserted into an episomal plasmid of the cell. Taking RNA polymerase and DNA polymerase as examples for illustration, in some preferred modes, the cell extract contains endogenously expressed RNA polymerase and/or DNA polymerase.

Endogenous integration of the coding sequence or genes encoding the heterologous proteins into the cell from which the cell extract is derived (also referred to as an engineered strain) may allow the engineered strain to endogenously express the heterologous proteins, which may include, but are not limited to: RNA polymerase, DNA polymerase, etc. Means and methods for endogenous integration of the coding sequence or gene encoding the heterologous protein may be referred to the heterologous protein and its genetic engineering methods provided in the existing literature including, but not limited to, documents CN108690139A, CN109423496A, CN106978439A, CN110408635A, CN110551700A, CN110093284A, CN110845622A, CN110938649A, CN2018116198190, "Molecular and Cellular Biology,1990,10(1): 353-360" and the like, and the references cited therein, specifically including, but not limited to: insertion of coding sequences into intracellular episomal plasmids, insertion of coding genes into the genome of a cell, in situ replacement of portions of a gene of the genome of a cell with a coding gene, and the like, as well as combinations thereof.

In some preferred modes, the source cell of the cell extract is endogenously integrated with the coding gene of RNA polymerase, can endogenously express the RNA polymerase, can perform in-vitro cell-free protein synthesis under the condition of not adding exogenous RNA polymerase, replaces an exogenous addition mode, simplifies a formula, improves the operation convenience and saves the cost. Implementations of the endogenous integrated RNA polymerase include, but are not limited to: inserting a gene encoding RNA polymerase into a cellular plasmid or into the genome of a cell, replacing a portion of a gene or sequence of the genome of a cell with a gene encoding RNA polymerase in situ (i.e., comprising a step of knocking out the portion of the gene or sequence), knocking out the portion of the gene and inserting a gene encoding RNA polymerase, and combinations thereof. Further, in some preferred modes, the source of the cell extract is yeast. Further, in some preferred embodiments, the source of the cellular extract is kluyveromyces lactis. In some embodiments, the gene encoding T7 RNA polymerase is integrated into the genome of kluyveromyces lactis that endogenously expresses T7 RNA polymerase, and the cell extract thus prepared contains endogenously expressed T7 RNA polymerase, without the additional addition of RNA polymerase in the in vitro cell-free protein synthesis system; can perform in vitro cell-free protein synthesis without additional addition of exogenous RNA polymerase. In other embodiments, the gene encoding RNA polymerase is inserted into an intracellular episomal plasmid, such as a Kluyveromyces lactis cell plasmid, to produce a cell extract. Refer specifically to the preparation method of CN 109423496A.

Other genetic modification methods can be adopted to modify the source cells (engineering strains) so as to improve the activity of cell extracts and better promote the in vitro protein synthesis. In some embodiments, certain gene fragments in the genome of the engineered strain are knocked out, expression of related proteins is inhibited, a cell extract is prepared by using the engineered strain subjected to the genetic modification, and the content of the related proteins in the cell extract is reduced or removed, so that the purpose of improving in vitro protein synthesis capacity is achieved, for example, knocked-out genes and related knocking-out methods adopted in documents such as CN108949801A, CN2018116083534, CN2019107298813 and the like. In some embodiments, the source cell is further genetically modified using methods of genetic modification such as CN109022478A, CN109423497A, CN109837293A, CN109971775A, CN110408636A, CN107574179A, CN110551745A, CN110819647A, CN2018112862093(CN111118065A), and the like.

The preparation method of the cell extract can adopt the reported technical means. In brief summary, the following steps may generally be included: providing sufficient amount of cells, quick freezing the cells with liquid nitrogen, breaking the cells, centrifuging and collecting supernatant to obtain cell extract. The means and method for disrupting the cells are not particularly limited, and some preferred means include high-pressure disruption, freeze-thaw (e.g., liquid nitrogen cryo-disruption), and the like. The preparation method of the cell extract can refer to documents such as CN106978349A, CN108535489A, CN108642076A, CN109593656A, CN109971783A, CN109321620A, CN110408512A and CN 110652780A. In some embodiments, the cell extract can be prepared by subjecting the seed cells to fermentation culture, centrifuging, removing the culture medium, and collecting a sufficient amount of cells. The extract product obtained according to the method for producing a cell extract may have a small or very small amount of intact cells remaining, and such extract products are also within the scope of the cell extract of the present invention. That is, the in vitro protein synthesis system of the present invention does not exclude the presence of intact cells.

The cell extract prepared by the method provided by the invention can ensure that the in vitro protein synthesis reaction is normally carried out, and contains necessary components required by protein synthesis such as ribosome, tRNA with an amino acid transfer function, aminoacyltRNA synthetase and the like. In some embodiments, the cell extract is exemplified by a yeast cell extract, and is prepared by a method comprising: (i) providing a source cell; (ii) washing the yeast cells to obtain washed yeast cells; (iii) performing cell breaking treatment on the washed yeast cells to obtain a yeast crude extract; and (iv) performing solid-liquid separation on the yeast crude extract, wherein the collected supernatant part is the cell extract. The yeast cell extract, in some preferred embodiments, is a kluyveromyces lactis cell extract.

In the invention, the content of protein contained in the cell extract is 20-100 mg/mL in some preferable modes. In other preferable modes, the concentration is 20-50 mg/mL. In other preferable modes, the concentration is 50-100 mg/mL. In other preferred embodiments, the concentration is any one of 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, or a range of concentrations between any two concentrations, inclusive. Methods for determining protein content include, but are not limited to, ultraviolet absorption, biuret, BCA, Lowry, Coomassie Brilliant blue, Kjeldahl, and the like. In some preferred embodiments, the method for determining the amount of protein is a coomassie brilliant blue assay.

The concentration of the cell extract in the in vitro protein synthesis reaction mixture system is not particularly limited. The volume ratio and the weight ratio can be adopted; unless otherwise specified, refer to the final volume ratio in the in vitro protein synthesis reaction mixture system. In some preferred modes, the concentration of the cell extract is 20% to 80% (v/v); in other preferred embodiments, the concentration of the cell extract is 20% to 70% (v/v); in other preferred embodiments, the concentration of the cell extract is from 30% to 60% (v/v); in other preferred embodiments, the concentration of the cell extract is from 40% to 50% (v/v); in other preferred embodiments, the concentration of the cell extract is 80% (v/v); all calculated according to the total volume of the in vitro protein synthesis system or the total volume of the in vitro protein synthesis reaction mixed system. Examples of the concentration of the cell extract also include, but are not limited to, any one of the following volume percentages, or a numerical range between any two of the following volume percentages (the numerical range may or may not include both of the following endpoints): 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%.

The cell extract is derived from a prokaryotic cell, a eukaryotic cell, or a combination thereof.

In some preferred embodiments, the cell extract is derived from a prokaryotic cell. Among these, some preferred ones are derived from escherichia coli (e.coli cells) or Bacillus.

In some preferred embodiments, the cell extract is derived from a eukaryotic cell.

The cell source of the cell extract may be one or more types of eukaryotic cells selected from the group consisting of: including but not limited to mammalian cells (e.g., rabbit reticulocyte, HF9 cell, CHO cell, HEK293 cell, human cell, etc.), plant cells (e.g., wheat germ cell, tobacco BY-2cell), yeast cells, insect cells, nematode cells, and combinations thereof. Examples of the human cells include Hela cells and K562 cells. Sources of such mammalian cells include, but are not limited to, murine, rabbit, monkey, human, ovine, porcine, bovine, canine, equine, and the like.

The cell source of the cell extract and the preparation method thereof can also be reported by reference to the prior documents, and the cell sources reported by the following documents are all taken as references and are included in the invention: "Nicole E.Gregorio, Max Z.Levine and Javin P.Oza.A. User's Guide to Cell-Free Protein Synthesis [ J ]. Methods protocol.2019, 2, 24", "Y Lu.Advances in Cell-Free biochemical technology [ J ]. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45" and the like and documents cited directly or indirectly. For example, prokaryotic sources include, but are not limited to, e.coli (e.coli); eukaryotic cell sources include, but are not limited to, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Streptomyces lividans (Streptomyces lividans), wheat germ cells (steamed gem), tobacco BY-2 cells (tobaco BY-2cell), Spodoptera frugiperda cells (Sffi cells or SF cells or Sf cells, for short, belonging to insect cells), Trichoplusia ni cells (insect cells), Rabbit reticulocyte (rabbitristocellulare), CHO cells (Chinese hamster ovary cells), human K562 cells, HEK293 cells, HeLa cells, mouse fibroblast (mouse fibroblast), Lehmania tarsa cells (Lehmania germ cells, yeast cells, et al, unicellular organisms.

In some preferred embodiments, the yeast cell is saccharomyces cerevisiae, pichia pastoris, kluyveromyces, or a combination thereof. Further, in some preferred embodiments, the Kluyveromyces is Kluyveromyces lactis (k.lactis), Kluyveromyces lactis var. drosophilatus, Kluyveromyces lactis var. siamensis, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus var. siamensis, Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces maruyveri var. vannudenii, Kluyveromyces crispatus (Kluyveromyces bzhanskiii), Kluyveromyces hainanensis (Kluyveromyces), Kluyveromyces kluyveri (Kluyveromyces), kluyverykluyveromyces kluyveris, Kluyveromyces lactis, kluyverykluyveromyces, kluyverykluyverykluyveromyces (Kluyveromyces), kluyverykluyverykluyveromyces lactis, kluyverykuryomyces, kluyverykluyverykulare, kluyverykulare, kluyverykluyverykulare, or the like; references include, but are not limited to, the following: EP1197560A1, "Marc-Andre Lachance, the Yeast (Fifth edition), Chapter 35, Kluyveromyces van der Walt (1971) 2011, Pages 471-.

Kluyveromyces (Kluyveromyces) is a species of ascosporogenous yeast, and among them, Kluyveromyces marxianus (Kluyveromyces marxianus) and Kluyveromyces lactis (Kluyveromyces lactis) are industrially widely used yeasts. In comparison with other yeasts, kluyveromyces lactis has many advantages such as superior secretion ability, better large-scale fermentation characteristics, a level of food safety, and also the ability of post-translational modification of proteins. The genome of the wild-type strain of Kluyveromyces lactis does not contain a gene encoding T7 RNA polymerase.

In some preferred embodiments, the source of the cellular extract is kluyveromyces lactis, and any one or a combination of the following gene sequences is endogenously integrated: a gene encoding RNA polymerase and a gene encoding DNA polymerase. In some preferred embodiments, the endogenous integration is into an episomal plasmid or into the genome of the cell.

In some preferred embodiments, the source of the cellular extract is kluyveromyces lactis, and any one or a combination of the following gene sequences is endogenously integrated: a gene encoding T7 RNA polymerase and a gene encoding phi29 DNA polymerase. In some preferred embodiments, the endogenous integration is into an episomal plasmid or into the genome of the cell.

In some preferred embodiments, the cell extract may be selected from any one of the following sources: escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, nematode cells, pathogens, combinations thereof. In some preferred embodiments, the yeast cell is kluyveromyces, saccharomyces cerevisiae, saccharomyces pichia pastoris, or a combination thereof. In some preferred embodiments, the Kluyveromyces is Kluyveromyces lactis var. drosophilarium, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus var. marxianus, Kluyveromyces marxianus var. vannundeii, Kluyveromyces multibuyveri, Kluyveromyces marinus, Kluyveromyces williamsii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensurae, Kluyveromyces lactis, or a combination thereof.

The cell extract is in some preferred forms a yeast cell extract; in some preferred modes, the extract is a Kluyveromyces lactis cell extract, and in other preferred modes, the extract is a Kluyveromyces marxianus cell extract or a Kluyveromyces lactis cell extract.

In some preferred embodiments, the cell extract may be selected from any one of the following sources: escherichia coli, kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda cells (Sf cells, an insect cell), leishmania tarentolae cells, rabbit reticulocyte, chinese hamster ovary cells (CHO cells), african green monkey kidney COS cells, african green monkey kidney VERO cells, baby hamster kidney cells (BHK cells), human Hela cells, human Hybridoma cells (human Hybridoma cells), human fibrosarcoma HT1080 cells, and combinations thereof.

1.1.1.2. Exogenous addition of translation-related elements

The translation-related elements in the in vitro protein synthesis system may be provided or supplemented by means of exogenous additions.

In some preferred modes, the exogenously added translation-related element is obtained by purification after secretory expression by an engineered strain. The kind of the engineering strain includes but is not limited to the cell type of the cell extract prepared in the above 1.1.1.1.

In some preferred forms, the exogenously added translation-related element is derived from E.coli.

In some preferred embodiments, the Protein synthesis Using Recombinant Elements (PURE) system developed by Japanese scientists is used to provide the various factors/various related Elements (derived from E.coli) required for the in vitro Protein synthesis process, respectively, rather than providing the various factors integrally by cell extraction. Reference is made to the introduction of the PURE system in the publications "Lu, Y.Advances in Cell-Free Biosynthetic technology.Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45.", "Y Shimizu, A Inoue, Y Tomari, et al.cell-Free transformed with purified components [ J ]. Nature Biotechnology,2001,19(8):751 755", et al.

In some preferred forms, the exogenously added translation-related element is derived from a eukaryotic cell.

In some preferred forms, the exogenously added translation-related element is derived from a combination of E.coli and a eukaryotic cell.

In some preferred forms, the exogenously added translation-related element is derived from a yeast cell, a mammalian cell, a plant cell, an insect cell, a nematode cell, a pathogen, or a combination thereof.

In some preferred embodiments, the exogenously added translation-related element is derived from kluyveromyces, saccharomyces cerevisiae, saccharomyces pichia pastoris, or a combination thereof.

In some preferred embodiments, the exogenously added translation-related element is derived from kluyveromyces lactis, kluyveromyces marxianus, kluyveromyces polybuvinus, kluyveromyces hainanensis, kluyveromyces williamsii, kluyveromyces fragilis, kluyveromyces hubeiensis, kluyveromyces polyspora, kluyveromyces siamensis, kluyveromyces hirsutus, or a combination thereof.

When two or more kinds of exogenously added translation-related elements are contained in the system, the cells from which the exogenously added translation-related elements are derived may be the same or different.

1.1.2. Exogenous RNA polymerase and exogenous DNA polymerase

The process of in vitro synthesis of proteins includes at least a translation process and optionally also a transcription process.

The transcription process to convert DNA into mRNA is not isolated from RNA polymerase. In this case, the corresponding in vitro protein synthesis system, in some preferred embodiments, further comprises an RNA polymerase, and the source of the RNA polymerase may be selected from: endogenously expressed RNA polymerase (provided via cell extract), exogenously added RNA polymerase, translation products of an exogenous nucleic acid template encoding RNA polymerase, and combinations thereof.

The endogenously expressed RNA polymerase is not added separately but is present in the cell extract.

When the genome of the cell from which the cell extract is derived does not contain the gene encoding RNA polymerase, nor does it endogenously integrate the coding sequence/gene encoding RNA polymerase, it is usually necessary to add an additional exogenous RNA polymerase to facilitate the reaction. For example, when a cell extract of a wild-type Kluyveromyces lactis strain is used, the T7 promoter cannot be recognized by the cell extract prepared from the wild-type Kluyveromyces lactis strain.

In order to achieve the inclusion of endogenously expressed RNA polymerase in the cell extract, some preferred modes are to integrate the coding sequence/genes encoding RNA polymerase into the host cell from which the cell extract is prepared; in particular, some preferred modes are realized by the following modes: the coding sequence of RNA polymerase is inserted into an isolated plasmid in the cell, or the coding gene of RNA polymerase is integrated into the genome of the cell, or a combination of the two methods is adopted to carry out strain modification, and then a cell extract is prepared. Such means of integrating the coding sequence/gene of the RNA polymerase into the genome of the cell include, but are not limited to: insertion into the genome of a cell, in situ replacement of portions of the genome, and combinations thereof.

The exogenously added or endogenously expressed RNA polymerase may each independently preferably be T7 RNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises a DNA polymerase, and the source of the DNA polymerase may be selected from the group consisting of: endogenously expressed DNA polymerase (provided via cell extract), exogenously added DNA polymerase, translation products of an exogenous nucleic acid template encoding the DNA polymerase, and combinations thereof.

The in vitro protein synthesis system optionally comprises an exogenous RNA polymerase or/and a nucleic acid template encoding an RNA polymerase.

The in vitro protein synthesis system, optionally includes an exogenous DNA polymerase or/and a nucleic acid template encoding a DNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises an exogenous RNA polymerase, an exogenous DNA polymerase. Reference CN 108642076A.

The addition of exogenous RNA polymerase to an in vitro protein synthesis system is a conventional technical approach. In vitro protein synthesis systems added with exogenous RNA polymerase reported in the prior art are all included in the invention and used as an optional mode of the in vitro protein synthesis system. For example, the in vitro protein synthesis system of Kluyveromyces lactis with exogenous RNA polymerase (such as T7 RNA polymerase) added in CN108535489A is included in the present invention as an alternative to the in vitro protein synthesis system of the present invention.

The in vitro protein synthesis system can also comprise at least one of the following components: exogenous RNA polymerase, exogenous nucleic acid template for coding RNA polymerase, exogenous DNA polymerase and exogenous nucleic acid template for coding DNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises an exogenous RNA polymerase and an exogenous DNA polymerase.

In some preferred embodiments, the in vitro protein synthesis system comprises exogenous T7 RNA polymerase and exogenous phi29DNA polymerase.

The exogenous RNA polymerase may be added directly, an exogenous nucleic acid template encoding RNA polymerase (translated into RNA polymerase upon protein synthesis) may be added, or a combination thereof. The coding sequence of RNA polymerase may be constructed together with the nucleic acid template encoding the target protein, or may be constructed separately from the target protein.

Similarly, the DNA polymerase can be added directly, or a foreign nucleic acid template containing its coding sequence (translated as DNA polymerase when protein synthesis is performed), or a combination thereof. Either a nucleic acid template encoding the protein of interest or a separate foreign nucleic acid template.

When the nucleic acid template for encoding the target protein is a DNA template, the amplification process of the DNA can be included, or the amplification process of the DNA can not be included; if the in vitro protein synthesis reaction also includes a DNA amplification process, especially when the amount of the DNA template is insufficient, the system needs to contain endogenously expressed or/and exogenously added DNA polymerase, for example, exogenous phi29DNA polymerase is added to CN 108642076A. In some embodiments, after in vitro amplification of the DNA encoding the target protein mmefp, the amplification product is added to the reaction system as a foreign DNA template, and the in vitro protein synthesis reaction may not need to include a DNA amplification process. When a DNA polymerase is added to the system, that is, when the in vitro reaction process includes a DNA amplification process, it is usually necessary to add a substrate for synthesizing DNA.

The DNA polymerase may be a polymerase derived from a eukaryote or a prokaryote. Examples of eukaryotic polymerases are any one or any combination of the following: pol- α, pol- β, pol- δ, pol- ε, and the like, fragments of any of the foregoing, and variants of any of the foregoing (including variants of any of the foregoing fragments). Prokaryotic polymerases are exemplified by any one or any combination of the following: coli (e.coli) DNA polymerase I (e.g., Klenow fragment), e.coli DNA polymerase II, e.coli DNA polymerase III, e.coli DNA polymerase IV, e.coli DNA polymerase V, bacteriophage T4 DNA polymerase, Bacillus stearothermophilus (Bacillus stearothermophilus) polymerase I, Phi29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, Staphylococcus aureus (Staphylococcus aureus) Pol I, etc., a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, a variant of any of the foregoing (including variants of any of the foregoing fragments). Such variants include, but are not limited to, mutants. In some preferred embodiments, the variant is a homolog.

In some preferred embodiments, the polymerase (exogenous RNA polymerase, exogenous DNA polymerase) is a polymerase capable of performing normal temperature amplification, and the normal temperature may be from room temperature to 37 ℃, specifically, may be in a temperature range of 20 ℃ to 37 ℃, 25 ℃ to 37 ℃, and the like. The polymerase capable of carrying out normal-temperature amplification can be selected according to an exogenous nucleic acid template; the room temperature amplification polymerases that can be used in vitro cell-free systems are all included as reference in the scope of the present invention, and include, but are not limited to, phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, exo-klenow DNA polymerase, Bsu DNA polymerase, Pol III DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, T5 RNA polymerase, etc., partial domains of any of the foregoing polymerases, subunits or fragments of any of the foregoing, variants of any of the foregoing, and any combination of the foregoing polymerases and partial domains, subunits, fragments, variants (including, but not limited to, mutants) thereof. The present invention may also employ other DNA polymerases such as Taq DNA polymerase, Pfu DNA polymerase, Pol I DNA polymerase, Pol II DNA polymerase, and the like.

In some preferred embodiments, the DNA polymerase has a strand displacement function.

In some preferred embodiments, the DNA polymerase lacks 3 '-5' exonuclease activity.

The amplification techniques, particularly the normal temperature amplification method, which can be used in the present invention are not particularly limited, and the normal temperature amplification techniques which can be used in vitro cell-free systems are all included in the scope of the present invention by reference.

1.1.3. Energy system/energy regeneration system

An energy system/energy regeneration system is used to provide the energy required for the protein synthesis process.

Energy systems/energy regeneration systems reported for use in vitro cell-free protein synthesis systems can provide energy for the in vitro protein synthesis systems of the invention. Including but not limited to the literature: CN109988801A, CN108535489A, CN110551785A, CN2018116198186, CN2018116198190, US20130316397A, US20150376673A, "MJ Anderson, JC Stark, CE Hodgman and MC Jewett. engineering environmental Cell-Free protein synthesis with glucose synthesis [ J ]. FEBS Letters,2015,589(15): 1723. 1727", "Y Lu. Advances in-Free biosynetic Technology [ J ]. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45", "P research, MT and BC, Cell-Free amino acid synthesis with energy regeneration [ J ]. the present invention is incorporated by reference into the patent literature, see patent literature 34, publication No. 9, et al.

In some preferred embodiments, the energy system is a sugar (e.g., a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide) and phosphate energy system, a sugar and phosphocreatine energy system, a phosphocreatine and phosphocreatine enzyme system, a phosphocreatine and phosphocreatine kinase system, a glycolytic pathway and its intermediate energy system (a monosaccharide and its glycolytic intermediate energy system, a glycogen and its glycolytic intermediate energy system), or a combination thereof. Specifically, the phosphate refers to an inorganic phosphate, and may be orthophosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof. The polysaccharide may be selected from polysaccharides including, but not limited to, starch (soluble starch in some preferred forms), glycogen, dextrins (e.g., maltodextrin, corn dextrin, cyclodextrin), and the like. Examples of the disaccharide include sucrose, maltose and the like. The monosaccharide can be a six-carbon sugar or a five-carbon sugar. Examples of such monosaccharides include: glucose, mannose, lactose, and the like. The glycolytic pathway and its intermediate energy systems include, but are not limited to, glucose-based energy systems.

In some preferred embodiments, the energy system is a sugar and phosphate energy system, and the sugar may be selected from the group including, but not limited to: glucose, fucose, mannose, galactose, lactose, xylose, arabinose, sucrose, maltose, starch, glycogen, dextrins (such as maltodextrin, corn dextrin, cyclodextrin), and any combination thereof.

The concentration of each component in the energy system is not particularly limited, including but not limited to, the use of the presently reported protocols and equivalents thereof. In some preferred examples, the energy system used is a complex energy system consisting of monosaccharides (glucose), polysaccharides (maltodextrin or corn dextrin) and phosphates.

1.1.4. Substrate for RNA synthesis

The substrate for RNA synthesis refers to a starting material capable of providing a structural unit of RNA. In some preferred embodiments, the substrate of the synthetic RNA is a mixture of nucleotides. In some preferred embodiments, the substrate for the synthetic RNA is a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof. In some preferred embodiments, the substrate for the synthetic RNA is a mixture of Nucleoside Triphosphates (NTPs). In some preferred modes, the nucleoside triphosphate mixture is a mixture of adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate or/and uracil nucleoside triphosphate; in other preferred embodiments, the nucleoside triphosphate mixture is a mixture of the four nucleoside triphosphates. In the present invention, the concentration of each single nucleotide is not particularly limited, and may be measured as the nucleotide required for synthesizing the protein. In some preferred embodiments, the concentration of each mononucleotide is 0.5 to 5 mM. In other preferred embodiments, the concentration of each mononucleotide is 1.0 to 2.0 mM. The concentration of each single nucleotide is each independently exemplified by any one of the following concentrations, or a range of concentrations between any two of the following concentrations (the range of concentrations includes both endpoints): 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0 mM. The above concentrations refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

1.1.5. Substrate for DNA synthesis

In the case of DNA amplification or in vitro protein synthesis reactions involving DNA replication, it is often necessary to add substrates for DNA synthesis. The substrate for synthesizing DNA refers to a raw material capable of providing a structural unit of DNA. The substrate for the synthetic DNA is in some preferred forms a mixture of deoxynucleotides; in some preferred forms, mixtures of deoxynucleoside triphosphates (dNTPs).

When the in vitro protein synthesis system contains a DNA polymerase, it preferably also contains a substrate for synthesizing DNA.

1.1.6. Substrates for synthetic proteins

The substrate of the synthetic protein refers to a raw material capable of providing amino acid units constituting the protein. In some preferred forms, the substrate of the synthetic protein is a mixture of amino acids. In some preferred modes, the amino acids required for synthesis of the protein are metered in.

The concentration of each amino acid is independent. When the substrate of the synthetic protein contains different kinds of amino acids, the amounts of any two kinds of amino acids may be the same or different independently from each other.

The concentration of any amino acid is preferably 0.01 to 5mM in one embodiment, and 0.1 to 1mM in another embodiment. The concentration of each amino acid is, independently, exemplified by any one of the following concentrations, or a range of concentrations between any two of the following concentrations (the range of concentrations includes both endpoints): 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.2mM, 1.5mM, 1.8mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0 mM. The above concentrations refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

The amino acid mixture at least comprises amino acid mixtures required by the process for synthesizing the target protein, and is selected from the group consisting of but not limited to: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, combinations thereof. In some preferred forms, the amino acid mixture is a mixture of the aforementioned twenty amino acids. The amino acid mixture required in the process of synthesizing the target protein not only comprises the amino acid forming the primary sequence of the target protein, but also comprises other amino acids involved in the synthesis process.

The amino acid mixture can include natural amino acids, unnatural amino acids, or combinations thereof.

The amino acid mixture may comprise_L-an amino acid,_D-amino acids, or combinations thereof.

The amino acid mixture may include, in addition to natural amino acids, unnatural amino acids,_D-amino acids, radioisotope labelled amino acids, modified amino acids, etc. The unnatural amino acid is not particularly limited and may be selected from the group consisting of: including but not limited to unnatural amino acids reported or cited in the following documents: "Y Lu. cell-free synthetic biology Engineering in an open world [ J ] ].Synthetic and Systems Biotechnology,2017,2,23-27”、“W Gao,E Cho,Y Liu and Y Lu.Advances and challenges in cell-free incorporation of unnatural amino acids into proteins[J]Frontiers in pharmacology,2019,10:611 ", and the like, and documents cited directly or indirectly. The radioisotope-labeled amino acid is not particularly limited, and includes, but is not limited to, isotopic labeling employed in the reported field of protein synthesis. The modified amino acid is not particularly limited, including but not limited to modification by amino acid side groups.

In some preferred forms, the amino acid mixture is a mixture of natural amino acids.

In some preferred forms, the amino acid mixture is a mixture of twenty natural amino acids.

1.1.7. Other exogenously added components

The in vitro protein synthesis system can also comprise any one or the combination of the following exogenous addition components: DNA amplification related elements, RNA amplification related elements, rnase inhibitors, crowding agents (in some preferred forms polyethylene glycol and/or the like), magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, buffers, aqueous solvents. Reference may be made to WO2016005982A1, US20060211083A1, "L Kai, V

R Kaldenhoff and F Bernhard.Artificial environments for the co-translational stabilization of cell-free expressed proteins[J]PloS one,2013,8(2): e56637 ", US20030119091a1, US20180245087a1, US5665563, WO2019033095a1, US9410170B2, US9528137B2 and the like and documents cited directly or indirectly thereof.

The in vitro protein synthesis system optionally includes exogenously added translation-related elements. When the cell extract is insufficient to provide all the translation-related elements necessary for the synthesis of the target protein (in deficient species and/or in deficient amounts), it is also possible to add missing or deficient translation-related elements by means of exogenous addition. In particular, when the endogenous secretion product of the source strain is deficient in a component for the expression of the heterologous protein, it can be supplemented by exogenous addition. For example, the purified translation-related element may be selected from, including but not limited to, any one of the following groups or a combination thereof: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors. The translation-related enzymes include, but are not limited to, various aminoacyl-tRNA synthetases, peptidyl transferases, RNA polymerases, and the like, or combinations thereof. The exogenously added translation-related element, in some preferred forms, is a purified translation-related element.

When the protein synthesis process involves DNA amplification, elements associated with DNA amplification may be added by exogenous means in addition to the means provided endogenously. The DNA amplification-related elements may include, in addition to DNA polymerase, other factors such as helicase (HDA amplification), recombinase and single-stranded DNA binding protein (RPA amplification), DNA ligase, and the like, depending on different amplification mechanisms.

When the protein synthesis process involves RNA amplification, elements associated with RNA amplification may be added by exogenous means in addition to the means provided endogenously. Depending on the amplification mechanism, other factors such as reverse transcriptase, RNA ligase, etc. may be included in addition to RNA polymerase.

The RNA inhibitor may function to stabilize RNA.

In some preferred embodiments, the in vitro protein synthesis system further comprises crowding agents (crowding agents) for mimicking the crowded macromolecular environment within the cell. The structure of the crowding agent is not particularly limited, and may be linear or non-linear, and the non-linear structure includes, but is not limited to, branched, multi-armed, cyclic, comb-shaped, tree-shaped, star-shaped, and other structural types. In some preferred examples, the crowding agent may be selected from the group consisting of: polyethylene glycol, polyvinyl alcohol (PVA), polystyrene (polystyrene), dextran (dextran), sucrose polymers (e.g., Ficoll sucrose polymers, such as Ficoll-400), polyvinylpyrrolidone (PVP, poly (vinylpyrrolidone), albumin, the like, any combination thereof. Sources of albumin include, but are not limited to: human serum albumin, bovine serum albumin, porcine serum albumin, and combinations thereof; in some preferred embodiments, the albumin is human serum albumin (human serum albumin). The crowding agents can also be referred to as crowding agents disclosed in the following documents: the document "X Ge, D Luo and J xu. cell-free protein expression under macromolecular growth conditions [ J ]. PLoS One,2011,6(12): e 28707" and references cited therein. In some preferred embodiments, the concentration of crowding agent in the in vitro protein synthesis reaction mixture is sufficient to increase the amount of protein synthesis.

In some preferred embodiments, the crowding agent has a molecular weight of no more than 400 kDa. In some preferred embodiments, the crowding agent has a molecular weight of no more than 200 kDa. Generally, the molecular weight specification preferably has a molecular weight distribution of. + -. 10% or less. In a preferred embodiment, the amount of the crowding agent is selected from 0.5% to 15%, and further from 1% to 12%, in terms of the weight percentage (wt%) or volume percentage (% (v/v)) or mass-to-volume concentration (% (w/v)) of the crowding agent in the in vitro protein synthesis reaction mixture system; for example, any one of the following concentration values, or a concentration range between any two of the following concentration values (the concentration range includes both endpoints): 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%. The amount of the crowding agent may be adjusted to be reduced appropriately according to the amount of the protein fixation system. Particularly, when the protein immobilization system comprises a polymer linker, the amount of the crowding agent may be appropriately adjusted to increase the amount of the protein synthesized, depending on the content of the polymer linker in the reaction purification mixture system for in vitro protein synthesis, the hydrodynamic radius, and the like.

In some preferred embodiments, the in vitro protein synthesis system further comprises polyethylene glycol and/or analogs thereof, which act as crowding agents. Such as polyethylene glycol, among others, can also adjust the system viscosity. Polyethylene glycol having CH₂CH₂Repeating unit of O(EO units), commonly referred to as PEG (polyethylene glycol), PEO (poly (ethylene oxide), POE (polyoxyethyleneene). Analogs of the polyethylene glycol include, but are not limited to, copolymers rich in EO units, polyethylene glycol derivatives, other polyoxyalkylenes that can act as crowding agents (e.g., polyoxypropylene, POP) and their derivatives, and the like; the derivatives, taking polyethylene glycol derivatives as examples, include, but are not limited to, chemical modifications (such as methoxy polyethylene glycol, amino modifications, carboxyl modifications, etc.), amino acid modifications, polypeptide modifications, protein modifications, block polymers containing polyethylene glycol blocks, polymers containing polyethylene glycol side chains, etc. The concentration of polyethylene glycol or an analog thereof is not particularly limited, and generally, the concentration of polyethylene glycol or an analog thereof is 0.1% to 10%, in some preferred manners 0.1% to 8%, in other preferred manners 0.5% to 4%, and in other preferred manners 1% to 2%, in terms of mass volume concentration (% (w/v)) in the in vitro protein synthesis reaction mixture system or in terms of total weight (wt%). Unless otherwise specified, the present invention refers to the mass volume concentration in% (w/v), e.g., 2%, which means 2% (w/v), corresponding to 2g/100mL, 20 mg/mL. In some preferred embodiments, the molecular weight of the polyethylene glycol and/or the analog thereof is no more than 40000Da, and representative molecular weights are, for example, any one of the following molecular weights or a numerical interval between any two of the following molecular weights (inclusive): 200. 400, 500, 600, 750, 800, 1000, 1200, 1400, 1450, 1500, 1600, 1800, 2000, 2500, 3000, 3350, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 15000, 16000, 18000, 20000, 25000, 30000, 35000, 40000; unit Da; each of the above numbers is numerically equal to the weight average molecular weight or the number average molecular weight. In some preferred embodiments, the molecular weight distribution is ± 10% or less with respect to the molecular weight specification. The molecular weight of the polyethylene glycol and/or the analogue thereof is 200Da to 10000Da in some preferred modes, 3000Da to 10000Da in other preferred modes, 200Da to 8000Da in other preferred modes, 2000Da to 8000Da in other preferred modes, and 3000Da in other preferred modes 8000 Da. In the present invention, the molecular weight of polyethylene glycol or the like refers to the weight average molecular weight M unless otherwise specified_w. Representative PEGs are selected from the group consisting of: PEG200, PEG400, PEG1000, PEG1500, PEG2000, PEG3000, PEG3350, PEG5000, PEG6000, PEG8000, PEG10000, and the like, combinations thereof; wherein the number of 3350 and the like is numerically equal to the weight average molecular weight.

The magnesium ion is derived from a magnesium ion source, which may be selected from the group consisting of, but not limited to: magnesium aspartate (which may preferably be magnesium L-aspartate), magnesium gluconate, magnesium glutamate (which may preferably be magnesium L-glutamate), other magnesium amides, magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, and combinations thereof. In some preferred embodiments, the concentration of the magnesium ion is in the range of 0.1 to 50 mM. In other preferred embodiments, the concentration is in the range of 0.5 to 20 mM. In other preferred embodiments, the concentration is in the range of 1 to 10 mM. In some preferred forms, the source of magnesium ions is selected from any one, any two, any three or all of magnesium aspartate, magnesium gluconate, magnesium glutamate and magnesium acetate.

The potassium ion is derived from a potassium ion source, which may be selected from the group consisting of, but not limited to: potassium acetate, potassium glutamate (potassium L-glutamate may be preferred), other potassium amino acids, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, and combinations thereof. In some preferred embodiments, the concentration of the potassium ion is in the range of 0 to 500 mM. In other preferred embodiments, the concentration is in the range of 1 to 250 mM. In other preferred embodiments, the concentration is in the range of 5 to 200 mM. In other preferred embodiments, the concentration is in the range of 10 to 100 mM. In some preferred forms, the source of potassium ions is selected from any one, any two, any three, or all of potassium aspartate, potassium glutamate, potassium acetate, and potassium phosphate.

The cation of the soluble amino acid salt may include, but is not limited to, magnesium ion, potassium ion, sodium ion, zinc ion, calcium ion, ammonium ion, etc., and the amino acid residue portion thereof may be selected from any of the amino acids (natural, non-natural, derivatives, etc.) and combinations thereof described herein. Examples of such soluble amino acid salts include, but are not limited to, magnesium aspartate, magnesium glutamate, potassium aspartate, potassium glutamate, and may also include, but are not limited to, sodium aspartate, sodium glutamate, sodium tryptophan, potassium glycinate, potassium leucine, and the like.

The optimization and optimization of polyethylene glycol, magnesium ions, potassium ions and the preferred mode thereof reported in WO2016005982A1 are optionally incorporated herein by reference.

The antioxidant, which may also be referred to as a reducing agent. May include, but is not limited to, Dithiothreitol (DTT), 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced Glutathione (GSH), tricarboxymethylphosphonic acid (TCEP), 3-mercapto-1, 2-propanediol (MPD), and the like. In some preferred forms dithiothreitol. DTT is used in the conventional concentration, such as 0.5-10 mM in some embodiments; in other embodiments, a concentration of 0 to 1.7mM is used.

The anti-freezing agent may be selected from anti-freezing agents used for solid formulations including, but not limited to, WO2018138195A1 and references cited therein. As the antifreeze, trehalose, dimethyl sulfoxide (DMSO), glycerol and the like are exemplified. The purpose of adding the antifreezing agent is mainly to enable the whole system or partial subpackaged components of the system to be stored at low temperature, particularly when the components are stored in a kit mode. The added antifreezing agent allows the function of regulating the in vitro protein synthesis reaction.

Some types of cryoprotectants, including but not limited to trehalose, may also be a constituent component of the energy system.

The reaction promoter includes, but is not limited to, a reaction promoter (e.g., aluminum salt) as provided in CN 109971783A. In some preferred embodiments, the reaction promoter is an aluminum salt, an aluminum oxide (e.g., alumina), an iron salt, an iron oxide, a calcium salt, or a combination thereof.

Such anti-foaming agents are exemplified by those provided in CN1934276A and its cited references. Specific examples include, but are not limited to, alkyl polyoxyalkylene glycol ethers (alkyl polyoxyalkyllene glycol ethers), esters, silicones, polysiloxanes, sulfites, sulfonates, fatty acids and derivatives thereof, and the like.

The alkane may function to provide a hydrophobic interface or to mimic a hydrophobic environment. The relevant content of patent application CN202010179689.4 is incorporated herein by reference. Examples of said alkanes are C_6～44Pure or mixed alkanes, such as cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetradecane, vaseline, etc.

The buffer is mainly used for maintaining the pH environment of the system. In some preferred modes, the compound is selected from any one or a combination of the following: Tris-HCl, Tris base, HEPES (4-hydroxyethyl piperazine ethanesulfonic acid).

The aqueous solvent, in some preferred forms, is a buffer.

It is to be noted that any one of the components of the in vitro protein synthesis system according to the present invention allows for the addition of functions or purposes other than those previously described.

Any one of the components of the in vitro protein synthesis system to which the present invention relates allows two or more functions to be performed. For example, some sugar components may serve as both components of the energy system and functional components such as crowding agents, freezing point depressants, and the like.

1.1.8. Specific embodiments of the in vitro protein Synthesis System

The concentrations of the components in the following embodiments are final concentrations (relative to the mother liquor) corresponding to the initial concentrations in the in vitro protein synthesis reaction mixture.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract, an endogenously expressed RNA polymerase (contained in the cell extract) or an exogenously added RNA polymerase, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a crowding agent, magnesium ions, potassium ions, a buffer, and optionally any one of the following exogenous components: an exogenous nucleic acid template (which may independently be a preferred DNA template), an endogenously expressed DNA polymerase or an exogenously added DNA polymerase, an exogenous nucleic acid template (which may independently be a preferred DNA template) encoding a DNA polymerase, other DNA amplification related elements, substrates for DNA synthesis, translation related elements, RNA amplification related elements, rnase inhibitors, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, aqueous solvents. The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a yeast cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract (the cell source has been modified by a strain to integrate a gene encoding an RNA polymerase into the genome of the cell or inserted into an episomal plasmid) and further comprises one or more exogenous components selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K), Tris or a salt thereof (e.g., hydrochloride), potassium acetate, potassium glutamate (optionally potassium L-glutamate), potassium chloride, magnesium acetate, magnesium glutamate (optionally magnesium L-glutamate), magnesium aspartate (optionally magnesium L-aspartate), magnesium gluconate, soluble amino acid salts, nucleoside triphosphate mixtures (NTPs), amino acid mixtures, creatine phosphate, creatinase phosphate, creatine phosphate kinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, phosphate (e.g., potassium phosphate), DNA amplification-related elements, deoxynucleoside triphosphate mixtures, RNA amplification-related elements, RNase inhibitors, polyethylene glycol, Tris, and mixtures thereof, Dextran, sucrose polymer, Dithiothreitol (DTT). The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a yeast cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract and further comprises one or more exogenous components selected from the group consisting of: HEPES-K, Tris or a salt thereof, potassium acetate, potassium glutamate (may preferably be L-potassium glutamate), potassium chloride, magnesium acetate, magnesium glutamate (may preferably be L-magnesium glutamate), magnesium aspartate (may preferably be L-magnesium aspartate), magnesium gluconate, soluble amino acid salts, nucleoside triphosphate mixtures (NTPs), amino acid mixtures, creatine phosphate kinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, phosphate (e.g., potassium phosphate), exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, other DNA amplification-related elements, deoxynucleoside triphosphate mixtures, RNA amplification-related elements, RNase inhibitors, polyethylene glycol, dextran, sucrose polymers, glucose-containing polymers, and the like, Dithiothreitol. The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a yeast cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract (the source cell is optionally strain engineered, optionally incorporates a gene encoding an RNA polymerase into the genome of the cell or is inserted into an intracellular episomal plasmid) and further comprises one or more exogenous components selected from the group consisting of: HEPES-K, Tris (hydroxymethyl) aminomethane hydrochloride (Tris & HCl), potassium acetate, potassium glutamate (optionally, potassium L-glutamate), potassium chloride, magnesium acetate, magnesium glutamate (optionally, magnesium L-glutamate), magnesium aspartate (optionally, magnesium L-aspartate), magnesium gluconate, soluble amino acid salts, nucleoside triphosphate mixtures (NTPs), amino acid mixtures, phosphocreatine, phosphocreatinase, phosphocreatine kinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, potassium phosphate, RNase inhibitors, polyethylene glycol, dextran, sucrose polymers, dithiothreitol, trehalose, alumina promoters, antifoam agents, alkanes, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, and mixtures thereof, A DNA template coding for T7 RNA polymerase, a DNA template coding for phi29 DNA polymerase, other DNA amplification related elements, a deoxynucleoside triphosphate mixture, and RNA amplification related elements. The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a yeast cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

In some preferred embodiments, the in vitro protein synthesis system comprises a cell extract and further comprises one or more exogenous components selected from the group consisting of: HEPES-K, Tris & HCl (pH8.0), potassium acetate, potassium glutamate (preferably L-potassium glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably L-magnesium glutamate), magnesium aspartate (preferably L-magnesium aspartate), magnesium gluconate, glucose, L-arabinose, sucrose, maltose, maltodextrin, corn dextrin, cyclodextrin, nucleoside triphosphate mixture (four nucleoside triphosphate mixture, wherein the concentration of single nucleoside triphosphate can be the same or independent of each other), amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, glutamic acid, arginine, lysine, arginine, lysine, arginine, lysine, arginine, lysine, and/or a mixture thereof, Arginine and/or histidine; mixtures of twenty amino acids may be preferred; wherein, the concentrations of the single amino acids can be the same or independent of each other), potassium phosphate, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, other DNA amplification related elements, deoxynucleotide triphosphate mixture, RNA amplification related elements, RNase inhibitor, polyethylene glycol, dextran, sucrose polymer, dithiothreitol. The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a yeast cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

Specifically, in some preferred embodiments, the in vitro protein synthesis system comprises 50% to 80% (v/v) of the cell extract, and further comprises one or more components selected from the group consisting of: 9.78mM Tris-HCl (pH8.0), 20-80 mM potassium acetate, 2-10 mM magnesium acetate, 1.5-8 mM magnesium L-aspartate (more preferably 1.5-6 mM), 0-20 mM magnesium D-aspartate, 1.5-10 mM magnesium gluconate, 0.5-5 mM four nucleoside triphosphates (the concentrations of the single nucleoside triphosphates may be the same, such as 1.8mM, or may be independent of each other), 0.1-1 mM twenty amino acid mixtures (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, the concentrations of the single amino acids may be the same, such as 0.5mM, or may be independent of each other), 10-40 mM glucose, 5-110 mM L-arabinose, L-aspartic acid, lysine, arginine, and histidine, 200-400 mM maltodextrin (about 52mg/mL when measured as glucose monomer, such as 320 mM), 10-40 mM potassium phosphate, 0.5-5% (w/v) polyethylene glycol (such as 2% (w/v)), and 0.4-5 mM dithiothreitol (such as 0.44 mM). The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a yeast cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

One embodiment of the in vitro protein synthesis system further includes, but is not limited to, for example, the cell-free E.coli-based protein synthesis system described in WO2016005982A 1. Other citations of the present invention, including but not limited to in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, as described in direct and indirect citations thereof, are also incorporated herein as embodiments of the in vitro protein synthesis system of the present invention. For example, the in vitro Cell-Free protein synthesis system described in the "Lu, Y.Advances in Cell-Free biosynthestic technology. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45" section, including but not limited to the "2.1 Systems and Advantages" section, pages 27-28, can be used as an in vitro protein synthesis system for carrying out the present invention. For example (unless conflicting with the present invention, the following documents and cited documents are cited in full contents and for full purposes), documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A (CN2018108881848), CN110845622(CN2018109550734), CN109971775A (CN 2018131331300), CN109971775A (CN 2018123211477), CN109971775A (CN2018116083534), CN109971775A (CN 20116116198186), CN 1613672 (CN 201819898190), CN109971775A (CN 20191021619, CN 20191285672 (CN 201201918191023148), CN 20201091023 91023, CN 202010890108981813, CN 202010890108901089518, CN 2022693393, CN 202359181919891989, CN 20201091989, CN 2020109181918191989, CN 202010910108948, CN 202010890108901091819181918193393, CN 202010894, CN 20226933, CN 2023536933, CN. The methods for preserving the components, the forms of preservation in the kit and the dispensing means in the above-mentioned documents are also included in the present invention as alternatives.

1.2. Exogenous nucleic acid template

The foreign nucleic acid template of the present invention refers to a nucleic acid template encoding a target protein, unless otherwise specified. In addition, the exogenous nucleic acid template of the present invention, where indicated, may also include nucleic acid templates encoding protein factors or proteases required for in vitro protein synthesis processes, such as, for example, exogenous nucleic acid templates encoding RNA polymerase, exogenous nucleic acid templates encoding DNA polymerase.

If the synthesis system does not have a nucleic acid template encoding the target protein, the in vitro synthesis reaction of the target protein cannot be performed.

The nucleic acid template encoding the protein of interest in any embodiment of the invention may be independently a DNA template, an mRNA template, or a combination thereof.

The nucleic acid template encoding the protein of interest in any embodiment of the invention may independently preferably be a DNA template.

The nucleic acid template encoding the target protein serves as a direct template (mRNA), an indirect template (DNA), or a combination thereof for synthesizing the target protein.

The nucleic acid template encoding the protein of interest is allowed to include non-coding regions. The expression product can be polypeptide or protein, and can also be fusion protein. One translation (or transcription translation) process is performed on one nucleic acid template molecule, allowing the number of polypeptide or protein molecules synthesized to be 1, 2, or more.

The transcription and translation mode protein synthesis process uses a DNA template as an indirect template, and the translation mode protein synthesis process can use an mRNA template as a direct template.

In some preferred embodiments, the in vitro protein synthesis system of the present invention is an in vitro transcription and translation system, i.e., an IVTT system, using a DNA template as a nucleic acid template encoding a protein of interest.

The nucleic acid template encoding the target protein contains translation-related elements required for synthesis of the target protein.

In any embodiment of the invention, it may be independently preferred that the nucleic acid template encoding the protein of interest contains a promoter element that is recognized by the cell extract.

In some preferred embodiments, the nucleic acid template encoding the protein of interest comprises a promoter element recognized by the cell extract.

In some preferred embodiments, the nucleic acid template encoding the target protein contains a T7 promoter capable of initiating a gene transcription process of the target protein, i.e., the gene transcription process of the target protein is initiated by the T7 promoter on the nucleic acid template.

In some preferred embodiments, the nucleic acid template encoding the target protein comprises a T7 promoter capable of initiating a gene transcription process for the target protein (in which case the T7 promoter is located upstream of the coding sequence for the target protein in the nucleic acid template and the T7 promoter initiates the gene transcription process for the target protein), and the in vitro protein synthesis system comprises a cell extract comprising endogenously expressed T7 RNA polymerase.

In some preferred embodiments, the nucleic acid template encoding the protein of interest comprises a protein of interest translation system, a resistance gene translation system, a lac repressor translation system; the translation systems each include a corresponding promoter.

In some preferred embodiments, the nucleic acid template encoding the protein of interest further comprises a gene that controls plasmid copy number.

In some preferred embodiments, the nucleic acid template encoding the protein of interest further comprises a transcription enhancing element, such as a kozak sequence.

In some preferred embodiments, the nucleic acid template encoding the protein of interest further comprises a translation enhancing element, such as a translation enhancer element, an IRES element, a kozak sequence, and the like.

The exogenous nucleic acid template, including but not limited to the nucleic acid template encoding the target protein, may also adopt the nucleic acid template structure disclosed in the following documents: CN108690139A, CN109022478A, CN109423497A, CN109837293A, CN109971775A, CN110408635A, CN110408636A, CN110551700A, CN110551745A, CN110819647A, and CN 110845622A.

1.2.1. Exogenous DNA template (including DNA template encoding target protein)

The foreign DNA template of the present invention is, unless otherwise specified, specifically a DNA template encoding a target protein.

The exogenous DNA template of the present invention may be DNA, cDNA, methylated DNA, or a combination thereof. Wherein, the cDNA can be obtained by reverse transcription of RNA or miRNA. miRNA (MicroRNA) is a non-coding single-stranded RNA molecule which is coded by endogenous genes and has the length of about 20-25 nucleotides.

The DNA template for coding the target protein contains a coding sequence of the target protein.

In some preferred embodiments, the DNA template encoding the target protein contains a gene encoding the target protein.

The DNA template encoding the target protein is determined according to the amino acid sequence of the target protein.

The DNA template encoding the target protein may further contain other functional elements such as a promoter, a terminator, an enhancer (for example, enhancer elements described in documents CN109423497A, CN109022478A, CN109837293A (CN201711194355.9), CN109971775A and the like and cited documents thereof, such as an omega sequence and a homologous sequence thereof, a combined enhancer element), a kozak sequence (references CN109022478A, CN109837293A, CN109971775A and the like and cited documents thereof), an IRES element (internal ribosome entry sequence, references CN109022478A, CN109423497A and the like and cited documents thereof), a Multiple Cloning Site (MCS), a gene controlling the copy number of a plasmid, and the like. The DNA template encoding the target protein may further contain a coding sequence/coding gene encoding other amino acid chains such as a signal peptide (corresponding to a signal sequence), a leader peptide (corresponding to a leader sequence), a functional tag (e.g., a purification tag, a solubilization tag), a linker peptide, and the like. The DNA template encoding the target protein may further contain a 5 'untranslated sequence and a 3' untranslated sequence. The nucleic acid sequences of the solubilization tags disclosed directly or indirectly in patent application CN201911204796.1 and the cited documents are incorporated herein by reference.

In some preferred embodiments, the DNA template encoding the protein of interest comprises a purification tag sequence, and the translated expression product comprises a purification tag capable of specifically binding to a purification medium in the protein immobilization system, and capturing the translated protein product from the reaction mixture onto the protein immobilization system.

In some preferred embodiments, the DNA template encoding the protein of interest contains a promoter element. The promoter element is required to be recognized by the cell extract used or other components of the in vitro protein synthesis system; it may be a promoter recognized by a wild-type cell extract, or a strain from which a cell extract is derived may be modified to recognize the promoter. The promoter in the DNA template of the invention may be selected from the group consisting of: AOD1, MOX, AUG1, AOX1, GAP, FLD1, PEX8, YPT1, LAC4, PGK, ADH4, AMY1, GAM1, XYL1, XPR2, TEF, RPS7, T7, and combinations thereof. References include, but are not limited to, the following and citations thereof: "Cereghino G. applications of yeast in Biotechnology: protein production and genetic analysis. Current operation in Biotechnology,1999,10(5), 422-" 427 ".

In some preferred examples, the exogenous DNA template uses T7 promoter to start the transcription program of the target protein; the T7 promoter is a strong promoter capable of specifically reacting to T7 RNA polymerase.

In some preferred embodiments, the exogenous DNA template comprises a T7 promoter capable of initiating the gene transcription process of the protein of interest.

The concentration of the exogenous DNA template is selected according to factors such as the amount of target protein to be expressed in an experimental scheme, the protein synthesis rate and the like. In some preferred modes, the concentration of the exogenous DNA template is 1-400 ng/. mu.L. In other preferable modes, the concentration of the exogenous DNA template is 1-80 ng/. mu.L. In other preferable modes, the concentration of the exogenous DNA template is 5-50 ng/. mu.L. In other preferable modes, the concentration of the exogenous DNA template is 1-50 ng/. mu.L. In the present invention, the DNA template is added at a final concentration which is an initial concentration in a mixed system for in vitro protein synthesis reaction, unless otherwise specified.

The exogenous DNA template may be circular DNA or linear DNA. May be single-stranded or double-stranded. The gene encoding the protein of interest may be selected from the group including, but not limited to: genomic sequences, cDNA sequences, and combinations thereof. The exogenous DNA template may also contain a promoter sequence, a 5 'untranslated sequence, and a 3' untranslated sequence.

In some preferred embodiments, the exogenous DNA template further comprises any one or a combination of elements selected from the group consisting of: promoters, terminators, poly (a) elements, transport elements, gene targeting elements, selection marker genes, enhancers, IRES elements, kozak sequences, resistance genes, transposase-encoding genes, signal sequences (signal sequences), leader sequences (for example, as described in CN109022478A and cited therein), genes controlling plasmid copy number (rop genes), tags enhancing translation level (for example, polypeptide tags as described in CN 2019112066163), other functional tags (for example, purification tags, fluorescence tags, solubilization tags, etc.), and the like. Reference is made to US20060211083a1 et al.

The exogenous DNA template may also be constructed in an expression vector. One of ordinary skill in the art can use well-known methods to construct expression vectors containing genes encoding proteins of interest. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

For example, the nucleic acid construct of the "Z1-Z2" structure is inserted into the cloning site of a plasmid vector as plasmid DNA; wherein, Z1 is a promoter, "-" is a covalent bond or a nucleotide fragment, and Z2 is a coding sequence of a target protein. In some preferred modes, Z1 is the T7 promoter.

In some preferred embodiments, the exogenous DNA template is a circular DNA, and may be further preferably a plasmid DNA. The corresponding plasmid DNA is not particularly limited as long as it can react with a cell extract of the system to synthesize the target protein. Generally, the plasmid contains functional elements such as a promoter, a terminator, and an untranslated region (UTR). In some preferred embodiments the plasmid contains a promoter that is recognized by the in vitro protein synthesis system; in particular, in some preferred modes, the plasmid contains a promoter that is recognized by a cell extract. For example, plasmids containing the T7 promoter can in principle be used as foreign DNA templates or plasmid vectors. For example, pET series plasmids of Escherichia coli, pGEM series plasmids, and the like can be used to practice the present invention. In other preferred embodiments, the plasmid DNA contains a promoter that is recognized by the exogenously added component.

Taking the example of the transcription process of target protein initiated by exogenous DNA template using T7 promoter, the T7 promoter can be recognized by endogenously expressed T7 RNA polymerase in cell extract, or by exogenously added T7 RNA polymerase, or by translation product of gene encoding exogenously added T7 RNA polymerase.

Linear DNA can be obtained by in vitro nucleic acid amplification techniques. The amplification techniques that can be used are not particularly limited and include, but are not limited to, PCR amplification techniques, isothermal amplification techniques, room temperature amplification techniques, and the like. In some preferred modes, the isothermal amplification technology is a normal temperature amplification technology.

In some preferred embodiments, the exogenous DNA template is linear DNA and is a PCR linear fragment. The PCR linear fragment can be obtained by reported PCR technology.

In other preferred embodiments, the exogenous DNA template is linear DNA, and is double-stranded linear DNA obtained using an amplification system. The amplification system is not particularly limited, and may be selected from, but not limited to, existing commercial kits and amplification systems reported in the literature, as long as it can amplify the DNA template of the present invention encoding the target protein. Examples include, but are not limited to, commercial DNA amplification systems provided by Biomatch, Neta Scientific Inc., ABM, Thermo Fisher Scientific, Expedeon, Vivantis, and the like.

In other preferred embodiments, double-stranded DNA is used as the exogenous DNA template and is constructed in a circular plasmid vector. The plasmid vector used typically contains functional elements such as the T7 promoter, the T7 or LAC4 terminator, the 5 'UTR, and the 3' UTR.

As some specific embodiments, in examples 5-7, double-stranded DNA was used as a template for foreign DNA and constructed in a circular plasmid vector; these plasmids contain the T7 promoter as a promoter for initiating transcription and translation of the target protein; in examples 5-7, T7 RNA polymerase was endogenously expressed by the modified Kluyveromyces lactis, cell extracts were prepared from the modified strains, and an in vitro cell-free protein synthesis system was constructed in which the T7 promoter was suitable for cell-free in vitro expression of various proteins. The plasmid also contains LAC4 terminator, UTR and other functional elements.

In some embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a coding sequence of a target protein, a 3' non-coding region, a terminator, a replication initiation site (f1 ori), an AmpR promoter, an ampicillin resistance gene (AmpR gene), a high copy number replication initiation site (ori), a gene controlling the copy number of a plasmid (rop gene), a lacI promoter, a coding sequence of lacI.

In some embodiments, the plasmid DNA comprises at least the structural elements identified in table 1.

TABLE 1 description of the major structural elements of a plasmid DNA (pD2P-mEGFP)

In other embodiments, in addition to the functional elements of table 1, a purification tag, such as for example a polyhistidine tag (His-tag), is present between the 5' UTR and the coding sequence of the protein of interest.

In other embodiments, in addition to the functional elements of table 1, a kozak sequence is present downstream of the 5' UTR to increase translation levels.

In other embodiments, in addition to the functional elements of embodiment table 1, there is a coding sequence for a signal peptide (signal sequence) between the 5 'UTR and the coding sequence for the protein of interest, downstream of the 5' UTR.

In other embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a leader sequence, a coding sequence of a target protein, a 3' non-coding region, a terminator, a replication initiation site (f1 ori), an AmpR promoter, an AmpR gene, a high copy number replication initiation site (ori), a gene controlling the copy number of a plasmid (rop gene), a lacI promoter, a coding sequence of lacI.

In other embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a coding sequence of a signal peptide, a coding sequence of a target protein, a 3' non-coding region, a terminator, an ori of f1, an AmpR promoter, an AmpR gene, an ori, a rop gene, a lacI promoter, a coding sequence of lacI. Specifically, for example, the following functional elements are included in the plasmid DNA: a T7 promoter, a 5 'noncoding region, a coding sequence of a signal peptide, a coding sequence of a target protein mEGFP, a 3' noncoding region, a T7 terminator or LAC4 terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, a coding sequence of lacI.

In other embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a coding sequence for a signal peptide, a coding sequence for a purification tag, a Multiple Cloning Site (MCS), a coding sequence for a protein of interest, a 3' non-coding region, a terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, a coding sequence for lacI. Specifically, for example, the following functional elements are included in the plasmid DNA: a T7 promoter, a 5 'noncoding region, a coding sequence for a signal peptide, a coding sequence for a purification tag, MCS, a coding sequence for the protein of interest mffp, a 3' noncoding region, LAC4 terminator or T7 terminator, f1 ori, AmpR promoter, AmpR gene, ori, rop gene, lacI promoter, a coding sequence for lacI.

The basic structure of the plasmid and the method for inserting the coding gene of the target protein into the plasmid vector can adopt the conventional technical means in the field, and are not described in detail herein. For example, patent documents CN108690139A, CN107574179A, CN108949801A and the like can be referred to. For example, the basic structure of the plasmid can be referred to the attached drawings of the Chinese patent application CN 201910460987.8.

In the present invention, the concentration of the DNA template encoding a non-target protein can be determined in accordance with the desired expression amount of the non-target protein with reference to the amount of the above-mentioned DNA template encoding a target protein. The non-target protein refers to a translation product that is not a target expression protein but is synthesized to facilitate the reaction.

1.2.2. Exogenous mRNA template

The invention can also adopt exogenous mRNA template to replace exogenous DNA template, or adopt the mixture of exogenous mRNA template and exogenous DNA template, add into the above-mentioned in vitro protein synthesis system, carry on the synthetic reaction of in vitro protein, synthesize the target protein encoded by mRNA template.

1.2.3. In vitro nucleic acid amplification (in vitro nucleic acid amplification technique, in vitro nucleic acid amplification method)

"in vitro nucleic acid amplification" is the process of replicating nucleic acids in vitro.

The nucleic acid templates used in the in vitro protein synthesis system of the present invention, including nucleic acid templates encoding target proteins and optionally nucleic acid templates encoding other proteins, can be prepared independently using in vitro nucleic acid amplification techniques, or can be coupled to include nucleic acid amplification during in vitro protein synthesis reactions.

The in vitro nucleic acid amplification technique that can be used is not particularly limited, and may be non-isothermal amplification or isothermal amplification (also referred to as isothermal amplification). Including but not limited to Polymerase Chain Reaction (PCR) technology, isothermal amplification technology, room temperature amplification technology, etc. In some preferred modes, the isothermal amplification technology is a normal temperature amplification technology.

Among them, isothermal amplification techniques can be referred to those disclosed in the following documents: "J Kim et al, Isothermal DNA Amplification in biochemical analysis: templates and applications [ J ]. Bioanalysis,2011,3(2): 227-. Specifically, nucleic acid isothermal amplification methods that can be used in the technical means of the present invention include, but are not limited to: loop-mediated isothermal amplification method/loop-mediated isothermal amplification (LAMP), strand displacement amplification method/strand displacement amplification method (SDA), nucleic acid sequence-dependent amplification method (NASBA), rolling circle amplification method (RCA), nicking enzyme isothermal amplification of nucleic acids (nicking enzyme amplification reaction, NEAR), helicase-dependent isothermal amplification method (HDA), transcription-dependent amplification method, hybrid capture method, transcription-mediated amplification method (TMA), recombinase-mediated amplification method (RAA), recombinase polymerase amplification method (RPA), and the like. In some preferred embodiments, rolling circle amplification is used.

The in vitro nucleic acid amplification method, particularly the normal temperature amplification method, which can be used in the present invention is not particularly limited, and the normal temperature amplification techniques that can be used in the in vitro cell-free system in the prior art are all included in the scope of the present invention by reference, including but not limited to Rolling Circle Amplification (RCA), polymerase amplification with combinatorial enzymes (RPA), Strand Displacement Amplification (SDA), Helicase Dependent Amplification (HDA), 3SR (self-sustained sequence amplification), and the like. In vitro nucleic acid amplification methods (particularly, ambient temperature amplification methods) disclosed in the following references, including but not limited to: "Nicole E.Gregorio, Max Z.Levine and Javin P.Oza.A. User's Guide to Cell-Free Protein Synthesis [ J ]. Methods protocol.2019, 2, 24", "Y Lu.Advances in Cell-Free biosynthesis Technology [ J ]. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45", "Y Lu.cell-Free Synthesis biology: Engineering in an open world System [ J ]. Synthesis and Biotechnology,2017,2, 23-27" and the like and direct or indirect citations thereof.

In vitro nucleic acid amplification of the invention may also employ amplification techniques such as SMART amplification method (SMAP), Single Primer Isothermal Amplification (SPIA), exponential amplification reaction (EXPAR), thermostable HDA (tHDA), Multiple Displacement Amplification (MDA), restriction assisted RCA, and the like.

The in vitro nucleic acid amplification reaction of the present invention may be carried out continuously at a specific temperature or temperature range which is advantageous for the reaction. Any of the ambient amplification techniques of the invention also allows for performance under conditions of small temperature fluctuations. The reaction conditions of any one of the ambient amplification techniques of the invention are also allowed to fluctuate within an acceptable temperature range.

1.3. Target protein

The target protein suitable for use in the in vitro protein synthesis system of the present invention is not particularly limited as long as the target protein is capable of specifically binding to the protein immobilization system.

The target protein may not carry a purification tag, and in this case, the target protein itself should be capable of being captured by the purification medium in the protein immobilization system. For example, the term "target protein, purification medium" refers to a combination of "antibody, antigen", "antigen, antibody", "avidin or its analog, biotin or its analog", and the like.

In some preferred embodiments, the protein of interest carries a purification tag that is capable of specifically binding to the purification medium. One, two or more purification tags per target protein molecule; when two or more purification tags are contained, the kinds of the purification tags are one, two or more. It should be noted that, as long as the amino acid sequences of the tags are different, the tags are regarded as different types of tags.

The purification tag in the protein of interest may be selected from the following classes: a histidine tag, an avidin analog, a Streg tag (a tag comprising a WSHPQFEK sequence or a variant thereof), a tag comprising a WRHPQFGG sequence or a variant thereof, a tag comprising a RKAAVSHW sequence or a variant thereof, a FLAG tag, a C tag, a Spot tag, a GST tag, an MBP tag, a SUMO tag, a CBP tag, an HA tag, an Avi tag, an affinity protein, an antibody-based tag, an antigen-based tag, and combinations thereof. It may also be selected from the purification tags disclosed in US6103493B2, US10065996B2, US8735540B2, US20070275416a1, including but not limited to Streg tags and variants thereof.

The purification tag may be fused via the N-terminus or C-terminus.

The histidine tag typically contains at least 5 histidine residues, such as a 5 × His tag, a 6 × His tag, an 8 × His tag, and the like.

The octapeptide WRHPQFGG can specifically bind to core streptavidin (core streptavidin).

A Streg tag capable of forming a specific binding interaction with avidin or an analog thereof, said Streg tag comprising WSHPQFEK or a variant thereof. By way of example, WSHPQFEK- (XaaYaWaaZaa)_n-WSHPQFEK, wherein Xaa, Yaa, Waa, Zaa are each independently any amino acid, Xaa yaawazaa comprises at least one amino acid and (Xaa yaawazaa) _nAt least 4 amino acids, wherein n is selected from 1-15 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15); (XaaYaWaaZaa)_nSpecific examples of (G)₈,(G)₁₂,GAGA,(GAGA)₂,(GAGA)₃,(GGGS)₂、(GGGS)₃. Streg tags such as WSHPQFEK, WSHPQFEK- (GGGS)_n-WSHPQFEK、WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK、SA-WSHPQFEK-(GGGS)₂GGSA-WSHPQFEK, WSHPQFEK-GSGGG-WSHPQFEK-GL-WSHPQFEK, GGSA-WNHPQFEK-GGGSGSGGSA-WSHPQFEK-GS, GGGS-WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK, etc.

The FLAG tag includes a DYKDDDDK sequence or a variant thereof, such as DYKDDDDK, DYKDHD-G-DYKDHD-I-DYKDDDDK.

The Spot tag comprises the PDRVRAVSHWSS sequence or a variant thereof.

The C tag comprises an EPEA sequence or a variant thereof.

The GST tag refers to a glutathione S-transferase tag or a variant thereof.

The MBP tag refers to a maltose binding protein tag or a variant thereof.

The SUMO tag is a known Small molecule ubiquitin-like modifier (Small ubiquitin-like modifier), and is one of important members of the polypeptide chain superfamily of ubiquitin (ubiquitin). In the primary structure, SUMO has only 18% homology with ubiquitin, however, the tertiary structure and its biological function are very similar.

The CBP tag refers to a CBP comprising the KRRWKKNFIAVSAANRFKKISSSGAL sequence or a variant thereof.

The HA tag comprises a tag YPYDVPDYA sequence or a variant thereof. Is a hemagglutinin surface epitope derived from influenza virus, which comprises 9 amino acids.

The Avi tag, a known small tag consisting of 15 amino acid residues, is specifically recognized by biotin ligase BirA.

Antibody-based labels, including but not limited to the complete structure of an antibody (complete antibody), antibodies with/without side chain modifications, antibodies with/without glycosylation modifications, antibodies with/without fatty acid chain modifications, domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.), and the like.

Antigenic class tags include, but are not limited to, the complete structure of an antigen (complete antigen), domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., epitopes, etc.), and the like.

In some preferred embodiments, the target protein is linked to a purification tag at the N-terminus or C-terminus, or to both termini.

The source cell of the cell extract for expressing the target protein is not particularly limited as long as the target protein can be expressed in vitro. The exogenous proteins disclosed in the prior art and suitable for in vitro protein synthesis systems derived from prokaryotic cell extracts and eukaryotic cell extracts (yeast cell extracts can be preferred, and kluyveromyces lactis can be more preferred), or the endogenous proteins suitable for prokaryotic cell systems and eukaryotic cell systems (yeast cell systems can be preferred, and kluyveromyces lactis can be more preferred) synthesized in cells can be synthesized by using the in vitro protein synthesis system disclosed by the invention, or synthesized by using the in vitro protein synthesis system provided by the invention.

The application fields of the target protein include but are not limited to the fields of biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano engineering, cosmetics, food additives, nutritional agents, agriculture, feed, living goods, washing, environment, chemical dyeing, fluorescent labeling and the like.

The target protein can be a natural protein or an altered product thereof, and can also be an artificially synthesized sequence. The source of the native protein is not particularly limited, including but not limited to: eukaryotic cells, prokaryotic cells, pathogens; wherein 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 source can include, but is not limited to, murine (including rat, mouse, guinea pig, hamster, etc.), rabbit, monkey, human, pig, sheep, cow, dog, horse, etc. The pathogens include viruses, chlamydia, mycoplasma, etc. The viruses include HPV, HBV, TMV, coronavirus, rotavirus, etc.

The types of the target protein include, but are not limited to, polypeptides ("target protein" in the present invention 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, subunits or fragments of any of the foregoing, and variants of any of the foregoing. The "subunit or fragment of any one of the aforementioned proteins" includes a subunit or fragment of "a partial domain of any one of the aforementioned proteins". The "variant of any one of the aforementioned proteins" includes a variant of "a partial domain of any one of the aforementioned proteins, a subunit or fragment of any one of the aforementioned proteins". Such "variants of any of the foregoing proteins" include, but are not limited to, mutants of any of the foregoing proteins. In the present invention, the meanings of two or more "preceding" cases in succession in other positions are similarly explained.

The structure of the target protein can be a complete structure, and can also be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins and the like. Examples of incomplete antibody structures are nanobodies (heavy chain antibody lacking light chain, V) _HH, retains the full antigen binding ability of the heavy chain antibody), the heavy chain variable region, the Complementarity Determining Region (CDR), and the like.

For example, the target protein synthesized by the in vitro protein synthesis system of the present invention can be selected from the group consisting of, but not limited to, any one of the following proteins, fusion proteins in any combination, compositions in any combination, or mixtures: luciferase (e.g., firefly luciferase), Green Fluorescent Protein (GFP), enhanced green fluorescent protein (eGFP), Yellow Fluorescent Protein (YFP), aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, Catalase (Catalase, e.g., murine Catalase), actin, antibody, variable region of antibody (e.g., single chain variable region of antibody, scFV), single chain of antibody and fragment thereof (e.g., heavy chain of antibody, nanobody, light chain of antibody), alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and precursors thereof, glucagon-like peptide (GLP-1), interferon (including but not limited to interferon alpha, e.g., interferon alpha A, interferon beta, interferon gamma, etc.), interleukin (e.g., interleukin-1 beta, interleukin 2, interleukin 12, etc.),(s), Lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), transthyretin, tyrosinase, xylanase, beta-galactosidase (β -galactosidase, LacZ, such as e.g. e.coli β -galactosidase), etc., a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, or a variant of any of the foregoing (as defined above, including mutants, such as, for example, luciferase mutants, eGFP mutants, which may also be homologous). Examples of the aminoacyl tRNA synthetase include human lysine-tRNA synthetase (lysine-tRNA synthetase), human leucine-tRNA synthetase (leucine-tRNA synthetase), and the like. Examples of the glyceraldehyde-3-phosphate dehydrogenase include Arabidopsis glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Reference may also be made to patent document CN 109423496A. The composition or mixture of any combination may include any one of the proteins described above, and may also include a fusion protein of any combination of the proteins described above.

In some preferred embodiments, the protein synthesis ability of the in vitro protein synthesis system is evaluated by using a target protein having a fluorescent property, such as GFP, eGFP, an analogue thereof, or a mutant thereof.

1.4. Protein fixation system

The Protein Immobilization System (PIS) can capture a target protein product from a reaction mixture and can separate the target protein product from a reaction purification mixture, thereby releasing the captured target protein.

1.4.1. Structure and composition of protein fixation system

The protein-immobilized cells include at least a Solid Matrix (SM). The solid matrix is an essential supporting part of the protein fixing system, is insoluble in the in vitro protein synthesis system, and can be physically separated from the in vitro protein synthesis system. For example, magnetic microspheres are used as a solid matrix, and the physical separation of a protein immobilization system can be realized by utilizing the action of a magnet. For another example, separation from the liquid phase can be conveniently achieved by using a solid phase synthetic resin as the solid matrix.

In some preferred embodiments, the protein immobilization system comprises a solid substrate and a purification medium (PE) attached to an outer surface of the solid substrate.

1.4.1.1. Solid matrix (solid matrix, SM)

In the present invention, the solid matrix is preferably solid particles having a diameter size of 0.1 μm to 1000. mu.m. The smaller particle size helps the protein solid system to realize suspension in a reaction system for protein synthesis, more fully contact with a protein product and improve the capture efficiency and the binding rate of the protein product. In some preferred embodiments, the solid substrate has a diameter size of any one of the following particle size scales (the deviation may be ± 25%, ± 20%, ± 15%, ± 10%) or a range between any two of the particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 5 μm, 1 μm, 5 μm, and a, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm. Unless otherwise specified, the diameter size refers to an average size.

In some preferred modes, the diameter size of the solid matrix is selected from 0.1-10 μm.

In some preferred modes, the diameter size of the solid matrix is selected from 0.2-6 μm.

In some preferred modes, the diameter size of the solid matrix is 0.4-5 μm.

In some preferred modes, the diameter size of the solid matrix is selected from 0.5-3 μm.

In some preferred modes, the diameter size of the solid matrix is selected from 0.2-1 μm.

In some preferred modes, the diameter size of the solid matrix is selected from 0.5-1 μm.

In some embodiments, the solid substrate is an agarose resin.

In some preferred embodiments, the solid substrate is a magnetic microsphere.

In some preferred embodiments, the protein immobilization system comprises a solid substrate, a purification medium, and a polymer connecting the solid substrate and the purification medium.

In some embodiments, the solid substrate is an agarose resin and the purification medium is nickel ions that are attached to the agarose resin by NTA (nitrilotriacetic acid) or by NTA and other attachment elements.

In some preferred modes, the polymer has certain hydrophilicity and cannot be separated out of the solution, so that polymer chains can be fully stretched in the aqueous phase, cannot be separated out of the solution and cannot agglomerate, the activity space of the purification medium can be enlarged, the target protein can be captured by the purification medium more quickly and fully, and the efficiency of capturing the target protein and the binding rate of the target protein are improved.

In some preferred embodiments, the polymer has a branched structure, with more purification media being carried by multiple (at least 3) branches.

In some preferred modes, a branched polymer is connected to the outer surface of the solid matrix, one end of the branched polymer is covalently fixed on the outer surface of the solid matrix, and branches with independent ends are distributed outside the solid matrix; the purification medium is attached to the branched end of the branched polymer.

By "immobilized" is meant "immobilized" on the outer surface of a solid substrate by covalent attachment.

In some preferred embodiments, the branched polymer is covalently coupled to the outer surface of the solid substrate directly or indirectly through a linking element.

The structure of the branched polymer includes, but is not limited to, a branched structure, a comb structure, a tree structure, a hyperbranched structure, a cyclic branched structure, and the like, and a combination thereof.

In some preferred embodiments, the branched polymer has a linear backbone, and in this case, the polymer has both the high flexibility of the linear backbone and the advantage of high magnification of the number of branches, and can better achieve high-rate, high-throughput binding, high-efficiency, and high-ratio (high-yield) separation.

The number of linear backbones to which one binding site of the outer surface of the solid substrate may be covalently coupled may be 1 or more.

In some preferred embodiments, a binding site on the outer surface of the solid substrate leads out only one linear backbone, which provides a larger space for the linear backbone.

In other preferred embodiments, one binding site on the outer surface of the solid substrate leads out only two linear backbones, providing as much space as possible for the linear backbones to move.

In some preferred embodiments, the branched polymer has a comb-like structure. In this case, the comb structure has a linear main chain and at least 3 side branches; one end of the linear main chain is covalently fixed on the outer surface of the solid matrix, and the other end of the branched chain type polymer is distributed outside the solid matrix.

For a protein immobilization system adopting a branched chain type polymer to connect a purification medium, one end of the branched chain type polymer is covalently coupled to the outer surface of a solid matrix (such as a magnetic microsphere), the rest ends including all branched chains and all functional groups are dissolved in a solution and distributed in the outer space of the solid matrix, and a molecular chain can be fully stretched and swung, so that the molecular chain can be fully contacted with other molecules in the solution, and the capture of a target protein can be further enhanced. When the target protein is eluted from the protein fixing system, the target protein can directly get rid of the constraint of the protein fixing system and directly enter the eluent. In particular, for comb-shaped polymers, compared with polymers physically wound on the outer surface of the solid substrate or integrally formed with the solid substrate, the polymers covalently fixed through one end of the linear main chain (in some preferred modes, a single linear main chain of the polymers is covalently fixed, and in other preferred modes, the fixed end of the main chain is covalently led out of 2 or 3 linear main chains) can effectively reduce the stacking of the molecular chain, strengthen the stretching and swinging of the molecular chain in the solution, enhance the capture of the target protein, and reduce the retention ratio and the retention time of the target protein during elution.

In some preferred embodiments, the polymer has a linear backbone (e.g., a comb-like structure, or a comb-like structure) that is a polyolefin backbone or an acrylic polymer backbone.

In some preferred forms, the polymer has a linear backbone, which is a polyolefin backbone, and is provided by the backbone of an acrylic polymer.

In some preferred embodiments, the monomer unit of the acrylic polymer is an acrylic monomer molecule such as acrylic acid, acrylate, methacrylic acid, methacrylate, or a combination thereof. The acrylic polymer may be obtained by polymerization of one of the above monomers or by copolymerization of an appropriate combination of the above monomers.

In some preferred embodiments, the linear backbone of the polymer is a polyolefin backbone. Specifically, for example, the polyolefin backbone is a linear backbone provided by a polymerization product of one of acrylic acid, acrylate, methacrylic acid, methacrylate, or a combination thereof (a linear backbone provided by a copolymerization product thereof), or a linear backbone of a copolymerization product formed by polymerization of the above monomers. The polymerization product of the above monomer combination is exemplified by acrylic acid-acrylic ester copolymer, and also methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate copolymer. The above-mentioned monomers participate in the polymerization to form a copolymerization product, such as maleic anhydride-acrylic acid copolymer, for example.

In other preferred embodiments, the linear backbone of the polymer is an acrylic polymer backbone. The polyolefin main chain may be a linear main chain containing only carbon atoms, or may contain hetero atoms (hetero atoms are non-carbon atoms) in the linear main chain.

In other preferred forms, the linear backbone of the polymer is a block copolymer backbone comprising polyolefin blocks, for example, a polyethylene glycol-b-polyacrylic acid copolymer (within the scope of acrylic copolymers). It is preferable that the flexible swing of the linear main chain is smoothly exerted, that the accumulation of the branched chain is not caused, and that the residence time or/and the residence ratio is not increased.

In other preferred embodiments, the linear backbone of the polymer is a condensation-polymerized backbone. The condensation polymerization type main chain refers to a linear main chain which can be formed by condensation polymerization between monomer molecules or oligomers; the polycondensation main chain may be of a homo-type or a co-type. Such as polypeptide chains, polyamino acid chains, and the like. Specifically, for example, an epsilon-polylysine chain, an alpha-polylysine chain, gamma-polyglutamic acid, polyaspartic acid chain, etc., an aspartic acid/glutamic acid copolymer, etc.

The branched polymer has at least 3 branches.

Each branch end is independently bound or unbound to the purification medium.

When the purification medium is bound to the end of the branched chain, the number of the purification medium may be 1 or more.

In some preferred embodiments, at least 3 purification media are bound to one molecule of the branched polymer.

The number of branched chains in the branched polymer is related to factors such as the size of a solid matrix (such as magnetic microspheres), the type of the skeleton structure of the polymer, the chain density (particularly, the branched chain density) of the polymer on the outer surface of the solid matrix, and the like. In the case of comb polymers, the number of branches is related to the size of the solid substrate, the length of the polymer backbone, the linear density of the side branches grafted along the polymer backbone, the chain density of the polymer on the outer surface of the solid substrate, and other factors. The branched chain quantity of the polymer can be controlled by controlling the feeding amount of the raw materials.

1.4.1.2. Purification media (PE)

The purification medium is a functional element of a protein immobilization system for specifically capturing a target protein, i.e., the purification medium and the target protein molecule are capable of specific binding.

The purification media is located on the outer surface of the solid substrate and is bound to the outer surface of the solid substrate by the loading means described in section "1.4.1.3".

In some preferred embodiments, the captured protein molecule can also be released under suitable conditions.

The purification medium may contain, but is not limited to, metal ions, biotin-type tags, avidin-type tags, polypeptide-type tags, protein-type tags, immunological-type tags, or combinations thereof.

In some preferred embodiments, the metal ion is Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof.

In some preferred forms, the purification medium is: a metal ion, avidin, an avidin analog that can bind biotin or an analog thereof, biotin, a biotin analog that can bind avidin or an analog thereof, an affinity protein, an antibody, an antigen, DNA, or a combination thereof. Wherein the metal ion is Ca in some preferred modes²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof. The definition of antibody, antigen, refers to the term moiety, which is understood to also include, but is not limited to, domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.), epitopes (epitopes), epitope peptides, variants of any of the foregoing, and the like.

In the invention, the purification medium in the protein immobilization system and the target protein or a purification label carried in the target protein can mutually form specific binding action. Therefore, the target protein purification tag can be used as an alternative mode of a purification medium in a protein immobilization system; peptides or proteins used in the purification medium of the immobilization system may also be used as an alternative to the purification tag in the protein of interest.

In some preferred embodiments, the biotin-type tag is biotin, a biotin analog that binds avidin, or a combination thereof.

In some preferred embodiments, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

In some preferred modes, a branched chain type polymer is fixed on the outer surface of the magnetic microsphere body, and the tail end of a branched chain of the branched chain type polymer is connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

In some preferred embodiments, the avidin is any one of streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

Such avidin analogs, e.g., tamavidin 1, tamavidin2, and the like. Tamavidin 1 and Tamavidin2 are proteins found by Yamamoto et al in 2009 to have the ability to bind biotin (Takakura Y et al Tamavidins: Novel avidin-like biotin-binding proteins from the tamogitateke mushroom [ J ]. FEBS Journal,2009,276,1383-1397), which have a strong affinity for biotin similar to streptavidin. The thermal stability of Tamavidin2 is superior to that of streptavidin, and its amino acid sequence may be retrieved from relevant database, such as UniProt B9A0T7, or optimized with codon conversion and optimizing program to obtain DNA sequence.

Such as a WSHPQFEK sequence or a variant sequence thereof, a WRHPQFGG sequence or a variant sequence thereof, and the like.

In some preferred forms, the purification medium is: a polypeptide tag, a protein tag, or a combination thereof.

In some preferred forms, the purification medium is an affinity protein.

Affinity protein: specifically binds with target protein and has higher affinity binding force, such as protein A, protein G, protein L, modified protein A, modified protein G, modified protein L, etc.

In some preferred forms, the affinity protein is protein a, protein G, protein L, a modified protein of any of the foregoing, or a combination thereof.

Protein A: protein A, a 42kDa surface Protein, was originally found in the cell wall of Staphylococcus aureus. It is encoded by the spa gene, the regulation of which is controlled by the DNA topology, cellular osmolarity and a two-component system known as ArlS-ArlR. Due to their ability to bind immunoglobulins, have been used in research related to the field of biochemistry. Can be specifically combined with the Fc of the antibody, is mainly used for purifying the antibody and can be selected from any commercial products. The terms "Protein A", "SPA", and "Protein A" are used interchangeably herein.

Protein G: protein G, an immunoglobulin binding Protein, is expressed in group C and group G streptococci, similar to Protein a, but with different binding specificities. It is a 65kDa (G148 protein G) and 58kDa (C40 protein G) cell surface protein, which is primarily used for antibody purification by specific binding to antibodies or certain functional proteins, and can be selected from any commercially available product.

Protein L: protein L, is limited to those antibodies that specifically bind kappa (. kappa.) light chains. In humans and mice, most antibody molecules contain a kappa light chain and the remaining lambda light chain. Is mainly used for antibody purification and can be selected from any commercial product.

In some preferred embodiments, the immunological tag is any one of an antibody-type tag and an antigen-type tag.

The definition of antibody, antigen, refers to the term moiety, which is understood to also include, but is not limited to, domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.), epitopes (epitopes), epitope peptides, variants of any of the foregoing, and the like.

In some preferred forms, the polypeptide tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a tag comprising a WSHPQFEK sequence, a tag comprising a variant sequence of WSHPQFEK, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof.

In some preferred embodiments, the protein tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

In some preferred embodiments, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one.

In some preferred embodiments, the antibody-type tag is an anti-protein antibody.

In some preferred embodiments, the antibody-type tag is an anti-fluorescent protein antibody.

In some preferred embodiments, the antibody-type tag is an antibody against green fluorescent protein or a mutant thereof.

In some preferred embodiments, the antibody-type tag is a nanobody.

In some preferred embodiments, the antibody-type tag is a nanobody against a protein.

In some preferred embodiments, the antibody type tag is a single domain antibody against a protein.

In some preferred embodiments, the antibody-type tag is a single domain antibody against a protein.

In some preferred embodiments, the antibody type tag is an antibody VHH fragment of an anti-protein.

In some preferred embodiments, the antibody type tag is an anti-protein antibody scFV fragment.

In some preferred embodiments, the antibody-type tag is a nanobody against a fluorescent protein.

In some preferred modes, the antibody type tag is a nanobody against green fluorescent protein or a mutant thereof.

In some preferred embodiments, the antibody-type tag is an antibody Fab fragment.

In some preferred embodiments, the antibody-type tag is an antibody F (ab') 2 fragment.

In some preferred embodiments, the antibody-type tag is an antibody Fc fragment.

For example, the nanobody anti-eGFP shown in SEQ ID No. 6 is used as a purification medium.

In some preferred embodiments, the protein A-mEGFP-avidin fusion protein is bound to biotin-modified magnetic microspheres and forms biotin-avidin affinity complex linking elements. Examples of the ProteinA-mEGFP-avidin fusion protein include ProteinA-mEGFP-Streptavidin fusion protein and ProteinA-mEGFP-Tamavidin2 fusion protein.

In some preferred embodiments, the protein G-mEGFP-avidin fusion protein is bound to biotin-modified magnetic microspheres and forms biotin-avidin affinity complex linking elements. Examples of the ProteinG-mEGFP-avidin fusion protein include ProteinG-mEGFP-Streptavidin fusion protein and ProteinG-mEGFP-Tamavidin2 fusion protein.

In some preferred embodiments, the anti EGFP-avidin fusion protein is bound to biotin-modified magnetic microspheres and forms a biotin-avidin affinity complex linking element. In some more preferred embodiments, the anti EGFP-avidin fusion protein is an anti EGFP-mScarlet-avidin fusion protein. In some more preferred modes, the anti EGFP-avidin fusion protein is an anti EGFP-mScelet-Tamvavidin 2 fusion protein, a fusion protein of nanobodies.

The nucleotide sequence of ProteinA (protein A) is shown in SEQ ID No. 1.

The nucleotide sequence of ProteinG (SEQ ID No.:5) was from the group G Streptococcus (Streptococcus sp. group G), and the gene sequence of the antibody-binding region thereof was selected.

The mEGFP has a nucleotide sequence shown in SEQ ID No.:3 and an amino acid sequence shown in SEQ ID No.: 4.

Streptavidin, Streptavidin.

The anti-EGFP, namely the anti-eGFP, is a nano antibody with an amino acid sequence shown as SEQ ID No. 6 and used for resisting green fluorescent protein.

Tamavidin2, an avidin analog, having a nucleotide sequence shown in SEQ ID No. 2.

The mScarlet is a bright red fluorescent protein and the corresponding nucleotide sequence is SEQ ID No. 7.

1.4.1.3. Loading mode of purification medium in protein fixing system

The manner in which the purification medium is attached to the outer surface of the solid matrix of the protein immobilization system is not particularly limited.

In some embodiments, for example, the solid matrix (e.g., magnetic microspheres coated with aminated silica) coated with an aminated coupling agent is directly subjected to a condensation reaction with biotin to covalently link biotin with amide bonds, thereby obtaining a protein immobilization system using biotin as a purification medium.

In some preferred modes, the purification medium is attached to the outer surface of the solid matrix at intervals of the immobilized polymer to form a structural mode of 'solid matrix-polymer-purification medium'. One end of the polymer is covalently immobilized on the outer surface of the solid substrate, and this fixed position is also described as an immobilization site of the polymer.

In some preferred forms, the purification medium is attached to the outer surface of the solid substrate by a linear polymer. The length and flexibility of the linear polymer are utilized to enable the purification medium to be freely distributed outside the solid matrix, so that the movement space of the purification medium is increased, the efficiency of capturing the target protein is increased, and the binding amount is increased. For example, CN110498830A used linear polyethylene glycol to link a solid matrix (magnetic microspheres) and a purification medium (biotin).

In some preferred forms, the purification medium is attached to the outer surface of the solid substrate by a branched polymer. The branched polymer is described in section "1.4.1.1. solid matrix". For example, CN2019105401326, CN201911209156X and CN2019221117066 adopt a branched polyacrylic acid skeleton to connect a large amount of purification media.

The gel-like porous materials commonly used at present, such as agaroses, are adopted by most commercially available microspheres. The porous material has abundant pore structure, thereby providing large specific surface area and providing a large number of binding sites for purifying substrates, but correspondingly, when the protein is adsorbed, captured or eluted, the protein molecules are required to additionally enter or escape from the complicated pore channels in the porous material, more time is required, and the protein molecules are easier to retain. In contrast, a linear polymer or a branched polymer is connected with a purification medium to capture target protein, only the outer surface space of a solid matrix (such as magnetic microspheres) is utilized, and the target protein can be directly released into eluent without passing through a complex reticular channel during adsorption and elution, so that the elution time is greatly reduced, the elution efficiency is improved, the retention ratio is reduced, and the purification yield is improved.

In some preferred forms, the purification media is attached to the outer surface of the solid substrate by the following attachment elements: including, but not limited to, nucleic acids, oligonucleotides, peptide nucleic acids, aptamers, deoxyribonucleic acids, ribonucleic acids, leucine zippers, helix-turn-helix motifs, zinc finger motifs, biotin, avidin, streptavidin, anti-hapten antibodies, and the like, combinations thereof. Of course, the linking element may also be a double stranded nucleic acid construct, a duplex, a homo-or hetero-hybrid (a homo-or hetero-hybrid selected from DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA), or a combination thereof.

In some preferred embodiments, the type of binding force of the purification medium attached to the outer surface of the solid substrate (e.g., the end of a branch of a branched polymer) is: covalent bonding, dynamic covalent bonding, supramolecular interactions, or combinations thereof.

In some preferred embodiments, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

In some preferred forms, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In some preferred embodiments, the affinity complex selection criteria are: has good specificity,The affinity is strong, and a site for chemical bonding is provided, so that the affinity complex can be covalently connected or can be covalently modified to be covalently connected to the outer surface of the solid matrix, such as a binding site of the outer surface, a main chain terminal of a linear polymer and a branch chain terminal of a branched polymer. Such as a combination of: biotin and avidin, His tag and Ni²⁺Antigens and antibodies.

When the loading mode comprises dynamic covalent bonds and supermolecular interactions (especially affinity complex interactions), a reversible loading mode is formed, and the purification medium can be unloaded from the tail end of the branched chain under certain conditions, so as to be updated or replaced.

The regeneration of the purification medium corresponds to the regeneration of the protein fixation system, and the types of the purification medium before and after the regeneration are the same.

The change of the purification medium corresponds to the change of the protein fixation system, and the types of the purification medium are different before and after the change.

In some preferred embodiments, the branches of the polymer chain covalently bind the purification medium to the ends of the polymer branches via a functional group-based covalent bond. Can be generated by covalent reaction of functional groups contained in branched chains of polymer molecules on the outer surface of a solid matrix (such as magnetic microspheres) and a raw material of a purification medium. In some preferred embodiments, the functional group is a specific binding site.

The covalent bond based on the functional group refers to a covalent bond formed by the functional group participating in covalent coupling. In some preferred embodiments, the functional group is a carboxyl group, a hydroxyl group, an amino group, a thiol group, a salt of a carboxyl group, a salt of an amino group, a formate group, or a combination thereof. The salt form of the carboxyl group is in some preferred forms a sodium salt form such as COONa; the salt form of the amino group may be embodied in the form of an inorganic salt, or in the form of an organic salt, including, but not limited to, hydrochloride, hydrofluoride, and the like. The "combination of functional groups" refers to all branches of all polymer molecules of the outer surface of one solid substrate (e.g. magnetic microspheres) allowing for covalent bond formation based on participation of different functional groups; that is, all molecules of the purification medium (e.g., biotin) on the outer surface of a solid matrix can be covalently linked to different functional groups, respectively, but one molecule of the purification medium can be linked to only one functional group.

1.4.1.4. Mechanism of action of the purification Medium

The force of the purification medium for capturing the target protein molecule in the reaction-purification mixed system can be selected from: covalent bonds, dynamic covalent bonds, supramolecular interactions, combinations thereof.

In some preferred embodiments, the protein product of interest is bound to the protein immobilization system by: biotin-avidin binding, Streg tag-avidin binding, avidin-avidin binding, histidine tag-metal ion affinity, antibody-antigen binding, or a combination thereof. The Streg tag, which is a peptide tag developed by IBA that can specifically bind to avidin or its analogs, typically contains the WSHPQFEK sequence or its variant sequences.

1.4.2. Typical protein fixing system-magnetic microsphere system

In some preferred embodiments, the protein immobilization system is a magnetic microsphere system.

The magnetic microsphere system has a magnetic microsphere body.

The volume of the magnetic microsphere body can be any feasible particle size.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.1-10 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.2-6 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.4-5 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.5-3 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.2-1 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.5-1 μm.

In some preferred embodiments, the average diameter of the magnetic microsphere body is about 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, and the submultiples may be ± 25%, ± 20%, ± 15%, and ± 10%.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In some preferred modes, the diameter of the magnetic microsphere body is 1 μm, 10 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1000 μm, and the deviation range can be ± 25%, 20%, 15%, 10%.

Different magnetic materials can provide different types of activation sites, can create differences in the manner in which the purification media are bound, and can also differ in the ability to disperse and settle with a magnet, and can also create selectivity for the type of substrate being purified.

The magnetic microsphere body and the magnetic microsphere comprising the magnetic microsphere body can be quickly positioned, guided and separated under the action of an external magnetic field, and can be endowed with various active functional groups such as hydroxyl, carboxyl, aldehyde group, amino and the like on the surface of the magnetic microsphere by surface modification or chemical polymerization and other methods.

The polymer used for connecting the magnetic microspheres and the purification medium can be one or more selected from polyacrylic acid, polyacrylate, methyl polyacrylic acid, methyl polyacrylate and other polymers with carboxyl branched chains.

In some preferred forms, the solid matrix of the protein immobilization system is a magnetic microsphere body, the outer surface of the magnetic microsphere body is connected with a branched-chain polymer, one end of the branched-chain polymer is immobilized on the outer surface of the magnetic microsphere body, and branched chains with independent ends are distributed outside the magnetic microsphere body; the purification medium is attached to the branched end of the branched polymer.

One end of the branched polymer can be covalently fixed on the outer surface of the magnetic microsphere body in a direct mode or in an indirect mode through a connecting element.

In some preferred embodiments, the branched polymer has a linear main chain, and the linear main chain is directly covalently coupled to the outer surface of the magnetic microsphere body through one end, or indirectly covalently coupled to the outer surface of the magnetic microsphere body through a linking element.

The term "immobilized" refers to being "immobilized" on the outer surface of the magnetic microsphere body by covalent bonding.

In some preferred forms, the purification medium is biotin; the case where the purification substrate (target protein) is avidin or the like, or the case where the purification substrate contains a structure of avidin or the like can be applied. In this case, an affinity complex binding may be formed between the purification medium and the purification substrate. The avidin may be any one of streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

In some preferred forms, the purification medium is an affinity protein; can be widely used for purifying antibody substances. The affinity protein can be any one of protein A, protein G and protein L, or modified protein thereof.

In some preferred forms, the purification medium is an affinity protein, the affinity protein having a linking element for an affinity complex between the affinity protein and the end of a branch of the polymer; at this point, the purification medium can be renewed or replaced, at which point the replacement of the affinity protein can optionally be followed by reuse. In some preferred modes, the binding effect of the biotin-avidin affinity complex exists between the avidin and the tail end of the polymer branch chain, namely the avidin is connected to the tail end of the polymer branch chain in a biotin-avidin manner, and the magnetic microspheres can be reused by replacing the avidin-avidin.

And the regeneration of the magnetic microsphere system correspondingly updates the purification medium, and the types of the purification medium before and after updating are the same.

In some preferred modes, the magnetic microsphere body is SiO₂A wrapped magnetic material. Wherein, SiO₂The wrapping layer may include a silane coupling agent with its own active site.

In some preferred forms, the magnetic material is selected from: iron compounds (e.g., iron oxides), iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides, chromium oxides, and combinations thereof.

In some preferred embodiments, the iron oxide is, for example, magnetite (Fe)₃O₄) Maghemite (gamma-Fe)₂O₃) Or a combination of the two oxides, preferably ferroferric oxide.

In some preferred forms, the magnetic material is selected from: fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo、FeSi、FeAl、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃、PbO·6Fe₂O₃GdO, and combinations thereof. Wherein, the Re is a rare earth element, rhenium.

1.4.2. Method for preparing protein fixing system

The preparation methods of the protein immobilization system can refer to patent applications CN110498830A, CN2019105401326, CN201911209156X and CN2019221117066, and the preparation methods in these references are all incorporated into the scope of the present invention by reference. CN110498830A can provide magnetic microspheres linked to purification media (biotin) with polyethylene glycol; CN2019105401326, CN201911209156X, CN2019221117066 can provide magnetic microspheres with a branched polymer linked to a purification medium (nickel ions, biotin or affinity protein).

Protein immobilization systems having branched polymer linking elements whose branched polymer structure permits immobilization of the reactive center R at a site on the outer surface of a solid substrate_CThe monomer molecule is polymerized, and the prepared branched polymer can be directly and covalently coupled to the outer surface of the solid matrix. In the latter case, conventional chemical coupling reaction may be used, but when the molecular weight of the branched polymer is large, the coupling efficiency may be limited due to steric hindrance.

1.4.2.1. Protein immobilization system with branched polymer connected with purification medium

In some embodiments, the protein immobilization system is loaded with purification media via a branched polymer. Such protein immobilization systems (including but not limited to magnetic microsphere systems) may be obtained by polymerization methods including but not limited to the following:

step (1): providing a solid substrate, chemically modifying the surface of the solid substrate, and introducing a reactive group R₁；

Step (2): alternatively, by reaction with a reactive group R₁Covalent coupling reaction between the two, introducing a functional group R₂. The functional group R₂Can be used as a reactive center R_CStarting a polymerization reaction; if the reactive group R of step (1)₁Can be used as a reactive center R_CThe polymerization is started, and the step can be omitted and directly enters the step (3). For addition polymerization (radical, cationic, anionic), the reactive center R_CNamely the initiation center; for step-wise polymerization, the reactive center R_CCan also serve as a starting point for "propagation" of the polymer chain.

The reactive group refers to a group capable of undergoing a coupling reaction and forming a covalent bond.

And (3): addition of monomer molecule M₁With said reactive center R _CStarting from this, the monomer molecule M is carried out_GThe polymerization reaction of (1); monomer molecule M_GComprising at least one monomer molecule M capable of providing a functionalized branch end_B(ii) a The functional branch chain end refers to that the branch chain end is a functional group F₁Or modified to be able to convert into functional groups F₁. Forming a chain skeleton of the branched polymer by polymerization, and forming a reactive center R_CForm a point of attachment for covalently immobilizing the branched polymer.

The functional group refers to a functional group having a binding force capable of forming a covalent bond, a dynamic covalent bond, a supramolecular interaction, and the like.

The mechanism of the polymerization reaction is not particularly limited, and may be selected from, for example: radical polymerization, cationic polymerization, anionic polymerization, step-by-step polymerization.

The monomer molecule M_GCan be a single type of molecule, in which case homopolymerization can be performed; or a combination of different kinds of molecules, in which case copolymerization can be carried out; the monomer molecule M capable of providing functional branched chain_BFor example: acrylic monomer molecules (free radical polymerization is possible), epoxypropanol or 3-methyl-3-oxetanylcarbinol or 3-ethyl-3-oxetanylcarbinol (anionic polymerization is possible), amino acids and derivatives thereof (stepwise polymerization is possible), and the like.

And (4): functional group F using purification media and branch ends₁The purification medium is combined to the tail end of the branched chain through the interaction between the two to obtain the protein fixing system with the structure of 'solid matrix-branched polymer-purification medium'.

Said "functional group F at the end of the purification Medium and the branched chain₁Interactions between "such as covalent bonds, dynamic covalent bonds, supramolecular interactions (e.g. affinity complex interactions).

1.4.2.2. With SiO₂Protein immobilization system using encapsulated magnetic material as solid matrix

Some embodimentsIn (2), the protein fixing system adopts SiO₂The coated magnetic material is used as a solid matrix, and the preparation process comprises the following steps: providing SiO₂Coated magnetic microspheres (commercially available or self-made) and SiO₂Activated modification of (2) to form a reactive group R₁The purification media is attached by five means including, but not limited to, the following.

The mode i: attaching the purification Medium to the reactive group R₁To (3).

Mode ii: covalently linking a functionalized linear polymer to a reactive group R₁Functional group F for linking the purification medium to the other end of the linear polymer₂To (3).

Mode iii: covalently linking functionalized branched polymers to reactive groups R ₁Functional group F for linking the purification Medium to the end of the Branch of the branched Polymer₁To (3).

Mode iv: at the reactive group R₁Carrying out polymerization reaction to generate a linear polymer and generating a functional group F at the other end of the linear polymer₂Functional group F for linking the purification Medium to the other end of the Linear Polymer₂To (3).

Mode v: at the reactive group R₁A polymerization reaction is carried out to form a branched polymer, and a functional group F is formed at the end of the branch of the branched polymer₁Functional group F for linking the purification Medium to the end of the branched chain₁To (3).

In some preferred embodiments, the branched polymers in the above-mentioned embodiments iii and v have a linear main chain, one end of which is covalently fixed to the reactive group R₁And a plurality of pendant side chains distributed along the polymer backbone.

It should be noted that the above-mentioned links are not required to be completely isolated, and two or three links are allowed to be combined into one link, for example, activated silica-coated magnetic microspheres (commercially available or self-made) may be directly provided.

1.4.2.3. Protein fixing system using biotin as purification medium

In some embodiments, a protein immobilization system using biotin as the purification medium is employed.

In some embodiments, the protein immobilization system adopts a magnetic microsphere system, which takes magnetic microspheres as a solid matrix, covalently fixes a branched polymer on the outer surface of the solid matrix, and the tail end of a polymer branch is bonded with biotin, and can be prepared by adopting the following method:

step (1): providing a magnetic microsphere body, carrying out chemical modification on the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form the amino modified magnetic microsphere A.

In some preferred modes, the magnetic microsphere body is chemically modified by using a coupling agent.

In some preferred embodiments, the coupling agent is an aminosilicone coupling agent.

In some preferred modes, the magnetic microsphere body is SiO₂The wrapped magnetic material is prepared by chemically modifying a magnetic microsphere body by using a silane coupling agent; the silane coupling agent is in some preferred forms an amino silane coupling agent.

Step (2): covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B.

And (3): polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain a branched-chain acrylic polymer which has a linear main chain and contains a functional group F ₁The polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C. This step can be carried out without addition of a crosslinking agent.

In some preferred modes, the functional group F₁Is carboxyl, hydroxyl, amino, sulfhydryl, formate, ammonium salt, salt form of carboxyl, salt form of amino, formate group, or combination of the aforementioned functional groups; the "combination of functional groups" refers to the work contained in all the branches of all the polymers on the outer surface of one magnetic microsphereThe number of functional groups may be one or more than one.

In other preferred embodiments, the functional group is a specific binding site.

And (4): functional group F contained by a branch of the polymer₁And covalently coupling biotin to the tail end of the branched chain of the polymer to obtain the biomagnetic microsphere D (biotin magnetic microsphere). In the prepared protein fixing system, the branched chain type polymer is an acrylic polymer (with a polyacrylic acid skeleton) with a comb-shaped structure.

A typical structure of the biotin magnetic microsphere is shown in FIG. 1.

One specific embodiment of the preparation of the biomagnetic microspheres D is as follows, specifically, a branched acrylic polymer is taken as an example to provide a linear main chain and a large number of branches. The invention discloses a specific implementation mode, which comprises the following steps: taking ferroferric oxide magnetic beads coated by silicon dioxide as a body of the magnetic microsphere; firstly, chemically modifying a silicon dioxide-coated ferroferric oxide magnetic bead by using a coupling agent 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2, an aminated coupling agent, also a silane coupling agent, more specifically an aminated silane coupling agent), introducing amino to the outer surface of the magnetic bead to finish the SiO reaction ₂Activating and modifying to obtain amino modified magnetic microspheres A; then, covalently coupling immobilized molecules (acrylic acid molecules, providing a carbon-carbon double bond and a reactive group carboxyl) to the outer surface of the magnetic bead by utilizing a covalent reaction between carboxyl and amino, so that the carbon-carbon double bond is introduced to the outer surface of the magnetic bead to obtain a carbon-carbon double bond-containing magnetic microsphere B; then, polymerization reaction of carbon-carbon double bonds is utilized to carry out polymerization of acrylic monomer molecules (such as sodium acrylate), and the polymerization product is covalently coupled to the outer surface of the magnetic bead while the polymerization reaction is carried out, so that SiO is completed₂Connecting a polymer to obtain an acrylic polymer modified magnetic microsphere C; the immobilized molecules are acrylic acid molecules, one immobilized molecule only leads out one polymer molecule, and simultaneously only leads out one polymer linear main chain; taking sodium acrylate as an example of a monomer molecule, the polymerization product is sodium polyacrylate, the main chain of whichIs a linear polyolefin main chain, and is covalently connected with a plurality of side chain COONa along the main chain, and the functional group contained in the side chain is also COONa; in the polymerization reaction, a cross-linking agent such as N, N' -methylenebisacrylamide (CAS: 110-26-9) is not used, and molecular chains are prevented from being cross-linked with each other to form a network polymer, but a linear main chain is generated in the polymerization product under the condition of not adding the cross-linking agent. If the molecular chains are crosslinked into a network polymer, a porous structure is formed, and the elution efficiency of the target protein is influenced.

The outer surface of the magnetic microsphere can also adopt other activation modification modes besides amination. For example, the aminated magnetic microsphere (amino-modified magnetic microsphere a) may further react with acid anhydride or other modifying molecules to perform chemical modification of the outer surface of the magnetic microsphere by carboxylation or other activation methods.

The immobilized molecules are small molecules, one end of each polymer is covalently immobilized on the outer surface of the magnetic beads. Can be used for fixing not only branched polymers but also linear polymers. The immobilized small molecule is not particularly limited as long as one end of the immobilized small molecule is covalently coupled to the outer surface of the magnetic bead, and the other end of the immobilized small molecule can initiate polymerization reaction, including homopolymerization reaction, copolymerization reaction or polycondensation reaction, or copolymerization coupling of one end of a polymer. The end of the polymer immobilized by the immobilization molecule may be any end of a linear polymer, any end of a linear main chain of a comb-like polymer, an immobilization site of a cyclic polymer, an end of the lowest generation number of a tree-like structure, or the like.

In some preferred embodiments, the immobilized molecule allows for the extraction of only a single polymeric linear backbone, as well as two or more polymeric linear backbones, as long as it does not result in chain stacking and/or does not result in an increase in the retention ratio. Preferably, one immobilized molecule leads out only one polymer molecule and only one polymer linear backbone.

In other preferred embodiments, the acrylic monomer molecules as polymerized monomer units may be acrylic acid, acrylate, methacrylic acid, methacrylate-based monomers, or combinations thereof; more preferably, the acrylic monomer molecule is sodium acrylate. According to the comparative experiment of the applicant, the result shows that compared with acrylic acid as a polymerization monomer, sodium acrylate is adopted as the polymerization monomer, more purification media can be combined, the purification effect of the prepared purified glass beads is better, and the binding force to a target (taking a target protein as an example, specifically taking histidine-tagged eGFP as an example) can be improved by 5-10 times or even more.

In other preferred embodiments, the acrylic polymer may be replaced by another polymer. The criteria chosen were: the formed polymer has a linear main chain, a large number of side branched chains are distributed along the main chain, and functional groups are carried on the side branched chains for subsequent chemical modification; namely, aiming at a binding site on the outer surface of the magnetic microsphere, a large number of functional groups are provided through branched chains distributed at the side end of a linear main chain of the polymer. Such as epsilon-polylysine chains, alpha-polylysine chains, gamma-polyglutamic acid, polyaspartic acid chains, aspartic acid/glutamic acid copolymers, and the like.

A method of introducing the above-described polymer surrogate molecules to the outer surface of the magnetic microspheres: according to the chemical structure of the polymer substitute and the type of the side chain active group thereof, selecting a proper activation modification mode of the outer surface of the magnetic microsphere, the type of immobilized molecules and the type of monomer molecules, and carrying out proper chemical reaction to introduce a large amount of active groups positioned on the side chain into the outer surface of the magnetic microsphere.

Covalently coupling acrylic polymer molecules (such as sodium polyacrylate linear molecular chains) to the outer surface of the magnetic microsphere, and then providing an activation site by using a functional group at the tail end of a branched chain, or activating the functional group at the tail end of the branched chain of the polymer molecules according to reaction requirements before connecting a purification medium (such as biotin molecules) to ensure that the functional group has reaction activity and forms an activation site; covalently coupling 1, 3-propane diamine to an activation site of a polymer branched chain (each monomer acrylic acid unit structure can provide one activation site) to form a new functional group (amino group), and then covalently coupling a purification medium molecule to the new functional group at the tail end of the polymer branched chain by using amidation covalent reaction between carboxyl and amino groups to complete the covalent connection of the purification medium to the tail end of the branched chain of the polymer, thereby obtaining the magnetic microsphere modified by the purification medium. When biotin is used as the purification medium, a single biotin molecule can provide a specific binding site. Taking the functional group of the polymer branch chain as COONa as an example, in this case, sodium acrylate is used as a monomer molecule, and before the covalent reaction with 1, 3-propane diamine, carboxyl activation can be performed first, and the existing carboxyl activation method can be used, for example: EDC. HCl and NHS were added.

In some preferred modes, the amount of the acrylic acid used for preparing the magnetic microspheres B is 0.002-20 mol/L.

In some preferred modes, the amount of the sodium acrylate used for preparing the magnetic microspheres C is 0.53-12.76 mol/L.

In some preferred embodiments, the method for preparing the biomagnetic microspheres D comprises the following steps:

firstly, 0.5-1000 mL (20%, v/v) of aqueous solution of silicon dioxide-coated ferroferric oxide magnetic microspheres is measured, the magnetic microspheres are washed by absolute ethyl alcohol, 10-300 mL of ethanol solution (5% -50%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) is added into the washed magnetic microspheres, reaction is carried out for 2-72 hours, and the magnetic microspheres are washed by absolute ethyl alcohol and distilled water, so that amino modified magnetic microspheres A are obtained.

Removing 1.0X 10^-4About 1mol of acrylic acid is added into a solution X with the pH value of 4-6 (the solution X is an aqueous solution with the final concentration of 0.01-1 mol/L2-morpholine ethanesulfonic acid (CAS: 4432-31-9) and 0.1-2 mol/L NaCl), 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl, CAS: 25952-53-8) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS, CAS: 6066-82-6) are added, and the reaction is carried out for 3-60 min. And adding the solution into PBS buffer solution with the pH value of 7.2-7.5 mixed with 0.5-50 mL of magnetic microspheres A, reacting for 1-48 hours, and washing the magnetic microspheres with distilled water to obtain the carbon-carbon double bond modified magnetic microspheres B.

And (3) taking 0.5-50 mL of magnetic microsphere B, adding 0.5-200 mL of 5-30% (w/v) sodium acrylate solution, then adding 10-20 mL of 2-20% (w/v) ammonium persulfate solution and 1-1 mL of tetramethylethylenediamine, reacting for 3-60 minutes, and then washing the magnetic microsphere with distilled water to obtain the sodium polyacrylate modified magnetic microsphere C.

Transferring 0.5-50 mL of magnetic microsphere C into a solution X with the pH value of 4-6, adding 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS), and reacting for 3-60 min. Then adding PBS buffer solution with 0.0001-1 mol of 1, 3-propane diamine and pH7.2-7.5, and reacting for 1-48 hours. Washing with distilled water, adding PBS buffer solution, and converting COONa of a side branch chain of a polymer in the magnetic microsphere C into an amino functional group; weighing 1.0 × 10^-6～3.0×10^-4Adding biotin into the solution X in mol, adding 2.0X 10^-6～1.5×10^- ³mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2.0X 10^-6～1.5×10^-3And (3) mol of N-hydroxysuccinimide, and reacting for 3-60 min. And then adding the mixture into the cleaned magnetic microsphere solution, reacting for 1-48 hours, and cleaning with distilled water to obtain the biotin-modified magnetic microsphere D.

1.4.2.4. Protein fixing system using affinity protein as purification medium

In some embodiments, a protein immobilization system with affinity proteins as the purification medium is employed.

In some embodiments, the following step (5) is performed on the basis of the above-mentioned 1.4.2.3 biomagnetic microsphere D to obtain a magnetic microsphere system using affinity protein as a purification medium.

And (5): the binding avidin-avidin covalently links complex E. The avidin-avidin covalent linking complex E is bound to the end of the branched polymer chain by the specific binding between biotin and avidin or its analog, and the biotin and avidin or its analog form an affinity complex binding to obtain the biomagnetic microsphere F (avidin magnetic microsphere).

A typical structure of the affinity protein magnetic microsphere is shown in FIG. 2.

The avidin-avidin covalent connection compound E is added into a system of the biomagnetic microsphere D, the avidin is non-covalently connected to the tail end of a polymer branched chain on the outer surface of the magnetic bead by utilizing the extremely strong specific affinity between biotin and avidin (such as streptavidin) or analogues thereof, the avidin modified magnetic bead (biomagnetic microsphere F) which can be used for separating and purifying antibody substances is obtained, and the avidin is used as a purification medium to provide a binding site for capturing target protein.

Avidin-avidin covalent linkage complex E: also called as avidin-avidin complex E, a complex formed by covalent linkage, in which one end is avidin or its analog and the other end is avidin, and the two are directly linked by a covalent bond or indirectly linked by a covalent linking member. The covalent linker includes a covalent bond (e.g., an amide bond), a linker peptide, and the like. The avidin-avidin complex E is exemplified by streptavidin-bearing avidin, wherein the avidin is selected from the group consisting of, but not limited to, protein a, protein G, and/or protein L, and the like. Examples of avidin-avidin complexes E also include: Streptavidin-Protein a complex, Streptavidin-Protein a fusion Protein, Streptavidin-enhanced green fluorescent Protein-Protein a fusion Protein (Protein a-eGFP-Streptavidin), Protein a-eGFP-Tamavidin2, Protein a-eGFP-Tamavidin1, and the like; wherein the eGFP broadly comprises an eGFP mutant, Streptavidin is Streptavidin, and Tamavidin1 and Tamavidin2 are both avidin analogues.

In some embodiments, the process for preparing the biomagnetic microspheres F is as follows: the biomagnetic microspheres D are added to a fusion protein solution of an avidin-protein A linked complex E (e.g., ProteinA-eGFP-Streptavidin, ProteinA-eGFP-Tamavidin2), and mixed for incubation. The protein A is fixed on the terminal group of the polymer branch chain on the outer surface of the biomagnetic microsphere D by the specific binding of avidin or analogues thereof (such as Streptavidin or Tamavidin2) and biotin, so as to obtain the biomagnetic microsphere F combined with avidin-protein A. In the structure of the obtained biomagnetic microsphere F, the side chain of the acrylic polymer contains an affinity complex structure of biotin-avidin-protein A, the side chain is covalently connected to a branch point (such as a branch point of a linear main chain) of the polymer through a biotin end, a non-covalent strong specific binding effect of an affinity complex is formed between biotin and avidin or an analogue thereof, the avidin or an analogue thereof is covalently connected with the protein A, a fluorescent label can be inserted between the avidin or an analogue thereof and the protein A, and other connecting peptides can also be inserted.

Among them, avidin-protein A fusion proteins, such as ProteinA-eGFP-Streptavidin fusion protein and ProteinA-eGFP-Tamavidin2 fusion protein, can be obtained by in vitro cell-free protein synthesis through IVTT reaction. And mixing the supernatant obtained after the reaction of the biomagnetic microspheres D and the IVTT, and realizing the combination of the affinity protein A through the specific combination action between the biotin on the outer surfaces of the biomagnetic microspheres D and the avidin fusion protein in the solution.

The amount of affinity protein bound to the outer surface of the magnetic microspheres can be determined by: first, after the binding reaction between the solution of the affinity protein and the magnetic beads is completed, the biomagnetic microspheres bound with the affinity protein are adsorbed and settled by a magnet. The liquid phase is then collected by separation and is designated as flow-through or flow-through. At this time, the concentration of the affinity protein in the liquid phase decreases. The change condition of the eGFP fluorescence value in the supernatant obtained by IVTT reaction before and after the combination of the biomagnetic microspheres is measured, the fluorescence intensity of the eGFP combined on the biomagnetic microspheres is calculated, and the concentration of the affinity protein is obtained through conversion. When the concentration of the affinity protein in the flow-through solution is not substantially changed compared with the concentration of the affinity protein in the IVTT solution before the incubation of the biomagnetic microspheres, the adsorption of the biomagnetic microspheres D on the affinity protein is saturated, and the fluorescence value of the corresponding eGFP is not obviously changed. The pure product of the eGFP can be used for establishing a standard curve of the fluorescence value and the concentration of the eGFP so as to quantitatively calculate the content and the concentration of the avidin-avidin (such as streptavidin-protein A) bound on the biomagnetic microspheres.

The antibody is expressed by an in vitro protein synthesis system (taking bovine serum antibody as an example), and the biological magnetic microsphere F combined with the protein A is used for separating and purifying the antibody, and the antibody binding capacity can be calculated by the following method: and incubating the protein A modified magnetic beads and a bovine serum antibody solution, eluting the bovine serum antibody from the magnetic beads by using an elution buffer solution after the reaction is finished, and separating to obtain an eluent containing the bovine serum antibody. The concentration of bovine serum albumin in the eluate was determined by the Bradford method. Meanwhile, BSA is used as a standard protein to carry out enzyme-labeling instrument test, the standard protein is used as a reference, the protein concentration of the purified antibody can be calculated, and the yield of separation and purification are further calculated.

1.4.2.5. Position control of magnetic microspheres

After the biomagnetic microspheres D (1.4.2.3.) or biomagnetic microspheres F (1.4.2.4.) are prepared, the magnetic microspheres can be simply settled by using a magnet, a liquid phase is removed, and adsorbed foreign proteins or/and other impurities are removed by washing.

By controlling the size of the magnetic microspheres and the chemical and structural parameters of the polymer, the magnetic microspheres can be stably suspended in a liquid phase and can not settle within two days or even longer. And can be stably suspended in a liquid system without continuous stirring. On one hand, the magnetic microsphere can be controlled to be in a nanometer size of several micrometers or even less than 1 micrometer, on the other hand, the grafting density of the polymer on the outer surface of the magnetic microsphere can be adjusted, and the characteristics of the hydrophilicity, the structure type, the hydrodynamic radius, the chain length, the number of branched chains, the length of the branched chains and the like of the polymer can be adjusted, so that the suspension performance of the magnetic microsphere system in the system can be better controlled, and the full contact between the magnetic microsphere system and an in vitro protein synthesis reaction mixed system can be realized.

1.4.2.6. Protein fixing system using nickel ions as purification medium

In some embodiments, a protein immobilization system using nickel ions as the purification medium is employed.

In some embodiments, the following step (6) can be performed on the basis of the magnetic microsphere C of 1.4.2.3. above to prepare a magnetic microsphere system using nickel ions as a purification medium.

And (6): coupling acrylic polymer modified magnetic microsphere C with tricarboxyamine (such as NTA (nitrilotriacetic acid)), and N, N-bis (carboxymethyl) -L-lysineAmino acids, such as a combination of the two), and then complexing the Ni via the three carboxyl groups of the tricarboxyamine²⁺The magnetic microsphere system (also called nickel magnetic bead) modified by nickel ions is obtained, and can be used for separating His-labeled protein.

In some preferred modes, the tricarboxyamine is NTA, and the dosage of the NTA is 0.5-550 g/L.

In some embodiments, nickel magnetic beads are prepared by: and (3) transferring 0.5-50 mL of the magnetic microsphere C into the solution X, adding 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.001-0.5 mol of N-hydroxysuccinimide, and reacting for 3-60 min. Adding 0.0001-1 mol of N, N-bis (carboxymethyl) -L-lysine (CAS: 113231-05-3) in PBS buffer solution with pH of 7.2-7.5, and reacting for 1-48 h. And adding 0.0001-1 mol of nickel sulfate solid particles into the reaction system, and reacting for 5 minutes-24 hours. And washing the magnetic microspheres with distilled water to obtain the magnetic microspheres N with nickel ions as a purification medium.

1.4.3. Regeneration and reuse of protein fixation systems

When the purification medium in the protein immobilization system is connected to the outer surface of the solid matrix or the connecting element on the outer surface of the solid matrix in a reversible mode, the purification medium can be unbound under proper conditions, can be reutilized by being recombined with the purification medium, and can also be replaced by different kinds of purification media.

Take the biomagnetic microsphere F (affinity protein magnetic microsphere) prepared above as an example. The affinity protein as a purification medium is linked to the branched ends of the branched polyacrylic acid backbone by the affinity complex interaction of "biotin-avidin". The interaction between biotin and avidin is reversible, and proper eluent is adopted for elution, so that avidin-avidin can be unbound from biotin at the tail end of a branched chain of the biomagnetic microsphere D (the biotin magnetic microsphere) and eluted, and the regeneration of the biomagnetic microsphere D is realized. Repeating the step (5), and combining the affinity protein at the tail end of the branched chain in a biotin-avidin-affinity protein mode again, so that the reutilization of the biomagnetic microspheres D, the renewal of the purification medium and the regeneration of the biomagnetic microspheres F can be realized. When the step (5) is carried out, the types of the avidin can be changed, different types of affinity proteins are combined, and the replacement of a purification medium is realized.

The strong affinity between biotin and streptavidin is the binding action of a typical affinity complex, which is stronger than the action of common non-covalent bonds and weaker than the action of covalent bonds, so that the avidin can be firmly bound on the outer surface of a magnetic bead (for example, the tail end of a polymer branched chain bound on the outer surface of the magnetic bead), and the streptavidin can be eluted from the specific binding position of the biotin to realize synchronous separation of the avidin when the avidin needs to be updated and replaced, and then an activation site capable of being recombined with a new avidin-avidin covalent binding complex E (for example, the avidin with a streptavidin label) is released, thereby realizing the rapid recovery of the purification performance of the magnetic bead, and greatly reducing the separation and purification cost of an antibody. The process of eluting the biological magnetic microsphere F modified with the avidin and removing the avidin-avidin covalent linkage compound E so as to obtain the biotin-modified biological magnetic microsphere D again is called as the regeneration of the biotin magnetic microsphere (or called biological magnetic microsphere D). The regenerated biomagnetic microspheres D have released biotin active sites and can be recombined with avidin-avidin covalent connection complexes E to obtain avidin-modified biomagnetic microspheres F again (corresponding to the regeneration of the biomagnetic microspheres F), so that fresh avidin can be provided and new antibody binding sites can be provided. Thus, the magnetic microspheres can be regenerated for use, and can be reused after the affinity protein is renewed or replaced.

In some embodiments, the process of regenerating the biomagnetic microspheres F is performed as follows: adding a denaturing buffer solution (containing urea and sodium dodecyl sulfate) into the biomagnetic microspheres F, incubating in a metal bath at 95 ℃, eluting off avidin-protein A fusion proteins (such as SPA-eGFP-Tamavidin2) combined with biotin on the biomagnetic microspheres D to obtain regenerated biomagnetic microspheres D (releasing biotin active sites at the ends of polymer branched chains), adding a fresh solution of the avidin-protein A fusion proteins (such as SPA-eGFP-Tamavidin2 supernatant after IVTT reaction) into the regenerated biomagnetic microspheres D, allowing the released biomagnetic microspheres D to be recombined with new avidin-protein A (such as SPA-eGFP-Tamavidin2) at the biotin active sites, and forming noncovalent specific binding action between the biotin and avidin or analogues thereof (such as Tamavidin2), thereby realizing the replacement of the protein A and obtaining the regenerated biological magnetic microsphere F.

1.5. Integrated process for protein synthesis-co-purification

The integrated preparation method for in vitro synthesis and purification of the protein provided by the invention couples the protein synthesis process and the separation and purification process of a protein product, and is an integrated protein synthesis-co-purification process. In the process of in vitro protein synthesis reaction, the separation of target protein products is synchronously carried out, and the target protein is combined to the outer surface of the protein fixing system. Thus, after the in vitro protein synthesis reaction is completed, the conventional additional purification incubation step can be omitted.

The in vitro synthesis and purification integrated preparation method of the protein comprises the following steps of ii: carrying out incubation reaction on the reaction purification mixed system (the intePure system, also called a coPure system, or called a reaction-purification coupling system) constructed in the first step under a proper condition to obtain a target protein product; during the in vitro protein synthesis reaction in which incubation is performed, the protein product of interest is able to specifically bind to a protein immobilization system, resulting in a protein immobilization system (also denoted loaded-PIS) that binds the protein product of interest.

The incubation reaction, which refers to the in vitro protein synthesis reaction, includes at least a translation process (in which case the nucleic acid template may include only an mRNA template), optionally a transcription process, a nucleic acid replication process (of DNA or/and RNA).

In some preferred embodiments, a DNA template encoding the protein of interest is used, and accordingly, the incubation reaction includes both transcription and translation processes.

1.6. Post-treatment process

1.6.1. Protein immobilization system for extracting target protein product

Step iii: loaded-PIS (protein immobilization system bound to the target protein product) is isolated from the reaction purification mixture.

The reaction purification mixed system is a solid-liquid mixed system. The loaded-PIS is a solid phase system. The process of extracting loaded-PIS is a process of solid-liquid separation and can be realized by adopting a conventional technical means. The method for realizing this process is not particularly limited as long as it is compatible with the protein immobilization system used. Including but not limited to filtration, washing, and the like.

When the magnetic microsphere system is used as a protein immobilization system, the magnetic microspheres can be directly transferred by using a magnetic device (such as a magnet), or the liquid phase can be transferred after the magnetic microspheres are immobilized by using the magnetic device.

1.6.2. Obtaining an eluate containing the target protein

Step iv: eluting the target protein from the loaded-PIS separated in the step iii to obtain an eluent (a primary purified product solution of the target protein) containing the target protein. This process is a process for releasing specific binding between a purification substrate (target protein) and a purification medium on a protein immobilization system, and can be carried out in any suitable manner as reported.

Optionally further comprising the step v: separating the target protein from the eluent to obtain the eluent of the primary purified product of the target protein.

In the integrated preparation method for in vitro protein synthesis and purification, after the in vitro protein synthesis reaction is started, the step iv needs to be controlled at a proper time, and under the condition of saving the incubation step, the higher protein synthesis amount and the higher protein purification yield are realized.

In some preferred embodiments, the end point of the in vitro protein synthesis reaction is monitored as the amount of protein synthesis or the reaction time, and the timing of performing step iv is controlled. For example, the amount of protein synthesis can be measured in real time by a fluorescent labeling method; or a specific length of reaction time.

The eluate containing the primary purified product of the target protein may be used as it is, or the primary purified product of the target protein may be used after being dried, or may be used after being further purified as necessary.

1.6.3. Refining Process (step vi)

When the purified product (primary purified product) of the target protein or the eluent or the solution thereof obtained in 1.6.2 can not meet the purity requirement, further purification is needed to obtain the repurified product of the target protein.

In the refining process for further purification, one or more purification operations such as ultrafiltration membrane, column chromatography, ion exchange column, hydrophobic column, salting out, recrystallization, vacuum drying, freeze drying, and the like may be performed.

1.7. Detection (step vii)

The intePure method may optionally further comprise the step of detecting any one product of the target protein. The detection object may be a synthetic product at any synthesis reaction time point, or a purified product in any link, or may be a product free from a solution (e.g., a reaction solution or an eluate), or a product bound to a protein immobilization system, or may be a primary purified product obtained in step vi, or a secondary purified product obtained in step vi.

Wherein the execution time of step vii is not particularly limited, and may be performed at any stage of the preparation method; the sample to be tested is not particularly limited, and may be a solution, a liquid mixture or a solid-liquid mixture containing a protein product, a powder (such as wet powder or dry powder), a liquid, wet powder or dry powder sample containing no protein product as a reference, or a sample at the reaction zero point.

The detection method can be realized by adopting a conventional technical method.

By detecting and calculating, the results including but not limited to yield, purity, molecular weight, protein function and the like can be obtained.

(k1) any of the in vitro protein synthesis systems of the invention;

(k3) any one of the protein immobilization systems of the invention, or a component thereof; the constituent elements of the protein fixing system comprise any one or the combination of a solid matrix, a solid matrix wrapped by a branched chain type polymer, a reaction raw material of the branched chain type polymer and a purification medium;

(k4) Optionally, a position control member of a protein fixation system;

(k5) optionally, washing solution a for washing the protein immobilization system bound to the target protein;

(k6) optionally, an eluent B capable of eluting the target protein from the protein immobilization system;

(k7) optionally, a regeneration reagent for the protein immobilization system, to effect regeneration of the protein immobilization system;

(k8) optionally, the purification medium is replaced with a reagent, which enables replacement of the purification medium;

the (k1) can constitute an in vitro protein synthesis kit.

The (k1) and (k2) can jointly form an in vitro protein synthesis kit.

The nucleic acid template for coding the target protein can be provided by a user after matched design, and then is matched with an in-vitro protein synthesis system provided by the kit for use.

In some preferred embodiments, the nucleic acid template encoding the protein of interest serves as a reference control.

The protein immobilization system is provided with a purification medium.

In some preferred embodiments, the protein immobilization system in (k3) is a magnetic microsphere system, and in this case, (k4), (k5), (k6) and (k7) are as follows:

(k4) optionally, the position control member of the magnetic microsphere is used for controlling the position of the magnetic microsphere system, so that the separation of the magnetic microsphere system from the liquid phase system can be realized;

(k5) optionally, washing solution A for washing the magnetic microspheres bound with the target protein;

(k6) optionally, eluent B capable of eluting the target protein from the magnetic microspheres;

(k7) optionally, a reagent for regenerating the magnetic microspheres.

In some preferred forms, the (k3) comprises a magnetic microsphere system or a component thereof; the magnetic microsphere system comprises a magnetic microsphere body, magnetic microspheres with the outer surfaces coated with branched chain polymers, reaction raw materials of the branched chain polymers and a purification medium or a combination of the reaction raw materials and the purification medium.

In some preferred embodiments, the components of the in vitro protein synthesis purification kit are placed in one or more containers as a solid (e.g., dry powder), semi-solid, liquid, emulsion, suspension, or a combination thereof. In some preferred modes, the dry powder is freeze-dried powder or vacuum-dried powder. The liquid comprises a pure substance and a solution.

In some preferred forms, the (k1) and the (k2) are packaged separately.

In some preferred embodiments, the cell extract is contained in (k1) and is separately packaged in a container.

In some preferred modes, the translation related elements in (k1) are independently packaged together in a container.

In some preferred embodiments, the (k1) comprises purified translation-related elements or a combination thereof (i.e., comprises a purified translation-related element or a combination of purified translation-related elements), and are packaged together in a single container.

The solid, such as powder (or dry powder) or granules.

Such as a paste, a sludge, etc. Wet powders are also broadly included.

The liquid can be pure or a mixture.

The emulsion refers to a mixed system of incompatible liquid phases, and is also called emulsion.

The suspension refers to a mixed system of an incompatible liquid phase and a solid.

The in vitro protein synthesis purification kit can be used for carrying out in vitro protein synthesis reaction to synthesize target protein, and simultaneously carrying out separation and purification of protein products to combine the protein products to the protein fixing system from reaction liquid.

In some preferred embodiments, the components of the in vitro protein synthesis purification kit together comprise an aqueous solution. The kit includes a container for the aqueous solution.

In some preferred modes, the components of the kit are divided into two parts, namely dry powder (such as freeze-dried powder and vacuum-dried powder) and liquid reagent. The in vitro protein synthesis and purification kit comprises two containers, wherein one container is used for containing a dry powder component, and the other container is used for containing a liquid reagent component. The liquid reagent includes all systems containing liquid phase, and can be homogeneous system or mixed system, including but not limited to pure substance, solution, emulsion, suspension, and combination thereof.

In some preferred embodiments, the in vitro protein synthesis and purification kit comprises the following separate containers: (a) a cell extract; (b) an energy system; (c) optionally, a nucleic acid template; (d) a buffer solution; (e) optionally, a pH adjusting component; (f) optionally, a number of other solid components; (h) optionally, a number of other liquid components; (i) optionally, a purified translation-related element; (j) optionally, the relevant elements are amplified. Wherein, the components (a), (b), (c), (i) and (j) are respectively and independently packaged into dry powder or aqueous solution. Wherein components (c), (e), (f), (i), (j) are each independently present or absent. The components (a) to (j) may be separately packaged in one container, or may be further separately packaged in different containers. The "number" herein means 1, 2 or more. Wherein, the components (a), (i) and (j) can be separately packaged, and can also be packaged in the same container. Component (d) may be co-packaged with any other component or components. The substrate for the synthetic protein, the substrate for the synthetic RNA and/or the substrate for the synthetic DNA, the magnesium ion, the potassium ion, the crowding agent and the like may be each independently packaged in (f) and/or (h).

When the kit comprises a container of solid particles, powder (such as dry powder and wet powder) and other components, the components need to be dissolved or redissolved when in use, and the dissolution can be realized by adding a buffering agent. The buffer may be provided by (d). The buffer may be a buffer commonly used in vitro protein synthesis systems (including, but not limited to, 4-hydroxyethylpiperazine ethanesulfonic acid, tris, and combinations thereof).

When the kit contains a liquid reagent, it may be preferred to include a cryoprotectant component therein.

In some preferred modes, each component of the kit is separately packaged into dry powder, buffer solution, other liquid reagents, and optionally solvent water.

In some preferred modes, the following components can be respectively dispensed or dispensed in different containers in a proper combination mode: a cell extract (containing endogenously expressed RNA polymerase, optionally containing endogenously expressed DNA polymerase), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a crowding agent, an exogenous magnesium ion, an exogenous potassium ion, a buffer, and optionally a dispensing container comprising any one of the following exogenous components or suitable combinations thereof: a nucleic acid template encoding a protein of interest, an exogenously added RNA polymerase, an exogenously added DNA template encoding an RNA polymerase, an exogenously added DNA template encoding an RNA polymerase, other DNA amplification elements, substrates for synthesizing DNA, translation-related elements, RNA amplification-related elements, RNase inhibitors, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, aqueous solvents. In some preferred embodiments, the cell extract comprises transfer rna (trna), ribosome (ribosome). The RNA polymerases may each independently be more preferably T7 RNA polymerase. The DNA polymerases may each independently be more preferably phi29 DNA polymerase. When a DNA polymerase is provided, a substrate for synthesizing DNA may be provided endogenously, exogenously or in combination thereof, usually together. The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a kluyveromyces cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

In some preferred modes, the following components can be respectively dispensed or dispensed in different containers in a proper combination mode: cell extracts (the source cell has no endogenous integrated RNA polymerase coding sequence/coding gene, nor endogenous integrated DNA polymerase coding sequence/coding gene), exogenously added RNA polymerase, energy systems, substrates for RNA synthesis, substrates for protein synthesis, crowding agents, exogenous magnesium ions, exogenous potassium ions, buffers, and optionally further comprising any of the following exogenous components or suitable combinations thereof in a dispensing container: a nucleic acid template encoding a protein of interest, an exogenous DNA template encoding an RNA polymerase, an exogenously added DNA polymerase, an exogenous DNA template encoding a DNA polymerase, other DNA amplification elements, substrates for DNA synthesis, translation-related elements, RNA amplification-related elements, rnase inhibitors, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, aqueous solvents. In some preferred embodiments, the cell extract contains transfer RNA, ribosomes. The RNA polymerases may each independently be more preferably T7 RNA polymerase. The DNA polymerases may each independently be more preferably phi29DNA polymerase. The cell extract is in some preferred forms a eukaryotic cell extract, in some preferred forms a kluyveromyces cell extract, and in some preferred forms a kluyveromyces lactis cell extract.

(1) In some preferred embodiments, the nucleic acid template encoding the protein of interest comprises a promoter element that is recognized by the in vitro protein synthesis system.

(2) In some preferred embodiments, the nucleic acid template encoding the protein of interest comprises a promoter element that is recognized by a cell extract of the in vitro protein synthesis system. For example, the cell extract contains an endogenously expressed RNA polymerase which corresponds to the promoter element on the nucleic acid template.

(3) In some preferred embodiments, the nucleic acid template encoding the target protein contains a T7 promoter, and the in vitro protein synthesis system includes T7 RNA polymerase, an exogenous nucleic acid template encoding T7 RNA polymerase, or a combination thereof.

(4) In some preferred embodiments, the nucleic acid template encoding the protein of interest comprises a T7 promoter, and the in vitro protein synthesis system comprises a cell extract comprising endogenously expressed T7 RNA polymerase.

(5) In some preferred embodiments, the nucleic acid template encoding the target protein contains a T7 promoter capable of initiating a gene transcription process of the target protein, i.e., the gene transcription process of the target protein is initiated by the T7 promoter on the nucleic acid template.

(6) In some preferred embodiments, the nucleic acid template encoding the target protein comprises a T7 promoter capable of initiating a gene transcription process for the target protein, and the in vitro protein synthesis system comprises T7 RNA polymerase, an exogenous nucleic acid template encoding T7 RNA polymerase, or a combination thereof.

(7) In some preferred embodiments, the T7 promoter is located upstream of the coding sequence for the target protein of the nucleic acid template, and initiates the transcription process of the target protein, and the in vitro protein synthesis system comprises a cell extract containing endogenously expressed T7 RNA polymerase.

The (k1), optionally including (k2), may adopt the dispensing manner of documents CN109321620A, CN110551785A, etc.

in some preferred modes, the protein immobilization system is coupled with any one of the in vitro protein synthesis systems (or the in vitro protein synthesis kit) of the invention, and is used for constructing a reaction and purification mixed system integrating protein synthesis and purification.

In some preferred modes, the protein fixing system is used in any one of the in vitro protein synthesis and purification kits of the invention, and can be used for implementing the method for preparing the inteure.

In some preferred embodiments, the method is applied to protein production, or to protein synthesis-based assays.

The application fields of the in vitro protein synthesis aspect include but are not limited to the fields of biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano engineering, cosmetics, food additives, nutritional agents, agriculture, feed, living goods, washing, environment, chemical dyeing, fluorescent labeling and the like.

In some preferred embodiments, the protein immobilization system is selected from the group consisting of the magnetic microsphere systems of any of the present invention. In some preferred embodiments, the magnetic microsphere system carries the purification medium through the branched-chain polymer coated on the outer surface of the solid matrix, and the amount of the purification medium can be amplified by multiple times, ten times, tens of times, hundreds of times, or even thousands of times.

In some preferred modes, the application is applied to the in vitro protein synthesis of the antibody substances or the application of the in vitro synthesis and purification of the antibody substances.

4. The invention will be further illustrated with reference to the following specific examples and figures 1-15. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are preferably carried out according to, with reference to, the conditions as indicated in the specific embodiments described above, and may then be carried out according to conventional conditions, for example "Sambrook et al, molecular cloning: a laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), "A laboratory Manual for cell-free protein Synthesis" Experimental Manual for ethylene by Alexander S.Spirin and James R.Swartz.cell-free protein synthesis: methods and protocols [ M ].2008 ", etc., or according to the conditions recommended by the manufacturer.

Unless otherwise indicated, percentages and parts referred to in this invention are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

It should be noted that, all the contents of the embodiments of the chinese patent applications CN2019105401326, CN2019221117066 and CN201911209156X (including related drawings) are incorporated into the present invention.

In some of the following examples, a protein factor system based on an in vitro cell-free protein synthesis method (D2P technology) was used to provide a mixed system containing a target protein.

Example 1 preparation of acrylic Polymer modified magnetic microspheres C

Firstly, 50mL of aqueous solution of silicon dioxide-coated ferroferric oxide magnetic microspheres (the particle size of the magnetic microspheres is about 1 μm) with solid content of 20% (v/v) is measured, the magnetic microspheres are settled by a magnet, liquid phase is removed, 60mL of absolute ethyl alcohol is used for cleaning the magnetic microspheres each time, and the total cleaning is carried out for 5 times. 100mL of an excessive ethanol solution (25%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the washed magnetic microspheres, and the mixture was mechanically stirred in a water bath at 50 ℃ for 48 hours, then in a water bath at 70 ℃ for 2 hours, the magnetic microspheres were settled with a magnet, the liquid phase was removed, the magnetic microspheres were washed with 60mL of absolute ethanol each time, 2 times in total, then with 60mL of distilled water each time, and the washing was repeated 3 times to obtain magnetic microspheres A.

Secondly, 0.01mol of acrylic acid is transferred and added into 100mL of solution X (solution X: the final concentration of 0.1 mol/L2-morpholine ethanesulfonic acid (CAS: 4432-31-9) and 0.5mol/L NaCl aqueous solution respectively), 0.04mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04mol of N-hydroxysuccinimide (CAS: 6066-82-6) are added, stirred and mixed evenly at room temperature, stirred and reacted for 15min, NaHCO is used for reaction₃Adjusting pH of the solution to 7.2 with solid powder, adding the above solution into 100mL PBS buffer solution containing 10mL magnetic microsphere A, mechanically stirring in water bath at 30 deg.C for 20 hr, and mixing with the above solutionAnd (3) settling the magnetic microspheres by using a magnet, removing a liquid phase, washing the magnetic microspheres by using 60mL of distilled water each time, and repeatedly washing for 6 times to obtain the magnetic microspheres B.

And thirdly, taking 1mL of the magnetic microsphere B, adding 12mL of 15% (w/v) sodium acrylate solution, adding 450 muL of 10% ammonium persulfate solution and 45 muL of tetramethylethylenediamine, reacting for 30 minutes at room temperature, settling the magnetic microsphere by using a magnet, removing a liquid phase, washing the magnetic microsphere by using 10mL of distilled water each time, and washing for 6 times in total to obtain the magnetic microsphere C (the magnetic microsphere C modified by the acrylic polymer).

Example 2 preparation of Biotin-modified biomagnetic microspheres D

Transferring the synthesized magnetic microspheres C into 10mL of solution X, adding 0.004mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.004mol of N-hydroxysuccinimide, stirring and uniformly mixing at room temperature, stirring for reacting for 15min, settling the magnetic microspheres by using a magnet, removing a liquid phase, and washing 3 times by using 10mL of distilled water each time; removing 4.0X 10^-4Dissolving 1, 3-propanediamine mol in 10mL of PBS buffer solution, adding into the washed magnetic microspheres, mechanically stirring for 20 hours in a water bath at 30 ℃, settling the magnetic microspheres by using a magnet, removing a liquid phase, washing for 6 times by using 10mL of distilled water each time, and adding into 10mL of PBS buffer solution; weighing 2.5X 10^-4mol biotin, 10mL of solution X, 1.0X 10^-3mixing mol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.001mol N-hydroxysuccinimide at room temperature, stirring for reaction for 15min, and reacting with NaHCO₃Adjusting the pH value of the solution to 7.2, adding the solution into the washed magnetic microspheres containing 10mL of PBS buffer solution, mechanically stirring for 20 hours in a water bath at 30 ℃, settling the magnetic microspheres by using a magnet, removing a liquid phase, and washing for 10 times by using 10mL of distilled water each time to obtain the biotin-modified biomagnetic microspheres D.

Example 3 preparation of affinity protein-modified magnetic microspheres F

3.1. Synthesis of ProteinA-eGFP-avidin fusion proteins

DNA sequences consisting of three segments of genes of fusion proteins ProteinA-eGFP-Streptavidin and Protein A-eGFP-Tamavidin2 were constructed, respectively.

The sequence of protein A is derived from Staphylococcus Aureus, SPA for short. The amino acid sequence of SPA is 516 amino acid residues in total length, and amino acids 37-327 are selected as gene sequences used for constructing fusion protein, namely antibody binding domain of SPA. The nucleotide sequence is obtained after the sequence is optimized by an optimization program, and the nucleotide sequence is shown in SEQ ID No. 1.

Tamavidin2, an avidin analog, is a protein with biotin-binding ability, contains 141 amino acid units in total, and obtains its nucleotide sequence as shown in SEQ ID No. 2 by codon conversion and program optimization.

eGFP, the nucleotide sequence of which is shown as SEQ ID NO. 3, is an A206K mutant of the eGFP and is also marked as mEGFP; the amino acid sequence is shown as SEQ ID No. 4.

DNA templates of two fusion proteins, namely Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamavidin2, are respectively constructed and prepared by adopting a recombinant PCR method. Then, two fusion proteins, Protein a-eGFP-Streptavidin and Protein a-eGFP-Tamavidin2, were synthesized by an in vitro Protein synthesis method, and patent documents CN201610868691.6, WO2018161374a1, KR20190108180A, CN108535489B, and the like were referred to by a publicly known in vitro Protein synthesis method. The IVTT system is used for expressing SPA-eGFP-Streptavidin and SPA-eGFP-Tamavidin2 respectively. In general terms, the IVTT system comprises a cell extract (containing RNA polymerase expressed by genome integration), a DNA template, an energy system (such as phosphocreatine-phosphocreatine kinase system), magnesium ions, sodium ions, polyethylene glycol and the like, and the reaction is carried out at the temperature of 28-30 ℃. And finishing the reaction after 8-12 hours. The resulting IVTT reaction solution contains ProteinA-eGFP-avidin fusion protein corresponding to the DNA template.

IVTT reaction solutions in which the target proteins were ProteinA-eGFP-Streptavidin (FIG. 3 "1") and ProteinA-eGFP-Tamavidin2 (FIG. 3 "2"), respectively, were obtained, and the RFU values were measured, and the results are shown in "total" in FIG. 3.

The IVTT reaction liquid of protein A-eGFP-Streptavidin and protein A-eGFP-Tamavidin2 is respectively centrifuged for 10min at 4000rpm and 4 ℃, and the supernatant is reserved. Record as IVTT supernatant.

3.2. Preparation of protein A-binding biomagnetic microspheres F

The obtained IVTT supernatant of two fusion proteins, namely protein A-eGFP-Streptavidin and protein A-eGFP-Tamavidin2, is incubated with the biomagnetic microspheres D obtained in example 2 respectively, the reaction is carried out for 1 hour, the content of the fusion protein in the biomagnetic microspheres F is determined according to the method given above, the binding capacity of the two fusion proteins is compared, and the result is shown in the figure 3 'supernatatant'.

Aspirate 30. mu.L of a 10% (v/v) suspension of biomagnetic microspheres D and mix with binding/washing buffer (10mM Na)₂HPO₄pH 7.4，2mM KH₂PO₄140mM NaCl, 2.6mM KCl) for use after 3 washes.

And (3) taking 2mL of IVTT supernatant containing the avidin-protein A fusion protein and the biomagnetic microspheres D to perform rotary incubation for 1 hour at the temperature of 4 ℃, collecting unbound supernatant, namely flow-through liquid (also called flow-through liquid), and repeating the step three times to obtain three different flow-through liquids. Namely, the same batch of the biomagnetic microspheres D is incubated with the avidin-protein A for three times continuously. Each time 2mL of the IVTT supernatant described in step 3.1 was used, the corresponding flow-through obtained each time is numbered 1, 2, 3 in order. The fluorescent values of eGFP were measured in the IVTT supernatant and the three flowthrough using the fluorometry method, and the results are shown in fig. 4. The closer the RFU value of the triple flow-through and the IVTT supernatant, the more saturated the binding of biomagnetic microspheres D to avidin-avidin a, and the corresponding RFU values are shown in table 2 below.

RFU values of SPA-eGFP-Streptavidin and SPA-eGFP-Tamavidin2 bound to the biomagnetic microspheres D are shown in tables 3 and 4 below, respectively. Wherein for Streptavidin, the first binding is already saturating the protein A binding capacity of biomagnetic microsphere D. For Tamavidin2, the protein a binding capacity of biomagnetic microspheres D was substantially saturated after the second binding. The first binding capacity was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the flow-through 1, the second binding capacity was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the flow-through 2, and the third binding capacity was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the flow-through 3, as shown in tables 3 and 4 below. Wherein, the binding force refers to the protein A fusion protein/biomagnetic microspheres D, the mass-to-volume ratio, and the unit (mg/mL).

The concentration of protein per binding was calculated from a standard curve of fluorescence values versus mass concentration of protein, and the total amount of protein per binding was calculated from the incubation volume (2 mL). Drawing up a standard curve according to the purified eGFP to obtain a conversion formula between the RFU value and the protein mass concentration of the eGFP as follows:

Wherein, C_fusionProtein mass concentration (. mu.g/mL), FRU RFU fluorescence reading, M_eGFPMolecular weight of eGFP (26.7kDa), M_fusionIs the molecular weight of the fusion protein; the molecular weight of the fusion protein SPA-eGFP-Streptavidin is 77.3kDa, and the molecular weight of the fusion protein SPA-eGFP-Tamavidin2 is 79.4 kDa.

The mass concentration (. mu.g/mL) of the fusion protein SPA-eGFP-Streptavidin or SPA-eGFP-Tamavidin2 can be converted. The total protein content of the fusion protein can be obtained by multiplying the mass concentration of the corresponding fusion protein by the volume of the solution. And (3) subtracting the protein amount of the avidin-protein A in the flow-through liquid from the protein amount of the avidin-protein A in the IVTT supernatant to obtain a difference value, namely the protein amount W of the avidin-protein A combined by the biomagnetic microspheres. Dividing W by the bed volume of the magnetic microspheres, and calculating to obtain the mass of the avidin-protein A fusion protein combined by the magnetic microspheres in unit volume, namely the binding force in mg/mL.

The binding amount of 30. mu.L of 10% (v/v) biomagnetic microsphere D suspension used in the present example, converted to 1mL of 100% biomagnetic microsphere D, is shown in tables 3 and 4. Wherein the binding force of Tamavidin2 and biomagnetic microspheres D is slightly stronger than that of Streptavidin.

TABLE 2 comparison of eGFP fluorescence values of IVTT supernatants without biomagnetic microsphere D treatment and treated triple flow-through

TABLE 3 comparison of results of flow-through with SPA-eGFP-Streptavidin treated with biomagnetic microsphere D

Remarking: the volume of the beads was 30. mu.L 10% (v/v), corresponding to 3. mu.L.

TABLE 4 comparison of the results of the flow-through fluid treated with the biomagnetic microspheres D from SPA-eGFP-Tamavidin2

Example 4 preparation of magnetic microsphere N (Nickel magnetic bead) Using Nickel ion as purification Medium

The magnetic microsphere C synthesized in example 1 was transferred to 10mL of solution X, and 0.004mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.004mol of N-hydroxysuccinimide were added thereto, and stirred and mixed at room temperature for 30 min. The magnetic microspheres were precipitated with a magnet, the liquid phase was removed, and the column was washed 3 times with 10mL of distilled water each time. 0.002mol of N, N-bis (carboxymethyl) -L-lysine (CAS: 113231-05-3) was weighed and dissolved in 10mL of solution Y (PBS buffer solution with pH 7.2-7.5), the solution pH was adjusted to 7 with sodium bicarbonate solid powder, and the solution was added to the washed magnetic microspheres and mechanically stirred in a water bath at 30 ℃ for 20 hours. 0.02mol of nickel sulfate solid particles was added to the reaction system, and stirring was continued for 2 hours. And (3) settling the magnetic microspheres by using a magnet, removing a liquid phase, washing the magnetic microspheres by using 6mL of distilled water each time, and washing 10 times in total to obtain the magnetic microspheres N (also called nickel magnetic beads) taking nickel ions as a purification medium.

Example 5 preparation of protein PRTa-GFP (99.4kDa) by an Integrated Process for in vitro Synthesis and purification of proteins

IVTT-co-purification reaction (intePure system, or called coPure system, or called reaction-purification coupled system)

The main reagents are as follows: an in vitro protein synthesis system (D2P system); plasmid vector pD2P _1.06e vector (for construction of plasmid DNA encoding a protein of interest); nickel magnetic beads (protein immobilization system using nickel ions as purification medium, prepared as described in example 4).

5.1.1. Providing an in vitro protein synthesis system

The final concentrations of the components in the in vitro protein synthesis system are respectively as follows: 80% (v/v) Kluyveromyces lactis extract, 15mM glucose, 320mM maltodextrin (molar concentration measured as glucose monomers), 24mM tripotassium phosphate, 1.8mM nucleoside triphosphate mixture (a mixture of adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a final concentration of 0.7mM), magnesium L-aspartate, 80mM potassium, 2% (w/v) polyethylene glycol 8000, D-arginine, D-arginine, and D-arginine, 9.78mM Tris HCl buffer, pH8.0 (w/v), trehalose. Wherein the Kluyveromyces lactis extract comprises endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified in the following way: adopting a modified strain based on a Kluyveromyces lactis strain ATCC 8585; integrating a coding gene of T7 RNA polymerase into a genome of Kluyveromyces lactis by adopting the method described in CN109423496A to obtain a modified strain, so that the modified strain can endogenously express T7 RNA polymerase; culturing cell material with the modified strain, and preparing cell extract. The preparation process of the kluyveromyces lactis cell extract adopts conventional technical means, and refers to the method recorded in CN 109593656A. The preparation steps, in summary, include: providing appropriate amount of raw materials of Kluyveromyces lactis cells cultured by fermentation, quickly freezing the cells with liquid nitrogen, crushing the cells, centrifuging, and collecting supernatant to obtain cell extract. The protein concentration of the obtained kluyveromyces lactis cell extract is 20-40 mg/mL.

According to comparison of control experiments, under the condition of not adding any exogenous RNA polymerase, the lactate Kluyveromyces lactis system without endogenously integrating the coding gene of the T7RNA polymerase can hardly perform in-vitro protein synthesis reaction; after the endogenous integration and transformation, the high-efficiency expression of the target protein can be realized without adding any exogenous RNA polymerase, the exogenous integration and transformation can be used as a substitution mode of an exogenous addition mode, and the protein synthesis level of a traditional in-vitro protein synthesis system can be reached (in the traditional in-vitro protein synthesis system, a bacterial strain which is not subjected to endogenous transformation of T7RNA polymerase is adopted to prepare a cell extract, and exogenous T7RNA polymerase is added in the synthesis system). The method for preparing the intePure is also applicable to an in-vitro protein synthesis system of a strain (including but not limited to a Kluyveromyces lactis strain) which is not subjected to endogenous modification of T7RNA polymerase, and can obtain the same or similar optimization effect.

5.1.2. Preparation of DNA template encoding protein of interest

The plasmid vector pD2P _1.06e vector (also referred to as pD2P _1.06e-8His-eGFP vector) of kanji (shanghai) biotechnology limited, whose plasmid map is shown in fig. 5, includes functional elements such as T7 promoter, 5 'UTR, leader peptide coding sequence (leading peptide), 8 × His (histidine tag), MCS sequence (multiple cloning site, MCS, multiple cloning site), mlfp coding sequence (SEQ ID No. 3), 3' UTR, LAC4 terminator (not shown), f1 ori, AmpR promoter, AmpR gene, ori, lacI promoter, lacI coding gene, and the like. In the figure, D2P _1.06e _ F and D2P _1.06e _ R indicate the binding sites of the forward primer and the reverse primer, respectively.

Plasmid DNA was prepared. The ORF sequence of the encoded protein PRTa (molecular weight of about 75kDa) was inserted/substituted into the MCS sequence in the plasmid vector (pD 2P-1.06 e vector) by using a method such as an enzymatic ligation method or a homologous recombination method, thereby constructing a recombinant vector encoding the target protein. After the E.coli cloning host DH5 alpha is transformed by the recombinant vector, monoclonal is screened by AmpR resistance, PCR detection shows positive, and final sequencing confirmation is carried out. A plasmid DNA (pD2P-PRTa-mEGFP) encoding the target protein was obtained.

The target protein-encoding pD2P-PRTa-mEGFP was subjected to in vitro DNA amplification by an RCA amplification method using phi29 DNA polymerase to obtain a target protein-encoding DNA template.

The target translation product is marked as PRTa-GFP, has a molecular weight of 99.4kDa, is a fusion protein of PRTa and mEGFP, and is provided with an 8 × histidine tag (His-tag).

IVTT-co-purification reaction

Taking 1mL of the in vitro protein synthesis system prepared in 5.1.1, adding 33 muL of DNA template (the final concentration is 10 ng/muL) for coding PRTa-GFP to form an IVTT reaction system; then, 1. mu.L of the nickel magnetic beads prepared in example 4 was added to form a reaction-purification mixture system for IVTT-co-purification. And uniformly mixing the mixture at 30 ℃ in a shaking table, and incubating and reacting for 3 hours. The resulting reaction mixture was designated as Total.

5.1.4. Separation of

After finishing the IVTT-co-purification reaction, separating the nickel magnetic beads from the IVTT reaction system by using a magnetic frame, discarding the supernatant, and recording the corresponding supernatant as a breakthrough solution (Flow-through solution, also called Flow-through solution); and adding 1mL of cleaning fluid, carrying out vortex oscillation cleaning for 3min, separating magnetic beads by using a magnetic frame, discarding the supernatant, repeatedly cleaning by using the cleaning fluid under the same condition for three times in total to obtain three groups of cleaning fluids Wash 1, Wash 2 and Wash 3 respectively. Then 50. mu.L of eluent was added and vortexed for 5 seconds. Separating the magnetic beads, sucking out the clear liquid containing the fusion protein product, and obtaining the eluent containing the PRTa-GFP product and recording the eluent as Elution.

Cleaning solution components: 20mM Tris-HCl, 500mM NaCl, 40mM imidazole, pH 8.0.

Eluent components: 20mM Tris-HCl, 500mM NaCl, 250mM imidazole, pH 8.0.

5.1.5. Detection of protein products

30 mu L of eluent containing PRTa-GFP product is taken, 7.5 mu L of 5 × loading buffer containing 5% beta-mercaptoethanol is added, heating is carried out for 10min at 95 ℃, SDS-PAGE electrophoresis is carried out, Coomassie brilliant blue R-250 is used for dyeing, the overnight is carried out, and target protein bands are observed after decoloration.

5.2. Control group IVTT, purification was performed stepwise (control system)

5.2.1. In vitro protein Synthesis reactions

Taking 1mL of the in vitro protein synthesis system prepared in 5.1.1, adding 33 muL of DNA template (the final concentration is 10 ng/muL) for coding PRTa-GFP to form an IVTT reaction system. Mixing uniformly at 30 ℃ in a shaking table, and carrying out in-vitro protein synthesis reaction for 3 h. The reaction solution at the end of the reaction was designated as Total.

5.2.2. Separating and purifying

And (4) nickel magnetic bead incubation: after the in vitro protein synthesis reaction is finished, collecting reaction liquid, centrifuging for 10min at 4 ℃ and 4000rpm, collecting Supernatant, and marking the obtained Supernatant as Supernatant. To the resulting supernatant, 1. mu.L of nickel magnetic beads was added, and the mixture was incubated at 4 ℃ for 1 hour on a rotary instrument.

Separation and purification: after the incubation is finished, separating the magnetic beads from the in vitro protein reaction system by using a magnetic frame, discarding the supernatant, and taking the corresponding discarded liquid as a Flow-through liquid. Adding 1mL of cleaning solution, and carrying out vortex oscillation cleaning for 3 min; separating the magnetic beads by a magnetic frame, removing a supernatant, repeatedly cleaning with a cleaning solution under the same condition for three times in total to obtain three groups of cleaning solutions Wash 1, Wash 2 and Wash 3; add 50. mu.L of eluent, vortex and shake for 5 seconds, separate the magnetic beads, aspirate the supernatant containing the fusion protein product, and obtain an eluent (50. mu.L) containing the PRTa-GFP product, which was designated as Elution.

5.3. Test method

5.3.1. Fluorescence detection

The RFU value of the solution sample was tested by the ultraviolet absorption method under the conditions of 488nm for excitation wavelength (Ex) and 507nm for emission wavelength (Em). The conversion equation between the concentration of the fusion protein product contained and the RFU value is shown in section 3.2 of example 3, formula (I).

Detecting parameters: for the liquid samples collected in the above process, 10 μ L of samples were taken for 3 sample detections, respectively, with the detection wavelength of Ex/Em:488nm/507nm and the detection machine of InfiniteF 200.

SDS-PAGE electrophoretic detection

SDS-PAGE electrophoresis is carried out on the liquid to be detected, Coomassie brilliant blue R-250 is dyed, the obtained product is kept overnight and decolored, and then a target protein band is observed, so that whether the molecular weight of the target protein is correct or not and the purity of the target protein can be analyzed.

Detecting parameters: 30 mu L of eluent (precipitation) containing the fusion protein product is taken, 7.5 mu L of 5 × loading buffer containing 5% beta-mercaptoethanol is added, heating is carried out for 10min at 95 ℃, SDS-PAGE electrophoresis is carried out, Coomassie brilliant blue R-250 is used for staining, overnight, and after decoloration, the size and the purity of a target protein band are observed.

5.4. Test results

Compared with a control system, the inteure system saves the process of additionally incubating the IVTT reaction liquid and the nickel magnetic beads for 1 h.

(1) Purification yield. The amount of the purified product was calculated from the RFU value and volume of the eluate (Elution) in accordance with the conversion formula (I) of section 3.2 in example 3. The fluorescence test results of the intePure system are shown in FIG. 6. The results of the fluorescence test of the control system are shown in FIG. 7. The yield is improved by 146% by adopting the method for preparing the intePure, the yield of the product is 2.46 mu g by the intePure system, and the yield of the product is 1 mu g by the control system.

(2) And (4) purity. The results of SDS-PAGE electrophoresis of the intePure system and the control system are shown in FIG. 8. The product purity (90%) of the intePure system is much higher than that (10%) of the product obtained by the control system. Presumably, in the inteure preparation method, nickel ions on the magnetic beads preferentially bind to the histidine-tagged fusion protein product and weakly bind to the hetero protein.

Example 6 preparation of protein PRTb-GFP (49.0kDa) by an integrated Process for in vitro Synthesis and purification of proteins

Reference example 5 preparation method and detection method.

6.1. Experimental groups: IVTT-co-purification reaction (intePure system, or called coPure system, or called reaction-purification coupled system)

The ORF sequence encoding the protein PRTb (molecular weight about 22kDa) was inserted/substituted into the MCS sequence in the plasmid vector (pD 2P-1.06 e vector). The target translation product is marked as PRTb-GFP, has a molecular weight of 49.0kDa, is a fusion protein of PRTb and mEGFP, and is provided with an 8 × histidine tag.

The in vitro protein synthesis system provided in example 5, 1.1 was used.

Plasmid DNA encoding PRTb-GFP was prepared by the method of 5.1.2 in example 5, and a DNA template encoding PRTb-GFP was prepared by in vitro amplification.

Taking 10mL of prepared in-vitro protein synthesis system, adding 330 mu L of DNA template (the final concentration is 10 ng/mu L) for coding PRTb-GFP to form an IVTT reaction system; then, 10. mu.L of the nickel magnetic beads prepared in example 4 were added to form a reaction-purification mixture system for IVTT-co-purification. And uniformly mixing the mixture at 30 ℃ in a shaking table, and incubating and reacting for 3 hours. After the reaction, a reaction solution (Total) was obtained.

After the IVTT-co-purification reaction was completed, the eluate (Flow-through), three washing solutions (Wash 1, Wash 2, Wash 3) and the eluate (Elution, also denoted as E1) containing the PRTb-GFP product were obtained by separation as described in example 5, 5.1.4.

In the same manner, only the amount of nickel magnetic beads was changed, and 30. mu.L of the nickel magnetic beads prepared in example 4 was added to the reaction-purification mixture, and the other parameters were not changed, to obtain the corresponding eluate (labeled as E2) containing PRTb-GFP.

6.2. Control group: IVTT, purification step by step (control system)

An IVTT reaction system is formed by adding 330 mu L of PRTb-GFP coding DNA template into 10mL of prepared in-vitro protein synthesis system. Mixing uniformly at 30 ℃ in a shaking table, and carrying out in-vitro protein synthesis reaction for 3 h. The reaction solution at the end of the reaction was designated as Total.

After the in vitro protein synthesis reaction is finished, collecting reaction liquid, centrifuging for 10min at 4 ℃ and 4000rpm, collecting Supernatant, and marking the obtained Supernatant as Supernatant. To the resulting supernatant, 10. mu.L of nickel magnetic beads were added, and the mixture was incubated at 4 ℃ for 1 hour on a rotary instrument.

After the incubation is finished, separating the magnetic beads from the protein reaction system by using a magnetic frame, discarding the supernatant, and taking the corresponding discarded liquid as a permeate (Flow-through). Adding 1mL of cleaning solution, and carrying out vortex oscillation cleaning for 3 min; separating the magnetic beads by a magnetic frame, removing a supernatant, repeatedly cleaning with a cleaning solution under the same condition for three times in total to obtain three groups of cleaning solutions Wash 1, Wash 2 and Wash 3; add 500. mu.L of the eluate, vortex for 5 seconds, separate the magnetic beads, aspirate the supernatant containing the fusion protein product and obtain an eluate (500. mu.L) containing the PRTb-GFP product, which is designated as Elution.

6.3. Test method

The liquid samples obtained during the preparation were subjected to fluorescence detection and the eluate (elute) containing the fusion protein product was subjected to SDS-PAGE electrophoresis using the method of example 5.3.

6.4. Test results

The results of fluorescence measurement of the experimental group (intePure system) and the control group (control system) are shown in FIG. 9. The results of SDS-PAGE electrophoresis of the intePure system and the control system are shown in FIG. 10.

Compared with a control system, the intePure system saves the process of separately purifying and incubating nickel magnetic beads for 1h after the reaction is finished.

(1) The expression level. The RFU values of the Total reaction solutions were compared, and the protein expression levels were calculated by combining the conversion formula (I) of section 3.2 in example 3, and the levels of the inteure system and the control system were 1051.4. mu.g and 983.5. mu.g, respectively. By adopting the intePure preparation method, compared with a control system, the protein expression level is improved by 6.9 percent.

(2) Purification yield. The amount of the purified product was calculated from the RFU value and volume of the eluate (Elution) in accordance with the conversion formula (I) of section 3.2 in example 3. The inteure system gave 64.7. mu.g of product and the control system gave 20.4. mu.g of product. By adopting the intePure preparation method, the yield is improved by 217 percent compared with a control system.

(3) And (5) purifying and obtaining yield. The purification yield was divided by the expression amount to obtain the purification yield. The content of the inteure system and the content of the control system are respectively 6.15 percent and 2.07 percent, and the content of the inteure system is improved by 197 percent compared with the content of the control system.

(4) And (4) purity. The product purity of the intePure system is improved by 100 percent compared with that of the control system. According to FIG. 10, the purity of the product in the eluate is further improved after the amount of nickel magnetic beads is increased from 10. mu.L to 30. mu.L.

Example 7 preparation of protein PRTb-GFP (49.0kDa) by an integrated Process for in vitro Synthesis and purification of the protein

Reference example 6 was conducted for the preparation method and the detection method.

IVTT-co-purification reaction (intePure system, or coPure system, or reaction-purification coupled system)

The target translation product was designated as PRTb-GFP and was identical to that in example 6. Plasmid DNA encoding PRTb-GFP was prepared by the method of 5.1.2 in example 5, and a DNA template encoding PRTb-GFP was prepared by in vitro amplification.

The in vitro protein synthesis system provided in example 5, 5.1.1, was used as the base system, and the following components were also added to construct the following three groups:

PRTb-1: 0.2mM of PMSF (phenylmethylsulfonyl fluoride), a protease inhibitor, was also added and used in combination with 10. mu.L of the nickel magnetic beads prepared in example 4.

PRTb-2: 1mM protease inhibitor PMSF (phenylmethylsulfonyl fluoride) was also added and used in combination with 30. mu.L of the nickel magnetic beads prepared in example 4.

control: no protease inhibitor or nickel magnetic beads were added.

Taking 10mL of prepared in-vitro protein synthesis system, adding 330 mu L of DNA template (the final concentration is 10 ng/mu L) for coding PRTb-GFP to form an IVTT reaction system; then adding a corresponding amount of the nickel magnetic beads prepared in the example 4 to form an IVTT-co-purification reaction and purification mixed system. And uniformly mixing the mixture at 30 ℃ in a shaking table, and incubating and reacting for 3 hours. After the reaction, a reaction solution (Total) was obtained.

After the IVTT-co-purification reaction was completed, the eluate (Flow-through, also denoted as FT), three washing solutions (Wash 1, Wash 2, Wash 3, also denoted as W1, W2, W3) and the eluate (Elute, also denoted as E or Elute) containing the PRTb-GFP product were obtained by separation as described in example 5, 5.1.4.

And (3) cleaning liquid treatment: 1mL of a washing solution containing 20mM of imidazole was subjected to vortex oscillation washing for 3min, and washed three times.

Treating the eluent: 50 μ L of eluent containing 250mM imidazole.

7.2. Test method

The liquid sample obtained in the preparation was subjected to fluorescence detection and the eluate (elute, also designated as E) containing the fusion protein product was subjected to SDS-PAGE using the method of example 5.3.

7.3. Test results

The results of the fluorescence test are shown in FIG. 11, and the results of W3 are not shown. The results show that certain concentrations of protease inhibitors facilitate the purification of proteins in the D2P system.

(1) The expression level. The RFU values of Total in the reaction solutions were compared, and the protein expression levels were calculated by combining the equation (I) for conversion of part 3.2 in example 3, wherein the RFU values of PRTb-1, PRTb-2 and control were 1051.4. mu.g, 179.9. mu.g and 983.5. mu.g, respectively. Compared with control, the protease inhibitors with different concentrations have different effects on protein expression, and the protein expression level is reduced by 81.7% when the protease inhibitor is used at a high concentration (1 mM); the protein expression level was increased by 6.9% relative to control at a low concentration of protease inhibitor (0.2 mM).

(2) Purification yield. The amount of the purified product was calculated from the RFU value and volume of the eluate (Elution) in accordance with the conversion formula (I) of section 3.2 in example 3. The PRTb-1, PRTb-2 and control gave 64.7. mu.g, 31.6. mu.g and 20.5. mu.g, respectively. The yield of PRTb-1 and PRTb-2 was increased by 215.8% and 54.4% respectively for the system with the protease inhibitor added thereto, relative to the control.

(3) Purification yield. The purification yield was divided by the expression amount to obtain the purification yield. PRTb-1, PRTb-2 and control were 6.15%, 17.59% and 2.08%, respectively, and PRTb-1 and PRTb-2 added with protease inhibitor were improved by 195% and 744% respectively, relative to control.

(4) And (4) purity. Compared with the control system, the system PRTb-1 and PRTb-2 added with the protease inhibitor have the product purity improved by 100 percent and 150 percent.

Example 8 examination of the Effect of the magnetic microsphere size on the purification Effect

8.1. Preparation of silica-coated magnetic microspheres (also known as magnetic microsphere body, magnetic beads, glass beads)

20g of Fe₃O₄The microspheres are put into a mixed solvent of 310mL of ethanol and 125mL of water, 45mL of 28 percent (wt) ammonia water is added, 22.5mL of tetraethoxysilane is added dropwise, the mixture is stirred and reacted for 24 hours at room temperature, and the mixture is washed by ethanol and water after the reaction. The method comprises the steps of using ferroferric oxide microspheres with different particle diameters (about 1 mu m, 10 mu m and 100 mu m) as raw materials and controlling the obtained glass beadsThe particle size. The ferroferric oxide microspheres with different particle sizes can be prepared by a conventional technical means.

The magnetic microspheres produced are used as a base material for modifying purification media or connecting elements-purification media and are therefore also referred to as magnetic microsphere bodies.

The prepared magnetic microsphere has a magnetic core, can be subjected to position control under the action of magnetic force, and realizes operations such as movement, dispersion, sedimentation and the like, so that the magnetic microsphere is a generalized magnetic bead.

The prepared magnetic microsphere has a coating layer of silicon dioxide, so the magnetic microsphere is also called as glass bead, and can reduce the adsorption of the magnetic core on the following components or components: polymers, purification media, components of in vitro protein synthesis systems, nucleic acid templates, protein products, and the like.

8.2. Preparation of Nickel magnetic beads with different glass bead (silica-coated magnetic microspheres) sizes

The glass beads with the diameters of 1 μm, 10 μm and 100 μm prepared in the step 8.1 are used as magnetic microsphere bodies to prepare nickel magnetic beads Ni1, Ni10 and Ni100 by the method of example 4. The reaction parameters were the same except for the different glass bead raw materials.

And taking a picture by using a microscope, and observing the size and the shape of the prepared nickel magnetic beads. As a result, as shown in FIG. 12, glass beads having diameters of about 1 μm (FIGS. 12A and B), 10 μm (FIG. 12C) and 100 μm (FIG. 12D) were obtained. Here, fig. 12(a) is a photographing result in a stationary state, and fig. 12(B) is a capturing result in a flowing state.

8.3. Comparison of results of purification of IVTT reaction solution

8.3.1. Construction of in vitro protein Synthesis System (D2P System)

In vitro protein synthesis reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

The final concentration of each component is as follows: 9.78mM pH8.0 Tris-HCl, 80mM potassium acetate, 5mM magnesium acetate, 1.8mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a concentration of 1.8mM), 0.7mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a concentration of 0.1mM), 15mM glucose, 320mM maltodextrin (molar concentration calculated as glucose units, mass volume concentration 52mg/mL), 24mM tripotassium phosphate, 2% (w/v) polyethylene glycol 8000, finally adding 50% by volume of Kluyveromyces lactis cell extract; the Kluyveromyces lactis cell extract contains endogenously expressed T7 RNA polymerase.

The kluyveromyces lactis cell extract is prepared by the method described in CN 109423496A. In general, a gene encoding T7 RNA polymerase is integrated into the genome of kluyveromyces lactis, and the obtained genetically modified strain is cultured to obtain a suitable amount of cells, and then a cell extract is prepared.

8.3.2. Preparation of nucleic acid template (DNA template) encoding 8His-mEGFP

A DNA fragment containing an 8His-mEGFP (histidine-tagged mEGFP) coding gene is inserted into a plasmid vector by adopting a PCR amplification and homologous fragment recombination method to construct a plasmid vector for expressing the mEGFP. The plasmid was confirmed to be correct by gene sequencing. Referring to fig. 5, the components of the plasmid include functional elements such as T7 promoter, 5 'UTR, leader peptide coding sequence (leading peptide), 8 × His (histidine tag), mlfp coding sequence, 3' UTR, LAC4 terminator, f1 ori, AmpR promoter, AmpR gene, ori, lacI promoter, and lacI coding gene, but do not include MCS sequence.

And carrying out in-vitro DNA amplification by using the plasmid DNA for coding the 8His-mEGFP as a nucleic acid template by using a phi29 DNA polymerase by an RCA amplification method to obtain the DNA template for coding the 8 His-mEGFP.

8.3.3. Carrying out in vitro protein Synthesis reaction (IVTT reaction)

And adding the 8.3.2. prepared DNA template (with the final concentration of 15 ng/. mu.L) for coding the 8His-mEGFP into the 8.3.1. constructed in vitro protein synthesis system, uniformly mixing, reacting for 3 hours in a shaking table at room temperature (20-30 ℃) to obtain IVTT reaction liquid (a PC group and a raw protein liquid) after the reaction is finished.

8.4. Purification and RFU testing

And (3) purifying the IVTT reaction liquid by adopting nickel magnetic beads with different sizes prepared in step 8.2, and inspecting the influence of the size of the magnetic microsphere body on the purification effect.

mu.L, 5. mu.L and 1. mu.L of nickel magnetic bead Ni1(1 μm), 10. mu.L, 5. mu.L and 1. mu.L of nickel magnetic bead Ni10(10 μm), 10. mu.L, 5. mu.L and 1. mu.L of nickel magnetic bead Ni100(100 μm) were added to 1mL of the IVTT reaction solution, and they were incubated for 3 hours after they were mixed by rotation of a rotator. The magnetic beads and solution were separated with a magnet and the collected supernatant was recorded as the transudate. The transudate was subjected to fluorescence measurements and the RFU value reflected the amount of protein not bound to the beads. Sample treatment: at 4000 rpm, the mixture was centrifuged at 4 ℃ for 1 minute. The sample to be detected is placed in a microplate reader, and the relative fluorescence unit value (RFU) is determined by adopting excitation wavelength/emission wavelength (Ex/Em):488nm/507 nm.

8.5. Analysis of purification results

FIG. 13 shows the results of purification of Ni1(1 μm) and Ni10(10 μm) magnetic beads. FIG. 14 shows the results of purifying nickel magnetic bead Ni1(1 μm) and nickel magnetic bead Ni100(100 μm). Wherein, the PC group is 8.3.3, and the RFU test result of the IVTT reaction liquid is obtained. The volume of the nickel magnetic beads added is represented by 10. mu.L, 5. mu.L, and 1. mu.L, respectively.

And subtracting the RFU value of the transudate from the RFU value of the PC group, and estimating the protein binding rate corresponding to the protein binding amount in the magnetic beads.

In FIG. 13, the concentration of the protein product 8His-mEGFP in the IVTT reaction solution was 97.4. mu.g/mL. The protein binding rates of nickel magnetic bead Ni1(1 μm) were 98.2%, 97.6%, and 53.8% at volume dosages of 10 μ L, 5 μ L, and 1 μ L, respectively. The protein binding rates of nickel magnetic bead Ni10(10 μm) were 96.9%, 51.9%, and 24.7% at volume dosages of 10 μ L, 5 μ L, and 1 μ L, respectively. The protein binding rates of the nickel magnetic beads Ni1(1 μm) were respectively improved by 1.3%, 46.8% and 54.1%.

In FIG. 14, the concentration of the protein product 8His-mEGFP in the IVTT reaction solution was 57.9. mu.g/mL. The protein binding rate of the nickel magnetic bead Ni1(1 μm) is higher than 98% (98.3% -98.9%) when the volume dosage is three, and the protein binding rate of the nickel magnetic bead Ni100(100 μm) when the volume dosage is 10 μ L, 5 μ L and 1 μ L is 87.5%, 46.9% and 15.2% respectively. The protein binding rates of the nickel magnetic beads Ni1(1 μm) were increased by 11.5%, 52.5% and 84.6%, respectively.

Example 9 examination of the influence of the chain Length of the Polymer

9.1. Preparation of magnetic microspheres with different polymer chain lengths

Magnetic microspheres using nickel ions as the purification medium were prepared by the method of example 4, wherein the concentrations of the polymerized monomers sodium acrylate were 15% (w/v) and 18% (w/v), respectively, and are denoted as L15 and L18.

9.2. Purification and RFU testing

The PC solution (IVTT reaction solution obtained at the end of IVTT reaction) was prepared by the method of 8.3 in example 8. Dilution was carried out at different ratios to obtain stock protein solutions containing different concentrations of the protein product (8 His-mEGFP).

Negative controls (NC group) were set: referring to the PC group, the same operation was performed without adding a DNA template, and the obtained "IVTT reaction solution" was the NC group.

Purification was carried out by the method of 8.4 in example 8. 1mL of the original protein solution with different dilution ratios is added with 2 mu L of nickel magnetic beads for incubation reaction, and the eluate after magnetic bead separation and two groups of eluents (eluent 1 and eluent 2) are respectively collected.

RFU testing was performed as described in example 8, 8.4 (tables 5 and 6). SDS-PAGE was also performed as described in example 5, 5.3.2 (spectrum not shown).

TABLE 5.15% (w/v) comparison of the purification results of the nickel magnetic beads polymerized with 18% (w/v) sodium acrylate

The NC group is a diluted solution of NC group 'IVTT solution' with the same dilution ratio as the original protein solution.

TABLE 6.15% (w/v) comparison of the purification results of nickel magnetic beads polymerized with 18% (w/v) sodium acrylate (2. mu.L of each bead volume)

The concentration of the protein product was calculated by using the conversion formula (I) of the 3.2 part in example 3.

Protein binding rate (eluent 1+ eluent 2)/original protein solution, calculated as protein.

The amount of protein bound per bead volume (amount of eluent 1 protein + amount of eluent 2 protein)/bead volume, also referred to as binding capacity, is in units of μ g/μ L or mg/mL.

In Table 5, the protein binding ratio was determined by the following equation 1- (RFU)_ft-RFU₀)/(RFU_ori-RFU₀) And (4) calculating. Wherein, the volume of the original protein liquid is the same as that of the NC group liquid with the corresponding dilution ratio, and the volume of the transudate liquid is basically the same as that of the original protein liquid and the NC group liquid with the corresponding dilution ratio. As can be seen from Table 5, the capture rate (binding rate) of the protein product by the nickel magnetic beads increases with the increase of the polymer chain length. L15 (15% sodium acrylate) RFU₃₂₀And L18 (18% sodium acrylate) RFU₃₁₀The protein binding rates of (a) and (b) are 85% and 99%, respectively; the protein binding rate of L18 was increased by 16.5%.

In Table 6, the protein binding rates of L15 (15% sodium acrylate) and L18 (18% sodium acrylate) were 41.78% and 55.55%, respectively, and the protein binding amounts per unit volume of magnetic beads were 40.0. mu.g/. mu.L and 61.7. mu.g/. mu.L, respectively, using the high concentration protein solutions. The protein binding rate of L18 with longer polymer chains and more branched chains is higher, and the protein binding amount per unit volume of the magnetic beads is respectively increased by 33.0% and 54.5%.

In addition, SDS-PAGE electrophoresis of the eluate containing the protein product showed that the purity of L18 (18% sodium acrylate) was also improved compared to L15 (15% sodium acrylate).

Example 10 preparation of protein 8His-GFP (about 30.7kDa) by Integrated Process for in vitro Synthesis and purification of proteins

Reference example 5 preparation method and detection method.

The plasmid DNA shown in FIG. 15 was used, and no MCS site was provided, as compared with the plasmid vector shown in FIG. 5 used in example 5, 5.1.2. The DNA template encoding 8His-eGFP was obtained by RCA amplification method using phi29 DNA polymerase for in vitro amplification at a final plasmid DNA concentration of 1 ng/. mu.L at 37 ℃ for 1.5 hours. Wherein the target protein is an eGFP fluorescent protein with 8His-eGFP as a histidine tag, the N end of the fluorescent protein eGFP (the amino acid sequence is shown as SEQ ID No.: 4) is provided with an 8 × histidine tag, a linker is arranged between the 8His, and the molecular weight of the fusion protein is about 30.7 kDa.

The in vitro protein synthesis system provided in example 5, 1.1 was used.

Taking 3mL of prepared in-vitro protein synthesis system, adding 99 mu L of DNA template (the final concentration is 15 ng/mu L) for coding 8His-eGFP to form an IVTT reaction system; then, 30. mu.L of 10% (v/v) nickel magnetic beads prepared in example 4 (the volume of the nickel magnetic beads is 3. mu.L) was added to constitute a "IVTT-co-purification" reaction-purification mixed system. And uniformly mixing the mixture at 30 ℃ in a shaking table, and incubating and reacting for 3 hours. After the reaction, a reaction solution was obtained.

After completion of the IVTT-co-purification reaction, the eluate (Flow-through), three Wash solutions (Wash1, Wash2, Wash3), and the eluate (Elution) containing the 8His-eGFP product were separated as described in reference example 5, 5.1.4. Specifically, a magnetic frame is utilized to separate nickel magnetic beads from an IVTT reaction system, a supernatant is discarded, and the corresponding supernatant is recorded as a Flow-through liquid (also called a Flow-through liquid); and adding 1mL of cleaning solution, carrying out vortex oscillation cleaning for 5 seconds, and cleaning for three times in total to obtain three groups of cleaning solutions, namely Wash1, Wash2 and Wash 3. Then 100. mu.L of eluent was added and vortexed for 5 seconds. The beads were separated and the supernatant containing the fusion protein product was aspirated off to give an eluate containing the 8His-eGFP product, designated as Elution.

Cleaning solution components: 20mM Tris-HCl, 500mM NaCl, 5mM imidazole, pH 8.0.

Eluent components: 20mM Tris-HCl, 500mM NaCl, 250mM imidazole, pH 8.0.

10.2. Test method

The liquid samples obtained during the preparation were subjected to fluorescence detection and the eluate (elute) containing the 8His-eGFP product was subjected to SDS-PAGE using the method of example 5.3.

10.3. Test results

The results of the fluorescence measurements are shown in Table 7. The results of SDS-PAGE are shown in FIG. 16.

TABLE 7 RFU test results for different samples of IVTT-co-purification reactions to prepare 8His-eGFP

Components	RFU value	Concentration (μ g/mL)	Volume of	Amount of protein (. mu.g)
					Total	5803±275	461.77	3mL	1385.30
Flow-through	4836±223	377.53	3mL	1132.58
					Wash1	1070±22	76.12	1mL	76.12
wash2	242±66	14.87	1mL	14.87
					wash3	285±37	18.01	1mL	18.01
Elution	15103±3194	1483.28	100μL	148.33

Remarking:

1. the amount of protein was calculated according to the conversion formula (I) of 3.2 in example 3, and estimated using the RFU mean value, wherein the molecular weight of 8His-eGFP was about 30.7 kDa.

And 2, Total corresponds to an IVTT reaction system which is synchronously carried out and is not added with nickel magnetic beads, and RFU test of the reaction liquid is obtained after the reaction is finished. Total was used to approximate the purification yield.

From experimental data, the following results can be obtained:

(1) the expression level. From the RFU value of the Total of the reaction solution, 1385.3. mu.g of protein expression was calculated by using the conversion formula (I) of 3.2 in example 3.

(2) Purification yield. The amount of the purified product was calculated to be 148.33. mu.g from the RFU value and volume of the eluate (Elution) in accordance with the conversion formula (I) of 3.2 in example 3.

(3) And (5) purifying and obtaining yield. The purification yield was divided by the expression to give a purification yield of 10.7%.

(4) And (4) purity. The product purity according to fig. 15 was 91.89%.

Example 11 preparation of protein 8His-GFP (about 30.7kDa) by an integrated method of in vitro protein synthesis and purification

Reference example 10 preparation method and detection method.

coPure group: IVTT-co-purification reaction (intePure system, or called coPure system, or called reaction-purification coupled system)

A DNA template encoding 8His-eGFP was prepared as described in 10.1 of example 10.

The in vitro protein synthesis system provided by the method of 10.1 of example 10 was used.

Taking 2mL of prepared in-vitro protein synthesis system, adding 66 mu L of DNA template (the final concentration is 15 ng/mu L) for coding 8His-eGFP to form an IVTT reaction system; then, 40. mu.L of 10% (v/v) nickel magnetic beads prepared in example 4 (the volume of the nickel magnetic beads is 4. mu.L) was added to form a "IVTT-co-purification" reaction-purification mixed system. And (3) uniformly mixing the mixture at 30 ℃ in a shaking table, and incubating for reaction for 3 hours. After the reaction, a reaction solution was obtained.

After completion of the IVTT-co-purification reaction, the eluate (Flow-through), three Wash solutions (Wash1, Wash2, Wash3), and the eluate (Elution) containing the 8His-eGFP product were separated as described in reference example 10.1. Specifically, a magnetic frame is utilized to separate nickel magnetic beads from an IVTT reaction system, a supernatant is discarded, and the corresponding supernatant is recorded as a Flow-through liquid (also called a Flow-through liquid); and adding 1mL of cleaning solution, carrying out vortex oscillation cleaning for 5 seconds, and cleaning for three times in total to obtain three groups of cleaning solutions, namely Wash1, Wash2 and Wash 3. Then 70. mu.L of the eluent was added and vortexed for 5 seconds. The beads were separated and the supernatant aspirated to give an eluate containing the 8His-eGFP product, designated as Elution.

Cleaning solution components: 20mM Tris-HCl, 500mM NaCl, 5mM imidazole, pH 8.0.

Eluent components: 20mM Tris-HCl, 500mM NaCl, 250mM imidazole, pH 8.0.

11.2. Control group: IVTT, purification step by step (control system)

An IVTT reaction system was constructed by taking 2mL of the prepared in vitro protein synthesis system and adding 66. mu.L of DNA template encoding 8His-eGFP (final concentration of 15 ng/. mu.L). Mixing uniformly at 30 ℃ in a shaking table, and carrying out in-vitro protein synthesis reaction for 3 h. The reaction solution at the end of the reaction was designated as Total.

After the in vitro protein synthesis reaction is finished, collecting reaction liquid, centrifuging for 10min at 4 ℃ and 4000rpm, collecting Supernatant, and marking the obtained Supernatant as Supernatant. To the resulting supernatant, 40. mu.L of 10% (v/v) nickel magnetic beads prepared in example 4 was added, and the mixture was incubated at 4 ℃ for 1 hour on a rotator.

After the incubation is finished, separating the magnetic beads from the protein reaction system by using a magnetic frame, discarding the supernatant, and taking the corresponding discarded liquid as a permeate (Flow-through). Adding 1mL of cleaning solution, and carrying out vortex oscillation cleaning for 5 seconds; separating the magnetic beads by a magnetic frame, removing a supernatant, repeatedly cleaning with a cleaning solution under the same condition for three times in total to obtain three groups of cleaning solutions Wash 1, Wash 2 and Wash 3; add 70. mu.L of eluent, vortex for 5 seconds, separate the beads, aspirate the supernatant, and obtain an eluent containing the 8His-eGFP product, which is designated as Elution.

11.3. Test method

11.4. Test results

The results of the fluorescence measurements are shown in Table 8. The results of SDS-PAGE are shown in FIG. 17.

TABLE 8 RFU test results of IVTT-co-purification reaction and stepwise preparation of different samples of 8His-eGFP

Remarking: the amount of protein was calculated according to the conversion formula (I) of 3.2 in example 3, and estimated using the RFU mean.

From experimental data, the following results can be obtained:

(1) purification yield. The amounts of the purified products of the coPure group and the control group were calculated to be 251.03 μ g and 144.53 μ g, respectively, based on the RFU value and volume of the eluate (Elution) in accordance with the conversion formula (I) of 3.2 in example 3. Compared with the traditional step method (a control group), the method of the invention improves the coPure method by 73.7 percent.

(2) And (4) purity. According to FIG. 16, the purity of the products obtained from the coPure group and the control group were 89.46% and 58.81%, respectively. Compared with the traditional step method (a control group), the method of the invention improves the coPure method by 52.1 percent.

The foregoing is only a part of the embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made which will achieve the same technical effects within the spirit or scope of the invention and the scope of the invention is to be determined by the appended claims.

Sequence listing

<110> Kangma (Shanghai) Biotech Co., Ltd

<120> protein in-vitro synthesis and purification integrated preparation method, kit and application thereof

<130> 2020

<141> 2020-12-18

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aacgttttgg gcgaggcaca gaaattaaat gacagccagg cccccaaggc cgacgcccag 180

cagaacaact tcaacaagga tcagcagagt gccttttacg aaatattgaa catgcccaac 240

ctaaacgagg cacagagaaa tgggttcatc cagagtttaa aagatgatcc cagtcagagt 300

accaacgttt tgggggaggc caaaaagtta aacgaaagcc aggcgcccaa agctgataac 360

aacttcaaca aggagcagca gaacgccttc tacgagattc taaacatgcc caacttgaac 420

gaggagcaga ggaacggctt catccagagt ttgaaagacg atcccagtca gagtgcaaac 480

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aacaaggagc agcagaacgc tttctatgaa atccttcatt tgccaaattt gaatgaggaa 600

cagaggaacg gcttcatcca gagtttgaaa gatgacccga gtcagagtgc caacttgttg 660

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gagcagcaga atgcctttta cgagatcttg cacttgccca acttgactga ggaacaaagg 780

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115

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gtttcaaagg gtgaagctgt tattaaggag tttatgagat tcaaagtgca tatggaaggt 60

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attaagatgg ctttgagatt gaaagatggt ggtagatatt tggctgattt caagactact 540

tataaggcta agaagccagt tcaaatgcca ggtgcttaca atgttgatag aaaattggat 600

atcacctctc ataatgaaga ttatactgtt gttgagcaat acgaaagatc tgaaggtaga 660

cattctactg gtggtatgga tgaattgtat aag 693

Claims

1. An integrated preparation method for in vitro synthesis and purification of protein is characterized by at least comprising the following steps:

wherein the in vitro protein synthesis system is capable of providing translation-related elements required for synthesis of the protein of interest in combination with the nucleic acid template encoding the protein of interest; the protein of interest is capable of specifically binding to the protein immobilization system;

step ii: carrying out incubation reaction under a proper condition to obtain a target protein product; during the incubation reaction, the protein product of interest is capable of specifically binding to the protein immobilization system;

step iv: eluting the target protein from the protein immobilization system combined with the target protein product obtained by separation in the step iii to obtain an eluent containing the target protein;

optionally further comprising the step v: separating the target protein from the eluent to obtain a purified product of the target protein;

further optionally comprising step vi: further purifying the target protein to obtain a repurified product of the target protein;

2. The integrated preparation method for in vitro synthesis and purification of protein according to claim 1, wherein the protein fixation system is provided with a purification medium; the protein of interest is capable of specifically binding to the purification medium;

preferably, the protein immobilization system comprises a solid substrate and a purification medium attached to an outer surface of the solid substrate;

preferably, the solid matrix has a diameter size selected from any one of the following particle size scales or a range between any two of the following particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 5 μm, 1 μm, 5 μm, and a, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm; the diameter sizes are averages;

Preferably, the diameter size of the solid matrix is selected from 0.1-10 μm;

preferably, the diameter size of the solid matrix is selected from 0.2-6 μm;

preferably, the diameter size of the solid matrix is selected from 0.4-5 μm;

preferably, the diameter size of the solid matrix is selected from 0.5-3 μm;

preferably, the diameter size of the solid matrix is selected from 0.2-1 μm;

preferably, the diameter size of the solid matrix is selected from 0.5-1 μm;

preferably, the protein immobilization system comprises a solid substrate, a purification medium, a polymer connecting the solid substrate and the purification medium;

preferably, the polymer has hydrophilicity and does not precipitate from a solution;

preferably, the outer surface of the solid matrix is connected with a branched-chain polymer, one end of the branched-chain polymer is covalently fixed on the outer surface of the solid matrix, and a branched chain with an independent terminal is distributed outside the solid matrix; the purification medium is connected to the end of the branched chain type polymer;

preferably, the branched polymer is covalently coupled to the outer surface of the solid substrate directly or indirectly via a linking element;

Preferably, the branched polymer has a branched structure selected from: branched structures, comb structures, tree structures, hyperbranched structures, cyclic branched structures, and combinations thereof;

preferably, the branched polymer has a comb structure having a linear main chain and at least 3 side branches; one end of the linear main chain is covalently fixed on the outer surface of the solid matrix, and the other end of the branched chain type polymer is distributed outside the solid matrix;

preferably, at least 3 of said purification media are attached to one molecule of said branched polymer.

3. The integrated preparation method for in vitro synthesis and purification of protein according to claim 2, wherein the protein immobilization system is a magnetic microsphere system, and the magnetic microsphere system has a magnetic microsphere body;

preferably, the magnetic microsphere body constitutes a solid matrix of the protein immobilization system; the outer surface of the magnetic microsphere body is connected with a branched chain type polymer, one end of the branched chain type polymer is covalently fixed on the outer surface of the magnetic microsphere body, and branched chains with independent ends are distributed outside the magnetic microsphere body; the purification medium is attached to the branched end of the branched polymer.

4. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 2-3, wherein the polymer has a linear backbone, the linear backbone is a polyolefin backbone or an acrylic polymer backbone;

preferably, the linear backbone of the polymer is a polyolefin backbone and is provided by the backbone of an acrylic polymer;

more preferably, the monomer unit of the acrylic polymer is acrylic acid, acrylate, methacrylic acid, methacrylate ester or a combination thereof.

5. The integrated in vitro synthesis and purification process according to any one of claims 2 to 4, wherein the purification medium is attached to the branched ends of the branched polymer in a manner that: covalent bonding, dynamic covalent bonding, supramolecular interactions, or combinations thereof;

preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof;

preferably one, said supramolecular interaction is selected from: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof;

More preferably, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

6. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 2-5, wherein the purification medium comprises metal ions, biotin-type tags, avidin-type tags, polypeptide-type tags, protein-type tags, immunological-type tags, or a combination thereof;

in one of the preferred embodiments, the biotin-type tag is biotin, a biotin analogue capable of binding avidin analogue, or a combination thereof;

in one of the preferred embodiments, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof;

in a more preferred embodiment, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof;

in a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof;

In one of the preferred modes, the purification medium is selected from: a metal ion, avidin, an avidin analog that can bind biotin or an analog thereof, biotin, a biotin analog that can bind avidin or an analog thereof, an affinity protein, an antibody, an antigen, DNA, or a combination thereof;

in a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, said affinity protein is selected from the group consisting of: protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof;

in a preferred embodiment, the immunological label is any one of an antibody-type label and an antigen-type label;

in a preferred embodiment, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one;

in a preferred embodiment, the antibody type tag is an anti-protein antibody;

in a preferred embodiment, the antibody-type tag is an antibody against a fluorescent protein;

in a preferred embodiment, the antibody type tag is an antibody against green fluorescent protein or a mutant thereof;

In a preferred embodiment, the antibody-type tag is a nanobody;

in one preferred mode, the antibody type tag is a nanobody against a protein;

in a preferred embodiment, the antibody type tag is a single domain antibody against a protein;

in a preferred embodiment, the antibody-type tag is a single domain antibody against a protein;

in a preferred embodiment, the antibody type tag is an antibody VHH fragment of an anti-protein;

in a preferred mode, the antibody type tag is an antibody scFV fragment of an anti-protein;

in one preferred mode, the antibody type tag is a nanobody against fluorescent protein;

in one preferred mode, the antibody type tag is a nano antibody against green fluorescent protein or a mutant thereof;

in a preferred embodiment, the antibody type tag is an antibody Fab fragment;

in a preferred embodiment, the antibody-type tag is an antibody F (ab') 2 fragment;

in a preferred embodiment, the antibody-type tag is an antibody Fc fragment;

the metal ion is preferably Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof.

7. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 3 to 6, wherein the magnetic microsphere body is SiO₂A wrapped magnetic material;

Preferably, the magnetic material is selected from: iron oxide, iron compound, iron alloy, cobalt compound, cobalt alloy, nickel compound, nickel alloy, manganese oxide, manganese alloy, zinc oxide, gadolinium oxide, chromium oxide, or combinations thereof;

further preferably, the magnetic material is selected from: fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、AlNiCo、FeCrCo、FeCrMo、FeAlC、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo、FeSi、FeAl、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃、PbO·6Fe₂O₃GdO, or a combination thereof;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.1-10 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.2-6 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.4-5 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.5-3 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.2-1 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.5-1 μm;

in one preferred mode, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm;

8. The integrated in vitro protein synthesis and purification preparation method according to any one of claims 1 to 7, wherein the target protein product is bound to the protein immobilization system by a force selected from the group consisting of: biotin-avidin binding, Streg tag-avidin binding, avidin-avidin binding, histidine tag-metal ion affinity, antibody-antigen binding, or a combination thereof;

the Streg tag is a tag comprising a WSHPQFEK sequence or a variant thereof.

9. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 2-8, wherein the target protein is provided with a purification tag, and the purification tag can be specifically combined with the purification medium;

one, two or more purification tags per target protein molecule; when two or more purification tags are contained, the kinds of the purification tags are one, two or more;

preferably, the purification tag in the target protein is selected from the following classes: a histidine tag, avidin, a tag comprising a WSHPQFEK sequence or variant thereof, a tag comprising a WRHPQFGG sequence or variant thereof, a tag comprising a RKAAVSHW sequence or variant thereof, a FLAG tag, a Spot tag, a C tag, a GST tag, a MBP tag, a SUMO tag, a CBP tag, an HA tag, an Avi tag, an affinity protein, an antibody, an antigen, a combination thereof;

Preferably, the target protein is linked to a purification tag at the N-terminus or C-terminus, or to both termini.

10. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 1 to 9, wherein the end point of the in vitro protein synthesis reaction is monitored by the amount of protein synthesis or the reaction time, and the timing for performing step iv is controlled.

11. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 1 to 10, wherein the translation-related elements in the in vitro protein synthesis system are provided by cell extracts, exogenously added translation-related elements, or a combination thereof;

in a preferred embodiment, the translation-related elements of the in vitro protein synthesis system include: tRNA, ribosome, translation-related enzyme, initiation factor, elongation factor, termination factor;

in one preferred form, the exogenously added translation-related enzymes include aminoacyl tRNA synthetases, RNA polymerases, peptidyl transferases, and the like, or combinations thereof, and optionally also include transcriptases, DNA polymerases, or combinations thereof;

in one preferred embodiment, the in vitro protein synthesis system comprises a cell extract, optionally comprising exogenously added translation-related elements;

In a more preferred mode, the cell source of the cell extract is Escherichia coli;

in another more preferred mode, the cell source of the cell extract is eukaryotic cells;

in another more preferred mode, the cell source of the cell extract is a combination of Escherichia coli and eukaryotic cells;

in a further more preferred manner, the cell extract is derived from a yeast cell, a mammalian cell, a plant cell, an insect cell, a nematode cell, a pathogen, or a combination thereof;

in a further more preferred mode, the cell source of the cell extract is Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof;

in a further more preferred embodiment, the cellular source of the cellular extract is Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybuvinii, Kluyveromyces hainanensis, Kluyveromyces williamsii, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensis, Kluyveromyces lactis, or a combination thereof;

in another preferred mode, the translation-related elements in the in vitro protein synthesis system are provided by exogenous addition;

In a more preferred embodiment, the exogenously added translation-related element is a purified translation-related element or a combination thereof;

in a more preferred embodiment, the exogenously added translation-related element is derived from Escherichia coli;

in another more preferred mode, the exogenously added translation-related element is derived from a eukaryotic cell;

in another more preferred mode, the exogenously added translation-related element is derived from a combination of E.coli and a eukaryotic cell;

in a further more preferred form, the exogenously added translation-related element is derived from a yeast cell, a mammalian cell, a plant cell, an insect cell, a nematode cell, a pathogen, or a combination thereof;

in a further more preferred manner, the exogenously added translation-related element is derived from kluyveromyces, saccharomyces cerevisiae, saccharomyces pichia pastoris, or a combination thereof;

12. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 1-11, wherein the in vitro protein synthesis system is capable of recognizing a promoter in the nucleic acid template encoding the target protein;

preferably, the in vitro protein synthesis system comprises an RNA polymerase;

preferably, the source of the RNA polymerase is selected from any one of the following: a cell extract comprising an endogenously expressed RNA polymerase, an exogenous RNA polymerase, a translation product of an exogenous nucleic acid template encoding an RNA polymerase, or a combination thereof;

more preferably, the RNA polymerase is T7 RNA polymerase;

preferably, the nucleic acid template encoding the target protein comprises a T7 promoter capable of initiating a gene transcription process for the target protein, and the in vitro cell-free protein synthesis system comprises T7 RNA polymerase, an exogenous nucleic acid template encoding RNA polymerase, or a combination thereof;

more preferably, the in vitro cell-free protein synthesis system comprises a cellular extract comprising endogenously expressed T7 RNA polymerase;

preferably, the in vitro protein synthesis system further comprises an energy system, a substrate for RNA synthesis, a substrate for protein synthesis;

Preferably, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, and an exogenously added RNA polymerase; the cell extract optionally contains endogenously expressed RNA polymerase;

preferably, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis; the cell extract comprises endogenously expressed RNA polymerase;

preferably, the in vitro protein synthesis system comprises purified translation-related elements, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis; the purified translation-related elements include: tRNA, ribosome, aminoacyl tRNA synthetase, RNA polymerase, initiation factor, elongation factor, termination factor;

the in vitro protein synthesis system optionally comprises a DNA polymerase;

preferably, the source of the DNA polymerase is selected from any one of the following: a cell extract comprising an endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding a DNA polymerase, or a combination thereof;

preferably, the in vitro protein synthesis system comprises a cell extract, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, an exogenously added RNA polymerase, an exogenously added DNA polymerase, a substrate for DNA synthesis; the cell extract optionally contains endogenously expressed RNA polymerase and/or DNA polymerase;

Preferably, the DNA polymerase is phi29 DNA polymerase;

preferably, the in vitro protein synthesis system optionally comprises any one or a combination of the following exogenously added components: DNA amplification related elements, RNA amplification related elements, rnase inhibitors, crowding agents, magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, buffers, aqueous solvents;

preferably, the in vitro protein synthesis system further optionally comprises any one or a combination of the following exogenously added components: rnase inhibitors, crowding agents, magnesium ions, potassium ions, soluble amino acid salts, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoaming agents, alkanes, buffers, aqueous solvents, exogenous nucleic acid templates encoding RNA polymerase, DNA polymerase, exogenous nucleic acid templates encoding DNA polymerase, other DNA amplification related elements, substrates for synthesizing DNA, RNA amplification related elements.

13. The integrated in vitro synthesis and purification preparation method of protein according to any one of claims 1 to 12, wherein the nucleic acid template encoding the target protein is a DNA template, an mRNA template or a combination thereof.

14. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 1 to 13, wherein the target protein is selected from any one of the following proteins, fusion proteins in any combination, and mixtures in any combination: luciferase, green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody, variable region of antibody, single-chain and single-chain fragments of antibody, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and its precursor, glucagon-like peptide, interferon, interleukin, lysozyme, serum albumin, transthyretin, tyrosinase, xylanase, beta-galactosidase, a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, a variant of any of the foregoing;

preferably, the variant is a mutant;

preferably, the variant is a homologue.

15. The integrated preparation method for in vitro synthesis and purification of protein according to any one of claims 3-14, wherein the protein immobilization system is a magnetic microsphere system; the step i of providing the magnetic microsphere system comprises preparing the magnetic microsphere system, and optionally comprises regenerating the magnetic microsphere system or/and replacing a purification medium;

The regeneration of the magnetic microsphere system refers to the renewal of the purification media, wherein the types of the purification media before and after the renewal are the same;

and the types of the purification media before and after the replacement of the purification media are different.

16. An in vitro protein synthesis purification kit, comprising:

(k1) an in vitro protein synthesis system according to any one of claims 1 to 15;

(k3) the protein immobilization system of any one of claims 1-15, or a component thereof; the constituent elements of the protein fixing system comprise any one or the combination of a solid matrix, a solid matrix wrapped by a branched chain type polymer, a reaction raw material of the branched chain type polymer and a purification medium;

(k4) optionally, a position control member of a protein fixation system;

(k7) optionally, a regeneration agent for the protein immobilization system;

(k8) optionally, purifying the medium for reagent replacement;

Each of (k2), (k4), (k5), (k6), (k7) and (k8) is independently present or absent;

the in vitro protein synthesis system can provide translation related elements required for synthesizing the target protein together with the nucleic acid template for encoding the target protein;

the nucleic acid template encoding the target protein is a DNA template, an mRNA template, or a combination thereof;

the protein immobilization system is provided with a purification medium;

the protein of interest is capable of specifically binding to the purification medium;

preferably, the protein purification and immobilization systems in (k3), (k4), (k5), (k6) and (k7) are all magnetic microsphere systems;

preferably, said (k3) comprises a magnetic microsphere system or a component thereof; the magnetic microsphere system comprises a magnetic microsphere body, magnetic microspheres with the outer surfaces coated with branched chain polymers, reaction raw materials of the branched chain polymers and a purification medium or a combination of the reaction raw materials and the purification medium;

preferably, the components of the kit are placed in one or more containers as a solid, semi-solid, liquid, emulsion, suspension, or combination thereof;

preferably, said (k1) and said (k2) are packaged separately;

preferably, (k1) comprises the cell extract and is separately packaged in a container;

Preferably, the translation-related components in (k1) are independently packaged together in a container;

preferably, (k1) comprises purified translation-related elements or a combination thereof, and are packaged together in a single container;

preferably, said nucleic acid template encoding a protein of interest comprises a promoter element capable of being recognized by said in vitro protein synthesis system of (k 1);

preferably, the nucleic acid template encoding the target protein contains a T7 promoter capable of initiating a gene transcription process for the target protein, and the in vitro cell-free protein synthesis system comprises T7 RNA polymerase, an exogenous nucleic acid template encoding T7 RNA polymerase, or a combination thereof;

17. Use of a protein immobilization system as claimed in any one of claims 1 to 15 for in vitro protein synthesis,

preferably, the protein fixing system is coupled with the in vitro protein synthesis system of any one of claims 1 to 15 for use in a reaction and purification mixed system integrated with protein synthesis and purification;

Preferably, the use of said protein immobilization system in a kit for the in vitro purification of protein synthesis according to claim 16;

preferably one, for use in protein manufacture, or for use in assays based on protein synthesis;