CN112538469A

CN112538469A - Restriction endonuclease DpnI preparation and preparation method thereof

Info

Publication number: CN112538469A
Application number: CN201911204796.1A
Authority: CN
Inventors: 郭敏; 徐丽琼; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2019-09-20
Filing date: 2019-11-29
Publication date: 2021-03-23

Abstract

The invention provides a preparation of restriction endonuclease DpnI and a preparation method thereof. The preparation method comprises the following steps of preparing the restriction endonuclease preparation through in-vitro protein synthesis reaction, specifically, constructing an in-vitro cell-free protein synthesis system, adding a DNA template containing a coding gene of DpnI, carrying out in-vitro protein synthesis reaction, synthesizing a target protein with a DpnI structure, carrying out solid-liquid separation, collecting supernate, measuring enzyme activity, selecting supernate with restriction endonuclease activity, obtaining the restriction endonuclease DpnI preparation, and packaging or preparing into a storage solution and then packaging. The product provided by the invention has a simple preparation method, avoids the loss of protein content caused by the traditional separation and purification steps, has high enzyme digestion activity, and can be widely applied to the fields of molecular biology, bioengineering, food and the like.

Description

Restriction endonuclease DpnI preparation and preparation method thereof

Technical Field

The invention relates to the technical field of protease, in particular to the technical field of restriction enzyme, and specifically relates to a preparation of restriction enzyme DpnI and a preparation method thereof.

Background

DpnI, a restriction enzyme, specifically and efficiently recognizes G methylated in DNA^mATC sequence (5' -G)^m6ATC-3'), and can perform specific cleavage after the site of methylated adenine, but can not cut off the unmethylated GATC sequence, and is mainly used for degrading methylated plasmid template, in vitro gene site-directed mutagenesis, and the like, and the application fields include but are not limited to the fields of biological medicine, molecular biology, bioengineering, food, agriculture, feed, living goods, washing, environment, and the like. Reference "Weiguo Han, Miao Shi and Simon D Spivack. site-specific related reporter constructs for functional analysis of DNA methylation [ J]Epipedics, 8(11), 1176-1187 "and references cited therein.

Most of the commercial products of DpnI are sold as solutions of pure DpnI, such as Thermo Scientific^TMDpnI (10U/. mu.L), DMT enzyme from Beijing Quanjin Biotechnology Ltd (GD111),

DpnI (E026), MCLAB DpnI (DPNI-100, DPNI-200, DPNI-OEM, B-DB10), DpnI from Biovision (M1236-2000), DpnI from Takara (1235A, 1235B), and Quickcut^TMDpnI (1609), DpnI (R6231) from Promega, DpnI (ABIN3188191) from Genomics, DpnI (RK21109) from ABClonal, DpnI (EN-160L, EN-160S) from Jena Bioscience, and the like; the amount of enzyme per unit volume (1. mu.L) in the above commercial DpnI product is 10 to 20U. The reaction was carried out at 37 ℃ for 1 hour in a 50. mu.L reaction system, and the amount of enzyme that completely digested and decomposed 1. mu.g of pBR322 DNA (dam methylated) was defined as 1 activity unit, i.e., 1U. Stock solutions of the above-described commercial DpnI products, for example,

the DpnI (E026) stock solution consisted of 10mM Tris-HCl (pH 7.5), 300mM NaCl, 1mM DTT (dithiothreitol), 0.1mM EDTA (ethylenediaminetetraacetic acid), 500. mu.g/ml BSA and 50% (v/v) glycerol. Thermo Scientific^TMDpnI (10U/. mu.L) stock solutionThe components are as follows: 10mM Tris-HCl (pH 7.4, 25 ℃), 400mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/mL BSA, and 50% glycerol. For another example, the storage solution of DpnI products (product numbers MD07801 and MD07802) from Nzytech is a buffer solution, and the components thereof are: 25mM Nahepes (pH 7.5), 500mM NaCl, 150mM imidazole, 2.5mM CaCl₂50% (v/v) glycerol.

The commercial DpnI products described above are all derived from escherichia coli (intracellular synthetic protein) recombined with a plasmid containing the DpnI gene, and the purified DpnI protein is stored in a stock solution/stock solution, such as the DpnI products (product numbers MD07801, MD07802) from Nzytech, wherein the DpnI purity specification is > 90%; the DpnI purity of ABClonal company (RK21109) is greater than 95%.

The existing commercialized DpnI needs to be subjected to separation and purification steps after protein expression, then is dissolved in a storage solution again, and is stored in a split charging manner at the low temperature of-20 ℃, and the separation and purification steps inevitably cause and actually cause the loss of the yield of the protein, and possibly cause the reduction of the activity of the protein.

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, have been developed in the 1960 s, and synthesis of target proteins was achieved by artificially controlling and adding substances such as substrates, energy, transcription and translation-related protein factors, etc., which are required for protein synthesis, using exogenous mRNA or DNA as a protein synthesis template. The in vitro protein synthesis system is generally characterized in that a gene template (mRNA template or DNA template), RNA polymerase, amino acid, ATP and other components are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of exogenous proteins. The protein complex in vitro system is a relatively rapid, time-saving and convenient protein expression mode without plasmid construction, transformation, cell culture, cell collection and disruption steps (CN 109988801A; "Assenberg R, Wan PT, Geisse S, Mayr LM.Advances in recombinant protein expression for use in pharmaceutical research. Current Opinion in Structural biology.2013,23 (393) -402;" ane Zemella, Lena Thoring, Christian Hoffmeister and Stefan bick.cell-free protein synthesis: proteins and con of prokaryotic and eukaryotic systems. chem.Chem.16: 2420 ").

Disclosure of Invention

Aiming at the technical problems, the invention provides a restriction enzyme preparation and a preparation method thereof, in particular provides a restriction enzyme DpnI preparation and a preparation method thereof; the restriction endonuclease preparation is prepared by an in vitro cell-free protein synthesis system, is subjected to solid-liquid separation after in vitro expression synthesis, collects supernatant, and can be packaged or prepared into storage liquid (optionally, storage liquid capable of being stored at low temperature) and then packaged. The preparation method is simple, avoids the protein content loss caused by the traditional separation and purification steps, has high enzyme digestion activity, and can be widely applied to the fields of molecular biology, bioengineering, food, agriculture, feed, living goods, washing, environment and the like. The packaging mode includes but is not limited to packaging and subpackaging.

The preparation of restriction enzyme (specifically the preparation of restriction enzyme DpnI) is obtained by the following technical scheme.

The preparation of the restriction endonuclease DpnI can be obtained by a preparation method comprising the following three steps:

firstly, constructing an in vitro cell-free protein synthesis system, wherein the system at least comprises the following components: a gene template; the in vitro cell-free protein synthesis system can express the gene template into a target protein.

The gene template refers to an exogenous gene template.

The expression product of interest encoded by the gene template is also referred to as a protein of interest.

The gene template is a gene template for coding a target expression product and contains a gene sequence for coding a target protein.

In any embodiment of the present invention, the gene templates are preferably DNA templates independently of each other.

Specifically, the gene template contains a gene sequence encoding DpnI; the in vitro cell-free protein synthesis system can express the gene template into a target protein containing a DpnI structure. The DpnI structure is included, including the cases of "having", "being equal to" DpnI structure. That is, the target protein may be a DpnI protein, or the structure of the target protein may include a DpnI structure.

The DpnI includes native DpnI enzymes and also includes non-native DpnI enzymes. For the definition of non-native DpnI enzymes see the section "terms and nouns".

Secondly, carrying out in-vitro protein synthesis reaction to synthesize target protein coded by the gene template;

step three, finishing the in vitro protein synthesis reaction, carrying out solid-liquid separation, and collecting supernatant to obtain a solution containing the target protein; wherein the solution with restriction enzyme activity is the preparation of the restriction enzyme DpnI;

optionally, a packaging step is included.

The term "having a restriction enzyme activity" as used herein means having a restriction enzyme activity of DpnI (DpnI is understood in a broad sense and referred to the term "explained section"), which is also referred to as having a DpnI enzyme activity, and which is provided by a target protein having a DpnI structure.

The "having restriction endonuclease activity" can be qualitatively verified by enzyme digestion test, and can also be quantitatively determined by measuring enzyme activity value. Preferably, the number of units of enzyme activity per volume is determined, said volume being typically 1. mu.L.

In one preferred embodiment, the target protein encoded by the gene template is a DpnI protein, a DpnI fusion protein, or a combination thereof. Namely, the target expression product is DpnI protein or/and DpnI fusion protein. Preferably, the DpnI fusion protein is a fusion protein of DpnI and 2-50 peptides or a solubilization label-labeled DpnI fusion protein.

When a DpnI fusion protein is included in the expression product of interest, one of the preferred modes of the DpnI fusion protein is a solubilization tag-tagged DpnI fusion protein, i.e., a fusion protein of DpnI with a solubilization tag SeP.

In one preferred embodiment, the solubilization tag-labeled DpnI fusion protein is SeP-X-DpnI fusion protein.

In a further preferred embodiment, the SeP-X-DpnI fusion protein is a SeFP-X-DpnI fusion protein.

In another preferred embodiment, the SeP-X-DpnI fusion protein is an eGFP-X-DpnI fusion protein.

Wherein SeP-X-DpnI, SeFP-X-DpnI and eGFP-X-DpnI are DpnI fusion proteins which are sequentially recombinant proteins obtained by fusing DpnI with SeP, SeFP and eGFP respectively, and the DpnI is marked by the SeP, SeFP and eGFP respectively; wherein SeP is a solubilization tag; SeFP is the fluorescent protein solubilization label, eGFP is the enhanced green fluorescent protein. Wherein X is absent or is a linker peptide; and X, when a linker peptide, functions as a linker, X should be capable of being expressed by the in vitro cell-free protein synthesis system. Wherein "-" is a peptide bond or a linker peptide.

In one of the preferred modes, the SeP solubilization tag is selected from: thioredoxin, thioredoxin reductase, maltose binding protein, glutathione-S-transferase, glutathione reductase, streptococcal protein B1 domain, small ubiquitin-related modifier protein, HaloTag protein, disulfide bond forming protein, bacterial translation initiation factor, transcription anti-termination factor, ribosomal protein L23, eGFP, GFP, sfGFP, CFP, YFP, eYFP, RFP, BFP, mSacrlet, or combinations thereof.

In one preferred embodiment, the SeFP fluorescent protein solubilization tag is selected from the group consisting of: eGFP, GFP, sfGFP, CFP, YFP, eYFP, RFP, BFP, mCardet, or combinations thereof.

In another preferred embodiment, the fusion protein of DpnI is a fusion protein of DpnI and SeFP, which is a solubilization tag of fluorescent protein, and is more preferably SeFP-X-DpnI.

In another preferred embodiment, the fusion protein of DpnI is an eGFP fusion protein of DpnI, and more preferably eGFP-X-DpnI.

In the target protein coded by the gene template, when X exists, X is a connecting peptide and is the connecting peptide which can be expressed by the in vitro cell-free protein synthesis system.

When X is present, in one preferred embodiment, X is a cleavable linker peptide or a stable linker peptide; the cleavable linker peptide is further preferably selected from the group consisting of: a connecting peptide with self-cutting property and a connecting peptide which is cut under the action of exogenous enzyme.

When X is present, the number of amino acids is preferably 2 to 100 (2 to 100 peptides), more preferably 2 to 50(2 to 50 peptides), and further preferably 5 to 25 (5 to 25 peptides).

In a preferred embodiment, the target protein encoded by the gene template is: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI, or combinations thereof.

Wherein eGFP (enhanced green fluorescent protein), mScalet (a bright red fluorescent protein), eYFP (enhanced yellow fluorescent protein), MBP (maltose binding protein), and DsbA (disulfide bond forming protein) are all solubilization tags; wherein eGFP, mScalet and eYFP are also fluorescence protein solubilization labels.

Wherein 2A is a linker peptide having self-cleaving properties. TEV is a linker peptide that is specifically recognized and cleaved by TEV enzyme, which acts as a stable linker when not treated with TEV enzyme.

The gene template accordingly preferably encodes a protein of interest as described above.

In one preferred embodiment, the gene template is a gene template encoding a DpnI protein, a gene template encoding a DpnI fusion protein, or a combination thereof.

In a preferred embodiment, the gene template is a gene template encoding a DpnI protein.

In a preferred embodiment, the gene template is a gene template encoding a DpnI fusion protein.

In a preferred embodiment, the gene template is a gene template comprising a gene sequence encoding a fusion protein of SeP and DpnI; more preferably a gene template containing the gene sequence encoding SeP-X-DpnI.

In a preferred embodiment, the gene template is a gene template encoding a fusion protein of DpnI and SeP. Further preferably SeP-X-DpnI fusion protein.

In a preferred embodiment, the gene template is a gene template comprising a gene sequence encoding a fusion protein of SeFP and DpnI; more preferably a gene template comprising the gene sequence encoding SeFP-X-DpnI.

In a preferred embodiment, the gene template is a gene template encoding a fusion protein of DpnI and SeFP. Further preferably a gene template encoding SeFP-X-DpnI fusion protein.

In a preferred embodiment, the gene template is a gene template comprising a gene sequence encoding a fusion protein of eGFP and DpnI; more preferably a gene template containing a gene sequence encoding eGFP-X-DpnI.

In a preferred embodiment, the gene template is a gene template encoding a fusion protein of DpnI and eGFP. Further preferably a gene template encoding an eGFP-X-DpnI fusion protein.

In a preferred embodiment, the gene template comprises genes encoding the following proteins or a combination thereof: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI.

In a preferred embodiment, the gene template is selected from the group consisting of genes encoding the following proteins or a combination thereof: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI.

The supernatant obtained in the preceding step is a solution, also denoted as supernatant solution. The supernatant solution contains the target protein, and the target protein contains a DpnI structure, so the obtained supernatant solution is also referred to as a DpnI solution.

And screening the supernatant with the DpnI enzyme activity into the preparation of the restriction enzyme DpnI. The DpnI enzyme content of the obtained preparation of restriction enzyme DpnI is preferably at least 4U/. mu.L; more preferably at least 5U/. mu.L.

The DpnI solution with the restriction endonuclease activity can be packaged to obtain a packaged product, and can be further subpackaged to obtain a subpackaged product.

The enzyme activity value (for example, the unit number of enzyme activity per unit volume) of the supernatant can be measured, and the collected supernatant with the activity of the restriction enzyme is packaged or prepared into DpnI storage solution and then packaged, so that the preparation of the restriction enzyme DpnI can also be obtained. The packaging means includes, but is not limited to, packaging or dispensing.

The "formulated DpnI stock solution" is preferably formulated as a DpnI stock solution that can be stored at low temperatures.

One of the preferred embodiments of the low temperature conditions for low temperature storage is below-18 ℃; the low temperature is more preferably about-20 ℃ (for example, preferably-18 ℃ to-23 ℃). Another preferred embodiment of the low temperature is-80 ℃ (± 5 ℃).

When the packaging is carried out in a split charging mode, the content of the DpnI enzyme in a single container after split charging is preferably at least 4U/mu L; more preferably at least 5U/. mu.L; more preferably at least 8U/. mu.L, more preferably 10-20U/. mu.L, more preferably 10U/. mu.L, 15U/. mu.L, 20U/. mu.L.

The in vitro cell-free protein synthesis system preferably comprises a cell extract in addition to the gene template. The selection criteria of the cell extract are as follows: the gene template capable of expressing the target protein, namely the gene template capable of expressing the encoding gene containing DpnI, can be used for synthesizing the target protein containing the DpnI structure.

In any embodiment of the present invention, the gene templates used in the in vitro cell-free protein synthesis system may each be independently selected from DNA templates or mRNA templates.

In any embodiment of the present invention, the gene templates may each independently preferably be DNA templates.

In one preferred embodiment, the cell extract contains an endogenously expressed RNA polymerase; further preferably, the cellular genome of the cellular extract has a gene encoding RNA polymerase integrated therein, or the gene encoding RNA polymerase is inserted into a plasmid of the cell.

In another preferred embodiment, the in vitro cell-free protein synthesis system further comprises exogenous RNA polymerase, a gene template containing a gene encoding RNA polymerase, or a combination thereof, in addition to the gene template or the cell extract.

In another preferred embodiment, a gene template containing a gene encoding RNA polymerase, a gene template containing a gene encoding DNA polymerase, or a combination thereof is added to the in vitro cell-free protein synthesis system. The "gene template containing a gene encoding RNA polymerase" and the "gene template containing a gene encoding DNA polymerase" refer to a gene template containing a gene sequence encoding RNA polymerase and a gene template containing a gene sequence encoding DNA polymerase. The gene template may be a DNA template or an mRNA template. It may be a gene template containing the gene encoding DpnI, or an additionally added foreign gene template.

In a preferred embodiment, a gene template containing a gene encoding RNA polymerase is added to the in vitro cell-free protein synthesis system.

In another preferred embodiment, a gene template containing a gene encoding a DNA polymerase is added to the in vitro cell-free protein synthesis system. The gene sequence of the DNA polymerase may be encoded in the same gene template as the gene encoding DpnI or in a separate foreign gene template.

In another preferred embodiment, the in vitro cell-free protein synthesis system further comprises exogenous DNA polymerase in addition to the gene template and the cell extract.

The cell extract, one of the preferred embodiments of its origin, is selected from any one of the following sources: coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof. The yeast cell is further preferably selected from the group consisting of Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or combinations thereof. The Kluyveromyces is more preferably Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybuhitensis, Kluyveromyces hainanensis, Kluyveromyces williamsii, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensis, Kluyveromyces lactis, or the like, or a combination thereof.

Another preferred embodiment of the source of the cell extract is selected from any one of the following sources: coli, Kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda insect cells (sf insect cells), rabbit reticulocyte, CHO cell, COS cell, VERO cell, BHK cell, human fibrosarcoma HT1080 cell, or a combination thereof.

In another preferred embodiment, the source of the cellular extract is kluyveromyces lactis having a gene encoding RNA polymerase, a gene encoding DNA polymerase, or a combination thereof integrated into its genome.

In a preferred embodiment, the in vitro cell-free protein synthesis system comprises an energy system in addition to the gene template.

In a further preferred embodiment, the in vitro cell-free protein synthesis system comprises a cell extract and an energy system in addition to the gene template.

One of the preferred modes of the energy system is selected from the group consisting of a polysaccharide and phosphate energy system, a phosphocreatine and phosphocreatine enzyme system, a glycolytic pathway and its intermediate energy system, or a combination thereof.

In a preferred embodiment, the in vitro cell-free protein synthesis system comprises, in addition to the gene template, a substrate for the synthesis of the protein.

In a preferred embodiment, the in vitro cell-free protein synthesis system comprises a DNA template.

Further preferably, a substrate for synthesizing RNA is included in addition to the DNA template.

Further preferably, the in vitro cell-free protein synthesis system further comprises a substrate for RNA synthesis and a substrate for protein synthesis.

Further preferably, the in vitro cell-free protein synthesis system comprises a cell extract, a substrate for RNA synthesis and a substrate for protein synthesis besides a DNA template;

still more preferably, the in vitro cell-free protein synthesis system comprises, in addition to the DNA template, a cell extract, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis.

The substrate for the synthesis of RNA is a mixture of nucleotides selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof.

The substrate of the synthetic protein is an amino acid mixture including at least amino acids required for synthesizing the target protein, i.e., at least amino acids for synthesizing the target protein (DpnI protein or/and DpnI fusion protein), and can be 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, and histidine. Unnatural amino acids, D-amino acids, radioisotope-labeled amino acids, modified amino acids, and the like may also be included. The definition and preferred modes of the target protein and the DpnI fusion protein are consistent with those described above.

The in vitro cell-free protein synthesis system can also comprise at least one of the following components: crowding agent, magnesium ion, potassium ion, antioxidant, buffer, aqueous solvent.

One of the preferred modes of the crowding agent is polyethylene glycol, dextran, Ficoll or a combination thereof.

One of the preferred modes of the magnesium ion source is magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or a combination thereof.

One of the preferred modes of the potassium ion source is potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or a combination thereof.

One of the preferable modes of the antioxidant is dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol and the like.

The buffer is preferably selected from any one of the following: Tris-HCl, Tris base, HEPES, or a combination thereof.

The aqueous solvent is preferably a buffer.

The DpnI stock solution can also comprise the prior various commercially available Dp besides the collected supernatantOne or more components (such as Tri-HCl, Tris-acetate, NaHepes, NaCl, CaCl) in stock solutions of nI products (e.g., all-gold DMT enzyme)₂Dithiothreitol DTT, EDTA, BSA, imidazole, glycerol, magnesium acetate, sodium acetate, etc.).

Preferably, the DpnI stock solution further comprises at least glycerol.

The cryopreserved DpnI stock solution generally further includes at least glycerin so as to be storable at low temperatures. The low temperature is preferably about-20 deg.C (e.g., -18 deg.C to 23 deg.C) or about-80 deg.C (e.g., -80 + -5 deg.C).

The preparation method of the preparation of the restriction endonuclease DpnI can also adopt an exogenous mRNA template to replace the DNA template, or adopt a mixture of the mRNA template and the DNA template to carry out in-vitro protein synthesis reaction.

The invention can adopt solubilization labels (including water-soluble fluorescent protein solubilization labels) to carry out co-expression with DpnI, and after solid-liquid separation, the supernatant solution of the DpnI fusion protein is obtained or further prepared into DpnI storage solution, and the DpnI storage solution can be used as the technical scheme of the preparation of restriction enzyme DpnI provided that the DpnI storage solution can exert the enzyme digestion activity.

The invention also provides a preparation method of the preparation of the restriction endonuclease DpnI, which is described above and is not repeated. The active component of the restriction enzyme in the preparation of the restriction enzyme DpnI is DpnI protein or DpnI fusion protein. The definition and preferred modes of the DpnI fusion protein are consistent with those described above.

Advantageous effects

The restriction endonuclease DpnI preparation and the preparation method thereof provided by the invention can adopt any suitable in-vitro cell-free protein synthesis system as long as DpnI protein or/and DpnI fusion protein can be expressed; particularly, when a coexpression system of DpnI and a solubilization label is adopted, a preparation of DpnI protease with high concentration can be obtained, and a storage solution which can be stored under the condition of low temperature can be further prepared. Any suitable optimization method that has been reported can also be used to optimize the in vitro protein synthesis system. The preparation method of the restriction endonuclease DpnI preparation has simple steps and easy operation, avoids the protein content loss caused by the traditional separation and purification steps, has high enzyme digestion activity, can avoid the activity loss possibly caused by the traditional purification steps, and can be widely applied to the fields of molecular biology, bioengineering, food, agriculture, feed, living goods, washing, environment and the like. In addition, in the coexpression system of DpnI and the solubilization tag SeP, the solubilization tag may also be a fluorescent protein solubilization tag, such as eGFP, mScarlet, eYFP, etc., and at this time, the fluorescent tag also has a fluorescent labeling function, which can assist in quantification, and can also utilize the fluorescent function to label and track, and the DpnI preparation of the present invention is applied to the aspects of detection, quantitative analysis, etc.

Drawings

FIG. 1 shows a plate obtained by transforming Trans 5. alpha. after treating pUC19 plasmid with 1. mu.L and 0.3. mu.L, respectively, using a positive control of DpnI product (DMT enzyme, GD111, 10U/. mu.L) from Kimura corporation and a negative control of 50% (v/v) glycerol solution prepared in a centrifugal supernatant obtained after an in vitro protein synthesis reaction (here, IVTT reaction) in which eGFP is expressed without expressing DpnI and its eGFP fusion protein.

FIG. 2 shows a plate obtained by treating pUC19 plasmid with 1. mu.L and 0.3. mu.L of a 50% (v/v) glycerol solution (DpnI stock solution) prepared from a centrifugal supernatant obtained after an in vitro protein synthesis reaction of plasmid vectors expressing different DpnI, and then transforming Trans 5. alpha. into the treated plasmid. The plasmid vectors of different DpnI respectively contain gene sequences for coding eGFP-2A-DpnI, eGFP-TEV-DpnI, DpnI-2A-eGFP, DpnI-eGFP and DpnI.

FIG. 3, 1. mu.L of a negative control (50% (v/v) glycerol solution prepared from a centrifugal supernatant obtained after an IVTT reaction by expressing eGFP), 1. mu.L of a positive control (product of DpnI from King Kogyo Co., Ltd., 10U/. mu.L), 2.5. mu.L of a mixture with 3. mu.L of a DpnI stock (example 6), 10. mu.L of a DpnI stock (example 4) of a DpnI-2A-eGFP group, and 10. mu.L of a DpnI stock (example 5) of a DpnI-eGFP group, and a plate obtained by transforming Tran 5. alpha. after treating a pUC19 plasmid.

FIG. 4 shows the results of fluorescent protein activity assay, measured as Relative Fluorescence Units (RFU). The black legend corresponds to the test results of the reaction solution after the in vitro protein synthesis reaction, and the gray legend corresponds to the test results of the supernatant solution after the solid-liquid separation.

FIG. 5 shows the results of nucleic acid electrophoresis tests conducted after digestion of the pUC19 plasmid, and the effect of DpnI digestion was examined. Wherein the column "M" is a nucleic acid molecular weight marker.

FIG. 6 shows the effect of digestion tests on DpnI stock solutions prepared using DNA templates encoding eGFP-2A-DpnI, mScarlet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, and DsbA-2A-DpnI (all at 0.3. mu.L). The positive control was the product of whole gold DpnI (DMT enzyme, 10U/. mu.L, 0.3. mu.L), and the negative control used a DNA template encoding only eGFP and not DpnI (0.3. mu.L). The figure shows a plate obtained by transforming Trans5 α after treating pUC19 plasmid with the indicated volume.

FIG. 7 shows the results of electrophoresis of nucleic acids obtained by digesting the plasmid pUC19 with DpnI stock solutions prepared using DNA templates containing genes encoding eGFP-2A-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, and DsbA-2A-DpnI, respectively. The positive control is the product DpnI from Transgene (DpnI from Transgene) from the whole formula gold company, and the negative control (negative control) uses a DNA template encoding eGFP but not DpnI.

FIG. 8 shows a TranI plate obtained by treating a plasmid pUC19 with a DNA template (eGFP gene having a histidine tag at the 5' end and His-tag) encoding tag-eGFP-TEV-DpnI, subjecting the DNA template to IVTT reaction, subjecting the resulting solution to solid-liquid separation to obtain a DpnI solution (L1, L2, L3), adding a 50% (v/v) DpnI stock solution (L1-G, L2-G, L1-G) obtained by one volume of glycerol, purifying the solution after IVTT reaction to obtain a purified product (tag-eGFP-TEV-DpnI protein) to obtain a purified product solution having a concentration of 0.095mg/mL (P1, P2, P3; both 50% by volume of glycerol), and converting the product DpnI product (DMT enzyme, 10U/. mu.L, positive control) of Kimura into TranI 5. alpha. Wherein the final concentration of tag-eGFP-TEV-DpnI in groups L1 and L1-G, P1 is 0.285 μ g (corresponding to volumes of 1.815 μ L, 3.353 μ L and 3.000 μ L, respectively), the final concentration of tag-eGFP-TEV-DpnI in groups L2 and L2-G, P2 is 0.095 μ g (corresponding to volumes of 0.605 μ L, 1.118 μ L and 1.000 μ L, respectively), the final concentration of tag-eGFP-TEV-DpnI in groups L3 and L3-G, P3 is 0.029 μ g (corresponding to volumes of 0.185 μ L, 0.341 μ L and 0.305 μ L, respectively), the volume of the positive control group is 0.3 μ L, and the negative control group is not treated with DpnI. Wherein the final concentration of tag-eGFP-TEV-DpnI is an estimate of the standard curve according to example 15.

Description of nucleotide and/or amino acid sequence Listing

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, unless otherwise specified, "DpnI protein", "DpnI protease", "DpnI enzyme", "DpnI", the "DpnI" in "DpnI fusion protein", "DpnI in" SeP-X-DpnI "in" DpnI fusion protein "," DpnI "in" SeFP-X-DpnI ", the" DpnI "in" eGFP-X-DpnI "and" DpnI "in" DpnI structure "have the same meaning, and they are broadly understood. DpnI includes wild-type DpnI protein (native DpnI enzyme) as well as non-native DpnI enzyme. DpnI can be a complete structure, subunit, fragment. The non-natural DpnI enzyme includes but is not limited to protease substances obtained by any one mode or combination mode of gene recombination products, gene mutation products, amino acid side group based modified products, end group based modified products, structural analogs, functional analogs with higher amino acid sequence homology and the like, and can be complete structures, subunits and fragments. The non-native DpnI enzyme suitable for use in the present invention should have the same or similar active moieties as the native DpnI enzyme and be capable of exerting the same or similar restriction endonuclease activity.

The DpnI fusion protein refers to a fusion protein which contains an amino acid sequence of DpnI and is connected with other amino acid sequences at the N end or the C end. The number of amino acids in the other amino acid sequence fused with DpnI is not particularly limited, but the amino acid sequence has at least 2 amino acid units, and may be a small-molecule short peptide or polypeptide (2 to 50 amino acids) or a large-molecule protein (at least 50 amino acids). Preferably, the fused additional amino acid sequence has a labeling effect on DpnI, and functions as a tag such as a solubilization tag, a purification tag, a fluorescence tag, and the like, in which case the additional amino acid sequence is also referred to as a tag. The DpnI fusion protein of the present invention is preferably a DpnI fusion protein labeled with a solubilizing tag, and may be further labeled with a fluorescent tag. The solubilizing tag and the fluorescent tag can be the same amino acid sequence (e.g., fluorescent protein solubilizing tag) or different amino acid sequences (e.g., two tags fused, one for solubilizing and one for fluorescence). Preferably, the fluorescent tag carried by the DpnI fusion protein is capable of providing solubilization.

The term "DpnI" also includes the case where only one amino acid is bonded to the N-terminus or C-terminus of DpnI. However, when the N-terminus or C-terminus of DpnI is linked to 2 or more amino acids, it falls into the category of DpnI fusion proteins.

In the present invention, the "preparation of restriction enzyme DpnI" has the same meaning as that of the "product of restriction enzyme DpnI" in the prior application as a priority document. Wherein "DpnI" is as defined above. "DpnI" does not limit the expression protein of the in vitro protein synthesis system to DpnI protein only, as long as the target protein in the DpnI preparation (DpnI solution or DpnI stock solution) contains the active structure of DpnI, and can exert the enzyme cutting activity of DpnI, that is, can specifically and effectively recognize and cut G methylated by adenine in DNA^mATC sequence. The DpnI existing mode in the restriction endonuclease DpnI preparation includes but is not limited to complete structure, DpnI fusion protein, subunit, fragment, gene mutant, gene recombination product, modified structure and the like.

The "preparation of restriction enzyme DpnI" (described as "product of restriction enzyme DpnI" in the previous application) of the present invention defines a preparation having the restriction enzyme activity of DpnI; preferably, the DpnI enzyme content is at least 4U/. mu.L. After the in vitro protein synthesis system is used for reaction, the obtained supernatant solution or storage solution, if not having the restriction enzyme activity of DpnI, is not in the category of the 'restriction enzyme DpnI preparation' of the invention, and the corresponding in vitro protein synthesis system is naturally not in the implementable mode of the invention. Vice versa, if the in vitro protein synthesis system is reacted to obtain a supernatant solution or a stock solution having the restriction enzyme activity of DpnI, it can be used as an embodiment of the present invention. The present invention, by limiting the enzymatic activity of the "preparation of restriction enzyme DpnI", certainly excludes those solutions which do not have the enzymatic activity of DpnI.

In the present invention, "DpnI solution", unless otherwise specified, means that after the in vitro protein synthesis reaction is completed, the expression product in the reaction solution includes DpnI or the structure of the expression product contains DpnI, and the supernatant obtained after the solid-liquid separation of the corresponding reaction solution is referred to as DpnI solution. More broadly, the DpnI solution may also include the reaction solution before solid-liquid separation.

The "DpnI stock solution" of the present invention refers to a DpnI stock solution prepared based on the above-mentioned DpnI solution, unless otherwise specified. The DpnI stock solution can preferably be stored under the corresponding low temperature conditions according to the present invention.

The DpnI solution and the DpnI stock solution are solution and stock solution which can play the enzyme activity of the restriction endonuclease of the DpnI. The liquid formulation contains either free DpnI protein or a DpnI fusion protein that contains the DpnI structure. The DpnI portion in the free DpnI component or fusion protein component may be a complete structure identical to the natural structure of DpnI, a subunit or a fragment of the natural structure, an unnatural structure obtained by genetic mutation, an unnatural structure obtained by genetic recombination, a chemically modified unnatural structure, a structural analog, a functional analog having high amino acid sequence homology with natural DpnI, or the like, as long as the enzymatic cleavage activity of DpnI can be exerted.

The reaction solution after the in vitro protein synthesis reaction is a system which is not subjected to solid-liquid separation treatment after the in vitro protein synthesis reaction is finished. A clear solution may be present, and insoluble solids may also be present.

The reaction solution after the IVTT reaction is a reaction solution after the in vitro protein synthesis reaction is finished, and refers to a system which is not subjected to solid-liquid separation treatment after the IVTT reaction is finished.

The IVTT reaction liquid comprises a system in the IVTT reaction and a system which is not subjected to solid-liquid separation treatment after the reaction is finished.

Unit of enzyme activity U of DpnI. In the present invention, 1 activity unit, i.e., 1U, means the amount of enzyme required to completely digest 1. mu.g of methylated pBR322 DNA by reacting at 37 ℃ for 1 hour in a 50. mu.L reaction system. In the present invention, the activity unit and the vitality unit have the same meaning and may be used interchangeably. In the present invention, the enzyme activity value is preferably represented by the number of enzyme activity units.

The method for testing the activity of the DpnI enzyme can adopt the existing reported test methods, including but not limited to the following two methods: 1. DNA electrophoresis detection method. Digesting the plasmid by DpnI, and detecting the digestion effect by electrophoresis, wherein the undigested plasmid band is higher in position, and the digested plasmid band is represented as a plurality of segment bands with smaller molecular weight. 2. Transformation competent cell assay. The plasmid digested by DpnI is used to transform competent cells, and the digestion effect of DpnI is identified according to the number of clones grown.

In the present invention, "target protein", and "target expression protein" refer to "target expression product" with respect to the protein synthesis system. For the in vitro protein synthesis system of the present invention, the expression product of interest refers to a protein product that is expressed and synthesized in the in vitro protein synthesis system through a translation process or through a transcription-translation process.

The expression system of the present invention, the in vitro cell-free expression system, and the in vitro cell-free expression system may be used interchangeably, and refer to the in vitro protein expression system of the present invention, and other descriptions such as an in vitro protein synthesis system, a cell-free in vitro protein synthesis system, an in vitro cell-free protein synthesis system, a CFS system (cell-free system), a CFPS system (cell-free protein synthesis system), and the like may also be used. Including in vitro translation systems, in vitro transcription translation systems (IVTT systems), and the like. In the present invention, the IVTT system is preferred.

IVTT, in vitro transcription translation.

The in vitro cell-free protein synthesis system of the present invention is a system capable of expressing the gene template as a target protein containing a DpnI structure, and is also a system capable of obtaining a supernatant solution or a stock solution having a restriction enzyme activity of DpnI. When the in vitro cell-free protein synthesis system is constructed, the proportion mode of each component is based on the condition that the above conditions can be met.

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 IVTT reactions (in vitro transcription translation reactions). In the present invention, IVTT reaction is preferred.

The expression of a gene template as a target protein means that the target protein is synthesized by translation using an mRNA template or transcription and translation using DNA as a template.

"capable of expressing the gene template encoding the target protein" means that the gene template can be expressed as the target protein.

The fusion label is a conventional technical means in the technical field of gene recombination. Fusion tags can be divided into two categories according to the molecular weight of the fusion tag: large protein molecules (or protein domains and their derivatives, typically greater than 50 amino acids) and small polypeptide fragments (no more than 50 amino acids). The former may be referred to as a protein tag and the latter may be referred to as a polypeptide tag. Protein tags with large molecular weight tend to increase the solubility of the protein of interest.

The solubilization label (English: solubility-enhancing tag or solubility-enhancing fusion tag or solubility fusion tag) is noted SeP in the present invention. The solubilization tag is a fusion tag that solubilizes expression of the synthetic protein of interest. The N-terminal or C-terminal of a solubilizing tag is linked with other peptide fragments, so long as the solubilizing effect can be achieved, and the solubilizing tag is also included in the scope of the present invention.

The fluorescent protein solubilization tag, a solubilization tag, and also a fluorescent protein, is designated SeFP in the present invention.

SeP-X-DpnI, a solubilization-tag-labeled DpnI fusion protein, also known as SeP-X-DpnI fusion protein.

SeFP-X-DpnI, fluorescent protein solubilization tag-labeled DpnI fusion protein, also known as SeFP-X-DpnI fusion protein.

eGFP-X-DpnI, an enhanced green fluorescent protein-labeled DpnI fusion protein, also known as eGFP-X-DpnI fusion protein.

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

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.

Linker peptide refers to a peptide segment as a linker. The linker peptide of the present invention is not limited to function only as a linker, but also allows for other functions, such as a purification tag as a linker.

2-50 peptides: the peptide has a main chain with 2-50 amino acids. Other numbers are prefixed, meaning the same, and indicate the number of amino acids contained in the amino acid backbone (without pendant modifying groups).

Peptides or proteins linked with "-": indicating linkage by peptide bond or linker peptide. From left to right, corresponding to the N-terminal and C-terminal, respectively, unless otherwise specified or unless clearly indicated to the contrary.

The gene sequence is as follows: in the present invention, the gene encoding a certain peptide or a certain protein refers to a gene sequence encoding the peptide or the protein, and may be DNA or mRNA. For example, a gene encoding DpnI refers to a gene sequence encoding DpnI. The coding gene for RNA polymerase refers to a gene encoding RNA polymerase. The gene encoding DNA polymerase refers to a gene encoding DNA polymerase.

Gene template: refers to a gene template used as a raw material for in vitro protein synthesis reaction, and comprises a DNA template and an mRNA template. In any embodiment of the present invention, the gene templates may each independently be DNA templates, mRNA templates, or a mixture thereof. In any embodiment of the present invention, the gene templates may each independently preferably be DNA templates. In the present invention, a DNA template is preferred unless otherwise specified.

Solid-liquid separation and supernatant fluid: the "solid-liquid separation" in "the step of terminating the in vitro protein synthesis reaction, performing solid-liquid separation, and collecting the supernatant" means that solid insoluble substances may be precipitated or precipitated from the system solution as the reaction proceeds, and the solid insoluble substances may be removed by the "solid-liquid separation" to obtain the supernatant. In the present invention, the expressed and synthesized DpnI protease and its fusion protein are dissolved in a large amount in the system solution by the action of the solubilization tag. When the obtained supernatant had the restriction enzyme activity, the resulting supernatant was screened as a "preparation of restriction enzyme DpnI" of the present invention.

The supernatant, supernatant solution, may be used interchangeably in the present invention. The supernatant refers to the supernatant and a solution of insoluble solid matter, and the supernatant refers to the suspension of suspended solid matter. The restriction endonuclease DpnI preparation provided by the invention can be screened from a supernatant solution, and the supernatant solution can also be prepared into a stock solution.

The supernatant rate refers to the content ratio of the protein expressed by the system in the supernatant after solid-liquid separation.

Glycerol solution: "50% (v/v)" glycerol solution means a solution diluted with one volume of glycerol. For example, a solution of DpnI diluted with 1:1 volume of glycerol to give a clear solution, i.e., a solution of DpnI in a mixed solvent of water and glycerol, which can be recorded as a 50% (v/v) glycerol solution. It should be noted that, in the prior application of the priority claims, the term "glycerol suspension" is used as a misapplication, and actually means "glycerol solution". According to common knowledge, water and glycerol are mutually soluble, and the person skilled in the art can determine uniquely that a "solution" rather than a "suspension" is obtained after dilution with glycerol.

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

The packaging method in the present invention includes, but is not limited to, packaging method or dispensing method. "direct packaging" refers to packaging without a step of formulating a stock solution. "direct packaging" refers to a direct packaging process without a separate packaging step.

In the present invention, the cell extract, the cell lysate, the cell disruption product, and the cell lysate have the same meaning, and english can adopt 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.

An amino acid mixture refers to a mixture containing at least two or more 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 mixtures 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.

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

In the present invention, the preferred embodiments such as "preferred", "more preferred" and "most preferred" are not intended to limit the embodiments of the present invention, but merely to provide examples of embodiments with better technical effects.

In the description of the present invention, for the preferred modes such as "one of the preferred modes", "one of the preferred embodiments", "preferred example", "preferred", "preferably", "more preferred", "further preferred", "most preferred", etc., and for the exemplary illustrations such as "one of the embodiments", "one of the modes", "example", "specific example", "by way of example", "for example", "such as", etc., the specific features described in each mode are included in at least one specific embodiment of the present 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. The expression "combined in a proper way" means that an in vitro cell-free protein synthesis system can be constructed, in vitro protein synthesis reaction is carried out, target protein coded by a gene template is synthesized, and a supernatant solution with the activity of the DpnI restriction endonuclease can be obtained through solid-liquid separation. For example, if there are several preferred modes one for the in vitro cell-free protein synthesis system (CFS system) and the DpnI enzyme content standard of the obtained DpnI preparation, any preferred mode of the DpnI enzyme content standard can be used as a preferred mode when any CFS system is used; similarly, any of the preferable embodiments of the CFS system can be used as a preferable embodiment when any of the DpnI enzyme content standards is used. The CFS system may include a DNA template, and may further include components such as a cell extract, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a crowing agent (e.g., PEG), magnesium ions, potassium ions, an antioxidant (e.g., DTT), a buffer, and an aqueous solvent, and any preferred embodiment of the DNA template may be further preferred when any preferred embodiment of the cell extract is used as the CFS system, and may be further preferred when an appropriate combination of other preferred embodiments of the cell extract and the like are used as the CFS system.

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.

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

The invention provides a preparation of restriction endonuclease DpnI and a preparation method thereof; the preparation of the restriction endonuclease DpnI is prepared by an in vitro cell-free protein synthesis system, after in vitro protein synthesis, a target protein in a solution contains a DpnI structure, and a supernatant with DpnI enzyme activity can be collected to obtain the preparation of the restriction endonuclease DpnI, and the preparation can be packaged or prepared into a storage solution and then packaged; it can also be formulated into a storage liquid which can be stored at low temperature. The preparation method is simple, avoids the protein content loss caused by the traditional separation and purification steps, has high enzyme digestion activity, can also avoid the activity loss possibly caused by the traditional purification steps, and can be widely applied to the fields of molecular biology, bioengineering, food, agriculture, feed, articles for daily use, washing, environment and the like.

The preparation of the restriction enzyme (specifically the preparation of the restriction enzyme DpnI) is realized by the following technical scheme.

The restriction enzyme preparation can be obtained by the following preparation method:

The gene template refers to an exogenous gene template.

The gene template is a gene template containing a gene sequence encoding a target expression product.

The gene template is a gene template for coding a target expression product.

The gene template is preferably a DNA template.

Specifically, the gene template contains a gene sequence encoding DpnI; the in vitro cell-free protein synthesis system can express the gene template into a target protein containing a DpnI structure. The protein of interest may be a DpnI protein, or the protein of interest may contain a DpnI structure. The gene template contains a gene sequence for coding DpnI and also refers to a coding gene containing DpnI.

The DpnI comprises a complete structure, subunits and fragments.

One of the preferred modes of the gene template is selected from: a gene template encoding a DpnI protein, a gene template comprising a gene sequence encoding a DpnI fusion protein, or a combination thereof. The definition and preferred modes of the DpnI fusion protein are consistent with those described above.

And secondly, carrying out in-vitro protein synthesis reaction to synthesize the target protein coded by the gene template. The target protein is DpnI protein or/and DpnI fusion protein.

Step three, ending the in vitro protein synthesis reaction, carrying out solid-liquid separation, and collecting supernatant to obtain a solution (supernatant solution) containing target protein, wherein the solution contains DpnI protein or/and DpnI fusion protein and is marked as DpnI solution; the solution with the restriction enzyme activity is the preparation of the restriction enzyme DpnI. Optionally, a packaging step is included.

The "having restriction endonuclease activity" can be qualitatively verified by enzyme digestion experiment, and can also be quantitatively determined by measuring enzyme activity value. Preferably, the number of units of enzyme activity per volume is determined, said volume being typically 1. mu.L.

And (3) screening the solution with the DpnI enzyme activity from the supernatant obtained in the step, namely the preparation of the restriction enzyme DpnI. The DpnI enzyme content of the obtained preparation of restriction enzyme DpnI is preferably at least 4U/. mu.L; more preferably at least 5U/. mu.L.

On the basis of the above steps, the following steps may be further included: the preparation of the restriction enzyme DpnI can also be obtained by packaging the collected supernatant with the DpnI enzyme activity according to the measured DpnI enzyme activity value (such as the unit number of enzyme activity in unit volume) or packaging the supernatant after preparing the DpnI stock solution. The packaging means includes, but is not limited to, packaging or dispensing.

One of the preferred embodiments of the low temperature conditions for low temperature storage is below-18 ℃; the low temperature is more preferably about-20 ℃ (for example, -18 ℃ to-23 ℃). Another preferred embodiment of the low temperature is-80 ℃ (± 5 ℃).

When the packaging is carried out in a split charging mode, the content of the DpnI enzyme in a single container after split charging is preferably at least 4U/mu L; more preferably at least 5U/. mu.L; more preferably at least 8U/. mu.L. More specifically, one of the preferable modes is 10 to 20U/. mu.L, and more preferably 10U/. mu.L, 15U/. mu.L, or 20U/. mu.L.

The preparation of the restriction endonuclease DpnI contains an expression product generated by in-vitro protein synthesis reaction, wherein the expression product is free DpnI protein, DpnI fusion protein or a mixture thereof (so that the preparation of the restriction endonuclease DpnI can play the enzyme cutting activity of DpnI. the expression product of the DpnI fusion protein type contains a DpnI structure in a fusion structure.

The present invention also provides a method for preparing the preparation of the restriction enzyme DpnI, which is described schematically as follows, and comprises the following three steps:

firstly, constructing an in vitro cell-free protein synthesis system, wherein the system at least comprises the following components: a gene template encoding an expression product of interest (i.e., a gene template containing a gene of interest, such as a plasmid DNA template, a linear DNA template, etc.);

the second step is that: carrying out in-vitro protein synthesis reaction to obtain a target expression product (target protein coded by the gene template) corresponding to the gene template;

step three, finishing the in vitro protein synthesis reaction, carrying out solid-liquid separation, and collecting supernatant to obtain a solution (supernatant solution) containing a target expression product; screening to obtain the restriction enzyme DpnI preparation meeting the enzyme activity requirement. Optionally, a packaging step is included.

The enzyme activity requires, at least, restriction enzyme activity. More specifically, the DpnI enzyme content of the resulting preparation of restriction enzyme DpnI is preferably at least 4U/. mu.L; more preferably at least 5U/. mu.L.

The screening method can be qualitative screening, or quantitative screening after measuring enzyme activity value (such as measuring unit of enzyme activity in unit volume).

The DpnI solution meeting the requirement can be screened from the supernatant after the solid-liquid separation to obtain the preparation of the restriction enzyme DpnI to be provided by the invention, and the preparation can be packaged and stored.

The preparation method of the preparation of the restriction endonuclease DpnI can also comprise the following steps: and (3) packaging the DpnI solution with the restriction endonuclease activity obtained after solid-liquid separation according to the measured enzyme activity value (unit number of enzyme activity units in unit volume), or packaging the DpnI solution after preparing into a DpnI storage solution. The "formulated DpnI stock solution" is preferably formulated as a DpnI stock solution that can be stored at low temperatures. The relevant packaging means, preferably means, are in accordance with those described above.

Target expression product, target protein, and target protein

The target expression product of the invention is protein capable of exerting the enzyme digestion activity of DpnI. Thus, the gene template should contain the gene sequence encoding DpnI, i.e. should contain the gene encoding DpnI.

The gene template should be capable of being expressed as a DpnI protein or a DpnI fusion protein as long as the expression product is DpnI or the structure of the expression product contains DpnI. The expression "the structure contains DpnI" means that the structure includes a subunit or a fragment having the same complete structure as a natural structure, an unnatural structure obtained by genetic mutation, an unnatural structure obtained by genetic recombination, and an unnatural DpnI obtained by chemical modification. The DpnI obtained by chemical modification can carry a modification unit besides a DpnI structure; the modification unit can be an amino acid or a peptide segment; the number of the modification units can be 1 or more; when the number of the modification units is more than 1, the structures of the modification units can be the same or different; the attachment site of the modification unit may be located at the C-terminus, N-terminus, or a pendant group of the amino acid unit of DpnI. When the number of modified amino acids at the N-terminal or C-terminal of DpnI is more than 1, the DpnI fusion protein falls into the structural category of the DpnI fusion protein of the invention.

The expression product of interest is preferably a DpnI protein, a DpnI fusion protein, or a combination thereof.

The DpnI fusion protein is also marked as a fusion protein of DpnI.

The DpnI fusion protein may contain a solubilizing tag, and may also contain other amino acid sequences such as a leader peptide, a purification tag, and the like.

In one preferred mode, the DpnI fusion protein is a fusion protein of DpnI and 2-50 peptides, or a solubilization tag labeled DpnI fusion protein. The fusion protein of DpnI and 2-50 peptides is characterized in that a peptide segment with the length of 2-50 amino acids is further connected to the N terminal or the C terminal of DpnI along the main chain of the amino acid chain of the protein, and when the length of the peptide segment exceeds 50 amino acids, the solubility of an expression product of DpnI in a system can be influenced. The length of 2-50 amino acids does not include amino acids of amino acid side groups modified in the middle of the main chain. The amino acid in the middle of the main chain refers to the amino acid except the N terminal and the C terminal on the main chain of the amino acid chain. In a DpnI fusion protein, a solubilization tag is preferably present when amino acids other than DpnI exceed 50 amino acid units along the length of the backbone.

In one preferred mode, the DpnI fusion protein is a fusion protein of DpnI and SeP, i.e., a solubilization tag-labeled DpnI fusion protein.

The fusion protein of DpnI and SeP, more preferably SeP-X-DpnI.

In a preferred embodiment, the DpnI fusion protein is a fusion protein of DpnI and SeFP.

The fusion protein of DpnI and SeFP is SeFP-X-DpnI in one of more preferable modes.

One of the preferred modes of the DpnI fusion protein is the ednp eGFP fusion protein.

The eGFP fusion protein of DpnI, one of the more preferable modes is eGFP-X-DpnI.

Wherein the definitions of DpnI, SeP, SeFP, X, "-" are in accordance with the above, preferably in accordance with the above or/and below.

In a preferred embodiment, the protein of interest encoded by the gene template comprises the following structure: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI, or combinations thereof. In addition to the above structures, other amino acid sequences such as leader peptide and purification tag may be present, and they may be linked to the N-terminus, C-terminus, peptide bond or linker peptide.

In a preferred embodiment, the gene template encodes a protein of interest selected from the group consisting of: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI, or combinations thereof. In addition to the above structures, other amino acid sequences such as leader peptide and purification tag may be present, and they may be linked to the N-terminus, C-terminus, peptide bond or linker peptide.

Solubilization label and fluorescent protein solubilization label

In the invention, the solubilization label SeP and the fluorescent protein solubilization label SeFP (such as eGFP, mScarlet and eYFP) can solubilize DpnI, so that the solubility of an expression product in a reaction system is improved, and the content of DpnI in a supernatant obtained after an in vitro protein synthesis reaction is further improved. The invention adopts water-soluble fluorescent protein (such as eGFP, mScarlet, eYFP) as a solubilization label or adopts other solubilization labels, co-expresses with DpnI, and the expression amount of the solubilization label is positively correlated with the increase of the expression amount of the DpnI, so that the solubilization matched with the concentration of the DpnI is adaptively provided, and the precipitation loss of the DpnI protein is reduced. The obtained supernatant solution or storage solution of the DpnI fusion protein after solid-liquid separation can be used as a restriction enzyme DpnI preparation of the invention for standby as long as the DpnI enzyme digestion activity can be exerted. In this case, as one of the preferable modes, the expression product of the in vitro cell-free protein synthesis system is a DpnI fusion protein labeled with the solubilization tag SeP, including but not limited to SeP-X-DpnI (a DpnI fusion protein, a protein in which DpnI is fused with SeP). SeP is a solubilization label, and can solubilize DpnI to reduce or reduce precipitate of DpnI, SeP is preferably water-soluble protein or water-soluble polypeptide fragment; wherein X, DpnI, "-" are as defined above.

Typically, SeP (solubilization tag) that can be used for co-expression with DpnI in the present invention includes, but is not limited to, various solubilization tags or solubilization promotion tags (solubility-enhancing tag) reported in the prior art, such as Thioredoxin (TRX), TrxA (thioredoxin A ), thioredoxin reductase B (trxB), Maltose Binding Protein (MBP), glutathione-S-transferase/S-glutathione transferase (GST), glutathione reductase (gor), streptococcal G protein B1 domain (GB1), small related modified protein/small molecule ubiquitin-like modified protein (SUMO), HaloTag protein, disulfide forming protein (DsbA), DsNu distinguishirome C, bacterial translation initiation factor (IF2), transcription anti-bC factor (sA), ribosomal protein L23, ribosome protein L23, Skp (OmpH, Hlpa), Mistic, SP-MBP (MBP with signal peptide), etc.; reference may also be made to "study progress of Supeng, Gong Guo Li. optimization of exogenous protein expression in E.coli [ J ]. Biotechnology Notification, 2017,33(02): 16-23", "Mayue, who is good, and health, Congyongjie. strategy review of soluble expression of exogenous protein in E.coli [ J ]. world science research and development, 2015,37(05): 627. 630", "Cao Li Juan. superacid protein fusion tag on soluble expression of recombinant protein in E.coli [ D ]. university of east China, 2008", "study of soluble expression of recombinant protein in Suyu. E.coli [ D ]. university of east China, 2007", "jungle, san Juan, Zhang Shi thioredoxin and related proteins [ J ]. oceanic science, 2008,6: 79-84", "Hu SM, J WaTF and Wang. expression of proteins for protein production [ J ]. Life of science, 2007, DOI 10.1002/9780470015902.a0020210 "," P Chiarella, B Edelmann, VM Fazio and AM Sawyer. antichemical characteristics of protein carriers common used in immunological compositions [ J ]. Biotechnology Letters,2010,32(9):1215 1221 "," Kwon et Al. recombinant expression and functional analysis of protein from Streptococcus vulgaris, Bacillus anthrocus, and Yeast peptides [ J ]. Biochemical, 2011,12:17 "," A. Corezzo and P optic expression. 12:17 "," A. expression and P expression of the protein E.coli [ J ]. promoter ] expression, expression of protein, expression of protein [ J ]. Biochemical, III, IV. A. 7. promoter, III, IV. D. expression of protein, III, 2006,17(4): 353-358', US20120107876A1, etc., and the solubilization tags or solubilization promotion tags disclosed in the documents cited directly or indirectly.

One of the preferred modes of SeP is SeFP. Among them, SeFP is a fluorescent protein and can solubilize DpnI, i.e., a fluorescent protein solubilization tag.

Fluorescent proteins useful as fluorescent protein solubilization tags (SeFP), such as eGFP, mScarlet, eYFP, are desirably water soluble, capable of co-expression with DpnI to provide for simultaneous enhanced solubilization in vitro protein synthesis reactions, and further capable of fluorescent labeling and/or quantification, e.g., to calculate the supernatant of solid-liquid separation procedures. The fluorescent proteins reported in the fields of protein synthesis, genetic engineering, and the like, and related technical fields can be used to practice the present invention as long as the above conditions are met. Typically, fluorescent proteins that can be used as solubilizing tags for co-expression with DpnI include, but are not limited to, eGFP, GFP, sfGFP (reference "Single expression of Single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner [ J].Applied Microbiology and Biotechnology,2019,103:6071-6079”)、sGFP(Shifted green fluorescence protein，“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”)、CFP(Cyan Fluorescent Protein), YFP (yellow Fluorescent Protein), eYFP (enhanced YFP), RFP (Red Fluorescent Protein), mCardet (a bright red Fluorescent Protein), BFP (blue Fluorescent Protein) or combinations thereof (e.g., BFP/YFP, BFP/GFP) and the like as well as mutants or modified products of the above Fluorescent proteins. Can also be selected from: including but not limited to "Imaging neural subsets in transforming multiple spectral variants of GFP [ J].Neuron,2000,28(1):41-51”、“Using GFP in FRET-based applications[J].Trends in cell biology,1999,9(2):57-60”、“[5]Fluorescence correlation spectroscopy of GFP fusion proteins in living plant cells[J].Methods in enzymology,2003,361:93-112”、“Annett Dümmler,Ann-Marie Lawrence and Ario de Marco.Simplified screening for the detection of soluble fusion constructs expressed in E.coli using a modular set of vectors[J].Microbial Cell Factories,2005,4(34):1-10”、“CL Young,ZT Britton and AS Robinson.Recombinant protein expression and purification:a comprehensive review of affinity tags and microbial applications[J].Biotechnology journal,2012,7(5):620-634”、“Jean-Denis Pédelacq,Stéphanie Cabantous,Timothy Tran,Thomas C Terwilliger and Geoffrey S Waldo.Engineering and characterization of a superfolder green fluorescent protein[J]Nature Biotechnology volume,2006,24(1):79-88 "," Wangfei, Popula Tao, Wangzhao & Red fluorescent protein research progress [ J]Biotechnological report 2017,33(9):32-47 "," Daphne S Binders, Lindsay Haarbosh, Laura van Weeren et al mScelet a bright monomeric red fluorescent protein for cellular imaging [ J]Nature Methods,2017,14:53-56. "," Lindsay Haarbosch, Daphne Binders, Marten Postma et al mCardet, a novel high quality quaternary yield (71%) monomeric red fluorescent protein with enhanced properties for FRET-and super resolution microscopics.2016, https:// doi.org/10.1002/97835278465. EMC2016.8648 "and the fluorescent proteins and their genetically modified and chemically modified products disclosed therein, either directly or indirectly.

Among them, egfp (enhanced green fluorescence protein) is an enhanced green fluorescence protein (enhanced green fluorescence protein), and has better water solubility. The mScarlet is a shorthand of mScarlet-I, and is obtained by performing gene modification on the basis of the red fluorescent protein mScarlet, and the brightness, the quantum yield and the fluorescence lifetime of the mScarlet are obviously improved. The eYFP is obtained by performing gene mutation transformation on aequorea victoria GFP protein and has yellow fluorescence. Maltose Binding Protein (MBP), a native protein in e.coli responsible for uptake, breakdown and transport of maltodextrin, a carbohydrate, is a commonly used protein expression tag protein that can significantly enhance the solubility of a variety of downstream fused target proteins. DsbA is a bacterial disulfide oxidoreductase, a key component of the Dsb (disulfide bond) enzyme family. DsbA catalyzes the formation of intrachain disulfide bonds, which allow the polypeptide to enter the periplasm of the cell. Structurally, DsbA contains a thioredoxin domain with an inserted helical domain.

In addition, the fluorescent protein solubilization label, such as eGFP, mScarlet, eYFP and the like, also has a fluorescent labeling function, can assist in quantification, can also utilize the fluorescent function to carry out sexual labeling tracking, and applies the preparation to detection and quantitative analysis.

Regarding the solubilization of the solubilizing label. Examples 1-5, examples 10-12 were solubilized using fluorescent tags eGFP, mScarlet, eYFP fused to DpnI and examples 13-14 were also solubilized using non-fluorescent tags MBP, DsbA fused to DpnI, and the resulting DpnI solutions and DpnI stock solutions both had comparable or even better cleavage than the commercial products.

In example 6, in the gene expression system encoding DpnI, insoluble substances were precipitated from the system as the reaction proceeded. In the co-expression system of DpnI, the system precipitation is significantly reduced in examples 1-3 encoding eGFP-2A-DpnI, eGFP-DpnI and eGFP-TEV-DpnI, and in examples 10-14 encoding eGFP-2A-DpnI, mReclet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI and DsbA-2A-DpnI. Referring to FIG. 4 (examples 1-3), the supernatant rate reached 70%. The supernatant rate is the content ratio of the target protein expressed by the system in the supernatant after solid-liquid separation. The supernatant rate can be obtained by using a fluorescent protein solubilization tag SeFP (such as eGFP) as an indication, and specifically, the eGFP is taken as an example: and taking the RFU value of the reaction solution subjected to fluorescence test after the reaction of the in-vitro cell-free synthesis system as the total value of the eGFP, measuring the RFU value of the supernatant obtained after centrifugation to obtain the fluorescence value of the eGFP in the supernatant, and obtaining the ratio (in percentage) of the RFU value to the fluorescence value of the eGFP in the supernatant, namely the supernatant rate.

The above solubilization mechanism can also be confirmed by reference to the document "Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using super fusion green fluorescence protein as fusion partner [ J ]. Applied Microbiology and Biotechnology,2019,103: 6071-6079").

Linker peptide

In the solubilization tag-labeled DpnI fusion protein, the solubilization tag and the DpnI structure may be linked by a peptide bond or a linker peptide. When the solubilization tag and the DpnI structure are linked by a linker peptide, the linker peptide is a linker peptide suitable for expression by the in vitro cell-free protein synthesis system of the invention.

Solubilization tag-labeled DpnI fusion proteins can be designated SeP-X-DpnI or DpnI-X-SeP. Wherein "X" is nothing or a linker peptide; "-" is a peptide bond or a linker peptide.

In the target protein encoded by the gene template, X is absent or is a connecting peptide. When present, X is a linker peptide and is a linker peptide suitable for expression by the in vitro cell-free protein synthesis system of the invention.

When X is not existed, the gene template for coding SeP-X-DpnI corresponds to the gene template for coding SeP-DpnI, and can be used for expressing SeP-DpnI fusion protein; the gene template for coding SeFP-X-DpnI corresponds to the gene template for coding SeFP-DpnI and can be used for expressing SeFP-DpnI fusion protein; the gene template for coding the eGFP-X-DpnI corresponds to the gene template for coding the eGFP-DpnI and can be used for expressing the eGFP-DpnI fusion protein.

When X is present, X can function as a linker. The structure of X is not particularly limited, and it is sufficient that the expression of the target protein is not affected in the corresponding cell-free protein synthesis system. Linker sequences used in existing fusion proteins are all incorporated by reference herein. The linker used by the cell expression system for intracellular synthesis of fusion protein can also be taken as reference and included in the scope of the invention. X useful in the practice of the present invention include, but are not limited to, proteins disclosed in "Fusion proteins: properties, Design and function [ J ]. Advanced Drug Delivery Reviews,2013,65(10): 1357. minus 1369", "Design of the proteins in the library of biological Fusion proteins [ J ]. Protein Engineering, Design and Selection,2001,14(8): 529. 532", "LINKER: algorithm to production Protein sequences for Fusion proteins [ J ]. Protein Engineering, distribution Selection,2000,13(5): 309. 312. variant R. binding T. sample and function [ 5911. 12. and the like as well as the indirect Fusion proteins disclosed in" publication of the "12. P.S.: 5934. sub.S.: patent application of the" 12. sub.S., "Design and function of the" patent application of the invention; also specifically included, but not limited to, the linker sequence of fusion proteins used in existing cell-free protein synthesis systems.

When X is present, the number of amino acid units of the connecting peptide capable of being expressed in a corresponding in vitro cell-free system is not particularly limited, and is preferably 2 to 100, more preferably 2 to 50, and further preferably 5 to 25.

When X is present, X is selected from a cleavable linker peptide or a stable linker peptide in one of the preferred ways; the cleavable linker peptide is preferably selected from the group consisting of: a connecting peptide with self-cutting property and a connecting peptide which is cut under the action of exogenous enzyme.

The cleavable linker peptide may adopt a reported sequence structure, including but not limited to "Fusion protein linkers: properties, design and function [ J ]].Advanced Drug Delivery Reviews,2013,65(10):1357-1369”“Vega,M.C.(Ed.).Advanced technologies for protein complex production and characterization.Advances in Experimental Medicine and Biology.2016.”、“Schmoll,M.,&

(Eds.). Gene expression systems In furniture i: advances and applications. functional biology.2016. "," In Innovations In biotechnology. edited by edition C, edition Agbo C; 2012 "," Luke G A, Roulston C, Tilsner J and Ryan M D.growing uses of 2A in plant biotechnology,2015 "and the like and cleavable linkers (cleavable linkers) disclosed and cited in documents cited directly or indirectly, typically as the 2A sequence or the like. For example, when eGFP is used as a solubilizing label, when eGFP-X-DpnI is a cleavable linker, the fusion protein can be degraded under the self-cleavage effect or the exogenous enzyme effect after being expressed, so that the eGFP fusion protein of DpnI is released, the wrapping and steric hindrance effects of eGFP on DpnI are reduced, and the enzyme digestion effect of the DpnI solution is improved. The linker with self-shearing property can release DpnI without adding exogenous enzyme. For the connecting peptide which is sheared under the action of the exogenous enzyme, the DpnI can be released when the exogenous enzyme is added, so that enzymolysis can be carried out before use, and the homogeneity of a system after the DpnI is released can be maintained.

The connecting peptide having self-cleaving property is exemplified by a 2A sequence. The 2A sequence is a polylinker peptide from a virus (e.g., GATNFSLLKLAGDVELNPGP from Equisne rhinitis B virus 1 virus, 20 amino acids in length) that is self-cleaving. References to 2A sequences include, but are not limited to, the following and citations thereof: "Garry A.Luke, Helena estimate, PabloDe Felipe and Martin D.Ryan.2A to the for-Research, technology and applications [ J ]. Biotechnology and Genetic Engineering Reviews,2009,26:223 + 260", "Fu Yan.detail analysis of sequence retrieval with 2A translation instructions [ D ]. Newcastle University, 2013", "Tariana M Souza-Moreia, Clara Navarete, Xin chemistry et al.Scoring of 2A protocols for polymorphic gene expression in layer [ J ]. YeMS Yeast [ J ]. 038 (355). The cleavage site occurs before the last amino acid residue. Therefore, the protein located in front of 2A will eventually be fused to the first 19 amino acid residues of 2A at the C-terminus, and the protein located behind 2A will eventually be fused to the last amino acid residue of 2A at the N-terminus. The 2A sequence employed in example 1 was derived from the Equine rhinitis B virus 1 virus; 2A sequences derived from other viruses having self-splicing function can also be used in the practice of the present invention, and differences in amino acid sequence are allowed.

The connecting peptide which is cleaved by an exogenous enzyme is exemplified by TEV. TEV is a 7 amino acid-long oligo-linker peptide (ENLYFQG) that can be specifically recognized and cleaved by TEV enzymes. When not digested with TEV, the TEV sequence can be used as a linker (linker) for joining DpnI to SeP, SeFP, eGFP, etc.

The stable linker peptide, which is not self-cleaving nor does it present an enzyme that specifically recognizes and cleaves, is generally stable, but allows degradation by means other than enzymatic hydrolysis, such as hydrolysis, acid-sensitive degradation, base-sensitive degradation, temperature-sensitive degradation, and the like. The stable linker peptides may employ reported sequence structures including, but not limited to, the related sequences disclosed and referenced in "Fusion protein linkers: properties, design and function [ J ]. Advanced Drug Delivery Reviews,2013,65(10): 1357-.

The amino acid constituting the linker peptide X may be a natural amino acid, an unnatural amino acid, a radioisotope-labeled amino acid, a modified amino acid, or other amino acid derivative. Examples of the structure of X include, but are not limited to, a 2A sequence, a TEV sequence, a sequence consisting of Gly and Ser, a sequence consisting of Gly and Ala (see Clifford R. Robinson and Robert T. Sauer. optimizing the stability of single-chain proteins by linker length and composition mutagenesis [ J ]. PNAS,1998,95(11): 5929-.

In vitro cell-free protein synthesis system

The in vitro protein synthesis reaction is carried out in an in vitro cell-free protein synthesis system. The order of addition of the components thereof is not particularly limited. However, it is preferable to add other components than the gene template and finally the gene template encoding the target protein, and then start the in vitro protein synthesis reaction to synthesize the target protein through a transcription translation process or a translation process.

The in vitro cell-free protein synthesis system preferably comprises a cell extract in addition to the gene template. The cell extract is used to provide a structural or biological factor for protein transcription and translation. The selection criteria of the cell extract are as follows: can express the gene template of the coding target protein and synthesize the target expression product.

In a preferred embodiment, the cellular extract may have a gene encoding RNA polymerase integrated into the cellular genome.

In another preferred embodiment, the in vitro cell-free protein synthesis system further comprises a cell extract, and exogenous RNA polymerase or/and a gene template containing a gene sequence encoding RNA polymerase is further added.

In another preferred embodiment, the in vitro cell-free protein synthesis system further comprises a cell extract, and further comprises exogenous DNA polymerase or/and a gene template containing a gene sequence encoding DNA polymerase.

The in vitro cell-free protein synthesis system, in one preferred embodiment, comprises an energy system in addition to the gene template.

In the in vitro cell-free protein synthesis system, in a preferred mode, the gene template is a DNA template.

In a further preferred mode, the in vitro cell-free protein synthesis system comprises a DNA template, and further comprises a substrate for RNA synthesis and a substrate for protein synthesis.

In a further preferred mode, the in vitro cell-free protein synthesis system comprises a cell extract, a substrate for RNA synthesis and a substrate for protein synthesis in addition to a DNA template;

Gene template

The gene template contains a target gene. The target gene refers to a gene sequence encoding a target protein.

The target gene and the gene template are determined according to the target expression product. The gene template encodes an expression product of interest.

The gene template is a gene template for coding a target expression product and contains a gene sequence for coding the target expression product.

The gene template contains a gene sequence for encoding DpnI (namely an encoding gene containing DpnI), can be expressed into protein containing a DpnI structure, and can be expressed into DpnI protein or/and DpnI fusion protein by the in vitro cell-free protein synthesis system. The gene template may also contain other gene sequences selected from promoters, terminators, enhancers (e.g., CN109423497A), and the like. It may further contain a gene sequence encoding other amino acid chains such as a leader peptide and a functional tag.

The gene sequence for coding the DpnI and the gene sequence for coding the DpnI structure in the DpnI fusion protein can be natural gene sequences, optimized artificial recombination sequences, gene mutation sequences and other artificial sequences, complete structures, local structures such as subunits, fragments and the like, as long as expression products of the expressed DpnI protein, the gene recombination protein, the gene mutation protein, the DpnI fusion protein and the like can play the activity of the DpnI enzyme.

The expression product is preferably a DpnI protein, a DpnI fusion protein, or a combination thereof. Accordingly, the gene template preferably contains the following gene sequences: a gene sequence encoding a DpnI protein, a gene sequence encoding a DpnI fusion protein, or a combination thereof.

One of the preferred modes of expression of the product is the DpnI protein. Accordingly, the gene template is a gene template encoding a DpnI protein.

One of the preferred modes of expression products is a DpnI fusion protein. Accordingly, the gene template is a gene template encoding a DpnI fusion protein, and belongs to a gene template containing a gene sequence encoding a DpnI fusion protein.

One of the preferred modes of expression of the product is a fusion protein of DpnI with a solubilization tag SeP. Accordingly, the gene template is a gene template encoding a fusion protein of DpnI and the solubilization tag SeP.

One of the preferred modes of expression products is a fusion protein of DpnI and a fluorescent protein solubilization tag SeFP. Correspondingly, the gene template is a gene template of a fusion protein for coding DpnI and a fluorescent protein solubilization label SeFP.

One of the preferred modes of expression of the product is the edfp fusion protein of DpnI. Accordingly, the gene template is a gene template encoding a fusion protein of DpnI and eGFP.

One of the preferred modes of the expression product is containing SeP-X-DpnI structure. Accordingly, the gene template contains the gene sequence encoding SeP-X-DpnI fusion protein.

One of the preferable modes of the expression product is a SeFP-X-DpnI structure. Accordingly, the gene template contains a gene sequence encoding the SeFP-X-DpnI fusion protein.

One of the preferred modes of the expression product is that the expression product contains an eGFP-X-DpnI structure. Accordingly, the gene template contains a gene sequence encoding an eGFP-X-DpnI fusion protein.

One of the preferred modes of expression of the product is SeP-X-DpnI. Accordingly, the gene template is a gene template encoding SeP-X-DpnI fusion protein.

One of the preferred modes of expression products is SeFP-X-DpnI. Correspondingly, the gene template is a gene template for encoding SeFP-X-DpnI fusion protein.

One of the preferred modes of expression of the product is eGFP-X-DpnI. Accordingly, the DNA template is a gene template encoding eGFP-X-DpnI fusion protein.

In a preferred embodiment, the gene template comprises a gene sequence encoding the following proteins or a combination thereof: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI.

In a preferred embodiment, the gene template is selected from the group consisting of the gene sequences of the following proteins or mixtures thereof: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI.

The protein synthesis process in the transcription and translation mode is based on a DNA template, and the protein synthesis process in the translation only mode can be based on an mRNA template.

Preferably, the in vitro protein synthesis system of the present invention is an in vitro transcription translation system, i.e. the IVTT system.

Preferably, the gene template of the invention is a DNA template.

DNA template

The DNA template contains a gene sequence encoding a protein of interest.

The DNA template is determined according to the expression product of interest. The DNA template encodes the expression product of interest.

The DNA template is a DNA template for coding a target expression product and contains a gene sequence for coding the target expression product.

The DNA template contains a gene sequence for encoding DpnI, can be expressed into protein containing a DpnI structure, and can be expressed into DpnI protein or/and DpnI fusion protein by the in vitro cell-free protein synthesis system. The DNA template may also contain other gene sequences selected from promoters, terminators, enhancers (e.g., CN109423497A), and the like. It may further contain a gene sequence encoding other amino acid chains such as a leader peptide and a functional tag.

In one of the alternative forms, the DNA template is preferably selected from: a DNA template encoding a DpnI protein, a DNA template containing a gene sequence encoding a DpnI fusion protein (including a DNA template encoding a DpnI fusion protein), or a combination thereof.

In one preferred embodiment, the DNA template comprises a gene sequence encoding a fusion protein of DpnI with solubilization tag SeP. Contains both the gene coding for DpnI and the gene coding for SeP.

In one preferred embodiment, the DNA template comprises a gene sequence encoding a fusion protein of DpnI and SeFP, a fluorescent protein solubilization tag. Contains both the gene coding for DpnI and the gene coding for SeFP.

In one preferred embodiment, the DNA template comprises a gene sequence encoding a fusion protein of eGFP and DpnI.

In one preferred embodiment, the DNA template comprises the gene sequence encoding SeP-X-DpnI.

In one preferred embodiment, the DNA template comprises a gene sequence encoding SeFP-X-DpnI.

In one preferred embodiment, the DNA template comprises a gene sequence encoding eGFP-X-DpnI.

One of the preferred modes of the DNA template is a DNA template encoding a DpnI protein, a DNA template encoding a DpnI fusion protein, or a combination thereof.

One of the preferred modes of the DNA template is a DNA template encoding a fusion protein of DpnI and the solubilization tag SeP.

One of the preferred modes of the DNA template is a DNA template encoding a fusion protein of DpnI and the fluorescent protein solubilization tag SeFP.

One of the preferred modes of the DNA template is a DNA template encoding a fusion protein of DpnI and eGFP.

One of the preferable modes of the DNA template is a DNA template encoding SeP-X-DpnI.

One of the preferable modes of the DNA template is a DNA template encoding SeFP-X-DpnI.

One of the preferred modes of the DNA template is a DNA template encoding eGFP-X-DpnI.

In a preferred embodiment, the DNA template comprises the following gene sequences: a gene sequence encoding DpnI, a gene sequence comprising the gene encoding SeP-X-DpnI, or a combination thereof.

In a preferred embodiment, the DNA template comprises the following gene sequences: a gene sequence encoding DpnI, a gene sequence comprising a gene encoding SeFP-X-DpnI, or a combination thereof.

In a preferred embodiment, the DNA template comprises the following gene sequences: a gene sequence encoding DpnI, a gene sequence comprising a gene encoding eGFP-X-DpnI, or a combination thereof.

The definitions and preferred embodiments of X, including the preferred embodiments of the number of amino acids, are the same as those described above.

In a preferred embodiment, the DNA template comprises the gene sequences encoding the following proteins or a combination thereof: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI.

In a preferred embodiment, the DNA template is selected from the gene sequences of the following proteins or mixtures thereof: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI.

In a preferred embodiment, the DNA template contains DNA sequences encoding DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScarlet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI (example 6, examples 1 to 3, examples 11 to 14); it may also contain a combination of the above gene sequences, allowing the synthesis of two or more proteins having DpnI enzyme activity in the CFS system. Wherein, when eGFP is expressed at the N-terminal side of DpnI, the obtained DpnI solution and DpnI stock solution have better enzyme digestion activity than the expression at the C-terminal side, and are equivalent to or even better than commercial products.

The concentration of the DNA template is selected and determined by conversion according to the amount of protein to be expressed in the experimental protocol. In a preferred embodiment, the concentration of the DNA template is 0.3 to 80 ng/. mu.L. In another preferred embodiment, the concentration of the DNA template is 1 to 50 ng/. mu.L. In another preferred embodiment, the concentration of the DNA template is 5 to 50 ng/. mu.L.

The DNA template used in the in vitro cell-free synthesis system can be circular DNA or linear DNA. May be single-stranded or double-stranded. The gene sequence of the DNA template may be selected from the group including, but not limited to: genome sequence and cDNA sequence. It may further contain a promoter sequence, a 5 'untranslated sequence, and a 3' untranslated sequence.

In a preferred embodiment, the gene sequence of the DNA template further comprises an element selected from the group consisting of: promoters, terminators, poly (A) elements, transport elements, gene targeting elements, selectable marker genes, enhancers, resistance genes, transposase encoding genes. Reference may be made to US20060211083a1 and the like.

The 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 gene sequences encoding proteins of interest. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

In a preferred embodiment, the circular DNA is plasmid DNA. The corresponding DNA plasmid is not particularly limited as long as it can be expressed in a cell extract. Generally, the plasmid contains functional elements such as a promoter, a terminator, and an untranslated region (UTR). One of the preferable modes of the plasmid is to contain a promoter corresponding to a cell extract biological factor. For example, plasmids containing the T7 promoter can be theoretically used as expression systems of the DNA templates described in the examples, and for example, pET series plasmids of Escherichia coli, pGEM series plasmids, and the like can be used in place of the plasmid vectors of the Kluyveromyces lactis extract of the examples to practice the present invention; the T7 promoter is a strong promoter that is specifically responsive to T7RNA polymerase and is a sequence that initiates transcription of the T7 phage gene.

The linear DNA may be obtained by 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. Wherein the constant temperature amplification technology is preferably a normal temperature amplification technology.

One of the preferred embodiments of the linear DNA is a PCR linear fragment. The PCR linear fragment can be obtained by reported PCR technology.

Another preferred embodiment of the linear DNA is a double-stranded linear DNA obtained by an amplification system. The amplification system is not particularly limited and may be selected from the group consisting of, but not limited to, existing commercial kits, and literature reported amplification systems, as long as it is capable of amplifying the DNA template of the present invention used in the in vitro cell-free synthesis system. 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 a preferred embodiment, the DNA template is double-stranded DNA and is constructed in a circular plasmid vector. The plasmid vector used typically contains functional elements such as the T7 promoter, the LAC4 terminator and/or the 5 'and 3' UTR.

Specifically, in one preferred embodiment, double-stranded DNA is used as a DNA template and constructed in a circular plasmid vector. These plasmids contain the T7 promoter and allow for cell-free in vitro expression of a variety of proteins in Kluyveromyces lactis cell extracts, including but not limited to the DpnI protein and its fusion proteins, particularly including but not limited to the DpnI protein and the DpnI fusion protein (one of the preferred modes for the DpnI fusion protein is SeP-X-DpnI; one of the preferred modes for SeP-X-DpnI is SeFP-X-DpnI, and one of the other preferred modes is eGFP-X-DpnI). The plasmid also comprises functional elements such as a T7 promoter, a LAC4 terminator, a UTR and the like.

The basic structure of the plasmid and the method for inserting the gene encoding the target protein into the plasmid vector can be carried out by conventional techniques in the art, and are not described 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 found in CN 201910460987.8.

mRNA template

The restriction endonuclease DpnI preparation of the invention can also adopt exogenous mRNA template to replace DNA template, or adopt the mixture of mRNA template and DNA template to carry out in vitro protein synthesis reaction.

Means for providing factors required for the synthesis of proteins

In addition to providing various factors required for in vitro transcription and translation by means of cell extracts, the PURE system (Protein synthesis Using Recombinant Elements) developed by Japanese scientists can be used. 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.

Cell extract

The cell extract should be capable of expressing the gene template used in the cell-free protein synthesis system, i.e., the gene template encoding the protein of interest.

The cell extract is used to provide structural or biological factors for protein transcription/translation. In the case of yeast cell extracts, they are typically used to provide substances such as ribosomes, transfer RNAs, aminoacyl tRNA synthetases, initiation and elongation factors and stop release factors required for protein synthesis, and may also provide other enzyme substances such as polymerases (RNA polymerases and/or DNA polymerases); the cell extract preferably does not contain intact cells. The cell extract may also contain some other proteins, especially soluble proteins, originating from the cytoplasm of the cell. The cell extract, among others, may preferably contain various factors required for the synthesis of endogenously expressed proteins. The various factors provided by the cell extract can naturally exist in the cell genome, can integrate the related genes into the cell genome, and can insert the related genes into a cell plasmid. RNA polymerase and DNA polymerase are exemplified. In one preferred embodiment, the cell extract contains an endogenously expressed RNA polymerase and/or DNA polymerase. For example, in examples 1 to 23, since the gene of RNA polymerase is integrated into the genome of yeast cells and RNA polymerase can be supplied endogenously by using a cell extract, cell-free protein synthesis in vitro can be performed without adding exogenous RNA polymerase. In addition, the gene encoding RNA polymerase can be inserted into a cellular plasmid, such as a Kluyveromyces lactis cellular plasmid, to prepare a cellular extract. As a method for integrating RNA polymerase into the genome of a cell, a method disclosed in patent application publication CN109423496A or the like can be cited.

In a preferred embodiment, the cell extract is derived from kluyveromyces lactis having a gene encoding RNA polymerase, a gene encoding DNA polymerase, or a combination thereof integrated into its genome.

In the present invention, one of the preferable modes of the protein content of the cell extract is 20-100 mg/mL; more preferably 50-100 mg/mL. The method for determining the protein content can adopt a Coomassie brilliant blue determination method.

The concentration of the cell extract in the in vitro protein synthesis system is not particularly limited. In a preferred embodiment, the concentration of the cell extract is between 20% and 70% (v/v); in another preferred embodiment, the concentration of the cell extract is 30-60% (v/v); in another preferred embodiment, the concentration of the cell extract is from 40% to 50% (v/v); in another preferred embodiment, the concentration of the cell extract is 80% (v/v); based on the total volume of the in vitro cell-free protein synthesis system.

The cell source of the cell extract may be one or more types of cells selected from the group consisting of: including but not limited to E.coli, mammalian cells (e.g., rabbit reticulocytes, HF9, Hela, CHO, K562, HEK293), plant cells (e.g., wheat germ cells, tobacco BY-2 cells), yeast cells, insect cells, or combinations thereof.

The preparation method of the cell extract can adopt the reported technical means. In brief summary, the following steps may generally be included: quickly freezing the cells with liquid nitrogen, adding auxiliary reagent, crushing the cells, centrifuging and collecting supernatant to obtain the cell extract.

The Cell source of the Cell extract and the method for preparing the same can also be reported by reference to the existing literature, including but not limited to the literature of "Nicole E.Gregorio, Max Z.Levine and Javin P.Oza.A. User's Guide to Cell-Free Protein Synthesis [ J ]. Methods protocols.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 Cell sources reported in the direct citations or indirect citations are all incorporated by reference into the present invention. 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-2 cells), Spodoptera frugiperda insect cells (sf cells), Trichoplusia ni insect cells (Trichoplusia instect cells), rabbit reticulocytes (rabbitristocellulare), CHO cells (Chinese hamartovary cells), human K562 cells, HEK293 cells, HeLa cells, mouse fibroblast cells (mouse embryo fibroblast), Leishmania tarentum cells (Leisholana cells, monoclonal organisms), and the like.

The yeast cell is preferably selected from one of the embodiments, preferably from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; the Kluyveromyces is further preferably Kluyveromyces lactis (K.lactis), Kluyveromyces lactis var. drosophilarium, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus var. marxianus, Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces marxianus (Kluyveromyces dozshanii), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces marxianus (siamenses siamensius), Kluyveromyces lactis (Kluyveromyces), Kluyveromyces lactis Kluyveromyces, Kluyveromyces (Kluyveromyces), Kluyveromyces lactis, Kluyveromyces, 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.

In the present invention, the cell extract can express the DpnI protein or the fusion protein containing the DpnI structure in vitro.

The cell extract can correspondingly express the gene template as DpnI protein or/and DpnI fusion protein in vitro.

In a preferred embodiment, the cell extract is capable of expressing DpnI.

In a preferred embodiment, the cell extract is capable of expressing DpnI, together with the solubilization tag SeP.

In a preferred embodiment, the cell extract is capable of expressing DpnI and simultaneously expressing the fluorescent protein solubilization tag SeFP.

In a preferred embodiment, the cell extract is capable of expressing DpnI and at the same time eGFP.

In a preferred embodiment, the cell extract is capable of expressing a fusion protein of DpnI and SeP; a further preferred embodiment is one which is capable of expressing SeP-X-DpnI.

In a preferred embodiment, the cell extract is capable of expressing a fusion protein of DpnI and SeFP; in a further preferred embodiment, the expression of the coding sequence SeFP-X-DpnI is possible.

In a preferred embodiment, the cell extract is capable of expressing the edpni eGFP fusion protein; in a further preferred embodiment, eGFP-X-DpnI can be expressed.

Wherein the definitions and preferred embodiments of X are in accordance with the above. X is absent or is a linker peptide capable of being expressed by the in vitro cell-free protein synthesis system; when X is present, it serves as a linker for the fusion protein.

Cell extracts derived from the disclosed cell sources that express both DpnI and the solubilization tag SeP can be used in the practice of the present invention.

In a preferred embodiment, cell extracts derived from a cell source that has been disclosed as expressing both DpnI and the fluorescent protein solubilization tag SeFP can be used in the practice of the present invention.

In a further preferred embodiment, cell extracts from cell sources that have been disclosed as expressing both DpnI and eGFP may be used in the practice of the present invention.

Among them, most of the existing commercialized DpnI adopts an escherichia coli expression system, and reference may be made to an expression system disclosed in patent document US6277608B1 and the like. WO1998038205A1 discloses that expression of a restriction endonuclease gene (e.g., a DpnI gene) can be carried out in a prokaryotic cell system (most preferably an Escherichia coli cell), a eukaryotic cell system (preferably an animal cell, more preferably an insect cell, a nematode cell, a mammalian cell, etc., most preferably a human cell) to obtain a restriction endonuclease (e.g., a DpnI enzyme). WO1998038205A1 also discloses that expression of a restriction endonuclease gene (e.g., a DpnI gene) can be carried out in a prokaryotic cell system (most preferably an Escherichia coli cell), a eukaryotic cell system (preferably an animal cell, more preferably an insect cell, a nematode cell, a mammalian cell, etc., most preferably a human cell) to obtain a restriction endonuclease (e.g., a DpnI enzyme). It is further disclosed that the host cell may preferably be selected from the group consisting of a bacterium (most preferably an e.coli cell (e.coli cell) or a Bacillus cell), a yeast cell, a plant cell, an animal cell (more preferably an insect cell, a nematode cell or a mammalian cell, most preferably a CHO cell, a COS cell, a VERO cell, a BHK cell or a human cell). Further host cells expressing restriction endonuclease genes (e.g., the DpnI gene) disclosed in WO1998038205A1 are also incorporated herein by reference and can be used in the practice of the present invention, especially with reference to page 10, lines 5-12, page 12, lines 20-13, lines 8, page 26, lines 1-5, etc.

Among them, cell sources including, but not limited to, the above-mentioned documents and cited documents that can express in vivo or can synthesize in vitro the solubilization tag SeP, the fluorescent protein solubilization tag SeFP, and the enhanced green fluorescent protein eGFP can be used as candidates for the cell extract source of the present invention.

US20160152998a1 discloses the synthesis of eGFP fusion proteins using plant cell expression systems. The literature "Efficient cell-free expression with the endogenous E.coli RNA polymerase and sigma factor 70[ J ]. Journal of Biological Engineering,2010,4: 8" discloses cell-free in vitro protein synthesis of eGFP based on E.coli extracts. EP2216414a1 discloses cell-free in vitro protein synthesis of eGFP fusion proteins based on e. US20110117597a1 discloses cell-free in vitro protein synthesis of eGFP based on wheat germ cell extracts. US20110117597a1 discloses cell-free in vitro protein synthesis of eGFP based on extracts of rabbit reticulocytes (mammalian cells). "Giant cells functional expressing membrane receptors for an infection photomeone [ J ]. Chemical Communications,2014,50(22): 2958" discloses cell-free in vitro protein synthesis of eGFP fusion proteins based on insect cell extracts. "cloning a high hybridizing plasmid-based cell-free protein synthesis system [ J ]. Biotechnology and Bioengineering,2017,114(6): 1343-1353" discloses cell-free in vitro protein synthesis of eGFP based on Streptomyces cell extracts.

The document "Single-Cell Dynamics of Genome-Nuclear amine Interactions [ J ] Cell,2013,153(1): 178-192" discloses that human fibrosarcoma HT1080 cells (human fibrosarcoma Cell line HT1080, mammalian cells) can express a fusion protein of eGFP and DpnI. Embodiments 1-23 of the present invention employ an in vitro protein synthesis system of yeast cell extracts (kluyveromyces lactis cell extracts) to express DpnI proteins and fusion proteins thereof.

In a preferred embodiment of the cell extract according to the present invention, the cell extract may be selected from any of the following sources: coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof. More preferably, the yeast cell is selected from the group consisting of Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or combinations thereof; the Kluyveromyces is further preferably Kluyveromyces lactis var. drosophilarium, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, Kluyveromyces marxianus, Kluyveromyces marxianus vanduli, Kluyveromyces polybubali, Kluyveromyces amabilis, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hupehensis, Kluyveromyces polyspora, Kluyveromyces siae, Kluyveromyces lactis, or a combination thereof.

Among them, the most commonly used system for expressing DpnI is an Escherichia coli expression system, which can also express eGFP protein and fusion protein thereof, and is one of the preferred sources of cell extracts for synthesizing the preparation of the restriction enzyme DpnI of the present invention and performing in vitro protein synthesis reaction.

In another preferred embodiment, the cell extract is a yeast cell extract, more preferably a kluyveromyces marxianus cell extract or a kluyveromyces lactis cell extract.

In another preferred embodiment, the cell extract may be selected from any one of the following sources: coli, Kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda insect cells (sf insect cells), rabbit reticulocyte, CHO cell, COS cell, VERO cell, BHK cell, human fibrosarcoma HT1080 cell, or a combination thereof.

RNA polymerase, DNA polymerase

Before the in vitro protein synthesis reaction is carried out, at least one of the following components can be added into the cell-free in vitro protein synthesis system to optimize the reaction system: exogenous RNA polymerase and exogenous DNA polymerase.

The addition of RNA polymerase can promote the transcription process, and the addition mode can be any one or the combination of the following modes: exogenous RNA polymerase and exogenous DNA polymerase.

In the case where the cell genome from which the cell extract is derived does not contain or incorporate RNA polymerase, it is often necessary to additionally add exogenous RNA polymerase to facilitate the reaction.

Exogenous RNA polymerase may be added directly, or an exogenous gene template encoding RNA polymerase may be added, or a combination thereof. The coding gene of RNA polymerase may be constructed together with a gene template encoding a target protein, or may be constructed separately from an independent foreign gene template.

Similarly, the DNA polymerase may be added directly, or a foreign gene template containing the gene encoding the DNA polymerase may be added, or a combination thereof may be added. Can be a gene template for coding a target protein or an independent exogenous gene template.

The in vitro protein synthesis reaction of the second step may or may not include a DNA amplification process when a DNA template is used; if the in vitro protein synthesis reaction also includes a DNA amplification process, it is usually necessary to add an exogenous DNA polymerase. In examples 1 to 23 of the present invention, after the target gene was amplified, the amplified product was added to the reaction system as a DNA template; the in vitro protein synthesis reaction of the second step does not include a DNA amplification process.

The polymerase (exogenous RNA polymerase, exogenous DNA polymerase) is preferably a polymerase that can be amplified at room temperature, preferably room temperature to 37 ℃, specifically, preferably 20 ℃ to 37 ℃, and more preferably 25 ℃ to 37 ℃. The polymerase capable of performing normal temperature amplification can be selected according to a gene template, and normal temperature amplification polymerases which can be used in vitro cell-free systems are all included in the scope of the present invention as reference, including but not limited to phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, exo-klenow DNA polymerase, Bsu DNA polymerase, PolIII DNA polymerase, T7RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, T5 RNA polymerase, fragments of any of the foregoing polymerases, and any combination of the foregoing polymerases and fragments thereof.

The amplification technique, particularly the amplification method at room temperature, which can be used in the present invention is not particularly limited, and the amplification technique at room temperature, which can be used in vitro cell-free systems, is 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 (selected-amplified sequence) and the like. References include, but are 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 word [ J ]. Synthesis and Systems Biotechnology,2017,2, 23-27" and the like and direct or indirect citations thereof.

Energy system/energy regeneration system

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

The reported energy system/energy regeneration system for the cell-free in vitro protein synthesis system can provide energy for the in vitro synthesis of the restriction enzyme DpnI and the fusion protein thereof. Including but not limited to documents CN109988801A, US20130316397A, US20150376673A, "MJ Anderson, JC Stark, CE Hodgman and MC Jewett. energy synthesis with glucose measurement [ J ]. FEBS Letters,2015,589(15): 1723-.

In a preferred embodiment, the energy system is a polysaccharide and phosphate energy system, a phosphocreatine and phosphocreatine system, a glycolytic pathway and its intermediate energy system, or a combination thereof. Specifically, the phosphate is preferably selected from orthophosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof. The polysaccharide may be selected from polysaccharides including, but not limited to, starch, maltodextrin, corn dextrin, maltose, and the like. The glycolytic pathway and its intermediate energy systems include, but are not limited to, glucose-based energy systems.

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. The energy system used in examples 1-6 was a polysaccharide and phosphate energy system, wherein the polysaccharide was corn dextrin and the phosphate was potassium phosphate. Example 15 uses a complex system of glucose, maltodextrin, and tripotassium phosphate.

Substrate for RNA synthesis

The substrate for the synthetic RNA is a mixture of nucleotides, one embodiment, selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof. Preferably a nucleoside triphosphate mixture dNTP. The mixture of nucleoside triphosphates is preferably a mixture of adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uridine triphosphate. In the present invention, the concentration of each mononucleotide is not particularly limited, and it is measured as a nucleotide necessary for synthesizing a protein, and in one of the generally preferred embodiments, the concentration of each mononucleotide is 0.5 to 5mM, and in another preferred embodiment, the concentration of each mononucleotide is 1.0 to 2.0 mM.

Substrates for synthetic proteins

The substrate of the synthetic protein is an amino acid mixture. The amino acids required for the synthesis of the protein are metered in. The concentration of each amino acid is usually 0.01 to 5mM in one preferred embodiment, and 0.1 to 1mM in another preferred embodiment.

The amino acid mixture comprises at least amino acids for synthesizing the protein of interest, i.e. the amino acid mixture comprises at least amino acids for synthesizing a DpnI protein or/and a DpnI fusion protein, selected from the group comprising, 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, and histidine. Examples of the fusion protein of DpnI include tag-labeled DpnI fusion proteins such as SeP, SeFP, eGFP and the like.

The amino acid mixture may include natural amino acids, unnatural amino acids.

The amino acid mixture may include L-amino acids, D-amino acids, or mixtures thereof.

In addition to natural amino acids, the amino acid mixture may include unnatural amino acids, D-amino acids, radioisotope-labeled amino acids, modified amino acids, and the like. The unnatural amino acid is not particularly limited and may be selected from the group consisting of: including, but not limited to, references such as "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 catalysts in cell-free interaction of non-natural amino acids in proteins [ J ]. Frontiers in pharmaceuticals, 2019,10: 611", and the unnatural amino acids reported or cited in the references, 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.

Other additive Components

The in vitro cell-free protein synthesis system can also compriseAt least one of the following components: crowding agents (preferably polyethylene glycol and/or the like), magnesium ions, potassium ions, antioxidants, 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 cell-free protein synthesis system may also contain polyethylene glycol and/or analogs thereof, used to mimic the crowded macromolecular environment within a cell, used as propagating agents, where, for example, polyethylene glycol may also modulate system viscosity. Polyethylene glycol having CH₂CH₂Repeating units of O, commonly designated peg (polyethylene glycol), PEO (poly (ethylene oxide), poe (polyoxyethyleneene). The concentration of polyethylene glycol or an analog thereof is not particularly limited, and usually, the concentration of polyethylene glycol or an analog thereof is 0.1 to 10%, preferably 0.1 to 8%, more preferably 0.5 to 4%, more preferably 1 to 2%, in terms of a mass volume concentration (% (w/v)) of the protein synthesis system or in terms of the 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. The polyethylene glycol preferably has a molecular weight of 200Da to 10000Da, more preferably 3000Da to 10000 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: PEG3000, PEG8000, PEG6000, PEG3350, combinations thereof; wherein the number of 3350 and the like is numerically equal to the weight average molecular weight. The molecular weight of the polyethylene glycol may also be, for example, 200Da, 400Da, 1500Da, 2000Da, 4000Da, 6000Da, 8000Da, 10000Da, etc. Typically, the molecular gauge is preferably ± 10% or less than ± 10%. Other macromolecules which may act as growth agents include, for example, dextran (dex)tran), Ficoll (e.g., Ficoll-400), etc., and also references "X Ge, D Luo and J xu. cell-free protein expression under macromolecular viewing conditions [ J]PLoS One,2011,6(12): e28707 "and references cited therein.

The magnesium ion is derived from a magnesium ion source, which may be selected from the group consisting of, but not limited to: magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, combinations thereof. One of the preferred embodiments has a concentration in the range of 0.1 to 50 mM. Another preferred embodiment has a concentration in the range of 0.5-20 mM. Another preferred embodiment has a concentration in the range of 1-10 mM.

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 chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, and combinations thereof. One of the preferred embodiments has a concentration in the range of 0-500 mM. Another preferred embodiment has a concentration in the range of 1 to 250 mM. Another preferred embodiment has a concentration in the range of 5-200 mM. Another preferred embodiment has a concentration in the range of 10-100 mM.

The optimization and optimization of polyethylene glycol, magnesium ion and potassium ion reported in WO2016005982A1 are also incorporated herein by reference.

The antioxidant, which may also be referred to as a reducing agent. Optional materials include, but are not limited to, Dithiothreitol (DTT), 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, and the like. One of the preferred embodiments is dithiothreitol. DTT may be used in a concentration conventionally used therefor, for example, 0.5 mM-10 mM in one embodiment.

The buffer is mainly used for maintaining the pH environment of the system. One of the preferred embodiments is selected from any one or a combination of the following: Tris-HCl, Tris base, HEPES.

The aqueous solvent is preferably a buffer.

Specific embodiments of the in vitro protein Synthesis System

Preferred embodiments of cell-free in vitro synthesis systems for synthesizing preparations of restriction enzyme DpnI include, but are not limited to, reported in vitro protein synthesis systems and reaction conditions for expressing DpnI and fusion proteins thereof.

In a preferred embodiment, the in vitro protein synthesis system comprises a DNA template and one or more or all of the following components selected from the group consisting of: yeast cell extract, potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K) or Tris, potassium acetate, magnesium acetate, nucleoside triphosphate mixture (dNTP), amino acid mixture, creatine phosphate, Dithiothreitol (DTT), phosphocreatine kinase, rnase inhibitor, sucrose, glucose, starch, dextrin, corn dextrin, maltodextrin, phosphate (e.g., potassium phosphate).

In a preferred embodiment, the in vitro protein synthesis system comprises a DNA template and one or more or all of the following components selected from the group consisting of: yeast cell extract, potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K) or Tris, potassium acetate, magnesium acetate, nucleoside triphosphate mixture (dNTP), amino acid mixture, creatine phosphate, Dithiothreitol (DTT), phosphocreatine kinase, rnase inhibitor, sucrose, glucose, starch, dextrin, corn dextrin, maltodextrin, phosphate (e.g., potassium phosphate), exogenous T7RNA polymerase, exogenous phi29 DNA polymerase.

In a preferred embodiment, the in vitro protein synthesis system comprises a DNA template encoding a protein of interest, and further comprises one or more or all components selected from the group consisting of: yeast cell extract (RNA polymerase is integrated into genome or RNA polymerase encoding gene is inserted into plasmid), potassium acetate, magnesium acetate, glutaminase, HEPES-K, Tris-HCl, dNTP, amino acid mixture, DTT, sucrose, glucose, starch, dextrin, corn dextrin, maltodextrin, tripotassium phosphate, polyethylene glycol, DNA template encoding RNA polymerase, DNA template encoding DNA polymerase. The yeast cell extract is preferably a Kluyveromyces cell extract, more preferably a Kluyveromyces lactis cell extract. In another preferred embodiment, the system for in vitro protein synthesis comprises a DNA template, a yeast cell extract, and one or more or all of the following components: tris (hydroxymethyl) aminomethane (Tris-HCl, pH8.0), potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, glucose, dNTP (mixture of four nucleoside triphosphates, with the same concentration of a single nucleoside triphosphate), 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, wherein, with the same concentration of a single amino acid), tripotassium phosphate. Specifically, it further preferably contains a DNA template, a yeast cell extract (more preferably a Kluyveromyces cell extract), and one or more or all of the following components: 9.78mM Tris (Tris-HCl) pH8.0, 80mM potassium acetate, 5mM magnesium acetate, 0.44mM dithiothreitol, 2% (w/v) polyethylene glycol, 40mM glucose, 1.8mM four nucleoside triphosphates (1.8mM is the concentration of a single nucleoside triphosphate), 0.5mM 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, 0.5mM is the concentration of a single amino acid), 24mM tripotassium phosphate. The DNA template adopts 5-50 ng/. mu.L DNA template for coding DpnI protein or DpnI fusion protein. One of the preferred modes of fusion of DpnI described above is with solubilization tag SeP, more preferably SeP-X-DpnI. Another preferred embodiment is a fusion protein with the fluorescent protein solubilization tag SeFP, more preferably SeFP-X-DpnI. Another preferred embodiment is an eGFP fusion protein, more preferably eGFP-X-DpnI.

In vitro protein synthesis reaction and subsequent treatment

The conditions for carrying out the in vitro protein synthesis reaction are determined according to a specific in vitro cell-free protein synthesis system, and the reported reaction conditions can be referred to. Preferably, normal temperature conditions are used for in vitro protein synthesis. The normal temperature is preferably from room temperature to 37 ℃, and particularly preferably from 20 ℃ to 37 ℃. One of the preferred modes is 25 ℃ to 37 ℃. Another preferred embodiment is 20 ℃ to 30 ℃. The reported normal temperature amplification method or isothermal amplification method suitable for normal temperature conditions can be used for implementing the technical scheme of the invention.

After the in vitro protein synthesis reaction is finished, carrying out solid-liquid separation, and collecting supernatant to obtain a solution containing the DpnI protease or/and the DpnI protein fusion protein. Preferred modes of the DpnI fusion protein include, but are not limited to, any of the preferred modes described above.

It should be noted that, for the crude product after the in vitro protein synthesis reaction is finished, that is, the reaction solution without solid-liquid separation, when there is no special requirement for the doping content of other components in the application, it can also be directly used. For example, including, but not limited to, in the fields of food, agriculture, feed, and the like, particularly in the field of additives. This is because the raw material components added for the in vitro protein synthesis reaction are non-toxic.

The solid-liquid separation can adopt a reported solid-liquid separation method, and preferably adopts a mode of minimizing damage to the activity of the protein and minimizing loss of the protein content. Generally, a centrifugation method may be used to perform solid-liquid separation, discard the precipitate, and retain the supernatant. In a preferred embodiment, the centrifugation is performed at a low temperature (e.g., 4 ℃) and at a rotation speed of 3000 to 6000rpm for 5 to 10 min. In a specific preferred embodiment, the centrifugation is carried out for 10min at a rotation speed of 4000rpm and at a temperature of 4 ℃.

DpnI solution: after solid-liquid separation, the obtained supernatant is a DpnI solution. The DpnI solution contains DpnI protease or/and DpnI fusion protein corresponding to the gene template. Preferred modes of the DpnI fusion protein include, but are not limited to, any of the preferred modes described above.

The enzyme activity (enzyme activity value, enzyme activity unit number) of the DpnI solution collected above was tested. The test methods were as described above. The solution with the restriction enzyme activity is screened out to be the preparation of the restriction enzyme DpnI of the invention. A DpnI solution having a DpnI enzyme activity of at least 4U/. mu.L is preferable as the preparation of the restriction enzyme DpnI of the present invention. More preferably, the enzyme activity of the obtained preparation of restriction enzyme DpnI is at least 5U/. mu.L. The DpnI solution can be packaged and stored, can also be subpackaged and stored, and can also be prepared into DpnI storage solution to be packaged and stored (packaged or subpackaged).

DpnI stock solution: the DpnI stock solution can be added with one or more components in the stock solution of the existing commercialized DpnI product in the supernatant obtained after the solid-liquid separation. Among the existing commercial DpnI stock solutions, the components include but are not limited to: Tri-HCl (preferably pH7.5), Tris-acetate (preferably pH7.9), NaHepes (preferably pH7.5), NaCl, CaCl₂DTT (dithiothreitol), EDTA (ethylene diamine tetraacetic acid), BSA (bovine serum albumin), imidazole, glycerol, magnesium acetate, sodium acetate and the like. The amounts of the added components are also preferably referred to the compositional ratio of the commercial DpnI product. It is particularly preferred to contain glycerol (the content of glycerol is preferably not more than 70%, more preferably 40% to 60%, most preferably 50%, in v/v). When the glycerol concentration is 50% (v/v), the volume usage of plasmid digested with the DpnI enzyme is recommended to be not more than 10% of the total system, with reference to the product description of the commercial products mentioned above. When the number of enzyme activity units of the DpnI stock solution is relatively low, the volume dosage required for enzyme digestion is increased, and the glycerol content in the DpnI stock solution can be correspondingly reduced.

Another preferred embodiment of the DpnI stock solution is: on the basis of supernatant obtained by solid-liquid separation of reaction liquid after the in vitro protein synthesis reaction is finished, at least one of the following components is also added: Tri-HCl, Tris-acetate, NaHepes, NaCl, CaCl₂Dithiothreitol, EDTA, BSA, imidazole, glycerol, magnesium acetate, sodium acetate.

The DpnI stock solution is preferably a DpnI stock solution which can be stored at low temperature. One of the preferred embodiments of the storable, low temperature conditions is below-18 ℃; the low temperature is more preferably about-20 ℃ (for example, -18 ℃ to-23 ℃). Another preferred embodiment of the low temperature is-80 ℃ (± 5 ℃).

The cryopreserved DpnI stock solution generally includes at least glycerin so as to be storable at low temperatures. The low-temperature storage conditions are preferably about-20 ℃ (± 3 ℃, more preferably 18 ℃ to 23 ℃) or about-80 ℃ (± 5 ℃).

One of the preferred embodiments of the low temperature storage solution is shown to contain glycerol, more preferably 50% (v/v) glycerol.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as "Sambrook et al, molecular cloning: a laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), "A laboratory Manual for cell-free protein Synthesis" Edected by Alexander S.Spirin and James R.Swartz.cell-free protein synthesis: methods and protocols [ M ] 2007 ", or according to the conditions suggested by the manufacturer or as directed by, with reference to, the specific embodiments described above. Unless otherwise indicated, percentages and parts 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.

The invention takes Kluyveromyces lactis (K.lactis or kl) as a cell extract source in the embodiment, and the Kluyveromyces lactis strain ATCC8585 and different strains thereof subjected to genetic modification comprise modification of endogenous expression RNA polymerase. It should be noted that the same design and analysis and experimental methods are also applicable to other cell extract sources described herein, such as other yeast cells, eukaryotic cells (mammalian cells, plant cells, insect cells) such as animal cells, and prokaryotic cells (e.g., E.coli).

Examples 1 to 6

Constructing an expression vector for encoding the DpnI protein and the eGFP fusion protein thereof:

the artificially constructed plasmid vector designed aiming at the kluyveromyces lactis cell extract contains functional elements such as a T7 promoter, a LAC4 terminator, 5 'UTR and 3' UTR and the like. The plasmid vector can express a plurality of proteins in vitro in the kluyveromyces lactis cell extract, including but not limited to DpnI, eGFP and fusion proteins of the DpnI and the eGFP. It should be noted that the plasmid expression vector in this embodiment is only used to specifically illustrate the embodiment of the present invention, and is not intended to limit the scope of the present invention, as long as it can express DpnI or its fusion protein (especially, DpnI and solubilization tag fusion protein, in this embodiment, gfp fusion protein of DpnI) in a cell-free in vitro protein synthesis system, and can be used as the plasmid vector of the present invention; furthermore, the kluyveromyces cell extract in this embodiment is not limited to be used as a cell extract source of the cell-free in vitro protein synthesis system, and cell extracts from other sources may also be used to construct a cell-free in vitro protein synthesis system, such as cell extracts from prokaryotic systems (e.g., escherichia coli), other yeast cells, plant cells, insect cells, and mammalian cells, which are referred to above and will not be described again; other plasmid vectors useful in the practice of the present invention include, but are not limited to, common plasmid vectors commercially available at the present time, such as, for example: pET series plasmids, pGEM series plasmids, and the like.

In this example, the following six genes were inserted into plasmid expression vectors by PCR amplification and homologous fragment recombination, respectively, to construct expression vectors expressing the DpnI protein and its eGFP fusion protein. The accuracy of each plasmid was confirmed by gene sequencing.

(1)eGFP-2A-DpnI(SEQ ID No:2)；

(2)eGFP-DpnI(SEQ ID No:3)；

(3)eGFP-TEV-DpnI(SEQ ID No:4)；

(4) DpnI-2A-eGFP (composed of gene sequence of DpnI, gene sequence of 2A-containing octadecapeptide, and gene sequence of eGFP in sequence);

(5)DpnI-eGFP(SEQ ID No:5)；

(6)DpnI(SEQ ID No:1)。

wherein, the amino acid sequence of DpnI is shown as UniProt P0A460, and is a DNA sequence obtained by optimization of an optimization program, and the optimized gene sequence is shown as SEQ ID No. 1.

The gene sequence of 2A is from Equisne rhinitis B virus, and is shown as SEQ ID No. 6.

The gene sequence of TEV is shown in SEQ ID No. 7.

The gene sequence of the DpnI-2A-eGFP is characterized in that on the basis of the DpnI-eGFP (SEQ ID No:5), the gene sequences of 2A (SEQ ID No:6) and eighteen peptides are sequentially connected between the DpnI (SEQ ID No:1) and the eGFP gene sequence.

The six plasmid expression vectors constructed above correspond to example 1 (designated as eGFP-2A-DpnI experimental group), example 2 (designated as eGFP-DpnI experimental group), example 3 (designated as eGFP-TEV-DpnI experimental group), example 4 (designated as DpnI-2A-eGFP experimental group), example 5 (designated as DpnI-eGFP experimental group), and example 6 (designated as DpnI experimental group), respectively.

Preparing a cell extract: examples 1-6 all employ yeast cell extracts, specifically Kluyveromyces lactis (k.lactis), which are prepared by conventional techniques, which, in general terms, include: quickly freezing the cells with liquid nitrogen, adding auxiliary reagent, crushing the cells, centrifuging and collecting supernatant to obtain the cell extract. The yeast genome has integrated the T7RNA polymerase gene (if the RNA polymerase gene is not integrated, the corresponding exogenous RNA polymerase is preferably added, and not limited to T7RNA polymerase).

Constructing an in vitro cell-free protein synthesis system (without adding a DNA template for the moment): the volume of the reaction system is 300 mu L; Tris-HCl pH8.0, at a final concentration of 36mM, potassium acetate 49mM, magnesium acetate 3.7mM, nucleoside triphosphate mixture 1.8mM (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate, each at a concentration of 1.8mM), amino acid mixture 0.1mM (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), corn dextrin 0.087g/mL, potassium bicarbonate 2mM, polyethylene glycol 8000 (w/v), potassium phosphate 24mM, and finally the Kluyveromyces lactis cell extract 50% by volume.

Adding a DNA template, and carrying out in-vitro protein synthesis reaction: adding 15 ng/mu L of DNA template (SEQ ID No.:1) for encoding the DpnI protein and the DNA templates of the five DpnI fusion proteins into the reaction system respectively, mixing uniformly, and placing the mixture in an environment with the temperature of 25-30 ℃ for reaction for 7 h.

Negative control (negative control): the DNA template coding DpnI and the fusion protein thereof is not added, but the DNA template coding eGFP is added to carry out in-vitro protein synthesis reaction, namely the system does not express DpnI and the eGFP fusion protein thereof, but expresses eGFP.

And (3) finishing the reaction: and (3) centrifuging the reacted system at the rotation speed of 4000rpm at 4 ℃ for 10 minutes to perform solid-liquid separation, discarding the precipitate, and reserving the supernatant to obtain a DpnI solution (containing DpnI or eGFP fusion protein thereof, wherein the expression product corresponds to the DNA template).

Determining the enzyme activity of the DpnI solution: the unit number of the enzyme activity of the DpnI solutions of examples 1 to 6 per unit volume was 40U/μ L, 20U/μ L, 18U/μ L, 4U/μ L, 2U/μ L, and 8U/μ L, respectively. The centrifugation supernatants of examples 1-3, 4, 6 belong to the preparation of restriction enzyme DpnI of the present invention. The enzyme activity unit number of unit volume of the enzyme in the embodiments 4 and 5 is relatively low, and the enzyme digestion effect of 10-20U/mu L can be achieved only by adding solution with a large volume multiple; although the solution of DpnI of example 5 is not recommended as a preparation of the restriction enzyme DpnI of the present invention, it does not prevent its application for similar purposes, but the amount of raw materials is more required and, in addition, the content of glycerol can be suitably reduced when preparing a stock solution.

And (4) subpackaging and storing: adding glycerol of one volume to the supernatant solutions (DpnI solutions) of examples 1 to 6 respectively to obtain glycerol solutions with final concentration of 50% (v/v) as DpnI stock solutions, wherein the enzyme activity units per volume are respectively 20U/. mu.L, 10U/. mu.L, 9U/. mu.L, 2U/. mu.L, 1U/. mu.L and 4U/. mu.L; the DpnI stock solutions obtained in examples 1-3 and example 6 (preferably at least 4U/. mu.L) are preparations of the restriction enzyme DpnI of the present invention; at the same time, the DpnI storage solution which can be stored at low temperature can be stored at-20 ℃; if long-term storage is required, the temperature can be stored at-80 ℃. Likewise, while the DpnI stock solutions of examples 4 and 5 are not recommended as a formulation for the restriction enzyme DpnI of the present invention, they do not prevent their use for similar purposes, except that more raw materials are needed and it is recommended that the glycerol content of the stock solution be suitably reduced.

Example 7 cleavage Effect test of DpnI stock solution (including preparation of restriction enzyme DpnI)

And (3) testing a system: a10. mu.L reaction system was used containing a final concentration of 20mM Tris sulfate pH9.2, 40mM potassium chloride, 10mM ammonium sulfate, 5mM magnesium sulfate, 2% (v/v) glycerol, 1. mu.g of pUC19 plasmid, and either 1. mu.L or 0.3. mu.L or other volumes of the DpnI stock solutions of examples 1-6 above (i.e., 50% (v/v) glycerol solution).

Positive control (positive control, PC group): the product DpnI (product name: DMT enzyme, product number: GD111, 10000units/mL, concentration 10U/. mu.L) from Beijing Quanyujin Biotechnology Co., Ltd is hereinafter referred to as Quanyujin DMT enzyme.

Negative control (negative control, NC group): and (3) adding the DNA template coding the DpnI and the eGFP fusion protein without adding the DNA template coding the eGFP to carry out in-vitro protein synthesis reaction, carrying out solid-liquid separation on the obtained reaction liquid which does not express the DpnI and the eGFP fusion protein but expresses the eGFP after the IVTT reaction is finished, and preparing a 50% (v/v) glycerol solution which is a negative control.

The test method comprises the following steps: after incubation at 37 ℃ for 1 hour, 5. mu.L of the digest was taken and 50. mu.L of Trans 5. alpha. competent cells purchased from Quanyujin were transformed. The transformation products were all spread on LB plates and after overnight growth, the digestion effect was observed, and the results are shown in FIGS. 1 and 2. The left panel of FIG. 1 shows the experimental results of a positive control (positive control, DpnI from Transgene). The right panel of FIG. 1 shows the experimental results of a Negative control (Negative IVTT of eGFP), i.e., the experimental results without enzymatic digestion reaction. FIG. 2 shows the results of the DpnI stock solutions of examples 1-6.

And (3) testing results: from the results shown in FIGS. 1 and 2, pUC19 was treated with a DpnI stock solution (example 1) obtained from the reaction solution after completion of the IVTT reaction for expressing eGFP-2A-DpnI as it is, whereby a higher cleavage effect was obtained as compared with the product DpnI (positive control) of King Kogyo Co., Ltd. The effect of the eGFP-DpnI experimental group (example 2) is only inferior to that of the eGFP-2A-DpnI experimental group (example 1), and is approximately equivalent to that of the positive control whole gold company DpnI product. The eGFP-TEV-DpnI test group of example 3 also showed better digestion effect, and the effect was approximately equivalent to that of the positive control whole-formula gold DpnI product.

The DpnI stock solution of the DpnI experimental group (example 6) keeps other parameters of the test system unchanged, and when 3 volumes (3 muL) of the stock solution are adopted, the enzyme digestion effect equivalent to that of the positive control group (full-type gold DMT enzyme) can be achieved.

The DpnI stock solution of the DpnI-2A-eGFP test group (example 4) was 10. mu.L, which was equivalent to the positive control group (whole type gold DMT enzyme).

The DpnI stock solution of the DpnI-eGFP test group (example 5) was 10. mu.L, which was equivalent to the positive control group (whole gold DMT enzyme).

And (3) testing conditions are as follows: mu.L of a glycerol solution containing 50% (v/v) of a negative control (eGFP-expressing) and 1. mu.L of a positive control (product of DpnI from Korea Co., Ltd., whole gold DMT enzyme, 10U/. mu.L) and 2.5. mu.L of each of them were added to 10. mu.L of pUC19, and 3. mu.L of DpnI stock solution of example 6 (DpnI-expressing DpnI), 10. mu.L of DpnI-2A-eGFP (example 4) and 10. mu.L of DpnI stock solution of DpnI-eGFP (example 5) were incubated at 37 ℃ for 1 hour, and then the digestion effect was examined by transforming competent cells. The results are shown in FIG. 3, which shows the respective digestion effects.

Example 8 fluorescent protein Activity test results

Fluorescent protein activity assay: the sample to be detected is placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), the intensity of a Fluorescence signal is detected, and a Relative Fluorescence Unit (Relative Fluorescence Unit, hereinafter referred to as RFU) is used as a measurement Unit.

Fluorescence tests were carried out on each of the DpnI stock solutions (50% (v/v) glycerol solutions) of examples 1 to 6 and the 50% (v/v) glycerol solution prepared from the reaction solution after the IVTT reaction of the negative control group was completed. Testing parameters: after centrifuging at 4000rpm and 4 ℃ for 1 minute, the sample to be tested is placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), and the relative fluorescence unit value (RFU) is obtained by detection.

As shown in FIG. 4, the DpnI stock solutions of examples 1-3 also have good fluorescence effect and can be used for tracking or labeling. According to the definition of the supernatant rate, the supernatant rate can reach 70% by calculation according to the RFU value.

Example 9, the result of the nucleic acid electrophoresis test was conducted on a pUC19 plasmid-treated solution for testing the cleavage effect.

mu.L of the product obtained by treating 1. mu.g of pUC19 plasmid with DpnI stock solution of the experimental group of examples 1-6, DpnI product (10U/. mu.L) of the positive control group (PC group), and 50% (V/V) glycerol solution of the negative control group (NC group) at 37 ℃ for 1 hour was added to 1. mu.L of 6 XDNA loading buffer, and subjected to 1.5% agarose DNA gel electrophoresis at 150V for 15 minutes. The results are shown in FIG. 5. Wherein the column "M" is marker, the nucleic acid molecular weight marker. The result of the enzyme digestion effect is basically consistent with the result of the transformation.

Examples 10-14 fusion proteins of DpnI with other SeFP fluorescent protein solubilization tags, other SeP solubilization tags

Using the method of example 1(eGFP-2A-DpnI experimental group), an expression vector encoding eGFP-2A-DpnI was constructed again, and the gene sequence shown in SEQ ID No:2 was inserted into a plasmid expression vector designated as peGFP-2A-DpnI.

Expression vectors encoding mSacrlet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI and DsbA-2A-DpnI were also constructed by the method of example 1, and the following four genes were inserted into the plasmid expression vector by PCR amplification and homologous fragment recombination, respectively. The plasmid vectors are sequentially marked as pmScarlet-2A-DpnI, peYFP-2A-DpnI, pMBP-2A-DpnI and pDsbA-2A-DpnI.

(7) mScarlet-2A-DpnI (SEQ ID No:8), corresponding to the experimental group mScarlet-2A-DpnI.

(8) eYFP-2A-DpnI (SEQ ID No:9) corresponds to the eYFP-2A-DpnI experimental group.

(9) MBP-2A-DpnI (SEQ ID No:10) corresponds to the experimental group of MBP-2A-DpnI.

(10) DsbA-2A-DpnI (SEQ ID No:11), corresponding to the experimental group of DsbA-2A-DpnI.

Wherein mScarlet corresponds to a bright red fluorescent protein, eYFP corresponds to an enhanced yellow fluorescent protein, MBP corresponds to maltose binding protein, and DsbA corresponds to a disulfide bond forming protein; as in example 1, 2A corresponds to the linker sequence which undergoes self-cleavage at the mRNA level. eGFP, mScarlet and eYFP belong to the fluorescent protein solubilization tag (SeFP), MBP and DsbA to the non-fluorescent solubilization tag (SeP).

The accuracy of each plasmid was confirmed by gene sequencing.

Yeast cell extracts (Kluyveromyces lactis cell extracts, which were obtained in batches different from those of examples 1 to 6 and incorporated the gene encoding T7RNA polymerase into their genomes) were prepared by the method of example 1.

Constructing an in vitro cell-free protein synthesis system (except cell extracts and DNA templates, other reaction parameters are consistent with those of examples 1-6), and carrying out in vitro protein synthesis reaction experiments, wherein the total number of the five experimental groups is five; the reaction viability of the CFS system of this example was about 1/2 that was the same as the CFS system constructed from the batch of cell extract used in examples 1-6. The DNA templates used plasmid vectors pepGFP-2A-DpnI, pmScarlet-2A-DpnI, peYFP-2A-DpnI, pMBP-2A-DpnI and pDSbA-2A-DpnI respectively correspond to example 10, example 11, example 12, example 13 and example 14.

Negative control (negative control): in vitro protein synthesis reactions were performed without the addition of a DNA template encoding DpnI and its fusion protein, but with the addition of a DNA template encoding eGFP. The negative control group was identical to the negative control groups of examples 1-6.

After the reaction was completed, solid-liquid separation was performed by the method of example 1, and the supernatant was collected to obtain a DpnI solution containing DpnI fusion proteins eGFP-2A-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, and DsbA-2A-DpnI, respectively. And (3) measuring the enzyme activity of the DpnI solution. The DpnI solutions of examples 10 to 14 had the number of units of enzyme activity per unit volume of 20U/. mu.L, 16U/. mu.L, 18U/. mu.L, and 20U/. mu.L, respectively. One volume of glycerol is added to obtain glycerol solutions with the final concentration of 50% (v/v), and the enzyme activity units of the obtained DpnI stock solutions in unit volume are respectively 10U/muL, 8U/muL, 9U/muL and 10U/muL. The cellular extracts of this example constructed a CFS system with approximately one-half the reactivity of examples 1-6.

The digestion effects of the DpnI stock solutions obtained from the above five experimental groups, the DpnI stock solutions obtained from the negative control group, and the positive control group (all-type gold DMT enzyme, the number of enzyme activity units is 10U/. mu.L) were tested by the method of example 7. The pUC19 plasmid was treated with 0.3. mu.L. The test results are shown in fig. 6. The obtained DpnI stock solution has enzyme digestion effect basically consistent with that of a commercial product (positive control) by adopting eGFP as a fluorescent protein solubilization label, adopting Scarlet and eYFP as fluorescent protein solubilization labels and adopting MBP and DsbA as solubilization labels.

The results of the nucleic acid electrophoresis test using the procedure of example 9 on the five experimental groups of DpnI stock solutions, the negative control group of DpnI stock solutions, and the positive control group (all-gold DMT enzyme, 10U/. mu.L) with the pUC19 plasmid cleaved are shown in FIG. 7. The results show that even under the condition that the CFS system has relatively lower reactivity than that of the CFS system in examples 1-6, the DpnI stock solutions obtained by five experimental groups of eGFP-2A-DpnI, mSCarlet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI and DsbA-2A-DpnI can still effectively cut the pUC19 plasmid, and the effect is slightly different or even better compared with the commercial DpnI product. The difference in dispersion from the positive control band is presumably due to the influence of DNA migration by biological macromolecules derived from the IVTT reaction solution; the purification step of IVTT reaction liquid is omitted, and the exertion of the DpnI enzyme activity of the obtained restriction endonuclease DpnI preparation and the application thereof are not influenced.

Example 15 cleavage Effect test of DpnI solution

An expression vector encoding the tag-eGFP-TEV-DpnI target protein was constructed using the method of example 3. The target protein is also provided with a histidine tag (His-tag) at the N-terminal of the eGFP-TEV-DpnI, and tripeptide (GSG) is inserted between the histidine tag and the eGFP to serve as a connecting peptide; in the gene sequence of the target protein, the 5' end of the gene sequence shown in SEQ ID No. 4 is connected with the sequence shown in SEQ ID No. 12. Inserting the gene sequence of the coding target protein tag-eGFP-TEV-DpnI into a plasmid expression vector, and confirming the correctness of the plasmid through gene sequencing; preparing a yeast cell extract, constructing an in vitro cell-free protein synthesis system, and carrying out in vitro protein synthesis reaction (IVTT reaction); and finishing the reaction, wherein the obtained reaction solution after the IVTT reaction is finished contains the target protein of the fusion protein tag-eGFP-TEV-DpnI of DpnI.

The volume of the in vitro protein synthesis reaction system is 300 mu L, and the in vitro protein synthesis reaction system comprises the following components:

(1) 9.78mM final concentration of pH8.0 Tris-HCl, 80mM potassium acetate, 5mM magnesium acetate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate, each at 1.8mM concentration), 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 0.1mM concentration), 15mM glucose, 320mM maltodextrin, 24mM tripotassium phosphate, 2% (w/v) polyethylene glycol 8000, and finally 50% volume of the above Kluyveromyces lactis cell extract (polymerase also having T7RNA integrated in its genome) was added Encoding a gene).

(2) DNA template: 15 ng/. mu.L of plasmid template encoding tag-eGFP-TEV-DpnI.

The other reaction parameters of the IVTT reaction, including temperature, time, etc., in addition to the components present, are in accordance with example 3.

And (3) group L: after the IVTT reaction is finished, the supernatant obtained by solid-liquid separation is marked as the L group. In the absence of TEV enzyme, eGFP and DpnI exist as tag-eGFP-TEV-DpnI fusion protein, TEV is used as a connecting peptide, and the molar ratio of eGFP to DpnI is 1: 1. Drawing up a standard curve according to the purified eGFP, and calculating a conversion formula of the RFU value of the eGFP and the protein mass concentration as follows:

where X is the protein mass concentration (. mu.g/mL), Y is the RFU fluorescence reading (relative fluorescence units), A is the molecular weight of eGFP (26.7kDa), and B is the molecular weight of tag-eGFP-TEV-DpnI (60.6 kDa). Substituting the measured RFU fluorescence value to determine the mass concentration (mu g/mL) of the target protein tag-eGFP-TEV-DpnI. the mass concentration of tag-eGFP-TEV-DpnI fusion protein was 0.157mg/mL (157. mu.g/mL).

L-G group (50% (v/v) glycerol solution group): after the L group of DpnI solutions was obtained, 1/2 volumes of glycerol were added by one volume to obtain a glycerol solution with a final concentration of 50% (v/v), i.e., a DpnI stock solution, which was designated as L-G group. the mass concentration of tag-eGFP-TEV-DpnI fusion protein is 0.085 mg/mL.

Group P (purification group): the method comprises the following steps of carrying out nickel affinity purification on a supernatant (DpnI solution) obtained by carrying out solid-liquid separation after IVTT reaction by using nickel magnetic beads produced by the company, modifying a large number of nickel ions on the surfaces of the nickel magnetic beads, and realizing separation and purification by utilizing the affinity between the nickel ions and histidine tags carried by the DpnI. After 10mL of the supernatant after IVTT reaction was incubated at 4 ℃ for 1.5 hours with 50. mu.L of nickel magnetic beads by rotation, the nickel magnetic beads were washed with a washing solution (50mM Tris pH8.0, 500mM sodium chloride, 20mM imidazole) and then eluted with an eluent (50mM Tris pH8.0, 500mM sodium chloride, 250mM imidazole), and the obtained eluent contained the purified tag-eGFP-TEV-DpnI protein. Adding glycerol with one volume to prepare a 50% (v/v) glycerol solution to be tested for the enzyme digestion effect. Wherein, the mass concentration of tag-eGFP-TEV-DpnI fusion protein in 50% (v/v) glycerol diluent of the eluent to be used is 0.095 mg/mL.

The preparation method of the nickel magnetic beads adopts the method in the embodiment 1 of the invention application CN201910540132.6, and the method for purifying protein by using the nickel magnetic beads refers to the embodiments 2-3 of the invention application. The structure of nickel magnetic beads is described in detail in this application. In general terms, the outer surface of the magnetic microsphere is modified with a large amount of nickel ions; specifically, ferroferric oxide wrapped by silicon dioxide is used as a magnetic core material of a magnetic bead, a reaction residue (residual group after residual covalent reaction) of an amino silane coupling agent is used as a connecting group to covalently graft one end of a main chain of polyacrylic acid, and nickel ions are further chelated at a side group of the polyacrylic acid through a residue after the reaction of N, N-bis (carboxymethyl) -L-lysine and a side carboxyl group of the polyacrylic acid. The nickel magnetic bead greatly improves the loading capacity of nickel ions by utilizing a comb/brush structure formed by a linear main chain and a large number of side groups of a polymer.

Positive control group: the full-type gold DMT enzyme has the enzyme activity unit number of 10U/mu L.

And (5) carrying out enzyme digestion effect test. The digestion system is that 500ng of pUC19 plasmid and a metered volume of tag-eGFP-TEV-DpnI solution are added into 14 mu L of digestion system, digestion is carried out for 1 hour at 37 ℃, and 3.5 mu L of transformed Tans5 alpha competent cells are taken. Three different final concentrations of 0.285. mu.g (designated as L1, L1-G, P1, using volumes of 1.815. mu.L, 3.353. mu.L, and 3. mu.L), 0.095. mu.g (designated as L2, L2-G, P2, using volumes of 0.605. mu.L, 1.118. mu.L, and 1. mu.L), and 0.029. mu.g (designated as L3, L3-G, P3, using volumes of 0.185. mu.L, 0.341. mu.L, and 0.305. mu.L) were added to each of the above L group, L-G group, and P group. The amount of positive control added was 0.3. mu.L.

The negative control group was not subjected to DpnI treatment. To a volume of 14. mu.L, 500ng of pUC19 plasmid was added, digested at 37 ℃ for 1 hour, and 3.5. mu.L of transformed Tans 5. alpha. competent cells were taken.

The results of the enzyme digestion test are shown in FIG. 8. In a low concentration system with about 0.03 mu G of enzyme added into a 14 mu L system, the enzyme digestion effect of unpurified L3 and L3-G is better than that of the purified P3 group, which indicates that the purification process causes the activity loss of tag-eGFP-TEV-DpnI. The glycerol dilution of the L-G group (L1-G, L2-G, L3-G) diluted with 50% (v/v) glycerol resulted in no loss of enzyme activity compared to the L group (L1, L2, L3) at the same final concentration of tag-eGFP-TEV-DpnI protease. Under the condition that the volume of the enzyme preparation used is comparable to that of a positive control DMT enzyme product, the adding volumes of an L3 group, an L3-G group and a positive control group are respectively 0.18 mu L, 0.34 mu L and 0.3 mu L, an enzyme digestion test system is 10 mu L, the enzyme digestion effects are compared, and a DpnI solution group (supernate, L group) and a DpnI storage solution group (L-G group) have better enzyme digestion activity. Moreover, a cleavage effect substantially equivalent to that of the 0.3. mu.L DMT product was achieved using 0.6. mu. L L-group solution.

Examples 16-23 in addition to DpnI, SeP and the linker peptide X between them, the protein of interest contains other peptide fragments

A28 aa long polypeptide chain is added at the N-terminal of the protein coded by SEQ ID No. 1-4 and SEQ ID No. 8-11 respectively, and comprises a leading peptide-His-GSG. Wherein, the length of the leader peptide is 17aa (aa represents 1 amino acid), which can promote the reaction activity of the in vitro protein synthesis system of the used yeast cell extract and improve the protein expression efficiency and the expression quantity; his is a histidine tag, 8aa in length, and is a purification tag; GSG is a tripeptide as a linker peptide; "-" indicates a peptide bond.

The target protein: a28 aa long leader peptide, His-GSG, was attached to the N-terminus of eGFP-2A-DpnI (SEQ ID No:2), mScalet-2A-DpnI (SEQ ID No:8), eYFP-2A-DpnI (SEQ ID No:9), MBP-2A-DpnI (SEQ ID No:10), DsbA-2A-DpnI (SEQ ID No:11), eGFP-DpnI (SEQ ID No:3), eGFP-TEV-DpnI (SEQ ID No:4), and DpnI (SEQ ID No:1), respectively. Corresponding in turn to examples 16 to 23. "-" denotes a peptide bond or a linker peptide.

A gene encoding a protein of interest: the 3 'end of the gene sequence of the leader peptide-His-GSG is connected with the 5' end of the proteins coded by SEQ ID No. 1-4 and SEQ ID No. 8-11.

Plasmid expression vectors encoding the proteins of interest were constructed using the method of example 15 and the plasmids were confirmed to be correct by gene sequencing.

Preparing a yeast cell extract (kluyveromyces lactis cell extract with T7RNA polymerase integrated into its genome) by the method of example 1; constructing an in vitro cell-free protein synthesis system, and carrying out in vitro protein synthesis reaction (IVTT reaction); and (4) finishing the reaction, carrying out solid-liquid separation, and collecting supernatant to obtain the DpnI solution containing the target protein. One half volume of the solution was taken to prepare a 50% (v/v) glycerol dilution to obtain a DpnI stock solution. In examples 16-22, the "leader peptide-His-GSG" is linked to the N-terminus of the solubilization tag or/and the fluorescent tag, and still performs the solubilization function, and all of them still belong to the solubilization tag-labeled DpnI fusion protein.

Positive control group: the product DpnI of Beijing holotype gold biotechnology limited, DMT enzyme, has an enzyme activity unit number of 10U/muL.

Through tests, the enzyme activity unit number of the DpnI solution of all groups is more than 16U/mu L, and the DpnI stock solution of all groups is more than 8U/mu L. The enzyme activity unit number of the DpnI solution of the experimental group containing the eGFP-2A-DpnI structure is more than 30U/muL, and the enzyme activity unit number of the DpnI stock solution is more than 15U/muL.

Taking DpnI stock solutions of each group respectively, and performing enzyme digestion effect test by adopting the method of the embodiment 7, wherein the DpnI stock solutions have better enzyme digestion activity; the digestion efficiency of the experimental group with 2A as the linker peptide X (examples 16-20) was better than that of the experimental group with TEV linker peptide (examples 21-22) and no linker peptide (example 23). Particularly, the experimental group containing the eGFP-2A-DpnI structure has better experimental effect than the positive control. With the exception of example 23 (labeled DpnI), all groups used 2 times the volume of the positive control (0.6. mu.L: 0.3. mu.L) achieved or exceeded the cleavage efficiency of the positive control (whole-form gold DMT enzyme).

The above is only a part of the preferred 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> a preparation of restriction endonuclease DpnI and its preparation method

<130> 2019

<141> 2019-11-29

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 762

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

atggaattgc atttcaactt ggaattggtt gaaacttata agtcaaactc acaaaaggct 60

agaattttga ctgaagattg ggtttacaga caatcttatt gtccaaattg tggtaataac 120

ccattgaatc atttcgaaaa taacagacca gttgctgatt tctattgtaa tcattgttca 180

gaagagttcg aattgaagtc taaaaagggt aatttctctt caactatcaa cgatggtgct 240

tatgctacta tgatgaagag agttcaagct gataataatc caaatttctt cttcttgacc 300

tacactaaga atttcgaagt taataacttc ttggtgttgc caaagcaatt cgttactcca 360

aaatcaatta tccaaagaaa gccattggct ccaactgcta gaagagctgg ttggattggt 420

tgtaatattg atttgtctca agtgccatca aagggtagaa tttttcttgt tcaagatggt 480

caagttagag atccagaaaa agttactaaa gagtttaaac agggtttgtt tttgagaaaa 540

tcctcattgt catcaagagg ttggactatt gaaattttga attgtatcga caagatcgaa 600

ggttctgagt ttactttgga agatatgtat agattcgagt cagatttgaa aaacatcttt 660

gttaagaaca accacatcaa agaaaagatt agacaacaat tgcagatctt gagagataag 720

gaaattattg agtttaaagg taggggtaaa tacagaaaat tg 762

<210> 2

<211> 1536

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctccttc 300

aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc 480

atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtgca 720

acgaattttt cactattgaa attagccgga gatgtcgaac ttaacccagg tcccatggaa 780

ttgcatttca acttggaatt ggttgaaact tataagtcaa actcacaaaa ggctagaatt 840

ttgactgaag attgggttta cagacaatct tattgtccaa attgtggtaa taacccattg 900

aatcatttcg aaaataacag accagttgct gatttctatt gtaatcattg ttcagaagag 960

ttcgaattga agtctaaaaa gggtaatttc tcttcaacta tcaacgatgg tgcttatgct 1020

actatgatga agagagttca agctgataat aatccaaatt tcttcttctt gacctacact 1080

aagaatttcg aagttaataa cttcttggtg ttgccaaagc aattcgttac tccaaaatca 1140

attatccaaa gaaagccatt ggctccaact gctagaagag ctggttggat tggttgtaat 1200

attgatttgt ctcaagtgcc atcaaagggt agaatttttc ttgttcaaga tggtcaagtt 1260

agagatccag aaaaagttac taaagagttt aaacagggtt tgtttttgag aaaatcctca 1320

ttgtcatcaa gaggttggac tattgaaatt ttgaattgta tcgacaagat cgaaggttct 1380

gagtttactt tggaagatat gtatagattc gagtcagatt tgaaaaacat ctttgttaag 1440

aacaaccaca tcaaagaaaa gattagacaa caattgcaga tcttgagaga taaggaaatt 1500

attgagttta aaggtagggg taaatacaga aaattg 1536

<210> 3

<211> 1476

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctccttc 300

aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc 480

atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagatggaa 720

ttgcatttca acttggaatt ggttgaaact tataagtcaa actcacaaaa ggctagaatt 780

ttgactgaag attgggttta cagacaatct tattgtccaa attgtggtaa taacccattg 840

aatcatttcg aaaataacag accagttgct gatttctatt gtaatcattg ttcagaagag 900

ttcgaattga agtctaaaaa gggtaatttc tcttcaacta tcaacgatgg tgcttatgct 960

actatgatga agagagttca agctgataat aatccaaatt tcttcttctt gacctacact 1020

aagaatttcg aagttaataa cttcttggtg ttgccaaagc aattcgttac tccaaaatca 1080

attatccaaa gaaagccatt ggctccaact gctagaagag ctggttggat tggttgtaat 1140

attgatttgt ctcaagtgcc atcaaagggt agaatttttc ttgttcaaga tggtcaagtt 1200

agagatccag aaaaagttac taaagagttt aaacagggtt tgtttttgag aaaatcctca 1260

ttgtcatcaa gaggttggac tattgaaatt ttgaattgta tcgacaagat cgaaggttct 1320

gagtttactt tggaagatat gtatagattc gagtcagatt tgaaaaacat ctttgttaag 1380

aacaaccaca tcaaagaaaa gattagacaa caattgcaga tcttgagaga taaggaaatt 1440

attgagttta aaggtagggg taaatacaga aaattg 1476

<210> 4

<211> 1497

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctccttc 300

aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc 480

atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggagaac 720

ttatatttcc agggaatgga attgcatttc aacttggaat tggttgaaac ttataagtca 780

aactcacaaa aggctagaat tttgactgaa gattgggttt acagacaatc ttattgtcca 840

aattgtggta ataacccatt gaatcatttc gaaaataaca gaccagttgc tgatttctat 900

tgtaatcatt gttcagaaga gttcgaattg aagtctaaaa agggtaattt ctcttcaact 960

atcaacgatg gtgcttatgc tactatgatg aagagagttc aagctgataa taatccaaat 1020

ttcttcttct tgacctacac taagaatttc gaagttaata acttcttggt gttgccaaag 1080

caattcgtta ctccaaaatc aattatccaa agaaagccat tggctccaac tgctagaaga 1140

gctggttgga ttggttgtaa tattgatttg tctcaagtgc catcaaaggg tagaattttt 1200

cttgttcaag atggtcaagt tagagatcca gaaaaagtta ctaaagagtt taaacagggt 1260

ttgtttttga gaaaatcctc attgtcatca agaggttgga ctattgaaat tttgaattgt 1320

atcgacaaga tcgaaggttc tgagtttact ttggaagata tgtatagatt cgagtcagat 1380

ttgaaaaaca tctttgttaa gaacaaccac atcaaagaaa agattagaca acaattgcag 1440

atcttgagag ataaggaaat tattgagttt aaaggtaggg gtaaatacag aaaattg 1497

<210> 5

<211> 1476

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

atggaattgc atttcaactt ggaattggtt gaaacttata agtcaaactc acaaaaggct 60

agaattttga ctgaagattg ggtttacaga caatcttatt gtccaaattg tggtaataac 120

ccattgaatc atttcgaaaa taacagacca gttgctgatt tctattgtaa tcattgttca 180

gaagagttcg aattgaagtc taaaaagggt aatttctctt caactatcaa cgatggtgct 240

tatgctacta tgatgaagag agttcaagct gataataatc caaatttctt cttcttgacc 300

tacactaaga atttcgaagt taataacttc ttggtgttgc caaagcaatt cgttactcca 360

aaatcaatta tccaaagaaa gccattggct ccaactgcta gaagagctgg ttggattggt 420

tgtaatattg atttgtctca agtgccatca aagggtagaa tttttcttgt tcaagatggt 480

caagttagag atccagaaaa agttactaaa gagtttaaac agggtttgtt tttgagaaaa 540

tcctcattgt catcaagagg ttggactatt gaaattttga attgtatcga caagatcgaa 600

ggttctgagt ttactttgga agatatgtat agattcgagt cagatttgaa aaacatcttt 660

gttaagaaca accacatcaa agaaaagatt agacaacaat tgcagatctt gagagataag 720

gaaattattg agtttaaagg taggggtaaa tacagaaaat tggtgagcaa gggcgaggag 780

ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 840

ttcagcgtgc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc 900

atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 960

ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 1020

gccatgcccg aaggctacgt ccaggagcgc accatctcct tcaaggacga cggcacctac 1080

aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 1140

ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac 1200

agccacaacg tctatatcac ggccgacaag cagaagaacg gcatcaaggc gaacttcaag 1260

atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 1320

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag 1380

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 1440

gccgggatca ctctcggcat ggacgagctg tacaag 1476

<210> 6

<211> 60

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

ggtgcaacga atttttcact attgaaatta gccggagatg tcgaacttaa cccaggtccc 60

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

gagaacttat atttccaggg a 21

<210> 8

<211> 1515

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

gtttcaaagg gtgaagctgt tattaaggag tttatgagat tcaaagtgca tatggaaggt 60

tctatgaatg gtcatgaatt tgaaattgag ggtgaaggtg aaggtagacc atatgaaggt 120

actcaaactg ctaaattgaa ggttactaaa ggtggtccat tgccattctc atgggatatt 180

ttgtcaccac aattcatgta tggttctaga gctttcatta agcatccagc tgatattcca 240

gattactata agcaatcatt cccagaaggt ttcaagtggg aaagagttat gaattttgaa 300

gatggtggtg ctgttactgt tactcaagat acttcattgg aagatggtac tttgatctat 360

aaggttaagt tgagaggtac taatttccca ccagatggtc cagttatgca aaagaaaact 420

atgggttggg aagctagtac tgaaagattg tatccagaag atggtgtttt gaagggtgac 480

attaagatgg ctttgagatt gaaagatggt ggtagatatt tggctgattt caagactact 540

tataaggcta agaagccagt tcaaatgcca ggtgcttaca atgttgatag aaaattggat 600

atcacctctc ataatgaaga ttatactgtt gttgagcaat acgaaagatc tgaaggtaga 660

cattctactg gtggtatgga tgaattgtat aagggtgcaa cgaatttttc actattgaaa 720

ttagccggag atgtcgaact taacccaggt cccatggaat tgcatttcaa cttggaattg 780

gttgaaactt ataagtcaaa ctcacaaaag gctagaattt tgactgaaga ttgggtttac 840

agacaatctt attgtccaaa ttgtggtaat aacccattga atcatttcga aaataacaga 900

ccagttgctg atttctattg taatcattgt tcagaagagt tcgaattgaa gtctaaaaag 960

ggtaatttct cttcaactat caacgatggt gcttatgcta ctatgatgaa gagagttcaa 1020

gctgataata atccaaattt cttcttcttg acctacacta agaatttcga agttaataac 1080

ttcttggtgt tgccaaagca attcgttact ccaaaatcaa ttatccaaag aaagccattg 1140

gctccaactg ctagaagagc tggttggatt ggttgtaata ttgatttgtc tcaagtgcca 1200

tcaaagggta gaatttttct tgttcaagat ggtcaagtta gagatccaga aaaagttact 1260

aaagagttta aacagggttt gtttttgaga aaatcctcat tgtcatcaag aggttggact 1320

attgaaattt tgaattgtat cgacaagatc gaaggttctg agtttacttt ggaagatatg 1380

tatagattcg agtcagattt gaaaaacatc tttgttaaga acaaccacat caaagaaaag 1440

attagacaac aattgcagat cttgagagat aaggaaatta ttgagtttaa aggtaggggt 1500

aaatacagaa aattg 1515

<210> 9

<211> 1536

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtgca 720

acgaattttt cactattgaa attagccgga gatgtcgaac ttaacccagg tcccatggaa 780

ttgcatttca acttggaatt ggttgaaact tataagtcaa actcacaaaa ggctagaatt 840

ttgactgaag attgggttta cagacaatct tattgtccaa attgtggtaa taacccattg 900

aatcatttcg aaaataacag accagttgct gatttctatt gtaatcattg ttcagaagag 960

ttcgaattga agtctaaaaa gggtaatttc tcttcaacta tcaacgatgg tgcttatgct 1020

actatgatga agagagttca agctgataat aatccaaatt tcttcttctt gacctacact 1080

aagaatttcg aagttaataa cttcttggtg ttgccaaagc aattcgttac tccaaaatca 1140

attatccaaa gaaagccatt ggctccaact gctagaagag ctggttggat tggttgtaat 1200

attgatttgt ctcaagtgcc atcaaagggt agaatttttc ttgttcaaga tggtcaagtt 1260

agagatccag aaaaagttac taaagagttt aaacagggtt tgtttttgag aaaatcctca 1320

ttgtcatcaa gaggttggac tattgaaatt ttgaattgta tcgacaagat cgaaggttct 1380

gagtttactt tggaagatat gtatagattc gagtcagatt tgaaaaacat ctttgttaag 1440

aacaaccaca tcaaagaaaa gattagacaa caattgcaga tcttgagaga taaggaaatt 1500

attgagttta aaggtagggg taaatacaga aaattg 1536

<210> 10

<211> 1920

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc 60

gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg 120

gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc 180

ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc 240

ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac 300

ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat 360

ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa 420

gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg 480

attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac 540

gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa 600

aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc 660

gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg 720

aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc 780

gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcgaa agagttcctc 840

gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt 900

gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc 960

atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg 1020

tatgtcgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc 1080

ctgaaagacg cgcagactgg tgcaacgaat ttttcactat tgaaattagc cggagatgtc 1140

gaacttaacc caggtcccat ggaattgcat ttcaacttgg aattggttga aacttataag 1200

tcaaactcac aaaaggctag aattttgact gaagattggg tttacagaca atcttattgt 1260

ccaaattgtg gtaataaccc attgaatcat ttcgaaaata acagaccagt tgctgatttc 1320

tattgtaatc attgttcaga agagttcgaa ttgaagtcta aaaagggtaa tttctcttca 1380

actatcaacg atggtgctta tgctactatg atgaagagag ttcaagctga taataatcca 1440

aatttcttct tcttgaccta cactaagaat ttcgaagtta ataacttctt ggtgttgcca 1500

aagcaattcg ttactccaaa atcaattatc caaagaaagc cattggctcc aactgctaga 1560

agagctggtt ggattggttg taatattgat ttgtctcaag tgccatcaaa gggtagaatt 1620

tttcttgttc aagatggtca agttagagat ccagaaaaag ttactaaaga gtttaaacag 1680

ggtttgtttt tgagaaaatc ctcattgtca tcaagaggtt ggactattga aattttgaat 1740

tgtatcgaca agatcgaagg ttctgagttt actttggaag atatgtatag attcgagtca 1800

gatttgaaaa acatctttgt taagaacaac cacatcaaag aaaagattag acaacaattg 1860

cagatcttga gagataagga aattattgag tttaaaggta ggggtaaata cagaaaattg 1920

<210> 11

<211> 1389

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

gcgcagtatg aagatggtaa acagtacact accctggaaa aaccggtagc tggcgcgccg 60

caagtgctgg agtttttctc tttcttctgt ccgcactgct atcagtttga agaagttctg 120

catatttctg ataatgtgaa gaaaaaactg ccggaaggcg tgaagatgac taaataccac 180

gtcaacttca tgggtggtga cctgggcaaa gatctgactc aggcatgggc tgtggcgatg 240

gcgctgggcg tggaagacaa agtcactgtt ccgctgtttg aaggcgtaca gaaaacccag 300

accattcgtt cagcatctga tatccgcgat gtatttatca acgcaggtat taaaggtgaa 360

gagtacgacg cggcgtggaa cagcttcgtg gtcaaatctc tggtcgctca gcaggaaaaa 420

gctgcagctg acgtgcaatt gcgtggcgtt ccggcgatgt ttgttaacgg taaatatcag 480

ctgaatccgc agggtatgga taccagcaat atggatgttt ttgttcagca gtatgctgat 540

acagtgaaat atctgtccga gaaaaaaggt gcaacgaatt tttcactatt gaaattagcc 600

ggagatgtcg aacttaaccc aggtcccatg gaattgcatt tcaacttgga attggttgaa 660

acttataagt caaactcaca aaaggctaga attttgactg aagattgggt ttacagacaa 720

tcttattgtc caaattgtgg taataaccca ttgaatcatt tcgaaaataa cagaccagtt 780

gctgatttct attgtaatca ttgttcagaa gagttcgaat tgaagtctaa aaagggtaat 840

ttctcttcaa ctatcaacga tggtgcttat gctactatga tgaagagagt tcaagctgat 900

aataatccaa atttcttctt cttgacctac actaagaatt tcgaagttaa taacttcttg 960

gtgttgccaa agcaattcgt tactccaaaa tcaattatcc aaagaaagcc attggctcca 1020

actgctagaa gagctggttg gattggttgt aatattgatt tgtctcaagt gccatcaaag 1080

ggtagaattt ttcttgttca agatggtcaa gttagagatc cagaaaaagt tactaaagag 1140

tttaaacagg gtttgttttt gagaaaatcc tcattgtcat caagaggttg gactattgaa 1200

attttgaatt gtatcgacaa gatcgaaggt tctgagttta ctttggaaga tatgtataga 1260

ttcgagtcag atttgaaaaa catctttgtt aagaacaacc acatcaaaga aaagattaga 1320

caacaattgc agatcttgag agataaggaa attattgagt ttaaaggtag gggtaaatac 1380

agaaaattg 1389

<210> 12

<211> 33

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

caccaccatc accaccacca tcacgggagc ggc 33

Claims

1. A preparation of restriction enzyme DpnI, which is obtained by a preparation method comprising the steps of:

firstly, constructing an in vitro cell-free protein synthesis system, wherein the system at least comprises the following components: a gene template; the gene template contains a gene sequence for encoding DpnI; the in vitro cell-free protein synthesis system can express the gene template into a target protein containing a DpnI structure; the gene template is a DNA template;

secondly, carrying out in-vitro protein synthesis reaction to synthesize target protein;

step three, finishing the in vitro protein synthesis reaction, carrying out solid-liquid separation, and collecting supernatant to obtain a solution containing the target protein; wherein the solution with the restriction enzyme activity is the preparation of the restriction enzyme DpnI;

optionally, a packaging step is included.

2. The preparation of restriction enzyme DpnI according to claim 1, wherein the target protein encoded by the gene template is DpnI protein, DpnI fusion protein, or a combination thereof;

the DpnI fusion protein is preferably a fusion protein of DpnI and 2-50 peptides and a solubilization label-labeled DpnI fusion protein.

3. The preparation of restriction enzyme DpnI according to claim 2, wherein the solubilization tag-labeled DpnI fusion protein is SeP-X-DpnI fusion protein; wherein SeP is a solubilization tag; x is absent or is a linker peptide; "-" is a peptide bond or a linker peptide;

the solubilization label marked DpnI fusion protein is preferably SeFP-X-DpnI fusion protein; wherein SeFP is a fluorescent protein solubilization label;

the solubilizing label SeP is preferably: thioredoxin, thioredoxin reductase, maltose binding protein, glutathione-S-transferase, glutathione reductase, streptococcal G protein B1 domain, small ubiquitin-related modifying protein, HaloTag protein, disulfide bond forming protein, bacterial translation initiation factor, transcription anti-termination factor, ribosomal protein L23, eGFP, GFP, sfGFP, CFP, YFP, eYFP, RFP, BFP, mScarlet, or combinations thereof;

the solubilization label marked DpnI fusion protein is also preferably eGFP-X-DpnI fusion protein; wherein, the eGFP is enhanced green fluorescent protein;

the fluorescent protein solubilization tag SeFP is preferably: eGFP, GFP, sfGFP, CFP, YFP, eYFP, RFP, BFP, mCardet, or combinations thereof.

4. The preparation of restriction enzyme DpnI according to claim 3, wherein X is a linker peptide; x is selected from a cleavable linker peptide or a stable linker peptide; the cleavable linker peptide is preferably selected from the group consisting of: a connecting peptide with self-cutting property and a connecting peptide which is cut under the action of exogenous enzyme.

5. The preparation of restriction enzyme DpnI according to any one of claim 3, wherein X is a linker peptide; the X is preferably 2-50 peptides, and more preferably 5-25 peptides.

6. The preparation of restriction enzyme DpnI according to claim 1, wherein the gene template encodes a protein of interest selected from the group consisting of: DpnI, eGFP-2A-DpnI, eGFP-TEV-DpnI, mScalet-2A-DpnI, eYFP-2A-DpnI, MBP-2A-DpnI, DsbA-2A-DpnI, or combinations thereof.

7. The preparation of restriction enzyme DpnI according to any one of claims 1 to 6, wherein the resultant preparation of restriction enzyme DpnI has a DpnI enzyme content of at least 4U/. mu.L.

8. The preparation of the restriction enzyme DpnI according to any one of claims 1 to 6, wherein the preparation method of the preparation of the restriction enzyme DpnI further comprises the steps of:

packaging the collected supernatant with the activity of the restriction endonuclease, or packaging after preparing DpnI storage liquid to obtain a preparation of the restriction endonuclease DpnI;

the packaging mode comprises packaging or subpackaging;

the DpnI storage liquid is preferably a DpnI storage liquid which can be stored at low temperature; the low temperature is lower than-18 ℃;

when the packaging is carried out in a split charging mode, the content of the DpnI enzyme in a single container after split charging is preferably at least 4U/mu L; more preferably at least 5U/. mu.L, more preferably at least 8U/. mu.L, more preferably 10 to 20U/. mu.L, more preferably 10U/. mu.L, 15U/. mu.L or 20U/. mu.L.

9. The preparation of restriction enzyme DpnI according to any one of claims 1 to 6 and 7 to 8, wherein the components of the in vitro cell-free protein synthesis system further comprise cell extracts; the cell extract is capable of expressing the gene template;

the cell extract is preferably selected from any one of the following sources: escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof; the yeast cell is preferably selected from Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof;

the Kluyveromyces is further preferably Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybuhitensis, Kluyveromyces hainanensis, Kluyveromyces williamsii, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensis, Kluyveromyces lactis, or a combination thereof;

the cell extract is more preferably selected from any one of the following sources: coli, kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda insect cells, rabbit reticulocytes, CHO cells, COS cells, VERO cells, BHK cells, human fibrosarcoma HT1080 cells, or a combination thereof;

preferably, the kluyveromyces lactis has a gene encoding RNA polymerase, a gene encoding DNA polymerase, or a combination thereof integrated into its genome.

10. The preparation of restriction endonuclease DpnI of claim 9, wherein said cell extract contains an endogenously expressed RNA polymerase, or an exogenous RNA polymerase, a gene template containing a gene encoding the RNA polymerase, or a combination thereof is further added to said in vitro cell-free protein synthesis system.

11. The preparation of restriction endonuclease DpnI of claim 9 or 10, wherein the components of said in vitro cell-free protein synthesis system further comprise exogenous DNA polymerase, a gene template containing a gene encoding the DNA polymerase, or a combination thereof.

12. The preparation of restriction enzyme DpnI according to any one of claims 1 to 6, 7 to 11, wherein the components of the in vitro cell-free protein synthesis system further comprise an energy system;

the energy system, preferably selected from the group consisting of polysaccharide and phosphate energy systems, phosphocreatine and phosphocreatine enzyme systems, glycolysis pathway and its intermediate energy systems, or combinations thereof.

13. The preparation of restriction enzyme DpnI according to any one of claims 1 to 6, 7 to 12, wherein the components of the in vitro cell-free protein synthesis system further comprise a substrate for the synthetic protein; the substrate of the synthetic protein is preferably an amino acid mixture comprising at least the amino acids used for the synthesis of the DpnI protein and/or SeP-X-DpnI fusion protein;

preferably, the gene template is a DNA template; the in vitro cell-free protein synthesis system also comprises a substrate for synthesizing RNA; the substrate of the synthetic RNA is preferably a mixture of nucleotides selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof.

14. The preparation of restriction enzyme DpnI according to any one of claims 1 to 6 and 7 to 13, wherein said in vitro cell-free protein synthesis system further comprises at least one of the following components: crowding agents, magnesium ions, potassium ions, antioxidants, buffers, aqueous solvents;

the crowding agent is preferably polyethylene glycol, dextran, Ficoll or a combination thereof;

the magnesium ion source is preferably magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or a combination thereof;

the potassium ion source is preferably potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or a combination thereof;

the antioxidant is preferably dithiothreitol;

the buffer is preferably selected from any one of: Tris-HCl, Tris base, HEPES, or a combination thereof;

the aqueous solvent is preferably a buffer.

15. The formulation of restriction enzyme DpnI according to claim 8, wherein the DpnI stock solution further comprises at least one of the following components: Tri-HCl, Tris-acetate, NaHepes, NaCl, CaCl₂Dithiothreitol, EDTA, BSA, imidazole, glycerol, magnesium acetate, sodium acetate; preferably, the DpnI stock solution further comprises at least glycerol.

16. A preparation method of a preparation of restriction endonuclease DpnI is characterized by comprising the following steps:

firstly, constructing an in vitro cell-free protein synthesis system, wherein the system at least comprises the following components: a gene template; the gene template contains a gene sequence for encoding DpnI; the in vitro cell-free protein synthesis system can express the gene template into a target protein containing a DpnI structure;

the gene template is preferably a DNA template;

step three, finishing the in vitro protein synthesis reaction, carrying out solid-liquid separation, and collecting supernatant to obtain a solution containing the target protein; screening to obtain a restriction enzyme DpnI preparation with restriction enzyme activity;

optionally, a packaging step is included.

17. The method for producing a preparation of restriction enzyme DpnI according to claim 16, wherein the enzyme activity value of the obtained preparation of restriction enzyme DpnI is measured, and the preparation of restriction enzyme DpnI having a DpnI enzyme content of at least 4U/. mu.L is screened.

18. The method for preparing a preparation of restriction enzyme DpnI according to claim 16, wherein the preparation of restriction enzyme DpnI further comprises the steps of:

measuring the enzyme activity value of the supernatant obtained in the third step, packaging the collected supernatant with the activity of the restriction enzyme, or preparing the supernatant into DpnI storage solution and then packaging, and screening to obtain a preparation of the restriction enzyme DpnI;

the packaging mode comprises packaging or subpackaging;

19. The method for preparing a preparation of restriction enzyme DpnI according to any one of claims 16 to 18,

the in vitro cell-free protein synthesis system is selected from the group consisting of the in vitro cell-free protein synthesis systems of any one of claims 2-6 and 9-15.