CN113801877A

CN113801877A - Protein expression method

Info

Publication number: CN113801877A
Application number: CN202011518448.4A
Authority: CN
Inventors: 杨修竹; 庄淼; 杜静; 张忆恒; 贺亮; 其他发明人请求不公开姓名
Original assignee: Suzhou Perotin Biotechnology Co ltd
Current assignee: Suzhou Perotin Biotechnology Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-12-17

Abstract

The application relates to the field of molecular biology, in particular to a protein expression method. The DNA template comprises a reading frame of a target protein, an upstream 5 'untranslated region, a upstream 3' untranslated region and two side linkers, wherein the linkers are hairpin structures formed by annealing of complementary single-stranded DNA in a chain. The DNA template does not have open 3 'and 5' ends and is resistant to exonuclease degradation. The joint can contain chemically modified basic groups, so that the possibility of using the template is increased, the joint can be recycled, the reaction cost of CFPS is reduced, the introduced chemical modification is convenient for enriching the template, the local concentration of the template is improved, the expression quantity of protein is further improved, the operation is simpler, and the cost is reduced.

Description

Protein expression method

Technical Field

The application relates to the field of molecular biology, in particular to a protein expression method.

Background

With the development of scientific technology, the cell-free protein expression technology has the advantages of allowing control of reaction conditions, components of reactants, direct monitoring of reaction expression and the like due to the unique characteristics of an open system, and becomes a powerful protein expression method. The Cell-free Protein Synthesis (CFPS) system is an in vitro gene expression system which takes exogenous DNA or mRNA as a template, manually adds required raw materials and energy substances, and synthesizes Protein by taking Cell extracts as conditions, can break through Cell restriction and conveniently and quickly express various proteins. In detail, CFPS uses DNA as a template, and transcribes corresponding mRNA under the action of RNA polymerase, transcription factor and other components; using mRNA as a template, proteins are synthesized by translation using ribosomes, amino acid substrates, tRNA, energy substances, and the like in the system.

The CFPS has an advantage in that it has the ability to directly use linear DNA templates (LETs), and it is not necessary to construct a target gene on an expression vector, which significantly simplifies the workflow of CFPS and shortens the protein synthesis time. CFPS based on LETs is an effective tool for high-throughput production and protein screening, but due to the increased susceptibility of LETs to exonucleases in a cell-free reaction environment compared with plasmids, LETs are degraded by nucleases in a reaction system, and the protein yield is reduced.

To increase the lifetime of LETs in CFPS and the reaction yield of proteins, the prior art has adopted methods such as: 1) modifying strains, and mutating or knocking out genes such as exonuclease RecBCD, ribonuclease RNase, polynucleotide phosphorylase polynucleotide-phosphoriylase and the like; 2) adding Gams protein capable of inhibiting RecBCD activity; 3) double-stranded DNA binding protein, etc.

However, the above methods all have drawbacks, for example, the growth rate of the modified strains becomes slow, and the protein synthesis ability of lysates obtained from these strains is reduced, which makes it difficult to apply them widely; the method for adding the nuclease inhibitor obtains better effect, the degraded linear template in unit time is reduced, the protein yield is increased, but the added inhibitor only aims at one exonuclease, and other uninhibited exonucleases can still play a role; in addition, protein needs to be added additionally in the system, so that the reaction cost and the operation complexity are increased; recently, a report protein scCro can be combined at two ends of a linear template, so that the template is prevented from being degraded by exonuclease, the protein expression amount is obviously higher than that of the linear template, but is still far lower than that of a plasmid template, and the method not only needs to additionally add the scCro protein, but also needs to add an ORC sequence at two ends of the linear template; in addition, in practical application, the cost of the CFPS template is also an important factor to be considered, and if the template can be recycled, the cost of the total reaction system will also be reduced, and the problem of the template cost is not solved in the existing solutions.

Disclosure of Invention

The invention aims to provide a DNA template for cell-free protein expression.

Another object of the present invention is to provide a method for cell-free protein expression.

The DNA template for cell-free protein expression according to the invention comprises a reading frame of a target protein and required 5 'and 3' untranslated regions, and two sides of a linker, wherein the linker is a hairpin structure formed by annealing of complementary single-stranded DNA in a chain.

The DNA template for cell-free protein expression according to the present invention, wherein the single strand forming the linker contains at least one base complementary pairing region therein.

The DNA template for cell-free protein expression according to the present invention, wherein when the number of total bases of a single strand forming the linker is an odd number, the remaining bases are paired two by two except for the middle base of the sequence; when the total number of bases is an even number, all bases pair two by two.

The DNA template for cell-free protein expression according to the present invention, wherein the number of bases in the base complementary pairing region is at least 12 bp.

When the total base number of the joint is odd number, the other bases except the middle base of the sequence are pairwise paired, and when the total base number is even number, all the bases are pairwise paired.

The DNA template for cell-free protein expression according to the present invention, wherein one or more bases in the linker are linked to a chemical modification group.

The DNA template for cell-free protein expression is characterized in that the chemical modification group is biotin, amino, acrylamide, azide or sulfhydryl.

The DNA template for cell-free protein expression according to the present invention, wherein a spacer base sequence of an appropriate length is included upstream and downstream of the 5 'and/or 3' untranslated region to provide proper steric hindrance.

A method of constructing a DNA template for cell-free protein expression, the method comprising the steps of:

synthesizing the reading frame of the target protein and the required 5 'and 3' untranslated regions;

designing and synthesizing complementary single-stranded DNA in a strand, annealing to form a hairpin structure, obtaining a linker, pairing all bases except the middle base of the sequence when the total base number of the linker is odd, pairing all bases when the total base number of the linker is even,

the above-described synthesized linkers were ligated to the 5 'and 3' untranslated regions upstream and downstream of the reading frame of the protein, respectively, to obtain a DNA template for cell-free protein expression.

According to the method for constructing a DNA template for cell-free protein expression of the present invention, endonuclease sites of the same species as the linear template are introduced into the single-stranded DNA at the 5 'and 3' ends at positions corresponding to the positions of the linear template, so that the linear template can be cleaved by the endonuclease to form sticky ends, which are ligated to the 5 'and 3' ends of the desired gene fragment, respectively.

The method for constructing a DNA template for cell-free protein expression according to the present invention, wherein the endonuclease site is selected preferably from an endonuclease recognizing a non-palindromic sequence.

The cell-free protein expression method according to the present invention comprises a step of expressing a protein using the above-described DNA template for cell-free protein expression.

According to the cell-free protein expression method, a joint of a DNA template is connected with a chemical modification group, and the DNA template is fixed on the surface of a solid phase or prepared into hydrogel through the chemical modification group to perform cell-free protein expression.

According to the cell-free protein expression method of the present invention, the solid phase surface may be magnetic beads or other solid phase surfaces, preferably SA magnetic beads.

The cell-free protein expression method according to the present invention, wherein the DNA template is prepared into a hydrogel and then cell-free protein expression is performed.

The embodiment of the application adopts at least one technical scheme which can achieve the following beneficial effects:

(1) the DNA templates for cell-free protein expression of the present application do not have open 3 'and 5' ends and are resistant to exonuclease degradation.

(2) The joints additionally arranged at the two ends can conveniently provide various chemically modified bases for the template, so that the possibility of using the template is greatly increased, for example, the template is fixed on the surface of a solid phase or prepared into hydrogel, the template can be recycled, the reaction cost of CFPS (circulating fluid chromatography) is reduced, the introduced chemical modification is convenient for enriching the template, the local concentration of the template is improved, and the expression amount of protein is further improved.

(3) The technical scheme of the application does not need to reform the strains, does not influence the growth of the strains, does not need to additionally add protein in a reaction system, and is simpler in operation and lower in cost.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a schematic representation of a DNA template of the present invention for cell-free protein expression;

FIG. 2 is a schematic diagram of a method for preparing a linker at both ends of a DNA template for cell-free protein expression according to the present invention;

FIG. 3 is a schematic diagram showing the ligation of a target gene fragment and a double-ended linker via sticky ends;

FIG. 4 is a schematic view of a linear template containing sticky ends in example 1;

FIG. 5 is a schematic diagram showing the comparison of the structures of the general linear template and the template of the present invention in example 1, wherein both ends of the general linear template are not closed, and are easily degraded by exonuclease in the reaction system, thereby affecting the protein expression level; the template provided by the patent has two closed ends, the stability in the system is greatly improved, and chemically modified bases can be introduced at 5 'or 3' joints according to experimental needs;

FIG. 6 is a graph showing the comparison between the protein expression level of the template of the present patent and the protein expression levels of the conventional linear template and plasmid in example 1;

FIG. 7 is a graph showing the effect of the present invention on template exonuclease resistance in example 2, wherein 1: marker, 2: common linear template, 3: the general linear template was digested with ExoIII, 4: the common linear template is cut by RecBCD enzyme, 5: plasmid, 6: the plasmid was digested with ExoIII, 7: the plasmid was digested with RecBCD, 8: DNA template for cell-free protein expression of the invention, 9: the template of the invention is cut by ExoIII enzyme, 10: the template is subjected to RecBCD enzyme digestion;

FIG. 8 is a graph showing the effect of recycling the DNA template for cell-free protein expression of the present invention in example 3;

FIG. 9 is a graph showing the recycling effect of the conventional PCR product in example 3;

FIG. 10 is a graph comparing the expression level of the template protein provided in example 4 with that of a conventional linear template and a plasmid;

FIG. 11 is a graph showing the recycling effect of the DNA template for cell-free protein expression of the present invention in example 5 after preparing a hydrogel.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The technical solutions provided by the embodiments of the present application are described in detail below with reference to the accompanying drawings.

Most of the existing CFPS linear templates are DNA double strands with two open ends, and are easily degraded by exonuclease in a reaction system, so that the protein expression efficiency is low. The invention provides a double-stranded DNA template with two closed ends, namely a single-stranded circular DNA molecule.

As shown in FIG. 1, according to the technical scheme of the present invention, a linearly expressed DNA template is prepared first, then two-end linkers are prepared, and the linkers are connected to the linearly expressed template, thereby adding blocking linkers for both ends of the linearly expressed template, respectively.

Linear expression templates are often obtained by gene synthesis or PCR amplification, and are routine in the art. The template should contain the reading frame of the protein of interest and the required 5 'and 3' untranslated regions, and further, spacer base sequences of appropriate length can be placed upstream and downstream to provide proper steric hindrance. In order to connect with the two-end joints, enzyme cutting sites and protective bases are respectively introduced into the 5 'end and the 3' end of the linear expression template. Wherein the endonuclease site is preferably selected to be an endonuclease that recognizes a non-palindromic sequence.

As shown in FIG. 2, preparing two-end linkers, firstly designing a single-stranded DNA complementary in the strand, annealing to form a hairpin structure, wherein the 5 'and 3' ends of the single-stranded DNA are respectively positioned in such a way that the two-end linkers introduce endonuclease sites of the same kind as the linear template, so that the two-end linkers can be cut by the endonuclease to form sticky ends, and the sticky ends are respectively connected to the 5 'end and the 3' end of the target gene segment.

When the base sequences in the joint are designed to be in strand reverse complementarity, partial base non-complementarity is allowed to occur, and preferably, when the number of the total bases is odd, the other bases are pairwise paired except the middle base of the sequence (as shown in figure 2); when the total number of bases is an even number, all bases pair two by two.

The corresponding positions of the 5 'end and the 3' end of the single-stranded DNA are two-end joints, endonuclease sites are introduced into the joints, the joints contain sites which can be recognized and cut by a selected endonuclease after annealing, and sticky ends or flat ends are formed after cutting and are respectively connected to the 5 'end and the 3' end of a target gene segment.

The linker may comprise one or more chemically modified bases in its sequence; chemically modified bases can provide convenience for subsequent immobilization of the template. The chemical modification can be biotin, amino, acrylamide, azide or sulfhydryl. Chemically modified bases can provide a convenient means for subsequent access to different forms of template.

As shown in FIG. 3, the prepared target gene fragment containing sticky ends and the two-end adaptors are connected by DNA ligase, and the connected double-stranded DNA template with two closed ends is obtained. When the connection is carried out, preferably, the target gene fragment is firstly connected with one end of the 5 'or 3' end connector and then connected with the other end connector, so that the connection efficiency can be improved.

According to the cell-free protein expression method of the present invention, the above-described DNA molecule is used as a template for protein expression.

If the prepared template contains chemically modified base, the chemically modified base can be further fixed on the surface of a solid phase or used for preparing hydrogel, so that the utilization rate of the template is further improved, wherein the chemically modified base can be biotin modified, amino modified, sulfydryl modified and the like.

The DNA template for cell-free protein expression according to the present invention can be directly used for cell-free protein expression; or can be fixed on the surface of a solid phase for cell-free protein expression, wherein the surface of the solid phase can be magnetic beads or other solid phase surfaces, preferably SA magnetic beads; cell-free protein expression can also be performed after preparation into a hydrogel by itself or with other molecules.

When cell-free protein expression is performed using the single-stranded circular DNA molecule as a template for protein expression, the cell-free protein expression system includes a cell extract, a supplement system, according to the teaching of the prior art.

The supplemental system comprises: a first buffer or a functional equivalent thereof, a magnesium salt or a functional equivalent thereof, a potassium salt or a functional equivalent thereof, an oxalate or a functional equivalent thereof, an amino acid or a mixture of amino acids or a functional equivalent thereof, a mixture of nucleotides or a functional equivalent thereof.

The molar concentration of the first buffer solution is 2-100 mM; the molar concentration of magnesium ions contained in the magnesium salt is 0.5-20 mM; the molar concentration of potassium ions contained in the potassium salt is 100-350 mM; or/and the molar concentration of the oxalate is 0.5-10 mM; the molar concentration of the amino acid or the amino acid mixture is 0.5-3 mM; the molar concentration of the nucleotide mixture is 0.5-1.5 mM.

For example, the first buffer is a phosphate buffer; the magnesium salt is magnesium glutamate or/and magnesium acetate; the potassium salt is potassium glutamate or/and potassium acetate; the oxalate is potassium oxalate or/and sodium oxalate; the amino acid or the amino acid mixture is at least one or more of glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine; the nucleotide mixture is nucleoside triphosphate NTP or nucleotide NMP, wherein the nucleoside triphosphate NTP comprises any one of the following combinations:

adenosine triphosphate ATP, guanosine triphosphate GTP, uridine triphosphate UTP and cytidine triphosphate CTP; or

Adenosine triphosphate ATP and guanosine triphosphate GTP.

Wherein the nucleotide NMP comprises any combination of:

adenine nucleotide AMP, guanine nucleotide GMP, uracil nucleotide UMP, and cytosine nucleotide CMP; or

Adenine nucleotide AMP and guanine nucleotide GMP.

Wherein, the steps for preparing the cell-free protein expression system are as follows: and adding the template into a cell-free protein expression system for expression at the expression temperature of 15-40 ℃ for 0.5-24 h.

The cell extract is selected from any one or any combination of several of escherichia coli cell extract, yeast cell extract, wheat germ extract, insect cell extract, rabbit reticulocyte extract and CHO cell extract.

Example 1

1. Preparing a target gene fragment:

as shown in FIG. 4, the endonuclease BssS alpha I capable of recognizing the non-palindromic sequence at both ends of the expression cassette of the target protein (green fluorescent protein) was selected from the existing pID-1 plasmid; after enzyme digestion and purification, the target gene fragment with 5 'and 3' cohesive ends is obtained.

2. 5 'and 3' linker preparation:

as shown in FIG. 2, a 5 ' linker and a 3 ' linker were designed, respectively, the linkers were DNA single strands complementary to each other in the reverse direction within the strands, and annealed under appropriate conditions to form hairpin structures, and then digested and purified with the endonuclease BssS α I used for the above-mentioned target gene fragment to obtain 5 ' linkers and 3 ' linkers each having a cohesive end, wherein biotin-modified iBiodT was used as the base T in the middle of the 5 ' linker to improve the template utilization rate.

5' end joint:

5’-GACCACGAGAGGATTAATCCCCCAATGATTTTTCGGAATCCCCC/iBiodT/GGGGGATTCCGAAAAATCATTGGGGGATTAATCCTCTCGTGGTC-3’

wherein: ibodt is a biotin-modified base dT; the quantity of the bases at the two ends of the symbol "//" is the same, and the bases at the two ends are completely complementary and paired after forming a hairpin structure; underlinedCACGAGAndCTCGTGconstitute the recognition site of endonuclease BssSaI;

3' end joint:

5’-GACCTCGTGAGGATTAATCCCCCAATGATTTTTCGGAATCCCCC/T/GGGGGATTCCGAAAAATCATTGGGGGATTAATCCTCACGAGGTC-3’

3. joining the target Gene fragment to the 5 'and 3' linkers

And (3) connecting the target gene fragment with a 3 'adaptor by using T4 ligase, purifying, connecting the purified product with a 5' adaptor, and purifying to obtain the double-stranded DNA template with the adaptors added at both ends. The template has the capability of resisting the degradation of exonuclease, can be directly added into a CFPS system for reaction, and can also be fixed on the surface of a solid phase carrier through a chemically modified joint or prepared into hydrogel.

4. Cell-free protein expression

The cell-free expression system was composed as follows (template plus): 10mM phosphate buffer solution, 1.2mM ATP, 0.85mM UTP, 0.85mM CTP, 0.85mM GTP, 280mM potassium glutamate, 8mM magnesium glutamate, 2.7mM potassium oxalate, 2mM amino acid mixture, 25% by volume of Escherichia coli extract; adding 7.5ng/uL of the prepared template into the system, adding all the components into a PCR tube, uniformly mixing, placing in a ThermoMixer constant-temperature mixing machine, shaking for 16 hours at 30 ℃ and 1000rpm, and taking out. Measuring the expression quantity of the fluorescent protein by using a microplate reader under the conditions of 485nm of exciting light, 535nm of emitting light and 0.1s of exposure time; under the same conditions, the common linear template (PCR product) and plasmid template were added at equimolar concentrations, respectively.

Among them, the structural differences between the conventional linear template (PCR product) and the template of the present invention prepared as described above are shown in FIG. 5.

As shown in FIG. 6, the protein expression level of the template provided by the present invention is equivalent to that of the plasmid template, and is superior to the effect of adding the double-stranded binding protein sCco to the system reported in Zhu B et al 2020, and the protein yield is still much lower than that of the plasmid template even though the addition of sCco to the system is improved.

EXAMPLE 2 exonuclease Effect

In order to verify the effect of the template on resisting the degradation of the exonuclease, the exonuclease ExoIII and RecBCD are respectively used for processing various templates; in addition to the template of the present invention, controls, common linear templates-PCR products (susceptible to exonuclease degradation) and plasmids (not susceptible to degradation) were added. As shown in FIG. 7, the template of the present invention is the same as the plasmid, and the brightness of the bands before and after exonuclease treatment is unchanged, so that exonuclease degradation can be resisted, while the target bands of the common linear template after exonuclease treatment are obviously weakened, and dispersed bands appear, so that the degradation is serious.

Example 3 Recycling of templates of the invention immobilized on magnetic beads

Coating the template obtained in the above step on SA magnetic beads (MyOne)^TMStreptavidin T1); adding template-coated magnetic beads toIn the same cell-free expression system as in example 1, the mixture was uniformly mixed and then placed in a thermo mixer, and the mixture was shaken at 1000rpm and 30 ℃ for 16 hours and then taken out. Measuring the expression quantity of the fluorescent protein by using a microplate reader under the conditions of 485nm of exciting light, 535nm of emitting light and 0.1s of exposure time; washing the magnetic beads after the first reaction for 3 times by using a 1 XPBS solution, and then carrying out a second reaction without changing a reaction system; in the same manner, the third reaction was carried out. As shown in FIG. 8, the magnetic beads coated with the template of the present invention showed strong reusability, and the protein yield was still decreased less after 3 times of repeated use, which is much higher than the protein expression level using the common PCR product as the template.

The magnetic beads are coated with common PCR products, and RecBCD inhibitor Gams protein is additionally added into the reaction system, as in the above method. After the reaction under the same conditions, as shown in FIG. 9, the protein yield had decreased to one tenth of the original yield in the second reaction. It can be seen that the yield of the linear template can be improved by the Gams protein in a short time, but the linear template can be degraded by other exonuclease in the system after a long time, and the method for protecting the linear template by using the Gams protein has no good effect compared with the technical scheme of the invention.

Example 4

1. The target gene fragment was prepared as described in example 1;

2. the 5 'and 3' linkers were prepared as in example 1, differing only in sequence,

a) in linker set 1, the 5 'linker and the 3' linker each contain 12 pairs of complementary bases:

5' end joint: 5' -GACCACGAGAGG/T/CCTCTCGTGGTC-3’

3' end joint: 5' -GACCTCGTGAGG/T/CCTCACGAGGTC-3’

b) In linker set 2, the 5 'linker and the 3' linker each contain 12 pairs of complementary bases and also several unpaired bases:

5' end joint:

5’-GACCACGAGAGGATTAATCCCCCA/T/TCCCCCAATGATCCTCTCGTGGTC-3’

3' end joint:

5’-GACCTCGTGAGGATTAATCCCCCA/T/TCCCCCAATGATCCTCACGAGGTC-3’

3. the connection of the target gene fragment with the 5 'and 3' linkers and the subsequent cell-free protein expression are the same as those in example 1;

as shown in FIG. 10, the template provided in example 4 has a much higher protein expression level than the conventional linear template, and is comparable to the plasmid template.

Example 5 preparation of DNA composite hydrogel Using template of the present invention

The template can also be used for preparing hydrogel, so that the template is enriched, the local concentration of the template is effectively improved, the expression quantity of protein is further improved, the prepared hydrogel can be recycled, and the reaction cost of CFPS (circulating fluidized bed polystyrene) is reduced;

1. the target gene fragment was prepared as described in example 1;

2. the preparation method of the 5 'and 3' joints is the same as that of the example 1, and only the modified bases of the joints at the two ends are different, so that the templates with the 5 'end and the 3' end both containing sulfydryl modification are prepared;

5' end joint:

5’-GACCACGAGAGGATTAATCCCCCAATGATTTTTCGGAATCCCCC/HS-dT/GGGGGATTCCGAAAAATCATTGGGGGATTAATCCTCTCGTGGTC-3’

3' end joint:

5’-GACCTCGTGAGGATTAATCCCCCAATGATTTTTCGGAATCCCCC/HS-dT/GGGGGATTCCGAAAAATCATTGGGGGATTAATCCTCACGAGGTC-3’

3. preparation of DNA composite hydrogel

(1) Preparation of solution 1: dissolving clay by pure water to prepare a clay solution with the mass fraction of 4%;

(2) preparation of solution 2: adding a four-arm polyethylene glycol sulfydryl (with the number average molecular weight of 20000) into the solution 1, and uniformly mixing, wherein the mass fraction of the four-arm polyethylene glycol sulfydryl is 4%;

(3) preparation of solution 3: dissolving tetraarm polyethylene glycol acrylate (number average molecular weight is 20000) with 2 × PBS (pH ═ 7) to prepare a tetraarm polyethylene glycol acrylate solution with a mass fraction of 4%;

(4) preparation of solution 4: adding the prepared template containing the thiol modification into the solution 3, and uniformly mixing, wherein the final concentration of the template is 200 ng/. mu.L;

(5) respectively taking 5 mu L of the solution 2 and the solution 4, uniformly mixing at 4 ℃, and then reacting at 37 ℃ for 2h to obtain the DNA composite hydrogel.

4. Cell-free protein expression

Adding the DNA composite hydrogel prepared by the template containing the sulfhydryl modification into the cell-free expression system which is the same as that in the embodiment 1, uniformly mixing the template with the final concentration of 20ng/uL, placing the mixture into a thermo Mixer constant-temperature mixer, shaking the mixture for 16 hours at the temperature of 30 ℃ and the speed of 1000rpm, and taking out the mixture. Measuring the expression quantity of the fluorescent protein by using a microplate reader under the conditions of 485nm of exciting light, 535nm of emitting light and 0.1s of exposure time; washing the magnetic beads after the first reaction for 3 times by using a 1 XPBS solution, then carrying out a second reaction, adding a new reaction solution, and keeping the reaction system unchanged; in the same manner, the third reaction was carried out. As shown in FIG. 11, the hydrogel prepared by the template of the present application shows strong reusability, and the protein yield is not reduced after 3 times of repeated use, which is much higher than the protein expression level using the common PCR product as the template.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

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Claims

1. A DNA template for cell-free protein expression comprising a reading frame for a protein of interest and 5 'and 3' untranslated regions upstream and downstream of the reading frame, and linkers upstream and downstream of the 5 'and 3' untranslated regions, the linkers being hairpin structures formed by annealing single-stranded DNA.

2. The DNA template for cell-free protein expression of claim 1, wherein the single strand forming the linker comprises at least one base-complementary pairing region.

3. The DNA template for cell-free protein expression of claim 2, wherein the linker structure is: when the total number of the bases of the single strand forming the joint is odd, all the other bases except the middle base of the sequence are paired pairwise; when the total number of bases is an even number, all bases pair two by two.

4. The DNA template for cell-free protein expression according to claim 2, wherein the number of bases of the base complementary pairing region is at least 12 bp.

5. The DNA template for cell-free protein expression of claim 1, wherein one or more bases in the linker are attached to a chemical modifying group.

6. The DNA template for cell-free protein expression of claim 5, wherein the chemical modification group is biotin, amino, acrylamide, azide, or thiol.

7. A method of constructing a DNA template for cell-free protein expression, the method comprising the steps of:

synthesizing a reading frame of a target protein and 5 'and 3' untranslated regions upstream and downstream of the reading frame;

synthesizing complementary single-stranded DNA in the strand, annealing the formed hairpin structure to obtain a linker,

the above-mentioned synthesized linkers are ligated to the 5 'and 3' untranslated regions upstream and downstream of the reading frame of the target protein, respectively, to obtain a DNA template for cell-free protein expression.

8. The method for constructing a DNA template for cell-free protein expression according to claim 7, wherein endonuclease sites homologous to the reading frame of the target protein and its upstream and downstream 5 'and 3' untranslated regions are introduced at the 5 'and 3' end positions of the single-stranded DNA.

9. The method of constructing a DNA template for cell-free protein expression according to claim 8, wherein the endonuclease site is an endonuclease that recognizes a non-palindromic sequence.

10. A cell-free protein expression method comprising the step of expressing a protein using a DNA constructed as a template,

the above-mentioned synthetic linkers are ligated to the 5 'and 3' untranslated regions upstream and downstream of the reading frame of the target protein, respectively.

11. The method for cell-free protein expression according to claim 10, wherein the linker is linked to a chemical modification group, and the DNA template is immobilized on a solid phase surface or prepared into hydrogel by the chemical modification group to perform cell-free protein expression; or

And preparing the prepared DNA template into hydrogel and then carrying out cell-free protein expression.