CN115838745A

CN115838745A - Linear DNA template and system suitable for cell-free synthesis of restriction endonuclease BsaI and application thereof

Info

Publication number: CN115838745A
Application number: CN202210262366.0A
Authority: CN
Inventors: 李健; 刘晚秋; 季向阳; 卢屹聪
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2023-03-24

Abstract

The invention belongs to the technical field of biology, and discloses a linear DNA template and a system suitable for cell-free synthesis of restriction endonuclease BsaI, and application thereof. The linear DNA template suitable for cell-free synthesis of the restriction enzyme BsaI sequentially comprises a fragment containing a promoter, an encoding gene of the restriction enzyme BsaI and a fragment containing a terminator, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No.1, and the linear DNA template does not contain the enzyme cutting site of the restriction enzyme BsaI. The linear DNA template can effectively prevent the linear DNA template from being degraded by nuclease, can be suitable for any in vitro cell-free protein system and can be expressed smoothly, and by adopting the linear DNA template to carry out in vitro cell-free protein synthesis, the obtained restriction enzyme BsaI has high purity and specific activity.

Description

Linear DNA template and system suitable for cell-free synthesis of restriction endonuclease BsaI and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a linear DNA template and system suitable for cell-free synthesis of restriction enzyme BsaI, and application thereof.

Background

Protein synthesis is classified into conventional intracellular synthesis techniques and in vitro protein-free synthesis techniques. Conventional intracellular synthesis techniques refer to the expression of foreign genes by model organisms such as bacteria, fungi, plant cells or animal cells. The in vitro protein-free synthesis technology is to realize the synthesis of target protein by adding substances such as substrates and capacities required by protein synthesis, transcription and translation related protein factors and the like under artificial control by taking exogenous mRNA or DNA as a protein synthesis template. In vitro protein synthesis is free of the steps of plasmid construction, transformation, cell culture, cell collection, cell disruption and the like, and is a relatively rapid and convenient protein expression mode.

Restriction endonucleases refer to a class of enzymes that recognize a specific sequence of deoxyribonucleotides and cleave the phosphodiester bond between two deoxyribonucleotides at specific sites in each strand, and are referred to as restriction endonucleases or restriction enzymes for short. Restriction enzymes can be classified into four major classes, class I-IV, according to their functional properties, size and cofactors required for their reaction.

Class II Class restriction enzymes are the only four classes of enzymes that do not require ATP and only Mg ²⁺ Most of enzymes for realizing the cutting function have short palindromic sequences, the cutting sites are usually recognition sequences, and the enzymes are restriction enzymes which are most widely applied in biology, and currently, more than 630000 Class II restriction enzymes are found, and 623 enzymes are developed into commercial enzyme preparations. The Class II Type restriction enzyme is divided into several subclasses of Type IIe, type IIf, type IIg, type IIm, type IIs and Type IIt, wherein the Type IIs Type restriction enzyme has a monomer structure, has a size of 45-110kDa, recognizes a non-palindromic sequence, and has a cleavage site located at a position other than at least two bases of the cleavage site. The BsaI related to the invention belongs to Class II Type IIs restriction endonuclease, is derived from Bacillus stearothermophilus 6-55, has an enzyme cutting site as shown in figure 1, and is commonly used in Golden Gate technology.

When the restriction enzyme is over-expressed as a recombinant protein in a heterologous expression host, the nature of its cleavage of DNA severely inhibits the normal growth of the host and the expression of the recombinant protein, resulting in difficulty in obtaining the target protein with biological activity. For the expression of restriction endonuclease at home and abroad, a strategy of coexpressing corresponding methylase or constructing a methylase-containing expression host is usually selected, and the normal growth of the host is ensured by modifying enzyme cutting sites in host DNA with the corresponding methylase. The co-expression strategy solves the expression problem of the restriction enzyme BsaI to a certain extent, but the steps are relatively complicated, the protein yield is relatively low, and the current commercial BsaI is relatively high in price.

At present, no in vitro protein-free synthesis technology for synthesizing the restriction enzyme BsaI has been reported.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a linear DNA template suitable for cell-free synthesis of the restriction enzyme BsaI, a system and uses thereof.

One of the purposes of the invention is to provide a linear DNA template suitable for cell-free synthesis of a restriction enzyme BsaI, wherein the linear DNA template sequentially comprises a segment containing a promoter, a coding gene of the restriction enzyme BsaI and a segment containing a terminator, the nucleotide sequence of the coding gene is shown as SEQ ID No.1, and the linear DNA template does not contain the enzyme cutting site of the restriction enzyme BsaI.

In the invention, the linear DNA template does not contain the restriction site of the restriction enzyme BsaI, so that the leakage expression during the transformation can be effectively avoided, and the BsaI restriction site carried by the plasmid of the linear DNA template is cut.

In the invention, the coding gene is obtained by optimizing and removing a restriction enzyme site (NNNNN) according to an escherichia coli codon. The coding gene optimized by the codon is obtained by a large amount of artificial optimization and screening, and compared with the sequence obtained by conventional optimization methods such as codon optimization software and the like, the coding gene optimized by the codon can remarkably improve the expression level of BsaI protein while well ensuring the natural active structure of the restriction enzyme BsaI.

According to the technical scheme of the invention, the fragment containing the promoter does not contain the restriction enzyme cutting site of the restriction enzyme BsaI.

According to a specific embodiment of the present invention, the promoter-containing fragment includes a promoter and a promoter upstream sequence. Specifically, the promoter in the promoter-containing gene is T7. According to the technical scheme of the invention, the fragment containing the terminator does not contain the restriction site of the restriction enzyme BsaI.

According to a specific embodiment of the present invention, the terminator-containing fragment includes a terminator and a terminator downstream sequence. Specifically, the terminator in the gene containing the terminator is T7.

Preferably, the length of the promoter upstream sequence is 2bp-300bp.

Preferably, the length of the sequence downstream of the terminator is 2bp-300bp.

The promoter upstream sequence and the terminator downstream sequence in the present invention can prevent the encoded gene from being degraded by nuclease.

According to the technical scheme of the invention, the linear DNA template further comprises a label positioned at the upstream or downstream of the coding gene, and the label is operably connected with the coding gene.

According to a specific technical scheme of the invention, the tag comprises one or more of 6 XHis, SUMO, MBP, strep-tag and Cold-TF factor. Further preferably, the tag is 6 × His. The tag is located between the promoter-containing gene and the encoding gene.

According to a particular embodiment of the invention, the linear DNA template further comprises HRV 3C enzyme, TEV (tobaco Etch Virus) protease and ULP1 (Ubl specific protease 1) between the tag and the coding gene to cleave the fusion tag.

The second objective of the present invention is to provide the method for constructing the linear DNA template, comprising the following steps:

1-1) synthesizing a restriction endonuclease coding gene, and amplifying a non-expression vector connector to obtain the coding gene;

1-2) taking a plasmid containing a promoter as a template, and amplifying to obtain the fragment containing the promoter; taking a plasmid containing a terminator as a template, and amplifying to obtain a fragment containing the terminator;

1-3) fusing the coding gene obtained in the step 1-1), the promoter-containing fragment obtained in the step 1-2) and the terminator-containing fragment by a fusion PCR method to obtain the linear expression template.

According to the technical scheme of the invention, in the step 1-1), the non-expression vector is pUC-GW-Amp.

According to the technical scheme of the invention, in the step 1-1), during amplification, the amplification primer sequence comprises sequences shown as SEQ ID No.3 and SEQ ID No. 4.

According to the technical scheme, in the step 1-2), the plasmid containing the promoter is pET28a.

According to the technical scheme of the invention, in the step 1-2), when the gene containing the promoter is obtained by amplification, the amplification primer sequence comprises the sequences shown as SEQ ID No.5 and SEQ ID No. 6.

According to the technical scheme, in the step 1-2), the plasmid containing the terminator is pET28a.

According to the technical scheme of the invention, in the step 1-2), when the gene containing the terminator is obtained by amplification, the amplification primer sequence comprises the sequences shown as SEQ ID No.7 and SEQ ID No. 8.

According to the technical scheme of the invention, in the step 1-3), the primer sequence of the fusion PCR comprises the sequences shown as SEQ ID No.5 and SEQ ID No. 8.

It is a further object of the present invention to provide the use of a linear DNA template as described above for the construction of a system suitable for the cell-free synthesis of the restriction enzyme BsaI.

It is a fourth object of the present invention to provide a system for cell-free synthesis of the restriction enzyme BsaI, said system comprising a linear DNA template as described above.

According to the technical scheme, the system also comprises a cell extract, and the cell extract can express the linear DNA template.

According to a particular embodiment of the invention, the cell extract is selected from the group consisting of E.coli extracts.

According to a more specific embodiment of the present invention, the Escherichia coli is selected from one or more of BL21 Star (DE 3), BL21 Star (DE 3) + pG-KJE, BL21 Star (DE 3) + pGro7, BL21 Star (DE 3) + pG-Tf2, rosetta + pG-KJE, rosetta + pGro7, rosetta + pG-Tf 2. In a preferred embodiment, the escherichia coli is BL21 Star (DE 3).

According to the specific technical scheme of the invention, the concentration of the linear DNA template is 3-9 mu M based on the total mass of the system.

According to a specific embodiment of the present invention, the concentration may be 3 to 6. Mu.M, 5 to 7. Mu.M, or 6 to 9. Mu.M. In a preferred embodiment, the concentration is 6. Mu.M.

The fifth purpose of the invention is to provide a synthetic method of restriction enzyme BsaI, which is characterized by comprising the following steps:

the restriction enzyme BsaI is obtained by performing a synthesis reaction using at least the linear DNA template as described above or the system as described above.

According to the technical scheme of the invention, the reaction temperature is 15-37 ℃.

According to the specific technical scheme of the invention, the reaction temperature can be 15-25 ℃, also can be 20-35 ℃, and also can be 30-37 ℃. In a preferred embodiment, it is 30 ℃.

According to the technical scheme of the invention, the reaction time is 2-8 h.

According to the specific technical scheme of the invention, the reaction time is 2-6 h, also can be 3-6 h, also can be 5-8 h. In a preferred embodiment, it is 6h.

According to the technical scheme, the reaction is further followed by a purification step, and the purification step is selected from one or more of affinity chromatography purification, ion exchange chromatography purification and gel chromatography purification.

According to a specific technical scheme of the invention, the purification comprises affinity chromatography purification and gel chromatography purification.

The Ni-NTA affinity chromatography purification is a chromatography system which specifically separates specific biomacromolecules according to the principle of reversible specific combination of the biomacromolecules and ligands. Ni NTA Beads 6FF can be combined with target protein with 6 × His (histidine) label, low-concentration imidazole is used for removing hybrid protein which is not combined with Beads, high-concentration imidazole is used for eluting the target protein, and the purpose of separating and purifying restriction enzyme BsaI is achieved. According to the invention, the hybrid protein is removed by using 50mM of low-concentration imidazole, and the target protein is eluted by using 500mM of high-concentration imidazole, so that the primarily purified restriction enzyme BsaI can be more efficiently obtained.

Preferably, the buffer used for eluting the hybrid protein for affinity chromatography purification comprises the following components: 40mM Tris,500mM NaCl,50mM imidazole, pH7.5.

Preferably, the buffer used for eluting the protein of interest for the affinity chromatography purification comprises the following components: 40mM Tris,500mM NaCl,500mM imidazole, pH7.5.

Preferably, the buffer for eluting the target protein used for the gel chromatography purification comprises the following components: 20mM Tris,500mM NaCl,1mM DTT, pH7.5.

The BsaI protein obtained by the affinity chromatography purification and the gel chromatography purification has high purity, and the whole purification process only needs 5-6h, so that the reduction of enzyme activity caused by a long-time purification process is avoided, and the improvement of enzyme yield and activity is facilitated. After the purification, bsaI can meet the purity requirement of molecular biology experiments.

Compared with the prior art, the invention has the following beneficial effects:

1) The invention provides a linear DNA template suitable for cell-free synthesis of restriction endonuclease BsaI, which can be suitable for any in vitro cell-free protein system, can be smoothly expressed and can effectively prevent the linear DNA template from being degraded by nuclease; the preparation of the linear template is simple and rapid, and conventional plasmid construction is not needed, so that the linear DNA template is adopted to carry out in-vitro cell-free protein synthesis, and the obtained restriction enzyme BsaI has high purity and high specific activity.

2) The invention also provides a system suitable for cell-free synthesis of the restriction enzyme BsaI, which can improve the yield of the cell-free synthesis of the restriction enzyme BsaI and simultaneously improve the reaction rate of the synthesis of the restriction enzyme BsaI.

3) The invention also provides a method for synthesizing the restriction enzyme BsaI without cells, which well solves the problem of inhibiting the toxicity of the restriction enzyme BsaI on an expression host, has short synthesis reaction time and high efficiency, and the specific activity of the obtained restriction enzyme BsaI is similar to that of a commercial enzyme, thereby having wide application prospect.

Drawings

FIG. 1 shows a schematic diagram of the restriction sites of the restriction enzyme BsaI in example 1 of the present invention.

FIG. 2 shows a spectrum of the vector pUC-GW-Amp in example 1 of the present invention.

FIG. 3 shows a spectrum of plasmid pET28a in example 1 of the present invention.

FIG. 4 is a graph showing the results of Western Blotting detection of the restriction enzyme His-BsaI in the soluble expressed protein and the whole cell expressed protein in example 2 of the present invention. Wherein S represents a soluble expression protein; t represents the whole bacteria expression protein.

FIG. 5 shows a map of plasmid pJL1_ sfGFP in example 2 of the present invention.

FIG. 6 is a graph showing the results of activity measurement of the restriction enzyme BsaI in the cell-free reaction system in example 2 of the present invention. Wherein BsaI + sfGFP represents an experimental group, and the co-expression plasmid pJL1_ sfGFP and the linear DNA template T7promoter-BsaI-T7terminator; sfGFP represents a negative control group and only plasmid pJL1_ sfGFP was expressed.

FIG. 7 is a SDS-PAGE pattern of the flow-through, wash buffer and elution buffer after the restriction enzyme His-BsaI passed through the affinity chromatography column in example 3 of the present invention. Wherein FT (Flow through) is Flow through liquid, W (Washed sample) is impurity washing buffer liquid, eluate is elution buffer liquid, and M is Marker.

FIG. 8 is a SDS-PAGE pattern showing BsaI protein after gel chromatography purification in example 3 of the present invention. Wherein M is Marker; bsaI is BsaI protein after purification by gel chromatography.

FIG. 9 is an electrophoretogram showing an enzyme digestion experiment performed on commercial BsaI and BsaI proteins of various concentrations in example 3 of the present invention. Where NC represents plasmid pJL1_ sfGFP, PC represents commercial BsaI, and 1 represents concentration of 0.45X 10 ^-1 mg/mL BsaI, 2 represents 0.45X 10 ^-2 mg/mL BsaI,3 represents 0.45X 10 ^-3 BsaI and 4 for 0.45X 10 mg/mL ^-4 mg/mL BsaI。

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The invention is further illustrated by the following specific examples of the preparation of the specific restriction enzyme BsaI. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein. The experimental procedures without specifying the specific conditions in the following examples are generally carried out according to conventional conditions, such as molecular cloning in Sambrook, etc.: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the recommendations of the reagent and Instrument vendors. The reagents used are commercially available or publicly available reagents unless otherwise specified.

In the following examples of the invention, the buffers used were prepared as follows:

s30 buffer solution: 10mM triisopropylethanesulfonyl (Tris) -acetic acid, 14mM magnesium acetate, 60mM potassium acetate, 2mM dithiothreitol (DDT), pH =8.2.

And (3) an equilibrium buffer: tris 40mM, naCl 500mM, imidazole 0.5mM, pH7.5.

Washing with a miscellaneous buffer solution: tris 40mM, naCl 500mM, imidazole 50mM, pH7.5.

Elution buffer: tris 40mM, naCl 500mM, imidazole 500mM, pH7.5.

Desalting buffer solution: tris 40mM, naCl 500mM, dithiothreitol 1MM, pH7.5.

Example 1 construction of a Linear DNA template T7promoter-BsaI-T7terminator

In this example, a linear DNA template T7promoter-BsaI-T7terminator was constructed and obtained, including the following:

1. BsaI Gene Synthesis

The DNA sequence of the restriction enzyme BsaI is shown as SEQ ID No.1, the amino acid sequence thereof is shown as SEQ ID No.2, the DNA sequence of the restriction enzyme BsaI is obtained by optimizing and removing a restriction enzyme site (NNNNNN) according to an escherichia coli codon, and the schematic diagram of the restriction enzyme site is shown as figure 1.

2. Linear DNA template suitable for cell-free expression preparation of restriction endonuclease Bsal

Synthesizing the DNA sequence of the restriction enzyme BsaI obtained in the step 1 into a vector pUC-GW-Amp to obtain a vector pUC-BsaI-Amp containing a BsaI coding gene, wherein the map of the vector pUC-GW-Amp is shown in figure 2; further construct a linear DNA template suitable for cell-free synthesis of restriction enzyme BsaI. The method comprises the following steps:

1) Taking plasmid pUC-BsaI-Amp as a template, amplifying primer sequences such as SEQ ID No.3 and SEQ ID No.4 to obtain a BsaI coding gene, introducing a 6 xHis purification tag sequence and a protease HRV 3C enzyme cutting site sequence into the N end of the BsaI coding gene, cutting off the 6 xHis tag at the N end when the HRV 3C enzyme cutting site sequence is convenient to purify, and finally obtaining a BsaI fragment of which the N end is provided with the 6 xHis tag and the HRV 3C enzyme cutting site;

2) Amplifying by using plasmid pET28a as a template and primer sequences such as SEQ ID No.5 and SEQ ID No.6 to obtain a T7promoter upstream sequence + T7 promoter; wherein, the map of the plasmid pET28a is shown in figure 3; the upstream sequence is effective in preventing degradation of the linear DNA template by nucleases, and is not limited to the sequence in this example;

3) Taking a plasmid pET28a as a template, and amplifying primer sequences such as SEQ ID No.7 and SEQ ID No.8 to obtain a downstream sequence of a T7terminator and a T7terminator; the downstream sequence is effective in preventing degradation of the linear DNA template by nucleases, and is not limited to the sequence in this example;

4) Primer sequences such as SEQ ID No.5 and SEQ ID No.8 are used for fusing a BsaI fragment with a tag of 6 XHis and HRV 3C enzyme cutting sites at the N end obtained in the step 1), a T7promoter upstream sequence + T7promoter obtained in the step 2) and a T7terminator + T7terminator downstream sequence obtained in the step 3) by a fusion PCR method to obtain a linear DNA template suitable for cell-free synthesis of restriction enzyme BsaI, wherein the linear DNA template is marked as T7promoter-BsaI-T7terminator.

Example 2 cell-free expression and Activity identification of restriction enzyme BsaI

In this example, cell-free expression and activity identification of the restriction enzyme BsaI was performed, including the following:

1. preparation of cell extracts

Taking Escherichia coli BL21 Star (DE 3) strain as a host, activating, picking out a single colony in 20mL LB culture medium, and culturing at 32 ℃ and 220rpm overnight; transferring the strain into 1L 2 XYPTG culture medium with the initial OD600 ≈ 0.05, culturing at 34 deg.C and 220rpm until OD600 ≈ 0.8, adding 0.5mM IPTG to induce T7 RNA polymerase, and culturing for 2.5-3h until OD600>2.5; centrifuging at 4 deg.C and 5000g for 15min, collecting thallus, and washing thallus with S30 buffer solution for 3 times to obtain thallus.

Weighing thalli, adding 1g of thalli into 1mL of S30 buffer solution, and performing vortex resuspension to obtain a bacterial suspension; putting 1.4mL of the bacterial suspension into a 1.5mL centrifuge tube for ultrasonic crushing, wherein the crushing procedure is as follows: 50% power, 10s ultrasonic wave and 10s pause until the energy reaches 600-620J; centrifuging at 4 deg.C and 12000g for 10min, and collecting supernatant; centrifuging the supernatant at 4 deg.C and 10000g for 10min to obtain supernatant as cell extract. The cell extract was frozen at-80 ℃ for use.

2. Expression of restriction enzyme BsaI

Preparing 15 mu L of cell-free reaction system: 12mM magnesium glutamate, 10mM ammonium glutamate, 130mM potassium glutamate, 1.2mM Adenosine Triphosphate (ATP), 0.85mM Guanosine Triphosphate (GTP), 0.85mM Uridine Triphosphate (UTP), 0.85mM Cytidine Triphosphate (CTP), 34. Mu.g/mL folinic acid, 170. Mu.g/mL E.coli tRNA mixture, 2mM 20 essential amino acids (each at a concentration of 2 mM), 0.33mM Nicotinamide Adenine Dinucleotide (NAD), 0.27mM coenzyme A (CoA), 1.5mM spermidine, 1mM putrescine, 4mM sodium oxalate, 33mM Pyruvate Enol (PEP), 5. Mu.M linear DNA template constructed in example 1, 27% of the total volume of the cell extract obtained in step 1 of this example in a cell-free reaction system.

And reacting the prepared 15 mu L cell-free reaction system at 30 ℃ for 6h to obtain a reaction product. Two parallel control groups were set up, labeled experimental group 1 and experimental group 2.

3. Western Blotting protein identification

The reaction product obtained in the experimental group 1 is used as a whole bacterium expression protein (T, total); the reaction product obtained in experiment group 2 was centrifuged at 13000g for 10min to obtain the supernatant as a soluble expressed protein (S, soluble).

To the whole-cell expressed protein and soluble expressed protein, 15. Mu.L of 2 Xloading buffer (loading buffer composed of 10% glycerol, 2.5% sodium dodecyl sulfate SDS, 1% mercaptoethanol and 2% bromophenol blue) was added, respectively, heated at 98 ℃ for 10min, 10. Mu.L of the buffer was subjected to 10% polyacrylamide gel electrophoresis (SDS-PAGE), and subjected to 150V electrophoresis at constant pressure for 50min. The molecular weight of the target protein obtained by contrasting the molecular weight of the standard protein is 63.7kDa, SDS-PAGE gel at the position with the corresponding molecular weight is cut and transferred to a PVDF membrane, the membrane is transferred for 80min at 75V, the membrane is sealed in a sealing solution (5% skimmed milk powder is dissolved in 1 xTBS/T10 mM Tris,150mM NaCl,1% Tween 20 and pH7.5) for 1h after the membrane is transferred, a primary antibody (6 xHis-labeled mouse monoclonal antibody) prepared by TNET is added into the membrane after the milk is washed, and the membrane is placed in a shaking table at 4 ℃ to be incubated overnight. Washing the membrane with 1 XTNET solution every other day for 3 times, each time for 10min; a secondary antibody (HRP-goat anti-mouse IgG (H + L) antibody) prepared by TNET was added, incubated in a shaker at room temperature for 1H, and the membrane was washed with 1 XTNET solution 3 times for 10min each time. And finally, developing by using an Odyssey imaging system.

FIG. 4 is a diagram showing the results of Western Blotting detection of restriction enzyme BsaI in the soluble expressed protein and the whole cell expressed protein in this example. Wherein S represents a soluble expression protein; t represents the whole bacteria expression protein.

As can be seen from FIG. 4, both the whole-cell expressed protein and the soluble expressed protein were expressed by the restriction enzyme BsaI.

4. Detection of enzyme activity of restriction enzyme BsaI

The T7promoter region of plasmid pJL1_ sfGFP contains a BsaI cleavage site, so plasmid pJL1_ sfGFP is used as a substrate for restriction endonuclease BsaI. The map of plasmid pJL1_ sfGFP is shown in FIG. 5.

Co-expressing pJL1_ sfGFP and the linear DNA template T7promoter-BsaI-T7terminator obtained in example 1 in 15. Mu.L of a cell-free reaction system with 200. Mu.L of a centrifuge tube as a reaction vessel as an experimental group, which is marked as BsaI + sfGFP group; pJL1_ sfGFP alone was expressed in 15. Mu.L reaction system as a negative control, labeled sfGFP. Two groups of parallel experiments are set, reaction is carried out for 6 hours at 30 ℃ in a qPCR instrument, and the change of the sfGFP fluorescence value is detected in real time.

The difference between the 15. Mu.L cell-free reaction system of the experimental group and the cell-free reaction system of step 2 in this example is that 5. Mu.M plasmid pJL1_ sfGFP constructed in step 4 in this example was added, and the other steps were the same.

The 15. Mu.L cell-free reaction system of the negative control group was different from the cell-free reaction system of step 2 in this example in that 5. Mu.M of the linear DNA template was replaced with plasmid pJL1_ sfGFP of step 4, and the other steps were the same.

FIG. 6 is a diagram showing the results of activity measurement of the restriction enzyme BsaI in the cell-free reaction system in this example. Wherein BsaI + sfGFP represents an experimental group, and the co-expression plasmid pJL1_ sfGFP and the linear DNA template T7promoter-BsaI-T7terminator; sfGFP represents a negative control group, expressing only pJL1_ sfGFP plasmid.

As can be seen from FIG. 6, the fluorescence values of the experimental group were significantly lower than those of the negative control group, indicating that the restriction enzyme BsaI expressed without cells had the cleavage activity of the corresponding substrate.

EXAMPLE 3 Mass expression, purification, and determination of enzyme Activity of restriction enzyme BsaI

In this example, the expression, purification, and enzyme activity measurement of restriction enzyme BsaI were performed, including the following steps:

1. cell-free mass expression of restriction enzyme BsaI

Cell-free extract was obtained according to the method of step 1 in example 2; a5 mL cell-free reaction system was prepared according to the method of step 2 in example 2.

A50 mL nuclease-free centrifuge tube is used as a reaction container, 5mL cell-free reaction expression system is prepared according to the components in the example 2, wherein the cell extract is still 27% of the total volume of the expression system, the difference is that the final concentration of a linear expression template T7promoter-BsaI-T7terminator is 7 mu M, the unit level expression amount is improved by increasing the concentration of the linear DNA template, and then the reaction is carried out in a temperature-controlled water bath reactor at 30 ℃ for 6h to obtain the restriction endonuclease with a label of 6 × His, which is marked as His-BsaI.

2. Purification of restriction enzyme His-BsaI

(1) Affinity chromatography purification

The restriction enzyme His-BsaI obtained in step 1 of this example was centrifuged at 21000g at 4 ℃ for 40min, 5mL of the supernatant was taken, an equal volume of equilibration buffer (40mM Tris,500mM NaCl,0.5mM imidazole, pH 8.0) was added, cell debris was removed through a 0.45 μm filter, the filtrate was subjected to Ni-NTA affinity chromatography (Superflow Agarose,1mL pre-column, GE), and the flow-through after application to an affinity column was collected; removing unbound hetero-proteins with 10mL of a washing buffer (40mM Tris,500mM NaCl,50mM imidazole, pH 7.5), and collecting the washing buffer; eluting with 2mL of elution buffer (40mM Tris,500mM NaCl,500mM imidazole, pH 7.5), and collecting the elution buffer; collecting the target protein containing His label, and marking as target protein His-BsaI. 10. Mu.L of each of the flow-through solution, the impurity-washing buffer solution and the elution buffer solution was subjected to SDS-PAGE, and the results are shown in FIG. 5.

FIG. 7 is a SDS-PAGE of the flow-through, wash buffer and elution buffer after the restriction enzyme His-BsaI was passed through the affinity column in this example. Wherein FT (Flow through) is Flow through liquid, W (Washed sample) is impurity washing buffer liquid, eluate is elution buffer liquid, and M is Marker.

As can be seen from FIG. 7, the target protein His-BsaI was enriched in a large amount by Ni-NTA affinity chromatography.

The collected target protein His-BsaI is subjected to multiple dilution by desalting buffer solution (40mM Tris,500mM NaCl,1mM DTT, pH7.5), desalted and concentrated to 1mL by an ultrafiltration tube (molecular cut-off of 10 kDa) to obtain the desalted target protein His-BsaI, and the protein concentration is verified by SDS-PAGE and determined by A280nm absorbance (Nono Drop).

In this example, the concentration of the desalted target protein His-BsaI was 1mg/mL.

(2) 6 × His tag was excised and purified by gel chromatography

1mL of the target protein His-BsaI obtained in step 2 (1) in this example was added to a 2mL centrifuge tube, 20. Mu.L of 1U/. Mu.L protease HRV 3C was added, and an overnight reaction was carried out at 4 ℃ to cleave the N-terminal tag 6 XHis of the target protein His-BsaI, thereby obtaining a His-tag cleaved target protein labeled as the target protein BsaI.

The BsaI protein of interest was further purified by gel chromatography using an AKTA (GE healthcare) protein automatic purifier (column type: hiload 26/600 su)perdex 75 pg) and eluted with an elution buffer (20mM Tris,500mM NaCl,1mM DTT, pH 7.5), the purity of the eluted sample is verified by SDS-PAGE, the absorbance value is measured by A280nm after concentration, and the molar extinction coefficient (A) of the BsaI protein is combined ^1mg/ml _280nm = 1.5) final protein concentration to obtain purified BsaI protein. The purified BsaI protein was supplemented with glycerol to a final concentration of 10% and stored at-80 ℃ for further use.

FIG. 8 is an SDS-PAGE pattern of BsaI protein purified by gel chromatography in this example. Wherein M is Marker; bsaI is BsaI protein purified by gel chromatography.

As can be seen from FIG. 8, bsaI protein was obtained in high purity after purification by gel column chromatography, and the final concentration of BsaI protein was 0.45mg/mL.

3. Enzyme activity assay of purified restriction enzyme BsaI

The enzyme activity of the desalted and concentrated protein (purified protein BsaI) obtained in step 2 of this example was determined by enzyme digestion. The enzyme digestion experiment comprises the following steps: the purified protein BsaI was diluted with a buffer (20mM Tris,500mM NaCl,1mM DTT, pH 7.5) to give a diluted solution having a concentration of 0.45X 10 ^-1 mg/mL、0.45×10 ^-2 mg/mL、0.45×10 ^-3 mg/mL and 0.45X 10 ^-4 mg/mL, 1. Mu.g of plasmid pJL1_ sfGFP (containing a BsaI cleavage site) as a substrate, 20. Mu.L of a reaction system (BsaI 5. Mu.L, 10 Xbuffer (NEB) 2. Mu.L), reacted at 37 ℃ for 1 hour, and then the cleavage of the substrate was detected by 1% agarose electrophoresis.

Positive control group: the purified protein BsaI from the experimental group was exchanged for commercial BsaI (purchased from NEB), and subjected to digestion and electrophoresis.

Negative control group: and removing the BsaI reaction system on the basis of the experimental group, and carrying out enzyme digestion and electrophoresis.

The definition of enzymatic activity, i.e. specific activity, is: the required amount of enzyme was able to digest 1. Mu.g of linear DNA template (containing 1 corresponding restriction enzyme site) within 1h in a total volume of 20. Mu.L of reaction system at 37 ℃.

FIG. 9 shows the commercial BsaI plasmid pJL1_ sf in this exampleElectrophorograms of the cleavage experiments were performed with GFP and different concentrations of BsaI protein. Where NC represents plasmid pJL1_ sfGFP, PC represents commercial BsaI, and 1 represents concentration of 0.45X 10 ^-1 mg/mL BsaI, 2 represents 0.45X 10 ^-2 mg/mL BsaI,3 represents 0.45X 10 ^-3 mg/mL BsaI,4 represents 0.45X 10 ^-4 mg/mL BsaI。

As can be seen from FIG. 9, the specific activity of the purified protein BsaI obtained in this example after affinity chromatography and gel chromatography was 2.2X 10 ³ ～2.2×10 ⁴ U/mg。

The invention provides a linear DNA template suitable for cell-free synthesis of restriction endonuclease BsaI, which can be suitable for any in vitro cell-free protein system, can be smoothly expressed and can effectively prevent the linear DNA template from being degraded by nuclease; the preparation is simple and rapid, and the conventional plasmid construction is not needed, so the linear DNA template is adopted to carry out in-vitro cell-free protein synthesis, and the obtained restriction enzyme BsaI has high purity and high specific activity; the invention also provides a system suitable for synthesizing the restriction enzyme BsaI without the cell, which can improve the yield of the restriction enzyme BsaI synthesized without the cell and simultaneously improve the reaction rate of the restriction enzyme BsaI synthesis; the invention also provides a method for synthesizing the restriction enzyme BsaI without cells, the method well solves the problem of inhibiting the toxicity of the restriction enzyme BsaI to an expression host, the synthesis reaction time is short, the efficiency is high, the specific activity of the obtained restriction enzyme BsaI is similar to that of a commercial enzyme, and the method has wide application prospect. In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention is not limited to those specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

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Sequence listing

<110> Shanghai science and technology university

<120> a linear DNA template, system and use thereof suitable for cell-free synthesis of restriction enzyme BsaI

<160> 8

<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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atgggcaaaa aagcggaata tggccaaggc catccgattt ttctggaata tgcggaacag 60

attattcagc ataaagaata tcaaggcatg ccggacctgc gctacccgga cggccgcatt 120

cagtgggaag cgccgagcaa ccgcaaaagc ggcattttta aagataccaa cattaaacgc 180

cgcaaatggt gggaacagaa agcgatcagc attggcattg atccgagcag caatcagtgg 240

attagcaaaa ccgcgaaact gattcatccg accatgcgca aaccgtgcaa aaaatgcggc 300

cgcattatgg atctgcgcta tagctatccg accaaaaacc tgattaaacg cattcgcaaa 360

ctgccgtatg tggatgaaag ctttgaaatt gatagcctgg aacatattct gaaactgatt 420

aagcgcctgg tgctgcagta tggcgataaa gtgtatgatg atctgccgaa actgctgacc 480

tgcaaagcgg tgaaaaacat tccgcgcctg ggcaacgatc tggatacctg gctgaactgg 540

attgatagcg tgtatattcc gagcgaaccg agcatgctga gcccgggcgc gatggcgaac 600

ccgccggatc gcctggatgg ctttcatagc ctgaacgaat gctgccgcag ccatgcggat 660

cgcggccgct gggaaaaaaa cctgcgcagc tataccaccg atcgccgcgc gtttgaatat 720

tgggtggatg gcgattgggt ggcggcggat aaactgatgg gcctgattcg caccaacgaa 780

cagattaaaa aagaaacctg cctgaacgat aaccatccgg gcccgtgcag cgcggatcat 840

attggcccga ttagcctggg ctttgtgcat cgcccggaat ttcagctgct gtgcaacagc 900

tgcaacagcg cgaaaaacaa ccgcatgacc tttagcgatg tgcagcatct gattaacgcg 960

gaaaacaacg gcgaagaagt ggcgagctgg tattgcaaac atatttggga tctgcgcaaa 1020

catgatgtga aaaacaacga aaacgcgctg cgcctgagca aaattctgcg cgataaccgc 1080

cataccgcga tgtttattct gagcgaactg ctgaaagata accattatct gtttctgagc 1140

acctttctgg gcctgcagta tgcggaacgc agcgtgagct ttagcaacat taaaattgaa 1200

aaccatatta ttaccggtca gatcagcgaa cagccgcgcg ataccaaata taccgaagaa 1260

cagaaagccc gccgcatgcg cattggcttt gaagcgctga aaagctatat tgaaaaagaa 1320

aaccgcaacg cgctgctggt gattaacgat aaaattattg ataaaattaa cgaaattaaa 1380

aacattctgc aagatattcc ggatgaatat aaactgctga acgaaaaaat tagtgagcag 1440

tttaacagcg aagaagtgag cgatgaactg ctgcgcgatc tggtgaccca tctgccgacc 1500

aaagaaagcg aaccggcgaa ctttaaactg gcgcgcaaat atctgcaaga aattatggaa 1560

attgtgggcg atgaactgag caaaatgtgg gaagatgaac gctatgtgcg tcagaccttt 1620

gcggatctgg attaa 1635

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Gly Lys Lys Ala Glu Tyr Gly Gln Gly His Pro Ile Phe Leu Glu

1 5 10 15

Tyr Ala Glu Gln Ile Ile Gln His Lys Glu Tyr Gln Gly Met Pro Asp

20 25 30

Leu Arg Tyr Pro Asp Gly Arg Ile Gln Trp Glu Ala Pro Ser Asn Arg

35 40 45

Lys Ser Gly Ile Phe Lys Asp Thr Asn Ile Lys Arg Arg Lys Trp Trp

50 55 60

Glu Gln Lys Ala Ile Ser Ile Gly Ile Asp Pro Ser Ser Asn Gln Trp

65 70 75 80

Ile Ser Lys Thr Ala Lys Leu Ile His Pro Thr Met Arg Lys Pro Cys

85 90 95

Lys Lys Cys Gly Arg Ile Met Asp Leu Arg Tyr Ser Tyr Pro Thr Lys

100 105 110

Asn Leu Ile Lys Arg Ile Arg Lys Leu Pro Tyr Val Asp Glu Ser Phe

115 120 125

Glu Ile Asp Ser Leu Glu His Ile Leu Lys Leu Ile Lys Arg Leu Val

130 135 140

Leu Gln Tyr Gly Asp Lys Val Tyr Asp Asp Leu Pro Lys Leu Leu Thr

145 150 155 160

Cys Lys Ala Val Lys Asn Ile Pro Arg Leu Gly Asn Asp Leu Asp Thr

165 170 175

Trp Leu Asn Trp Ile Asp Ser Val Tyr Ile Pro Ser Glu Pro Ser Met

180 185 190

Leu Ser Pro Gly Ala Met Ala Asn Pro Pro Asp Arg Leu Asp Gly Phe

195 200 205

His Ser Leu Asn Glu Cys Cys Arg Ser His Ala Asp Arg Gly Arg Trp

210 215 220

Glu Lys Asn Leu Arg Ser Tyr Thr Thr Asp Arg Arg Ala Phe Glu Tyr

225 230 235 240

Trp Val Asp Gly Asp Trp Val Ala Ala Asp Lys Leu Met Gly Leu Ile

245 250 255

Arg Thr Asn Glu Gln Ile Lys Lys Glu Thr Cys Leu Asn Asp Asn His

260 265 270

Pro Gly Pro Cys Ser Ala Asp His Ile Gly Pro Ile Ser Leu Gly Phe

275 280 285

Val His Arg Pro Glu Phe Gln Leu Leu Cys Asn Ser Cys Asn Ser Ala

290 295 300

Lys Asn Asn Arg Met Thr Phe Ser Asp Val Gln His Leu Ile Asn Ala

305 310 315 320

Glu Asn Asn Gly Glu Glu Val Ala Ser Trp Tyr Cys Lys His Ile Trp

325 330 335

Asp Leu Arg Lys His Asp Val Lys Asn Asn Glu Asn Ala Leu Arg Leu

340 345 350

Ser Lys Ile Leu Arg Asp Asn Arg His Thr Ala Met Phe Ile Leu Ser

355 360 365

Glu Leu Leu Lys Asp Asn His Tyr Leu Phe Leu Ser Thr Phe Leu Gly

370 375 380

Leu Gln Tyr Ala Glu Arg Ser Val Ser Phe Ser Asn Ile Lys Ile Glu

385 390 395 400

Asn His Ile Ile Thr Gly Gln Ile Ser Glu Gln Pro Arg Asp Thr Lys

405 410 415

Tyr Thr Glu Glu Gln Lys Ala Arg Arg Met Arg Ile Gly Phe Glu Ala

420 425 430

Leu Lys Ser Tyr Ile Glu Lys Glu Asn Arg Asn Ala Leu Leu Val Ile

435 440 445

Asn Asp Lys Ile Ile Asp Lys Ile Asn Glu Ile Lys Asn Ile Leu Gln

450 455 460

Asp Ile Pro Asp Glu Tyr Lys Leu Leu Asn Glu Lys Ile Ser Glu Gln

465 470 475 480

Phe Asn Ser Glu Glu Val Ser Asp Glu Leu Leu Arg Asp Leu Val Thr

485 490 495

His Leu Pro Thr Lys Glu Ser Glu Pro Ala Asn Phe Lys Leu Ala Arg

500 505 510

Lys Tyr Leu Gln Glu Ile Met Glu Ile Val Gly Asp Glu Leu Ser Lys

515 520 525

Met Trp Glu Asp Glu Arg Tyr Val Arg Gln Thr Phe Ala Asp Leu Asp

530 535 540

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<213> Artificial Sequence (Artificial Sequence)

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ttagaggtcc tgtttcaggg acctatgggc aaaaaagcgg aa 42

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tgttagcagc cggtcgactt aatccagatc cgcaaa 36

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<212> DNA

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ccctgaattg actctct 17

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aggtccctga aacaggacct ctaacatatg gtgatgatga tg 42

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tcgggctttg ttagcagccg g 21

<210> 8

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ggttgagtgt tgttccagt 19

Claims

1. The linear DNA template suitable for cell-free synthesis of the restriction enzyme BsaI is characterized by sequentially comprising a fragment containing a promoter, an encoding gene of the restriction enzyme BsaI and a fragment containing a terminator, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No.1, and the linear DNA template does not contain the enzyme cutting site of the restriction enzyme BsaI.

2. The linear DNA template of claim 1, comprising at least one of the following technical features:

a1 The promoter-containing fragment includes a promoter and a promoter upstream sequence; preferably, the promoter in the promoter-containing fragment is T7;

a2 The terminator-containing fragment includes a terminator and a terminator downstream sequence; preferably, the terminator in the terminator-containing fragment is T7;

a3 The linear DNA template further contains a tag located upstream or downstream of the encoding gene, the tag being operably linked to the encoding gene; preferably, the tag is selected from one or more of a 6 × His tag, a SUMO tag, an MBP tag, a Strep-tag and a Cold-TF factor tag.

3. The method of constructing a linear DNA template according to claim 1 or 2, comprising the steps of:

1-2) taking a plasmid containing a promoter as a template, and amplifying to obtain the fragment containing the promoter; amplifying to obtain a fragment containing the terminator by taking a plasmid containing the terminator as a template;

1-3) fusing the coding gene obtained in the step 1-1), the fragment containing the promoter obtained in the step 1-2) and the fragment containing the terminator by a fusion PCR method to obtain the linear DNA template.

4. Use of the linear DNA template of claim 1 or 2 in the construction of a synthesis system suitable for the cell-free restriction enzyme BsaI.

5. A system for the cell-free synthesis of the restriction enzyme BsaI, characterised in that it comprises a linear DNA template according to claim 1 or 2.

6. The system of claim 5, further comprising a cell extract capable of expressing the linear DNA template.

7. The system of claim 6, wherein the cell extract is selected from the group consisting of an E.coli extract; preferably, the Escherichia coli is selected from one or more of BL21 Star (DE 3), BL21 Star (DE 3) + pG-KJE, BL21 Star (DE 3) + pGro7, BL21 Star (DE 3) + pG-Tf2, rosetta + pG-KJE, rosetta + pGro7, rosetta + pG-Tf 2.

8. Use of a linear DNA template according to claim 1 or 2 or of a system according to any one of claims 5 to 7 for the synthesis of the restriction enzyme BsaI.

9. A synthetic method of restriction enzyme BsaI is characterized by comprising the following steps:

performing a synthesis reaction using at least the linear DNA template of claim 1 or 2 or the system of any one of claims 5-7 to obtain the restriction enzyme.

10. The method of claim 9, wherein the temperature of the reaction is 15 to 37 ℃;

and/or the reaction time is 2-8 h.