CN118562766A

CN118562766A - Restriction endonuclease and preparation method and application thereof

Info

Publication number: CN118562766A
Application number: CN202410757098.9A
Authority: CN
Inventors: 李艳艳; 张利存
Original assignee: Nucleosome Beijing Biotechnology Co ltd
Current assignee: Nucleosome Beijing Biotechnology Co ltd
Priority date: 2024-06-12
Filing date: 2024-06-12
Publication date: 2024-08-30

Abstract

The application relates to a restriction enzyme, a preparation method thereof and application thereof in enzyme cutting of DNA fragments. The preparation method of the restriction enzyme comprises the following steps: preparing a nucleic acid construct encoding a restriction enzyme and hfq chaperone protein, the restriction enzyme being ApalI protease; introducing the nucleic acid construct as an exogenous nucleic acid into a host cell, culturing the host cell such that the host cell expresses the restriction enzyme; the host cells were disrupted and the restriction enzymes were collected. Compared with the existing ApalI protease preparation process, the correct folding rate and enzyme yield of ApalI protease can be improved.

Description

Restriction endonuclease, preparation method and application thereof

Technical Field

The application relates to the technical field of bioengineering, in particular to a restriction enzyme and a preparation method and application thereof.

Background

Restriction enzymes have high fidelity, recognize cleavage of specific target sequences, and can be used not only for conventional molecular cloning, but also for methylation status analysis, SNP detection and gene expression sequence analysis. The existing production process of type II restriction enzyme, such as ApalI restriction enzyme, has the problems of low yield and incapability of meeting industry requirements, both in low-density shake flask culture and large-scale fermentation in a fermentation tank.

Therefore, how to increase the yield of restriction enzymes is a problem to be solved by those skilled in the art.

Disclosure of Invention

In order to improve the yield of restriction enzymes, the application provides a restriction enzyme, a preparation method and application thereof.

The preparation method of the restriction enzyme provided by the invention for realizing the purpose comprises the following steps:

Preparing a nucleic acid construct encoding the restriction enzyme and hfq chaperone protein, the restriction enzyme being ApalI protease;

Introducing the nucleic acid construct as an exogenous nucleic acid into a host cell, culturing the host cell such that the host cell expresses the restriction enzyme;

Disrupting the host cell and collecting the restriction enzyme.

According to another aspect of the present application there is provided a restriction enzyme prepared by the method of preparing a restriction enzyme as described above.

According to another aspect of the present application there is provided the use of a restriction enzyme for cleaving a target sequence in a DNA fragment, the restriction enzyme being prepared by the method for preparing a restriction enzyme as described above.

Compared with the existing production and preparation process of the restriction enzyme ApalI protease, the application can improve the correct folding rate of the ApalI protease and the enzyme yield.

Drawings

FIG. 1 is a process flow diagram showing a method for producing a restriction enzyme according to an embodiment of the present application;

FIG. 2 shows an electrophoresis pattern of a wild-type ApalI protease and a restriction enzyme provided in the present application after heat treatment at 65℃and 95℃in example 2;

FIG. 3 is a graph showing the statistics of the gray scale values of agarose gel electrophoresis of the restriction enzymes provided in the present application in example 3, which cut at different enzyme dosages;

FIG. 4 shows agarose gel electrophoresis of plasmid pUC19 of example 4 subjected to restriction enzymes provided by the present application at various times;

FIG. 5 shows the protein electrophoresis pattern of restriction enzymes stored at 25℃and 4℃and-20℃for three months, respectively, in example 5.

FIG. 6 shows agarose gel electrophoresis of plasmid pUC19 digested with restriction enzymes stored at 25℃and 4℃and-20℃for three months in example 5.

Detailed Description

Restriction enzymes have high fidelity, recognize cleavage of specific target sequences, and can be used not only for conventional molecular cloning, but also for methylation status analysis, SNP detection and gene expression sequence analysis. In 1978, restriction enzymes were discovered and characterized, and thus, the nobel prize was obtained, which has raised the hot spot for studying restriction enzymes. However, over time, the rate at which the restriction enzyme is found gradually decreases. Currently, naturally occurring restriction enzymes have been difficult to find. This has presented the task of synthesizing restriction enzymes with new recognition sites based on existing restriction enzymes.

Among the restriction enzymes known so far, the type II restriction modification system is the largest and most diverse, and is dominant in life-based science. Typical type IIP restriction enzymes recognize the palindromic sequence of 4-8bp as a dimer and cleave the sequence within the duplex. The structure of the restriction enzyme shows a common structural core, comprising four beta sheets and one alpha helix.

However, the existing production process of the type II restriction enzyme of the enterprise still stays at the stage of low-density shake flask culture, has low yield and high cost, and cannot meet the industry requirements. After the shake flask fermentation process parameters are directly transplanted to the fermentation tank, the yield of almost all restriction enzymes is reduced, and few types cannot be expressed successfully.

Thus, according to one aspect of the present application there is provided a method of preparing a restriction enzyme comprising the steps of:

S100, preparing a nucleic acid construct encoding a restriction enzyme and hfq (host factor for RNA PHAGE Q beta replicase) chaperone protein, wherein the restriction enzyme is ApalI protease;

s200, introducing the nucleic acid construct into a host cell as an exogenous nucleic acid, and culturing the host cell to cause the host cell to express the restriction enzyme;

S600, crushing the host cells and collecting the restriction enzymes.

Chaperones, also called chaperones, help other polypeptide-containing structures to complete their correct assembly within the cell and to separate from it after assembly, and do not constitute components of these protein structures when they perform their functions. The main functions are to assist folding/deconvolution and assembly/disassembly of other macromolecular structures, and can effectively prevent the formation of incorrect folding intermediates and the incorrect aggregation of unassembled protein subunits, and assist the transmembrane transport of polypeptide chains and the assembly and disassembly of large multi-subunit proteins.

Wherein, hfq is an RNA molecular chaperone protein, which can influence the translation level and the stability of mRNA by participating in the interaction process of non-coding RNA and target mRNAs thereof, mainly by promoting the base pairing of sRNA and mRNA, thereby playing the role of regulating and controlling to assist the correct folding of ApalI protease and improving the correct folding rate of ApalI protease, so as to improve the yield of ApalI protease.

According to the application, the nucleic acid construct is introduced into the host cell, and in the culture process of the host cell successfully introducing the nucleic acid construct, the nucleic acid construct is transcribed, translated and expressed into hfq molecular chaperones and ApalI protease, so that the content of hfq in the host cell can be increased, the base pairing of sRNA and mRNA can be promoted, the translation level of ApalI protease and the stability of mRNA can be improved, and correct folding of ApalI protease can be assisted, thereby improving the expression quantity of ApalI protease. Compared with the existing production and preparation process of the restriction enzyme ApalI protease, the application can improve the yield of ApalI protease.

In one possible implementation, the nucleic acid construct has a nucleotide coding sequence set forth in SEQ ID No:1, the corresponding amino terminal sequence is shown as SEQ ID No: 2.

In one possible implementation, the nucleic acid construct includes an inducer, and after the nucleic acid construct is introduced into the host cell in S200, the cultured host cell is expanded in S500, and the inducer is added and the cultured host cell is continued to be induced to induce the host cell to express the restriction enzyme. Further, the culture time of the induction culture is 8 to 10 hours.

In one possible implementation, constructing the nucleic acid construct comprises the steps of:

designing a primer pair Hfq-ApalI-F and Hfq-ApalI-R according to a chaperone protein Hfq gene sequence;

the primer pair Hfq-ApalI-F and Hfq-ApalI-R are amplified by PCR, the Hfq-ApalI fusion fragment is recovered by agarose gel purification and PCR product recovery kit, the Hfq-ApalI gene target fragment is cloned into a plasmid vector pET-15b to construct a pET-15b-SUMO-Hfq-ApalI expression vector, and the pET-15b-SUMO-Hfq-ApalI expression vector is used as a nucleic acid construct to be introduced into host cells.

Further, the introduction of the nucleic acid construct into the host cell specifically comprises the steps of:

The host cells were competent cells of the expression strain ER2566, 1. Mu.L of the plasmid vector which was constructed in the construction step and verified to be correct by experimental sequencing was added to the competent cells of ER2566, ice-bath for 5min, followed by heat shock at 42℃for 45s, and then ice-bath for 5min. Placing in a constant temperature shaking table for shake culture, wherein the culture conditions are as follows: 37℃at 200rpm for 10min.

Further, the nucleic acid construct, i.e., the plasmid vector, is loaded with an antibiotic resistance marker gene. After the nucleic acid construct is introduced into the host cell in S200, a step of selecting a host cell for resistance in S300, and selecting a host cell having resistance is further included, followed by culturing the host cell having resistance in S400, S600 and subjecting it to disruption treatment to collect the restriction enzyme. The antibiotic resistance marker is preferably an ampicillin (hereinafter abbreviated as Amp) resistance marker.

The steps of the resistance screening are specifically as follows: host cells obtained by shake culture after transformation are evenly coated on an Amp-resistant solid LB culture plate, inverted and cultured for 12 hours at 37 ℃. Host cells into which the nucleic acid construct has not been successfully introduced are not Amp resistant and cannot survive; the host cells into which the nucleic acid construct was successfully introduced were resistant to the antibiotic resistance marker gene loaded, and were able to grow on solid LB plates containing Amp. Monoclonal colonies on solid LB plates were picked as resistant host cells from the resistance screen.

Specific steps for culturing a resistant host cell are:

Selecting monoclonal colonies on a solid LB culture plate, inoculating to 5mL of liquid LB culture medium, (in the process of expression culture, corresponding antibiotics can be added into the culture medium to reduce adverse effects of mixed bacteria growth on the purity and yield of restriction enzymes, for example, 5 mu L of Amp with the concentration of 100mg/mL is added into 5mL of liquid LB culture medium), and placing the inoculated liquid LB culture medium into a constant-temperature shaking table for shake culture, wherein the culture conditions are as follows: 37℃at 200rpm for 10h.

And (3) amplifying culture: all 5mL of bacterial liquid after shake culture is inoculated into 1L of liquid LB culture medium of a culture bottle (1 mL of Amp with the concentration of 100mg/mL can be added into 1L of liquid LB culture medium), and the culture medium is placed into a constant temperature shaking table for shake culture, wherein the culture conditions are as follows: the culture was carried out at 37℃and 200rpm until the OD ₆₀₀ was about 0.8 (culture time: about 3.5 hours).

Induction culture: the flask in the shaker was taken out, 0.5mL (final concentration: 0.5 mM) of a total of 1M IPTG solution was added thereto, and the culture medium to which IPTG had been added was put in the shaker for shake culture under the conditions of: 16 ℃,180rpm, 8-10 h.

In the process of collecting and purifying the target protein, the purification method is also subjected to process optimization. In the method for purifying protease, firstly, the impurity protein in the supernatant is removed, then the target protein is precipitated by using polyethylenimine and an organic solvent, and finally, the target protein is finally obtained by a suspension solution and resuspension method. The method has the main functions of improving the purity of the target protease and improving the economic benefit.

In one possible implementation, the host cells are cultivated by fermentation using a liquid medium;

Disruption of the host cell comprises the steps of: and (3) centrifuging the fermentation liquid to obtain precipitate thalli, adding a lysate to the precipitate thalli subjected to primary centrifugation for resuspension, adding 0.05% -5%, preferably 0.15% of polyethyleneimine solution to the resuspension solution, centrifuging again to obtain precipitate, and collecting restriction endonuclease in the precipitate subjected to secondary centrifugation.

Polyethyleneimine is a cationic polyamine polymer, and rich amino groups of polyethyleneimine can participate in capturing metal ions through chelation, ion exchange and the like, so that the effect of replacing target proteins is achieved. The invention mainly uses the action of amino groups of polyethyleneimine combined with anionic proteins to complete cross-linking polymerization with hydroxyl groups, and finally removes His labels through elution to obtain target proteins.

Proteins are relatively unstable in disrupting host cells in lysates, and protease inhibitors (PI, protease Inhibitors) need to be added to the lysates in order to increase the stability of the target proteases when disrupting cells. However, PI damages the properties of proteins to some extent, has a certain toxicity, can be used only in a small amount, and has a limitation on the stability protection effect of proteins. The application provides a novel way of lysing and disrupting host cells: on the premise of ensuring the stability of the protein, the concentration of the protein in the lysate is improved by utilizing sodium ions added in the lysate and adding a certain proportion of polyethyleneimine, and the combination of His tag to the protein is increased in the subsequent purification, so that the concentration of the target protease is improved.

In conclusion, the polyethyleneimine can be combined with anionic proteins, has the effect of resisting protein pollution, is beneficial to improving the purification effect, and has wide application prospect in the aspect of separating and purifying proteins.

The polyethyleneimine solution added to the pellet resuspension solution once centrifuged in the step of disrupting host cells means a reaction solution containing polyethyleneimine. In addition, in order to study the optimal concentration of the polyethyleneimine, the invention designs several reaction solutions with different polyethyleneimine concentrations to respectively carry out experiments. The final concentrations of polyethylenimine in the reaction solutions were varied from one experiment to the next, and the sodium ion concentration was 500mM. Each set of experiments was performed according to the following procedure:

Group 1 reaction solution composition: 20mM Tris-HCl (pH=7.5), 500mM NaCl solution, 5% glycerol. Group 2 reaction solution composition: 20mM Tris-HCl (pH=7.5), 500mM NaCl solution, 5% glycerol, 0.05% polyethylenimine. Group 3 reaction solution composition: 20mM Tris-HCl (pH=7.5), 500mM NaCl solution, 5% glycerol, 0.1% polyethylenimine. Group 4 reaction solution composition: 20mM Tris-HCl (pH=7.5), 500mM NaCl solution, 5% glycerol, 0.15% polyethylenimine. Group 5 reaction solution composition: 20mM Tris-HCl (pH=7.5), 500mM NaCl solution, 5% glycerol, 0.2% polyethylenimine. Group 6 reaction solution composition: 20mM Tris-HCl (pH=7.5), 500mM NaCl solution, 5% glycerol, 0.25% polyethylenimine.

In the step of disrupting host cells, the pellet resuspension solution of one centrifugation is subjected to ultrasonication. In the process of ultrasonic crushing, the resuspension solution (bacterial liquid) is always placed in ice bath, and the reaction liquid containing polyethyleneimine is slowly dripped into the resuspension solution while stirring. Then crushing treatment such as secondary centrifugation and separation and purification treatment such as chromatography are carried out. The concentration of polyethylenimine in the reaction solution is different in each group of experiments, and the other process conditions are kept consistent. The final purified proteins of each group were subjected to concentration determination, and the experimental results are shown in the following table:

The experimental results show that: when the concentration of polyethyleneimine in the reaction solution is 0.15%, the obtained target protein has the highest concentration and the highest protein purification efficiency. The amino group of the polyethyleneimine with a certain concentration is combined with the anion ApalI protein to complete the cross-linking polymerization with the hydroxyl, and finally the purer target protein can be obtained. In addition, if the concentration of polyethyleneimine exceeds 0.2%, the ability to entrap anionic proteins is affected, thereby reducing the concentration of proteins.

Further, the conditions of one centrifugation are: centrifuge at 10000rpm for 20min at 4 ℃.

The ratio of adding the lysate to the primary centrifuged precipitated cells for resuspension is as follows: 20 to 40mL of the lysate per 1L of the precipitated cell, preferably 30mL of the lysate per 1L of the precipitated cell. The vessel containing the resuspension solution was placed on ice to resuspension the solution in an ice bath. To the ice bath of the heavy suspension solution added 5% polyethylenimine solution, while stirring the heavy suspension solution drop by dropping the polyacetrimide solution, until the final concentration of the polyacetrimide (in the heavy suspension solution and the mixed solution of the polyacetrimide) is 0.15%. The mixture of the resuspension solution and the polyacetylimine was stirred for a further 20min. The conditions for the secondary centrifugation were: centrifuging at 10000rpm for 30min at 4deg.C, and removing the supernatant.

In one possible implementation, the specific steps of collecting the restriction enzyme in the pellet of the secondary centrifugation are: the secondary centrifuged cell pellet was washed with PBS solution, and the solvent was replaced. Specifically: the secondary centrifuged bacterial pellet is resuspended by using PBS solution, the obtained primary washing heavy suspension is centrifuged at 4 ℃ and 10000rpm for 2-5 min, and the supernatant is discarded to obtain the primary washing bacterial pellet. The number of times of washing the secondary centrifuged cell pellet with PBS is not particularly limited, but is preferably 2 to 4 times. Taking the primary cleaning thallus sediment as a secondary cleaning raw material, specifically, re-suspending the primary cleaning thallus sediment by using PBS solution, centrifuging at 4 ℃ and 10000rpm for 2-5 min, and taking the sediment as the secondary cleaning thallus sediment. The remaining several washes and so on. In addition, the solvent may be replaced with the remaining washing solution or chromatography buffer.

After the washing is finished, the chromatographic buffer solution can be used for resuspending the bacterial precipitate for N times (the last time) to be used as a stock solution for chromatographic purification, so that the obtained restriction enzyme has higher concentration and purity. The chromatographic buffer used for the bacterial cell precipitate obtained by the resuspension washing is potassium phosphate buffer comprising 0.2M KCl, beta-mercaptoethanol and EDTA, the precipitate is washed twice again by the chromatographic buffer (resuspension, centrifugation, supernatant taking, precipitate resuspension, centrifugation, supernatant taking again, and supernatant mixing twice to obtain a chromatographic purified stock solution), and the target protein is dissolved in the supernatant of the buffer.

And (3) chromatographic purification: the nucleic acid construct comprises a chromatographic binding tag, such as his tag commonly used in nickel column chromatography, and the like, and the elution is carried out by separating through nickel column, phosphocellulose and/or DEAE cellulose chromatography, and the eluted solution is a purer restriction enzyme solution. On the one hand, the property and activity of the protein can be stabilized, and in addition, the target protein can be separated from a complex protein mixture, and the purity is higher.

The restriction enzyme solution obtained by chromatography purification can be directly put into use or stored for standby. The preservation steps of the restriction enzyme are specifically as follows: the protease obtained by the chromatographic purification is dissolved in 50. Mu.L of a protein preservation solution, and the final concentration of the restriction enzyme in the protein preservation solution is 0.1-1mg/ml, preferably 0.5mg/ml or 0.8mg/ml. The protein preservation solution containing the restriction enzyme is preserved in a refrigerator at-80 ℃.

In one possible implementation, the lysate contains 50mM NaH ₂PO₄, 300mM NaCl, 10mM imidazole, 10% glycerol, 0.1% TrionX-100, 0.5mM PMSF and 2mg/ml lysozyme are added to the lysate before use. The PBS solution included 20mM Na ₂HPO₄, 20mM NaH ₂PO₄, 140mM NaCl. Chromatography buffers included beta-mercaptoethanol, EDTA, potassium phosphate buffer and KCl. The protein stock solution contained 0.1mM EDTA, 25mM Tris-HCl, 250mM NaCl, 0.2% NP-40, 50% glycerol, 0.2% Tween-20, and fresh 2mM DTT solution was added before the protein stock solution was used.

According to another aspect of the present application there is provided the use of a restriction enzyme in the enzymatic cleavage of a target sequence in a DNA fragment, the restriction enzyme being prepared by the method for preparing a restriction enzyme as described above.

In one possible implementation, the application of the restriction enzyme in the digestion of the target sequence in the DNA fragment, i.e. the method of using the restriction enzyme, comprises the steps of: the restriction enzyme provided by the application is added into a liquid reaction system containing DNA fragments, wherein the DNA fragments comprise the enzyme digestion target sequence of ApalI protease.

In one possible implementation, the restriction enzyme is added to the liquid reaction system containing the DNA fragment at 25-65℃and preferably 37 ℃.

Example 1: method for researching preparation and yield of recombinant protein type restriction enzyme

The preparation method comprises the following steps: primer pairs ApalI-F and ApalI-R are designed according to the wild ApalI protease gene sequence, and primer pairs Hfq-ApalI-F and Hfq-ApalI-R are designed according to the chaperonin Hfq gene sequence. ApalI-F has the sequence shown in SEQ ID No: 3. ApalI-R has the sequence shown in SEQ ID No: 4. The sequence of Hfq-ApalI-F is shown as SEQ ID No: shown at 5. The sequence of Hfq-ApalI-R is shown as SEQ ID No: shown at 6.

Amplifying the primer pair Hfq-ApalI-F and Hfq-ApalI-R by PCR, recovering the Hfq-ApalI fusion fragment by agarose gel purification and PCR product recovery kit, cloning the Hfq-ApalI gene target fragment into a vector pET-15b, and constructing to obtain a pET-15b-SUMO-Hfq-ApalI expression vector;

And (3) transforming the expression strain through a fusion type Hfq-ApalI expression vector, carrying out induced expression and purifying to obtain the recombinant protein type restriction enzyme. The steps of transforming the expression strain and inducing expression of the fusion type Hfq-ApalI expression vector are as follows:

Transformation of the expression strain: sucking 1 mu L of recombinant plasmid which is sequenced correctly in the previous experiment, adding the recombinant plasmid into a prepared competent cell expression strain ER2566 taken out of an ultralow temperature refrigerator at-80 ℃, carrying out heat shock for 45s at 42 ℃ after ice bath for 5min, then carrying out shake culture in a constant temperature shaking table for 5min, and culturing the recombinant plasmid under the conditions: 37℃at 200rpm for 10min. The transformed competent cells were all uniformly spread on Amp-resistant solid LB culture plates, inverted at 37 ℃ and cultured for 12h.

Culturing of the bacterial cells: monoclonal colonies on a solid LB culture plate are picked out in an ultra-clean workbench, 5mL of liquid LB culture medium is added, (5 mu L of ampicillin with the concentration of 100mg/mL is added), shake culture is carried out in a constant-temperature shaking table, and culture conditions are as follows: all 5mL of bacterial liquid after shake culture is inoculated into 1L of liquid LB medium (1 mL of ampicillin with the concentration of 100mg/mL is added) of a culture flask at 37 ℃ and 200rpm for 10h, and the culture conditions are that the bacterial liquid is placed into a constant temperature shaking table for shake culture: culturing at 37deg.C and 200rpm until OD ₆₀₀ is about 0.8 (about 3.5 h), taking out the flask from the shaker, adding 0.5mL (final concentration of 0.5 mM) of 1M IPTG solution, and shake culturing the medium with added IPTG in the shaker under the following conditions: and (3) at 16 ℃ and 180rpm, and inducing expression for 8-10 h.

After the induction, the bacteria were collected at 4℃and 4000rpm for 20min each. Adding 30mL of lysate into 1L of bacterial liquid, adding the lysate to thoroughly resuspend the bacterial liquid, placing on ice, slowly dripping 5% polyethylenimine until the concentration is 0.15% while stirring in the ice bath process, stirring for 20min, centrifuging at 4 ℃ and 10000rpm for 30min, collecting precipitate, and carefully discarding the supernatant. The collected precipitate is washed twice with PBS solution for 2-5 min each time. Finally, the obtained restriction enzyme is suspended by using a chromatographic buffer solution, so that the concentration and purity of the obtained restriction enzyme are ensured to be higher. Next, the pellet was resuspended in chromatography buffer, which required the addition of 0.2M KCl, beta-mercaptoethanol, EDTA, potassium phosphate buffer, and washed twice with the chromatography buffer and the protein of interest was dissolved in the supernatant of the buffer.

The next step is to purify the protein in the supernatant, mainly by a nickel column and eluting. In this process, DEAE cellulose chromatography may be used to separate and complete elution, and finally the protein may be dissolved in 50. Mu.L of protein preservation solution and preserved at-80 ℃.

Example 2: investigation of the thermostability of recombinant protein type restriction enzymes (i.e., restriction enzymes provided by the present application)

The optimum temperature for the reaction of a restriction endonuclease is typically at most 37 ℃. However, the optimum temperature for a part of the enzymes is 25℃or 65℃and the enzyme efficiency is reduced by 50% at this temperature although the enzymes still exhibit activity at 37 ℃. The enzyme prepared from the thermophilic bacteria needs to react at a temperature of 37℃or higher. In order to detect the heat resistance of the recombinant protein type restriction enzyme, the experiment detects the heat resistance of the wild type ApalI protease and the recombinant protein type restriction enzyme after heating at 65 ℃ for 2 hours and at a high temperature of 95 ℃ for 1 hour respectively. The results are shown in FIG. 2, lanes 1-4 of FIG. 2 are shown as: a protein map of wild-type ApalI protease heated at 65℃for 2 hours, a protein map of recombinant protein type restriction enzyme heated at 65℃for 2 hours, a protein map of wild-type ApalI protease heated at 95℃for 1 hour, and a protein map of recombinant protein type restriction enzyme heated at 95℃for 1 hour. The recombinant protein type restriction endonuclease has certain heat resistance, more stable property, more beneficial to the activity preservation and transportation of protease preparations and reduced transportation and preservation cost.

Example 3: study of optimal conditions and amounts of recombinant protein type restriction endonucleases

Experiments were performed with commercially available restriction enzymes from NEW ENGLAND Biolabs, NEB for short, new England Biotechnology (Beijing) Inc., as a control group. In the environment of 37 ℃,1 mu L of commercial enzyme is added into 20 mu L of reaction system as a control group to carry out enzyme digestion reaction for 5min, and 10 mu L of digested reaction product is taken to carry out 1% agarose gel electrophoresis. In the environment of 37 ℃, adding the restriction enzyme provided by the application into 20 mu L (same as a control group) of a reaction system, performing enzyme digestion reaction for 5min, setting the using volumes of the restriction enzyme provided by the application in each experimental group to be 0.25 mu L, 0.5 mu L, 1.0 mu L, 1.5 mu L and 2.0 mu L respectively, taking 10 mu L of enzyme-digested reaction products, and performing 1% agarose gel electrophoresis, wherein the electrophoresis conditions are consistent with those of the control group. Agarose gel electrophoresis patterns were obtained and analyzed for different band gray values using ImageJ software. Wherein, the enzyme activity value of the NEB endonuclease of 1 mu L is 1u, and the enzyme dosage and the enzyme activity of the recombinant protein type restriction endonuclease are determined by comparing the enzyme cutting efficiency of the commercial NEB restriction endonuclease.

In this example, the most suitable amount of recombinant protein type restriction enzyme was tested by agarose gel electrophoresis, and the result of agarose gel electrophoresis was analyzed by ImageJ software for the gray value of the band after enzyme digestion corresponding to the amount of different enzyme, as shown in fig. 3, the abscissa in fig. 3 represents the amount of enzyme, and the ordinate represents the gray value. In FIG. 3, the control and the restriction enzymes added with 0.25. Mu.L, 0.5. Mu.L, 1.0. Mu.L, 1.5. Mu.L, and 2.0. Mu.L of the NEB restriction enzyme provided by the present application had the gradation values of 28458.882, 31231.054, 31307.418, 29547.175, 29738.589, and 22659.711 in this order, respectively, of 1. Mu.L of NEB restriction enzyme. According to the gray value result, selecting the input amount with clear band and no impurity band and the gray value closest to NEW ENGLAND Biolabs as the optimal input amount and calibrating the optimal enzyme dosage and enzyme activity of the recombinant protein type restriction enzyme.

The result shows that the optimal enzyme dosage and enzyme activity of the recombinant protein type restriction enzyme in a 20 mu L PCR amplification system are as follows: 0.25 mu L-0.88u;0.5 mu L-1.22u;1.0 mu L-0.67u; 1.5. Mu.L-0.68 u;2.0 mu L-0.73u. The recombinant protein type restriction enzyme has higher enzyme activity and better working performance when the optimal enzyme dosage of 0.5 mu L is added into a 20 mu L enzyme cutting system.

Example 4: research on optimal reaction conditions for achieving enzyme digestion effect of recombinant protein type restriction enzyme

In order to study the cleavage efficiency of the protease of the present application, the following experiment was designed. The plasmid pUC19 is used as a template, and the plasmid contains 3 'G/TGCAC' sequences, and three fragments with different sizes can be respectively cut by using recombinant protein type restriction endonuclease, namely about 2480bp, 1200bp and 960bp. The enzyme digestion temperature is 37 ℃ and the reaction time is 5min, 10min, 15min, 20min and 30min respectively; the results of the enzyme assay performed in a defined reaction buffer are shown in FIG. 4, lanes 1-6 are shown as: the agarose gel electrophoresis pattern of the plasmid pUC19 which is not digested, the agarose gel electrophoresis pattern of the plasmid pUC19 which is digested by the recombinant protein type restriction enzyme for 5min, the agarose gel electrophoresis pattern of the plasmid pUC19 which is digested by the recombinant protein type restriction enzyme for 10min, the agarose gel electrophoresis pattern of the plasmid pUC19 which is digested by the recombinant protein type restriction enzyme for 15min, the agarose gel electrophoresis pattern of the plasmid pUC19 which is digested by the recombinant protein type restriction enzyme for 20min, and the agarose gel electrophoresis pattern of the plasmid pUC19 which is digested by the recombinant protein type restriction enzyme for 30 min. The results show that: the protease after chromatographic purification provided by the application can complete enzyme digestion in 5min, has higher enzyme digestion efficiency, and has shorter enzyme digestion time than ApalI protease of commercial NEB, thus improving the experimental efficiency and further improving the function of restriction endonuclease.

Example 5: study of stability of recombinant optimized restriction Endonuclease

In this example, the recombinant protein type restriction enzyme after chromatographic purification was stored at room temperature of 25℃and at 4℃and at-20℃for several months, and the enzyme activity was detected. On the one hand, the recombinant protein type restriction enzyme stored for three months at 25 ℃,4 ℃ and-20 ℃ is respectively subjected to protein detection; on the other hand, the plasmid pUC19 was used as a template, and the cleavage conditions were set to 37℃for 5min; the results of the enzyme assays performed in a defined reaction buffer are shown in FIGS. 5 and 6, and lanes 1-3, respectively, are shown in FIG. 5: protein map of recombinant protein type restriction enzyme stored at 25℃and 4℃and-20℃for three months. Lanes 1-4, as shown in FIG. 6, are shown as: agarose gel electrophoresis of plasmid pUC19, agarose gel electrophoresis of plasmid pUC19 after digestion with recombinant protein type restriction enzyme at 25℃and 4℃and-20℃respectively. The results show that the recombinant protein type restriction enzyme stored at 25 ℃,4 ℃ and minus 20 ℃ still keeps activity and has better protein stability, and the recombinant protein type restriction enzyme is stored at room temperature for at least three months.

Claims

1. A method for preparing a restriction endonuclease, comprising:

preparing a nucleic acid construct encoding the restriction endonuclease and the hfq chaperone protein, wherein the restriction endonuclease is ApalI protease;

Introducing the nucleic acid construct as an exogenous nucleic acid into a host cell, and culturing the host cell to allow the host cell to express the restriction endonuclease;

The host cells are disrupted and the restriction endonuclease is collected.

2. The method for preparing a restriction endonuclease according to claim 1, wherein the coding sequence of the nucleic acid construct is as shown in SEQ ID No: 1.

3. the preparation method of restriction endonuclease according to claim 1 is characterized in that, described nucleic acid construct comprises inducible gene, after described nucleic acid construct is imported into host cell, amplification culture host cell, add inducing agent and continue inducing culture host cell, induce host cell to express described restriction endonuclease.

4. The method for preparing a restriction endonuclease according to claim 3, wherein the incubation time of the induction culture is 8 to 10 hours.

5. The method for preparing a restriction endonuclease according to claim 1, characterized in that the host cells are cultured by fermentation in a liquid culture medium;

The host cell disruption comprises the following steps: centrifuging the fermentation liquid to obtain precipitated bacterial cells, adding a lysis solution to the precipitated bacterial cells obtained by the first centrifugation to resuspend them, adding polyethyleneimine to the resuspended solution, centrifuging again to obtain precipitates, and collecting the restriction endonuclease in the precipitates obtained by the second centrifugation.

6. The preparation method of restriction endonuclease according to claim 1 is characterized in that said nucleic acid construct comprises an antibiotic resistance marker, after nucleic acid construct is imported into host cell, also comprises resistance screening host cell, screening obtains the step of resistant host cell, cultivates resistant host cell and carries out fragmentation processing and collects said restriction endonuclease.

7. The method for preparing a restriction endonuclease according to claim 6, wherein the antibiotic resistance marker is an ampicillin resistance marker.

8. A restriction endonuclease, characterized in that the restriction endonuclease is prepared by the preparation method of any one of claims 1 to 7.

9. An application of a restriction endonuclease, characterized in that the restriction endonuclease is used to cleave a target sequence in a DNA fragment, and the restriction endonuclease is prepared by the method for preparing the restriction endonuclease according to any one of claims 1 to 7.