CN112813088B - Preparation method of recombinant DpnI restriction enzyme - Google Patents
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Abstract
The invention provides a preparation method of recombinant DpnI restriction enzyme, which comprises the following steps: step one, synthesizing a coding gene of DpnI, constructing a prokaryotic expression vector, and obtaining a recombinant vector: the amino acid sequence of DpnI is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene of DpnI is obtained through adding a stop codon and codon optimization; transferring the recombinant vector into a methylase-deleted escherichia coli competent cell to obtain recombinant bacteria; step three, identifying the sequence correctness of the target fragment in the recombinant bacterium through PCR and sequencing; fourthly, carrying out DpnI restriction enzyme expression on the monoclonal with the correct target fragment; and fifthly, purifying the DpnI restriction enzyme. Compared with the DE3 lysogeny expression method of special strains in the prior literature, the strain material used in the method does not need special treatment, the operation flow is simplified, and the success rate is high.
Description
Technical Field
The invention belongs to the fields of molecular biology and cell engineering, and in particular relates to a preparation method of recombinant DpnI restriction enzyme.
Background
DpnI is a restriction enzyme in pneumococci (Diplococcus pneumoniae) and is characterised in that DpnI only cleaves when its recognition site is methylated (see FIG. 1). The GATC sequence exists widely on genomes of various species, and DpnI is a restriction enzyme type with high practicability in genetic and genetic engineering. Since the commonly used cloning and expression strains include DH5a, top10, BL21 (DE 3), BL21 (DE 3 plyS) and the like all contain endogenous methylases, the expression vector and the host genome are methylated, and a DpnI recognition site widely exists, so that the DpnI is used for cutting the host genome and the expression plasmid, and the DpnI is constructed by using a conventional T7 promoter as a vector, although the expression of the base protein in the expression strains such as BL21 (DE 3) and the like can be seriously inhibited by normal proliferation of the host bacteria and replication of the expression vector, so that the protein cannot be normally expressed.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a preparation method of DpnI restriction enzyme. Firstly, the DpnI protein coding sequence is synthesized by the gene, and then the gene is constructed on a vector taking a promoter (tac, lacUV, trc and the like) which can be identified by the self RNA polymerase of the escherichia coli as a heterologous gene expression promoter by a recombination or connection mode. The amplified ligation or recombinant molecular cloning reaction system product is directly transformed into JM110 (methylase deletion) strain (DH 5a or Top10 is not used as intermediate host), the grown monoclonal is identified, and the monoclonal with correct sequencing is selected for direct expression of DpnI. And carrying out affinity and gel chromatography to obtain high-purity protein.
The specific technical scheme is as follows: a method for preparing recombinant dpnl restriction enzyme, comprising the steps of: step one, synthesizing a coding gene of DpnI, constructing a prokaryotic expression vector, and obtaining a recombinant vector: the amino acid sequence of DpnI is shown as SEQ ID NO. 1 or a similar coding amino acid sequence with the similarity of 95% or more, and the nucleotide sequence of the coding gene of DpnI is obtained by adding a stop codon and codon optimization; transferring the recombinant vector into a methylase-deleted escherichia coli competent cell to obtain recombinant bacteria; step three, identifying the sequence correctness of the target fragment in the recombinant bacterium through PCR and sequencing; fourthly, carrying out DpnI restriction enzyme expression on the monoclonal with the correct target fragment; and fifthly, purifying the DpnI restriction enzyme.
Further, the prokaryotic expression vector is a vector of which the escherichia coli self-polymerase can recognize a promoter tac, lacUV or trc; the nucleotide sequence of the coding gene of DpnI is shown as SEQ ID NO. 2.
Further, the prokaryotic expression vector is pGEX-6p-1 vector; the methylase deleted E.coli was JM110.
Further, the first step is specifically: amplifying DpnI coding genes by using high-fidelity enzymes, simultaneously cutting pGEX-6p-1 vectors by BamHI and SalI restriction enzymes, and inserting the genes into pGEX-6p-1 by a homologous recombination mode; ensures the correct reading frame and takes GST protein as a purification tag.
Further, in the second step, 200-400 mu l JM110 competent cells and 40-80 mu l of recombination reaction products obtained by the recombination reaction system are mixed and transferred into a recombination vector to obtain recombinant bacteria;
40-80 μl of the recombination reaction system comprises: 200-400ng pGEX-6p-1 vector, 50-100ng DpnI encoding gene, 2-4. Mu.l Exnase recombinase and 8-16. Mu.l 5 Xbuffer.
Further, the second step is specifically: taking 200-400 μl JM110 competent cells out at-80deg.C, standing on ice for 10min, adding recombinant reaction product, slowly blowing, mixing, standing on ice for 15min, heating in a water bath at 42deg.C for 45s, taking out, standing on ice for 10min, adding 1ml LB, and culturing at 37deg.C for 1hr based on shaking table 220 rpm; finally 10000g of supernatant is removed by centrifugation, and thalli are uniformly coated on an ampicillin-resistant LB plate; the recombination reaction product is a product obtained by reacting 40-80 μl of the recombination reaction system at 37deg.C for 25 min;
the third step is as follows: selecting a monoclonal strain, culturing at 220rpm 37 ℃ in a 2.5ml test tube for 10hr, performing bacterial liquid PCR, and performing PCR reaction by using universal sequencing primers at two ends of a carrier multiple cloning site; sequencing the PCR identification of the correct monoclonal strain by censoring, and comparing the clone strain with the correct sequence; the fourth step is specifically as follows: the monoclonal is selected and inoculated into 10ml of LB culture medium with corresponding resistance of the vector, shake-cultured overnight, inoculated into 1L of LB shake flask with double antibody the next day, shake-cultured until OD 0.6-0.8 at 220rpm and induced by adding IPTG with final concentration of 0.5mM, and expressed for 21 hours.
Further, the fifth step is specifically: 10000rcf centrifugal force is used for centrifugation for 5min, bacterial cells are collected, a supernatant culture medium is removed, pre-cooled 30ml suspension bacterial cells containing 100mM KCl and 25mM Tris pH7.5 buffer solution are used, then high pressure 650bar is used for carrying out bacterial breaking for 5min, protease inhibitor is added into the suspension bacterial cells simultaneously, 14000rcf centrifugal force is used for removing sediment after the bacterial cells are simultaneously added into the suspension bacterial cells, corresponding affinity purification media are added into the supernatant for combination, specific site protease mediated on-column label cutting or direct elution without cutting is carried out, elution protein is subjected to AKTA mediated elution separation process, gel chromatography is carried out, and the purity of the obtained recombinant DpnI restriction endonuclease is determined.
Further, the method also comprises the step six of measuring the enzyme activity of the DpnI restriction enzyme.
Further, the sixth step is specifically: dpnI of different dilution ratios was added to the reaction system, 1. Mu.g of plasmid DNA was digested at 37℃for one hour, and finally the specific activity of the enzyme was confirmed by agarose electrophoresis.
Further, in the first step, the fusion expression protein constructed in the prokaryotic expression vector is one of MBP, GST, and 6×his.
In the second step, the methylase-deficient E.coli used is one of JM110 and ER 2925.
Though the strain sources such as JM110, ER2925 and the like can be used for obtaining the DpnI recombinant protein, the steps are complicated, the period is long and the success rate is low. The invention does not need to be dissolved and sourced, and has simpler operation. Compared with the DE3 lysogeny expression method of special strains in the prior literature, the strain materials used in the method are easier to obtain (commercially purchased), and the operation flow is simpler. The purity obtained by affinity purification using GST, MBP, etc. is higher and the purification procedure requires only two steps to pass through the column with little loss (Siwek, W. Et al 2012Nucleic acids research 40, 7563-7572; mierzejewska, K. Et al 2014Nucleic acids research 42, 8745-8754; gibson, D. G. Et al 2009Nature methods 6, 343-345).
The present invention will be further described with reference to the accompanying drawings, in order to fully explain the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 shows the cleavage sites of DpnI restriction enzyme.
FIG. 2 is a flowchart showing the operation of one embodiment of the method for producing a recombinant DpnI restriction enzyme according to the present invention.
FIG. 3 shows the monoclonal strain obtained in step 2 as identified by agarose gel electrophoresis.
The target amplified gene is about 850bp, and 8 selected monoclone are all correct cloning length.
FIG. 4 is a SDS-PAGE gel of DpnI restriction enzyme after affinity purification of a metal ion chelating agarose gel medium and further purification by gel chromatography.
After gel chromatography purification, the protein peak different effluent is sampled and run from the gel chart, and the purity reaches more than 95%. A is GST-DpnI; b is DpnI after GST cleavage.
FIG. 5 is a graph of agarose gel electrophoresis of a DpnI cut obtained by the present invention of a commercial pFastbac vector constructed with a fragment of interest. T represents restriction enzyme purchased from ThermoFisher Scientific.
Detailed Description
The reagents and consumables according to the invention are commercially available from Shanghai Baisai Biotechnology Co., ltd, except for the specific labels.
Example 1
In this example, the process flow of the preparation method of the recombinant DpnI restriction enzyme according to the present invention is shown in FIG. 2.
The amino acid sequence of DpnI is shown in SEQ ID NO. 1 or a similar coded amino acid sequence with the similarity of 95% or more. The nucleotide sequence of the coding gene obtained by adding the stop codon and optimizing the codon is shown as SEQ ID NO. 2, and the nucleotide sequence obtained by adding the stop codon and optimizing the codon can be different from the nucleotide sequence shown as SEQ ID NO. 1 under other specific embodiments. The coding gene of DpnI is synthesized according to the nucleotide sequence shown as SEQ ID NO. 2.
The construction to pGEX-6p-1 (Addgene) is described below as an example:
the DpnI coding gene is amplified by using high-fidelity enzyme, and the amplification primer pair is DpnI F (shown as SEQ ID NO: 3) and DpnI R (shown as SEQ ID NO: 4). Meanwhile, pGEX-6p-1 vector was digested with BamHI and SalI restriction enzymes, and then inserted into pGEX-6p-1 vector by homologous recombination (Gibson assembly method). Ensures the correct reading frame and takes GST protein as a purification tag.
And 2, transferring a product obtained by the recombination or ligation reaction into JM110 competent cells.
JM110 competent cells were purchased from the indigenous organism. Because JM110 has low transformation efficiency, the recombination reaction system needs to be enlarged by 2-4 times to obtain more positive clones, and taking recombination reaction (purchased from Vazyme) as an example:
the reaction conditions were 37℃for 25min.
To improve the efficiency of the transformation, 3 volumes of JM110 competent cells (300. Mu.l) were used under conventional conditions, and the competent cells were taken out, placed on ice for 10min, then added with the recombinant reaction product, gently blown and mixed, placed on ice for 15min, then heated in a 42℃water bath for 45s, taken out, placed on ice for 10min, and then added with 1ml of LB medium based on a shaking table 220rpm at 37℃for 1hr. Finally 10000g of the supernatant is removed by centrifugation, and the thalli are uniformly coated on an ampicillin-resistant LB plate.
The corresponding monoclonal strain is selected in a 2.5ml test tube, cultured for 10 hours at 220rpm and 37 ℃, bacterial solution PCR is carried out, and universal sequencing primers at two ends of a vector polyclonal site are used for carrying out PCR reaction, so that the result shown in figure 3 is obtained, and the primer pair is Verf F (shown as SEQ ID NO: 5) and Verf R (shown as SEQ ID NO: 6). And (3) delivering and comparing the PCR identified correct monoclonal bacterial liquid to determine the correct clone bacterial liquid, wherein the primer pair is Seq F (shown as SEQ ID NO: 7) and Seq R (shown as SEQ ID NO: 8).
Taking 1L expression as an example:
the monoclonal is selected and inoculated into 10ml of LB culture medium with corresponding resistance of the vector, shake-cultured overnight, inoculated into 1L of LB shake flask with double antibody the next day, shake-cultured until OD 0.6-0.8 at 220rpm and induced by adding IPTG with final concentration of 0.5mM, and expressed for 21 hours.
10000rcf centrifugal force is used for centrifugation for 5min to collect thalli, supernatant culture medium is removed, precooler (100mM KCl,25mM Tris pH7.5) is used for 30ml of suspended thalli, then high pressure 650bar is used for breaking bacteria for 5min, protease inhibitor is added during breaking bacteria, 14000rcf centrifugal force is used for 30min to remove sediment, corresponding affinity purification medium is added into supernatant for combination (Ni, glutethione, dextrin and the like), then specific site protease mediated direct elution is carried out on column label cutting or no cutting, and eluted proteins are subjected to AKTA (GE healthcare) mediated elution separation process, and gel chromatography is carried out. The final SDS-PAGE determines purity as shown in FIG. 4. Finally, protein concentration was performed, and the final protein concentration was determined by combining the absorbance of a280 with the extinction coefficient.
one unit is defined as the amount of enzyme required to digest 1. Mu.g of methylated plasmid DNA at 37℃for 1 hour, with a total reaction volume of 50. Mu.l.
DpnI of different dilution ratios was added to the reaction system, and 1. Mu.g of plasmid DNA was digested at 37℃for one hour, and finally the specific activity of the enzyme was confirmed by agarose electrophoresis, as shown in FIG. 5. Finally, the specific activity is determined to be more than 5x 10U/mg.
The reaction conditions are as follows: 50 μl reaction system, 10U DpnI,10U DpnI (T) buffer system: 100mM Tris-HCl,50mM NaCl,10mM MgCl 2 Mu.g of 0.025% Triton X-100 (pH 7.5@25℃) plasmid were reacted at 37℃for 10min. T represents DpnI restriction enzyme purchased from ThermoFisher Scientific, control reaction was performed, and DpnI (T) was used as a positive control. The Negative group is the DpnI restriction endonuclease gene on the original plasmid which is not recombined.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Sequence listing
<110> Shanghai Yongke biotechnology Co., ltd
<120> preparation method of recombinant DpnI restriction endonuclease
<130> CN100-20002PICN
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ccgctgaacc acttcgaaaa caaccgtccg gttgcggatt tctactgcaa ccactgcagc 180
gaagaattcg aactgaaaag caaaaaaggc aacttcagca gcaccatcaa cgacggcgcg 240
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tacaccaaaa acttcgaagt taacaacttc ctggtgctgc cgaaacagtt cgttaccccg 360
aaaagcatca tccagcgtaa accgctggcg ccgaccgcga accgtgctgg ctggatcggt 420
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Claims (1)
1. A method for preparing recombinant dpnl restriction enzyme, comprising the steps of:
step one, synthesizing a coding gene of DpnI, constructing a prokaryotic expression vector, and obtaining a recombinant vector: the amino acid sequence of the DpnI is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene of the DpnI is obtained through adding a stop codon and codon optimization; the prokaryotic expression vector is pGEX-6p-1 vector;
the first step is specifically as follows: amplifying DpnI coding genes by using high-fidelity enzymes, simultaneously cutting pGEX-6p-1 vectors by BamHI and SalI restriction enzymes, and inserting the genes into pGEX-6p-1 by a homologous recombination mode; ensuring the correct reading frame, and taking GST protein as a purification tag;
transferring the recombinant vector into a methylase-deleted escherichia coli competent cell to obtain recombinant bacteria; the escherichia coli deleted by the methylase is JM110;
in the second step, 200-400 mu l JM110 competent cells and a recombination reaction product obtained by a 40-80 mu l recombination reaction system are mixed and transferred into the recombination carrier to obtain the recombinant bacterium;
the 40-80 μl recombination reaction system comprises: 200-400ng pGEX-6p-1 vector, 50-100ng DpnI coding gene, 2-4 μl Exnase recombinase and 8-16 μl 5X buffer;
the second step is specifically as follows: taking 200-400 mu l JM110 competent cells out at-80 ℃, placing the competent cells on ice for 10min, adding a recombination reaction product, slowly blowing and uniformly mixing, placing the competent cells on ice for 15min, placing the competent cells on a water bath at 42 ℃ for heat shock 45s, taking out the competent cells and placing the competent cells on ice for 10min, adding 1ml of LB for culturing, and culturing for 1hr based on a shaking table at 37 ℃ and 220 rpm; finally 10000g of supernatant is removed by centrifugation, and thalli are uniformly coated on an ampicillin-resistant LB plate; the recombination reaction product is a product obtained by reacting the 40-80 mu l recombination reaction system at 37 ℃ for 25 min;
step three, identifying the sequence correctness of the target fragment in the recombinant bacterium through PCR and sequencing;
the third step is specifically as follows: selecting a monoclonal strain, culturing at 220rpm and 37 ℃ in a 2.5ml test tube for 10hr, performing bacterial liquid PCR, and performing PCR reaction by using universal sequencing primers at two ends of a vector polyclonal site; sequencing the PCR identification of the correct monoclonal strain by censoring, and comparing the clone strain with the correct sequence; the fourth step is specifically as follows: selecting a monoclonal and inoculating to 10ml of LB culture medium with corresponding resistance of the vector, performing shake culture overnight, inoculating to 1L of double-antibody LB shake flask the next day, shake culturing at 220rpm 37 ℃ until OD is 0.6-0.8, adding IPTG with final concentration of 0.5mM for induction, and expressing for 21 hours;
fourthly, carrying out DpnI restriction enzyme expression on the monoclonal with the correct target fragment;
step five, purifying DpnI restriction enzyme;
the fifth step is specifically as follows: 10000 Centrifuging with rcf centrifugal force for 5min to collect thalli, removing a supernatant culture medium, suspending thalli with precooled buffer solution 30ml containing 100mM KCl and 25mM Tris pH7.5, then carrying out bacterium breaking with high pressure 650bar for 5min, simultaneously adding protease inhibitor into the thalli, centrifuging with 14000rcf for 30min to remove precipitate, adding corresponding affinity purification medium into the supernatant for combination, then carrying out specific site protease mediated on-column label cleavage or direct elution without cleavage, carrying out gel chromatography on eluted proteins by using AKTA mediated elution separation process, and determining the purity of the obtained recombinant DpnI restriction endonuclease;
and step six, measuring the enzyme activity of the DpnI restriction enzyme.
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