CN112280763A - Production method of recombinant nicking restriction enzyme in escherichia coli - Google Patents
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Abstract
The invention discloses a production method of recombinant nicking restriction endonuclease in escherichia coli, which clones NT.BstNBI nicking enzyme and GST label into pACYCDuet-1 expression vector containing His label through a recombinant mode, methylase thereof is cloned into pBAD-HisA containing arabinose inducing elements, methylase expression is carried out by utilizing arabinose to protect thallus DNA, and the expression quantity reaches 35% under the low temperature condition. The high-activity recombinant NT.BstNBI nickase is obtained through combined tag protein expression, Ni column affinity chromatography and Superdex 200 gel filtration chromatography, the protein is purified through Superdex 200 molecular sieve chromatography, the purification process is groped and conditions are optimized, the whole purification process only needs 6 hours, the enzyme digestion time is not more than 20 hours, and the purification process and time are greatly simplified.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a production method of recombinant nicking restriction enzyme in escherichia coli.
Background
Restriction endonucleases are enzymes that recognize specific nucleotide sequences and cleave the phosphodiester bond between two nucleotides at specific positions in each strand, for short restriction enzymes, which can be classified into three types, i.e., type I, type II, and type III, according to their structures, cofactor requirements, cleavage and mode of action, with the type II restriction and modification system accounting for up to 93%.
The first type restriction enzyme has the functions of modification and recognition cutting; in addition to the ability to recognize specific base sequences on DNA, cleavage sites are usually as far as several kilobases from the recognition site, for example: EcoB, EcoK.
The third type restriction enzyme has the functions of modification and recognition cutting, and can recognize short asymmetric sequences, and the cutting position is about 24-26 base pairs away from the recognition sequence, for example: hinf III.
The second type restriction enzyme has only the function of recognizing the cutting, the modification is carried out by other enzymes, and the recognized position is mostly a short palindromic sequence; the base sequence to be cleaved is usually a recognized sequence, and is a restriction enzyme species having high utility in genetic engineering, for example: BanHI, Hind III, of which the type II enzymes are the simplest to speak, recognize palindromic sequences, cut DNA within or near the palindromic sequence to produce DNA products bearing 3 '-hydroxyl and 5' -phosphate groups, recognition sequences of predominantly 4-6bp, or longer and of two-fold symmetry, but a few recognize longer or degenerate sequences, the positions of cleavage being enzyme-specific and somewhat spaced apart, and a specific restriction endonuclease, which recognizes a specific sequence.
Restriction enzyme typeII has four subtypes: IIP, IIS, IIC and IIT. In most cases, IIPs used in the laboratory have only one protein domain with the ability to recognize and cleave within the sequence at the same time, while IIS has two domains, one of which is responsible for the recognition sequence and the other for cleavage, resulting in a protein cleavage site outside the recognition sequence due to the distance between the two domains. Nicking endonucleases have two domains similar to type IIS, and unlike type IIS, nicking endonucleases can only nick one strand of the double strand of DNA to form a nick.
With the development of biotechnology, the application field of nicking endonuclease is more extensive, for example, nicking endonuclease-based nucleic acid signal amplification technology, nicking endonuclease-mediated isothermal amplification technology and the like all utilize the characteristic that nicking endonuclease cuts single chain, NT.BstNBI is used as a special restriction endonuclease, related article descriptions are provided for property characteristics and functional mechanisms of NT.BstNBI protein, the current commercialized NT.BstNBI protein is expensive, only NEB company sells at present, the invention can greatly reduce the production cost, reduce the cost of subsequent downstream products, and also reduce the scientific research production cost of customers.
Disclosure of Invention
The invention aims to provide a method for producing recombinant nicking restriction enzyme in Escherichia coli.
The technical problems to be solved by the invention are as follows:
the method for inducing expression and purifying recombinant protein in escherichia coli is provided, and the yield and preparation efficiency of NT.BstNBI protein are increased by recombining a target gene into an expression vector and transforming the expression vector into an escherichia coli expression strain for induced expression.
The purpose of the invention can be realized by the following technical scheme:
a method for producing a recombinant nicking restriction enzyme in Escherichia coli, comprising the steps of:
step S1: constructing an expression plasmid pACYCDuet-GST-NT.BstNBI;
step S2: constructing a protective plasmid pBAD-M.NT.BstNBI;
step S3: expression, purification and identification of nt.
Further, the specific steps of step S1 are as follows:
step S11: gene synthesis of bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare the NT.BstNBI gene;
step S12: gene synthesis of GST tag:
according to the gene sequence after codon optimization, directly carrying out gene synthesis to prepare a GST tag;
step S13: gibson recombinant ligation:
linearizing pACYCDuet-1 by PCR, and seamlessly cloning the linearized product and the product NT.BstNBI gene prepared in the step S11 to prepare a recombinant ligation product;
step S14: transformation of recombinant ligation products:
adding 20 mu L of the recombinant ligation product prepared in the step S13 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium A plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S15: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S14, inoculating the recombinant clones into an LB liquid culture medium B, carrying out overnight culture under the conditions that the temperature is 37 ℃ and the rotating speed of a shaking table is 200r/min, extracting plasmids and sequencing to obtain an expression plasmid pACYCDuet-GST-NT.
Further, the LB liquid medium a described in step S14 is prepared by the steps of: mixing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 900mL of distilled water until the tryptone, the yeast extract and the sodium chloride are completely dissolved, adjusting the pH value of the solution to 7.0 and fixing the volume to 1L to prepare the LB liquid culture medium.
Further, the LB solid medium A described in step S14 was prepared by the following steps: mixing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, chloramphenicol and 900mL of distilled water, adding 15g of agar powder, and fixing the volume to 1L, wherein the dosage of the chloramphenicol is 50 mu g/mL.
Further, the LB liquid medium B described in step S14 was supplemented with chloramphenicol in an amount of 50. mu.g/mL, as compared with LB liquid medium A.
Further, the specific steps of step S2 are as follows:
step S21: gene synthesis of m.nt.bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare an M.NT.BstNBI gene;
step S22: gibson recombinant ligation:
linearizing pBAD-HisA by PCR, and seamlessly cloning the linearized product, the product M.NT.BstNBI gene prepared in the step S21 and the GST tag prepared in the step S12 to prepare a recombinant ligation product;
step S23: recombinant ligation product transformation
Adding 20 mu L of the recombinant ligation product prepared in the step S22 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium B plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S24: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S23, inoculating the recombinant clones into an LB liquid culture medium C, carrying out overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, extracting plasmids and sequencing to prepare the protective plasmid pBAD-M.NT.BstNBI.
Further, the LB solid medium B described in step S23 was prepared by the following steps: mixing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, ampicillin and 900mL of distilled water, adding 15g of agar powder, and fixing the volume to 1L, wherein the using amount of the ampicillin is 50 mu g/mL.
Further, the LB liquid medium C described in step S23 was supplemented with ampicillin in an amount of 50. mu.g/mL as compared with the LB liquid medium A.
Further, the specific steps of step S3 are as follows:
step S31: transforming the recombinant plasmid into escherichia coli:
transferring the positive expression plasmid pACYCDuet-GST-NT.BstNBI prepared in the step S15 and the protective plasmid pBAD-M.NT.BstNBI prepared in the step S24 into a competent cell BL21(DE3), coating the competent cell BL21 on an LB solid culture medium C plate, and performing inversion overnight culture at the temperature of 37 ℃ to prepare a cultured thallus;
step S32: IPTG and arabinose induced protein expression:
inoculating the cultured thalli prepared in the step S31 into an LB liquid culture medium D, after overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, inoculating the overnight thalli into the LB liquid culture medium D again according to the inoculation ratio of 1:100, continuing to culture for 6h until OD600nm reaches 0.6, adding IPTG (isopropyl thiogalactoside) to 0.4mM, after overnight induction protein expression at the temperature of 12 ℃, centrifuging for 10min at the rotation speed of 8000r/min, removing supernatant to obtain thalli, and preserving for later use at the temperature of-20 ℃;
step S33: purification of bstnbi protein:
adding the frozen thallus prepared in the step S32 into a bacterial lysate, performing ultrasonic bacteria breaking, centrifuging for 15min at the rotation speed of 16000r/min, taking the supernatant, placing the supernatant into a50 mL centrifuge tube, adding a 2mL Ni column, incubating for 2h at the temperature of 4 ℃, washing the column with the lysate for 100mL, eluting with the lysate containing 20mM imidazole for 30mL, and eluting with the lysate containing 500mM imidazole for 5mL to obtain NT.BstNBI protein;
step S34: removal of bstnbi protein tag:
the nt.bstnbi protein obtained in step S33 was expressed as 100:1, performing enzyme digestion overnight at the temperature of 4 ℃, performing reverse screening by using a GST column, wherein the effluent eluent is NT.BstNBI protein without the GST tag, using NT.BstNBI storage solution as a mobile phase to pass through a molecular sieve Superdex 200, collecting eluent containing target protein according to an ultraviolet absorption value, and detecting the target protein solution by using 4-20% polyacrylamide gradient gel.
Further, compared with the LB solid medium B, the LB solid medium C described in step S31 was added with chloramphenicol in an amount of 50. mu.g/mL, and the LB liquid medium D described in step S32 was added with chloramphenicol, ampicillin, and arabinose in an amount of 50. mu.g/mL, ampicillin in an amount of 50. mu.g/mL, and arabinose in an amount of 0.25. mu.g/mL, respectively, compared with the LB liquid medium A.
The invention has the beneficial effects that: the invention relates to a method for producing recombinant Sac I restriction endonuclease in escherichia coli, wherein the NT.BstNBI endonuclease belongs to nicking endonucleases, and the recognition sites of the nicking endonucleases are as follows: 5 '-GAGTCNNNN ^ -3' and 3 '-CTCAGNNNN-5', after cutting GAGTC, the 3 'end is provided with phosphate group, the 5' end is provided with hydroxyl, a restriction endonuclease R-M system is utilized to firstly utilize arabinose to induce methylase expression to methylate the expression strain so as to protect DNA of the expression strain from being cut, then the expression of endonuclease NT.BstNBI is carried out, meanwhile, a His-GST combined label is used for increasing the yield of protein, protein is purified by using the Superdex 200 molecular sieve chromatography, after the exploration of the purification process and the optimization of conditions, the whole purification process only needs 6 hours, the enzyme cutting time is not more than 20 hours, the purification process and the time are greatly simplified, the enzyme activity is ensured, the improvement of the enzyme yield is facilitated, and the purified NT.BstNBI is identified by the enzyme activity and the purity, the specific activity is 2.3 multiplied by 106U/mg, the purification is 30 times, the yield is 35%, the yield reaches 6 multiplied by 108Units/g wet cell, the yield and the efficiency are greatly improved compared with the previous reports, and the new method of the purification process lays a foundation for researching and developing other type II restriction endonucleases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the SDS-PAGE detection result of the target protein Ni column eluate;
FIG. 2 is a SDS-PAGE detection result of a reverse-screening sample passing through a molecular sieve Superdex 200 sample after the target protein is digested;
FIG. 3 shows the results of plasmid detection by digestion of the collected liquid.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for producing recombinant nicking restriction enzyme in Escherichia coli specifically comprises the following steps:
step S1: constructing an expression plasmid pACYCDuet-GST-NT.BstNBI;
step S11: gene synthesis of bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare the NT.BstNBI gene;
step S12: gene synthesis of GST tag:
according to the gene sequence after codon optimization, directly carrying out gene synthesis to prepare a GST tag;
step S13: gibson recombinant ligation:
linearizing pACYCDuet-1 by PCR, and seamlessly cloning the linearized product and the product NT.BstNBI gene prepared in the step S11 to prepare a recombinant ligation product;
step S14: transformation of recombinant ligation products:
adding 20 mu L of the recombinant ligation product prepared in the step S13 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium A plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S15: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S14, inoculating the recombinant clones into an LB liquid culture medium B, carrying out overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, extracting plasmids and sequencing to prepare an expression plasmid pACYCDuet-GST-NT.BstNBI;
step S2: constructing a protective plasmid pBAD-M.NT.BstNBI;
step S21: gene synthesis of m.nt.bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare an M.NT.BstNBI gene;
step S22: gibson recombinant ligation:
linearizing pBAD-HisA by PCR, and seamlessly cloning the linearized product, the product M.NT.BstNBI gene prepared in the step S21 and the GST tag prepared in the step S12 to prepare a recombinant ligation product;
step S23: recombinant ligation product transformation
Adding 20 mu L of the recombinant ligation product prepared in the step S22 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium B plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S24: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S23, inoculating the recombinant clones into an LB liquid culture medium C, carrying out overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, extracting plasmids and sequencing to prepare the protective plasmid pBAD-M.NT.BstNBI.
Step S3: expression, purification and identification of NT.BstNBI;
transferring the positive expression plasmid pACYCDuet-GST-NT.BstNBI prepared in the step S15 and the protective plasmid pBAD-M.NT.BstNBI prepared in the step S24 into a competent cell BL21(DE3), coating the competent cell BL21 on an LB solid culture medium C plate, and performing inversion overnight culture at the temperature of 37 ℃ to prepare a cultured thallus;
step S32: IPTG and arabinose induced protein expression:
inoculating the cultured thalli prepared in the step S31 into an LB liquid culture medium D, after overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, inoculating the overnight thalli into the LB liquid culture medium D again according to the inoculation ratio of 1:100, continuing to culture for 6h until OD600nm reaches 0.6, adding IPTG (isopropyl thiogalactoside) to 0.4mM, after overnight induction protein expression at the temperature of 12 ℃, centrifuging for 10min at the rotation speed of 8000r/min, removing supernatant to obtain thalli, and preserving for later use at the temperature of-20 ℃;
step S33: purification of bstnbi protein:
adding the frozen thallus prepared in the step S32 into a bacterial lysate, performing ultrasonic bacteria breaking, centrifuging for 15min at the rotation speed of 16000r/min, taking the supernatant, placing the supernatant into a50 mL centrifuge tube, adding a 2mL Ni column, incubating for 2h at the temperature of 4 ℃, washing the column with the lysate for 100mL, eluting with the lysate containing 20mM imidazole for 30mL, and eluting with the lysate containing 500mM imidazole for 5mL to obtain NT.BstNBI protein;
step S34: removal of bstnbi protein tag:
the nt.bstnbi protein obtained in step S33 was expressed as 100:1, performing enzyme digestion overnight at the temperature of 4 ℃, performing reverse screening by using a GST column, wherein the effluent eluent is NT.BstNBI protein without the GST tag, using NT.BstNBI storage solution as a mobile phase to pass through a molecular sieve Superdex 200, collecting eluent containing target protein according to an ultraviolet absorption value, and detecting the target protein solution by using 4% polyacrylamide gradient gel.
Example 2
A method for producing recombinant nicking restriction enzyme in Escherichia coli specifically comprises the following steps:
step S1: constructing an expression plasmid pACYCDuet-GST-NT.BstNBI;
step S11: gene synthesis of bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare the NT.BstNBI gene;
step S12: gene synthesis of GST tag:
according to the gene sequence after codon optimization, directly carrying out gene synthesis to prepare a GST tag;
step S13: gibson recombinant ligation:
linearizing pACYCDuet-1 by PCR, and seamlessly cloning the linearized product and the product NT.BstNBI gene prepared in the step S11 to prepare a recombinant ligation product;
step S14: transformation of recombinant ligation products:
adding 20 mu L of the recombinant ligation product prepared in the step S13 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium A plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S15: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S14, inoculating the recombinant clones into an LB liquid culture medium B, carrying out overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, extracting plasmids and sequencing to prepare an expression plasmid pACYCDuet-GST-NT.BstNBI;
step S2: constructing a protective plasmid pBAD-M.NT.BstNBI;
step S21: gene synthesis of m.nt.bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare an M.NT.BstNBI gene;
step S22: gibson recombinant ligation:
linearizing pBAD-HisA by PCR, and seamlessly cloning the linearized product, the product M.NT.BstNBI gene prepared in the step S21 and the GST tag prepared in the step S12 to prepare a recombinant ligation product;
step S23: recombinant ligation product transformation
Adding 20 mu L of the recombinant ligation product prepared in the step S22 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium B plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S24: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S23, inoculating the recombinant clones into an LB liquid culture medium C, carrying out overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, extracting plasmids and sequencing to prepare the protective plasmid pBAD-M.NT.BstNBI.
Step S3: expression, purification and identification of NT.BstNBI;
transferring the positive expression plasmid pACYCDuet-GST-NT.BstNBI prepared in the step S15 and the protective plasmid pBAD-M.NT.BstNBI prepared in the step S24 into a competent cell BL21(DE3), coating the competent cell BL21 on an LB solid culture medium C plate, and performing inversion overnight culture at the temperature of 37 ℃ to prepare a cultured thallus;
step S32: IPTG and arabinose induced protein expression:
inoculating the cultured thalli prepared in the step S31 into an LB liquid culture medium D, after overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, inoculating the overnight thalli into the LB liquid culture medium D again according to the inoculation ratio of 1:100, continuing to culture for 6h until OD600nm reaches 0.6, adding IPTG (isopropyl thiogalactoside) to 0.4mM, after overnight induction protein expression at the temperature of 12 ℃, centrifuging for 10min at the rotation speed of 8000r/min, removing supernatant to obtain thalli, and preserving for later use at the temperature of-20 ℃;
step S33: purification of bstnbi protein:
adding the frozen thallus prepared in the step S32 into a bacterial lysate, performing ultrasonic bacteria breaking, centrifuging for 15min at the rotation speed of 16000r/min, taking the supernatant, placing the supernatant into a50 mL centrifuge tube, adding a 2mL Ni column, incubating for 2h at the temperature of 4 ℃, washing the column with the lysate for 100mL, eluting with the lysate containing 20mM imidazole for 30mL, and eluting with the lysate containing 500mM imidazole for 5mL to obtain NT.BstNBI protein;
step S34: removal of bstnbi protein tag:
the nt.bstnbi protein obtained in step S33 was expressed as 100:1, performing enzyme digestion overnight at the temperature of 4 ℃, performing reverse screening by using a GST column, wherein the effluent eluent is NT.BstNBI protein without the GST tag, using NT.BstNBI storage solution as a mobile phase to pass through a molecular sieve Superdex 200, collecting eluent containing target protein according to an ultraviolet absorption value, and detecting the target protein solution by using 20% polyacrylamide gradient gel.
Example 3
1. Bacterial strain
Host bacteria Escherichia coli DH5 alpha, competent cell BL21(DE3) and expression vectors pACYCDuet-1 and pBAD-HisA.
2. Reagent
The restriction enzyme is NEB (Beijing) product; pACYCDuet-1 and pBAD-HisA are self-owned by general biosystems (Anhui) Inc.; the DNA polymerase is self-produced by the company; the gel recovery and plasmid minipump kit is an AXYGEN product; ni NTA Beads 6FF and Glutathieone Beads 4FF were purchased from Changzhou Tiandi and Biotech Inc.; arabinose was purchased from Sigma; t4 DNA ligase is a product of the company.
3. Protease gene synthesis
BstNBI protease and methylase genes are directly synthesized by the gene part of general biological systems (Anhui) GmbH according to gene sequences, and 5 'and 3' respectively have GST protein genes and pACYCDuet-1 homology arms.
4. GST tag protein Gene Synthesis
The GST tag protein gene is directly synthesized by the gene part of general biological systems (Anhui) GmbH according to the gene sequence, and 5 'and 3' respectively have the homologous arms of pACYCDuet-1 and NT.BstNBI protease genes.
5. Construction of pACYCDuet-GST-NT. BstNBI expression vector
Carrying out homologous recombination on the NT.BstNBI protease gene, the linearized vector pACYCDuet-1 and the GST tag protein gene by using a recombinase, transforming an escherichia coli DH5 alpha by using a recombinant product, and carrying out sequencing verification on an extracted plasmid by a sequencing department of the company.
6. Methylase M.NT.BstNBI gene synthesis
The methylase M.NT.BstNBI gene was directly synthesized by the gene part of general biological systems (Anhui) Ltd based on the gene sequence, and 5 'and 3' each had the homology arm of pBAD-HisA.
7. Construction of pBAD-M.NT.BstNBI expression vector
Methylase M.NT.BstNBI gene and a sexual vector pBAD-HisA are subjected to homologous recombination by using recombinase, a recombinant product is transformed into escherichia coli DH5 alpha, and an extracted plasmid is subjected to sequencing verification by a sequencing department of the company.
8. BstNBI enzyme expression and product purification
The two correctly sequenced vector plasmids pACYC Duet-GST-NT.BstNBI and pBAD-M.NT.BstNBI were co-transferred into E.coli BL21(DE3), and a single colony was transformed into 30mL of LB liquid medium containing chloramphenicol (50ug/mL), ampicillin (40ug/mL) and arabinose (0.25ug/mL), and after overnight incubation at 37 ℃ the mixture was mixed in the following manner (1: 100 to 800mL of a liquid medium containing kanamycin, culturing at 37 ℃, adding IPTG (final concentration of 0.4mM) when OD600 reaches 0.6, and inducing overnight at 12 ℃ to obtain cells;
adding thalli into lysate for resuspension, carrying out centrifugation after ultrasonication, centrifuging for 15min under the condition that the rotating speed is 16000r/min, placing supernatant into a clean 50mL centrifuge tube, adding a 2mL Ni column, incubating for 2h under the condition that the temperature is 4 ℃, washing the column by using the lysate, eluting by using the lysate containing 20mM imidazole for 30mL, eluting by using the lysate containing 500mM imidazole for 5mL, adding the obtained NT.BstNBI protein into PreScission Protease containing a GST label according to the proportion of 100:1, carrying out enzyme digestion overnight under the condition that the temperature is 4 ℃, carrying out reverse screening by using the GST column, taking the effluent eluent as a mobile phase to pass through a molecular sieve Superdex 200, collecting the eluent containing the target protein according to an ultraviolet absorption value, and detecting the target protein liquid by using 20% polyacrylamide gradient gel.
10. And carrying out enzyme digestion verification on the purified product of the target protein.
The SDS-PAGE detection of the target protein Ni column eluate is shown in figure 1;
m in FIG. 1 is Protein Marker; 1 is a crushed precipitate; 2, crushing the supernatant; 3 is an effluent liquid; 4 is 500mM imidazole impurity wash; 5 is 500mM imidazole impurity wash;
SDS-PAGE detection of the reverse-screened sample of the digested target protein by a molecular sieve superdex 200 sample is shown in figure 2;
the collected liquid enzyme digestion plasmid detection is shown in FIG. 3;
m in FIG. 3 is DL5000 Marker; 1 is pUC57-NT site original plasmid; BstNBI control generated by NEB; and 3 is a molecular sieve protein sample.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (10)
1. A method for producing a recombinant nicking restriction enzyme in Escherichia coli, comprising: the method comprises the following steps:
step S1: constructing an expression plasmid pACYCDuet-GST-NT.BstNBI;
step S2: constructing a protective plasmid pBAD-M.NT.BstNBI;
step S3: expression, purification and identification of nt.
2. The method for producing a recombinant nicking restriction enzyme according to claim 1 in Escherichia coli, wherein: the specific steps of step S1 are as follows:
step S11: gene synthesis of bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare the NT.BstNBI gene;
step S12: gene synthesis of GST tag:
according to the gene sequence after codon optimization, directly carrying out gene synthesis to prepare a GST tag;
step S13: gibson recombinant ligation:
linearizing pACYCDuet-1 by PCR, and seamlessly cloning the linearized product and the product NT.BstNBI gene prepared in the step S11 to prepare a recombinant ligation product;
step S14: transformation of recombinant ligation products:
adding 20 mu L of the recombinant ligation product prepared in the step S13 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium A plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S15: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S14, inoculating the recombinant clones into an LB liquid culture medium B, carrying out overnight culture under the conditions that the temperature is 37 ℃ and the rotating speed of a shaking table is 200r/min, extracting plasmids and sequencing to obtain an expression plasmid pACYCDuet-GST-NT.
3. The method for producing a recombinant nicking restriction enzyme according to claim 2 in Escherichia coli, wherein: the LB liquid medium A described in step S14 is prepared by the following steps: mixing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 900mL of distilled water until the tryptone, the yeast extract and the sodium chloride are completely dissolved, adjusting the pH value of the solution to 7.0 and fixing the volume to 1L to prepare the LB liquid culture medium.
4. The method for producing a recombinant nicking restriction enzyme according to claim 2 in Escherichia coli, wherein: the LB solid medium A described in step S14 is prepared by the following steps: mixing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, chloramphenicol and 900mL of distilled water, adding 15g of agar powder, and fixing the volume to 1L, wherein the dosage of the chloramphenicol is 50 mu g/mL.
5. The method for producing a recombinant nicking restriction enzyme according to claim 2 in Escherichia coli, wherein: compared with the LB liquid culture medium A, the LB liquid culture medium B in the step S14 is added with chloramphenicol of 50 mug/mL.
6. The method for producing a recombinant nicking restriction enzyme according to claim 1 in Escherichia coli, wherein: the specific steps of step S2 are as follows:
step S21: gene synthesis of m.nt.bstnbi:
directly carrying out gene synthesis according to the gene sequence after codon optimization to prepare an M.NT.BstNBI gene;
step S22: gibson recombinant ligation:
linearizing pBAD-HisA by PCR, and seamlessly cloning the linearized product, the product M.NT.BstNBI gene prepared in the step S21 and the GST tag prepared in the step S12 to prepare a recombinant ligation product;
step S23: recombinant ligation product transformation
Adding 20 mu L of the recombinant ligation product prepared in the step S22 into DH5 alpha competent cells, uniformly mixing on ice, standing for 15min, thermally shocking for 120S at 42 ℃, standing on ice for 5min, adding 500 mu L of LB liquid culture medium A, performing shake culture for 45min at 37 ℃ and 200r/min of shaking table rotation speed, coating the mixture on an LB solid culture medium B plate, and performing overnight culture at 37 ℃ to prepare recombinant clones;
step S24: identification of recombinant clones:
randomly picking 3 recombinant clones prepared in the step S23, inoculating the recombinant clones into an LB liquid culture medium C, carrying out overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, extracting plasmids and sequencing to prepare the protective plasmid pBAD-M.NT.BstNBI.
7. The method for producing a recombinant nicking restriction enzyme according to claim 1 in Escherichia coli, wherein: the LB solid medium B described in step S23 is prepared by the following steps: mixing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, ampicillin and 900mL of distilled water, adding 15g of agar powder, and fixing the volume to 1L, wherein the using amount of the ampicillin is 50 mu g/mL.
8. The method for producing a recombinant nicking restriction enzyme according to claim 1 in Escherichia coli, wherein: ampicillin was added to the LB liquid medium C described in step S23 in an amount of 50. mu.g/mL as compared with LB liquid medium A.
9. The method for producing a recombinant nicking restriction enzyme according to claim 1 in Escherichia coli, wherein: the specific steps of step S3 are as follows:
step S31: transforming the recombinant plasmid into escherichia coli:
transferring the positive expression plasmid pACYCDuet-GST-NT.BstNBI prepared in the step S15 and the protective plasmid pBAD-M.NT.BstNBI prepared in the step S24 into a competent cell BL21(DE3), coating the competent cell BL21 on an LB solid culture medium C plate, and performing inversion overnight culture at the temperature of 37 ℃ to prepare a cultured thallus;
step S32: IPTG and arabinose induced protein expression:
inoculating the cultured thalli prepared in the step S31 into an LB liquid culture medium D, after overnight culture at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min, inoculating the overnight thalli into the LB liquid culture medium D again according to the inoculation ratio of 1:100, continuing to culture for 6h until OD600nm reaches 0.6, adding IPTG (isopropyl thiogalactoside) to 0.4mM, after overnight induction protein expression at the temperature of 12 ℃, centrifuging for 10min at the rotation speed of 8000r/min, removing supernatant to obtain thalli, and preserving for later use at the temperature of-20 ℃;
step S33: purification of bstnbi protein:
adding the frozen thallus prepared in the step S32 into a bacterial lysate, performing ultrasonic bacteria breaking, centrifuging for 15min at the rotation speed of 16000r/min, taking the supernatant, placing the supernatant into a50 mL centrifuge tube, adding a 2mL Ni column, incubating for 2h at the temperature of 4 ℃, washing the column with the lysate for 100mL, eluting with the lysate containing 20mM imidazole for 30mL, and eluting with the lysate containing 500mM imidazole for 5mL to obtain NT.BstNBI protein;
step S34: removal of bstnbi protein tag:
the nt.bstnbi protein obtained in step S33 was expressed as 100:1, performing enzyme digestion overnight at the temperature of 4 ℃, performing reverse screening by using a GST column, wherein the effluent eluent is NT.BstNBI protein without the GST tag, using NT.BstNBI storage solution as a mobile phase to pass through a molecular sieve Superdex 200, collecting eluent containing target protein according to an ultraviolet absorption value, and detecting the target protein solution by using 4-20% polyacrylamide gradient gel.
10. The method for producing a recombinant nicking restriction enzyme according to claim 9 in Escherichia coli, wherein: compared with the LB solid medium B, the LB solid medium C in the step S31 is added with chloramphenicol of 50 mug/mL, compared with the LB liquid medium A, the LB liquid medium D in the step S32 is added with chloramphenicol, ampicillin and arabinose, the chloramphenicol is 50 mug/mL, the ampicillin is 50 mug/mL, and the arabinose is 0.25 mug/mL.
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