CN116694584A - Preparation method of T4DNA ligase - Google Patents
Preparation method of T4DNA ligase Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
- C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
- C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to a preparation method of T4DNA ligase. The preparation method of the T4DNA ligase comprises the following steps: performing anion exchange chromatography on the crude enzyme solution containing the T4DNA ligase to obtain a first eluent; performing hydrophobic chromatography or compound mode chromatography on the first eluent to obtain a second eluent; and carrying out affinity chromatography on the second eluent to obtain the T4DNA ligase. The crude enzyme liquid containing the T4DNA ligase is subjected to three-step chromatography, so that various impurities in the crude enzyme liquid can be removed, and the prepared T4DNA ligase has higher activity. On the premise of equivalent connection efficiency with the commercial T4DNA ligase, the connection efficiency of the T4DNA ligase obtained by the preparation method of the T4DNA ligase after 100-400 times dilution is higher than that of the commercial T4DNA ligase, and the T4DNA ligase has higher enzyme activity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of T4DNA ligase.
Background
T4DNA ligase is a single-stranded polypeptide (GenBank serial number: X00039.1) encoded by the T4 phage gene and having a molecular weight of 55.23kDa, which catalyzes the formation of a phosphodiester bond between the 5 '-P-and 3' -OH-ends of cohesive or blunt-ended double-stranded DNA or RNA with the assistance of in vitro ATP. The flow column which is now a molecular biological reagent market is widely applied to in vitro nucleic acid analysis, recombinant plasmid construction, in vitro DNA connection and the like. In recent years, along with development of genetic engineering, production of T4DNA ligase can be realized by means of heterologous expression, and compared with the T4DNA ligase produced by fermentation of wild bacteria, the recombinant T4DNA ligase produced by fermentation of genetic engineering bacteria has higher connection efficiency after separation and purification by means of crude purity, medium purity and fine purity, wherein most of purification methods commonly used for the T4DNA ligase involve ammonium sulfate fractional precipitation. It is reported in the literature that high salts inhibit the enzyme activity of T4DNA ligase and therefore methods using ammonium sulphate fractional precipitation during pretreatment and hydrophobic chromatography during subsequent purification are not readily scalable. At present, development of a high-activity purification preparation process is needed, the defect of development of T4DNA ligase in China is overcome, and the application scene of the T4DNA ligase is enlarged.
Disclosure of Invention
Based on this, it is necessary to provide a method capable of preparing a highly active T4DNA ligase.
A method for preparing T4DNA ligase, comprising the following steps:
performing anion exchange chromatography on the crude enzyme solution containing the T4DNA ligase to obtain a first eluent;
performing hydrophobic chromatography or composite mode chromatography on the first eluent to obtain a second eluent;
and carrying out affinity chromatography on the second eluent to obtain the T4DNA ligase.
In the preparation method of the T4DNA ligase, the crude enzyme liquid containing the T4DNA ligase is subjected to three-step chromatography, including anion exchange chromatography, composite hydrophobic chromatography and affinity chromatography, so that various impurities in the crude enzyme liquid can be removed, and the prepared T4DNA ligase has higher activity. Experiments prove that on the premise of being equivalent to the connection efficiency of the commercial T4DNA ligase, the connection efficiency of the T4DNA ligase obtained by the preparation method of the T4DNA ligase after 100-400 times dilution is higher than that of the commercial T4DNA ligase, and the T4DNA ligase has higher enzyme activity.
In one embodiment, the chromatographic medium used in the anion exchange chromatography is selected from any one of Q Sepharose FF, Q Sepharose 4FF, Q Sepharose XL, Q Sepharose HP, Q Sepharose Big Beads, capto Q, QAE Sephadex A-25, QAE Sephadex A-50, DEAE Sepharose FF, DEAE Sepharose CL-6B, DEAE Sephadex, capto DEAE, and Anx Sepharose 4 FF.
In one embodiment, the step of subjecting the crude enzyme solution to anion exchange chromatography comprises:
a counter-ion exchange column;
the electric conductivity of the crude enzyme liquid is regulated to be lower than 3mS/cm, and then the crude enzyme liquid is loaded to the balanced anion exchange column;
washing impurities of the loaded anion exchange column;
eluting the anion exchange column subjected to impurity washing treatment by adopting a first eluting buffer solution to obtain the first eluting buffer solution, wherein the pH value of the first eluting buffer solution is 7.0-8.0 and the first eluting buffer solution comprises 10-50 mM Tris-HCl, 200-300 mM NaCl and 1-3 mM DTT.
In one embodiment, the pH of the equilibration buffer used to equilibrate the anion exchange column is 7.0 to 8.0 and comprises 10mM to 50mM Tris-HCl, 1mM to 3mM DTT.
In one embodiment, the pH of the wash buffer used to wash the loaded anion exchange column is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl, 50 mM-100 mM NaCl,1 mM-3 mM DTT.
In one embodiment, in the step of subjecting the first eluate to the composite mode chromatography, the chromatography medium used is selected from any one of Capto MMC ImpRes and Capto MMC.
In one embodiment, the step of subjecting the first eluate to hydrophobic chromatography or to complex mode chromatography comprises:
a balanced hydrophobic chromatography column or a composite mode chromatography column;
after the electric conductivity of the first eluent is regulated to be below 5mS/cm, loading the first eluent into the balanced hydrophobic chromatography column or the composite mode chromatography column;
and eluting the loaded hydrophobic chromatography column or the composite mode chromatography column by adopting a second eluting buffer solution to obtain the second eluting solution.
In one embodiment, the second elution buffer has a pH of 7.0 to 8.0 and comprises 10mM to 50mM Tris-HCl, 300mM to 500mM NaCl.
In one embodiment, the pH of the equilibration buffer used to equilibrate the hydrophobic chromatography column or the complex mode chromatography column is between 7.0 and 8.0 and comprises between 10mM and 50mM Tris-HCl.
In one embodiment, the T4DNA ligase carries a His tag and the second eluent is affinity chromatographed by Ni-column affinity chromatography.
In one embodiment, the affinity chromatography medium is selected from any one of Ni Sepharose HP, ni Sepharose 6FF, TALON Superflow, ni Sepharose excel.
In one embodiment, the pH of the equilibration buffer used in the affinity chromatography medium is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl,10 mM-20 mM imidazole.
In one embodiment, the wash buffer used in the affinity chromatography medium has a pH of 7.0 to 8.0 and comprises 10mM to 50mM Tris-HCl, 300mM to 500mM NaCl, 30mM to 50mM imidazole.
In one embodiment, the pH of the elution buffer used for the affinity chromatography medium is 7.0 to 8.0 and comprises 10mM to 50mM Tris-HCl, 300mM to 500mM NaCl,100mM to 200mM imidazole.
In one embodiment, before the step of subjecting the crude enzyme solution to anion exchange chromatography, the method further comprises the step of preparing the crude enzyme solution: crushing the zymocyte sludge for producing the T4DNA ligase, adding a flocculating agent or a water-soluble cationic high molecular polymer with the final volume concentration of 0.1-1%, standing, carrying out solid-liquid separation, and collecting the supernatant to obtain the crude enzyme liquid.
In one embodiment, the step of subjecting the second eluent to affinity chromatography further comprises the step of subjecting the affinity chromatography eluent to ultrafiltration.
Drawings
FIG. 1 is a SDS-PAGE chart of a one-step anion exchange chromatography of a crude enzyme solution containing T4DNA ligase in example 3;
FIG. 2 is a SDS-PAGE diagram of the collected components of the anion exchange chromatography of example 4 by a two-step composite hydrophobic chromatography process;
FIG. 3 is a SDS-PAGE diagram of the affinity chromatography process of the composite hydrophobic chromatography elution collection fraction of example 4;
FIG. 4 is a SDS-PAGE chart of the collected fractions of the anion exchange chromatography of example 5 by a two-step hydrophobic chromatography process;
FIG. 5 is a SDS-PAGE diagram of the hydrophobic chromatography elution collection fraction of example 5 for use in affinity chromatography;
FIG. 6 is a SDS-PAGE map of the finished T4DNA ligase obtained in example 7;
FIG. 7 is a result of quality detection of the finished T4DNA ligase obtained in example 7;
FIG. 8 is a graph showing the activity of the final T4DNA ligase obtained in example 7;
FIG. 9 is a graph showing the reproducibility of the final T4DNA ligase obtained in example 7 with commercially available T4DNA ligase;
FIG. 10 is a graph showing the detection of the enzymatic thermostability of the finished T4DNA ligase obtained in example 7 and a commercially available T4DNA ligase;
FIG. 11 is a graph showing the freeze-thaw stability of the final product of T4DNA ligase obtained in example 7 and a commercially available T4DNA ligase.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention can be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
An embodiment of the present study provides a method for producing T4DNA ligase, which can obtain T4DNA ligase having high activity.
Specifically, the preparation method of the T4DNA ligase comprises the following steps S110-S130:
s110, performing anion exchange chromatography on the crude enzyme solution containing the T4DNA ligase to obtain a first eluent.
The anion exchange chromatography can remove most of host self-impurity proteins in the crude enzyme liquid, and improve the purity and activity of the T4DNA ligase.
In some embodiments, the chromatographic medium used in the anion exchange chromatography is selected from any one of Q Sepharose FF, Q Sepharose 4FF, Q Sepharose XL, Q Sepharose HP, Q Sepharose Big Beads, capto Q, QAE Sephadex A-25, QAE Sephadex A-50, DEAE Sepharose FF, DEAE Sepharose CL-6B, DEAE Sephacel, capto DEAE, anx Sepharose 4 FF.
In some of these embodiments, the step of subjecting the crude enzyme solution to anion exchange chromatography comprises S111-S114:
s111, balancing an anion exchange column.
Wherein the anion exchange column is equilibrated with an equilibration buffer. Further, the pH of the equilibration buffer used to equilibrate the anion exchange column is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl, 1 mM-3 mM DTT. DTT is Dithiothreitol, and is defined herein below without further description. Further, the anion exchange column is equilibrated with 5CV to 10CV (CV is column volume, and hereinafter, all are defined as such, and will not be described in detail) equilibration buffer.
Wherein, before the step of balancing the anion exchange column, the method comprises the following steps: the anion chromatography medium was washed with 2CV to 3CV of 2MNaCl and 2CV to 3CV of 0.5M NaOH, and then with 5CV to 10CV of deionized water to a pH neutral.
S112, adjusting the electric conductivity of the crude enzyme solution to be less than 3mS/cm, and loading the crude enzyme solution to an anion exchange column after balancing.
The conductivity of the crude enzyme liquid containing the T4DNA ligase is regulated to be less than 3mS/cm, and then the crude enzyme liquid is loaded, so that the T4DNA ligase in the crude enzyme liquid is more favorably combined on a chromatographic medium, and the yield of the T4DNA ligase is improved. Specifically, deionized water or ultrapure water is adopted for conducting adjustment of crude enzyme liquid.
Wherein the column volume of the anion exchange column is 25mL, and the sample loading flow rate is 6 mL/min-10 mL/min.
S113, performing impurity washing treatment on the loaded anion exchange column.
The impurity-washing treatment can remove the impurity protein which is combined with weaker on the chromatographic column, improve the purity of the T4DNA ligase, avoid the influence of the impurity protein on the activity of the T4DNA ligase, and ensure that the T4DNA ligase has higher activity.
Wherein, the pH of the impurity washing buffer solution used for the impurity washing treatment of the loaded anion exchange column is 7.0-8.0, and the buffer solution comprises 10 mM-50 mM Tris-HCl, 50 mM-100 mM NaCl and 1 mM-3 mM DTT. Further, the anion exchange column after sample loading is subjected to impurity washing treatment by adopting 5-10 CV of impurity washing buffer solution.
S114, eluting the anion exchange column subjected to impurity washing treatment by adopting a first eluting buffer solution to obtain a first eluting solution, wherein the pH value of the first eluting buffer solution is 7.0-8.0 and comprises 10-50 mM Tris-HCl, 200-300 mM NaCl and 1-3 mM DTT.
By adopting the first elution buffer solution, the T4DNA ligase connected to the chromatographic column can be eluted thoroughly, impurity elution of impurity proteins and the like can be avoided, and the purity and activity of the T4DNA ligase are improved.
Wherein, the anion exchange column after the impurity washing treatment is eluted by adopting a first elution buffer solution with the concentration of 5CV to 10CV.
In some of these embodiments, the step of subjecting the crude enzyme solution to anion exchange chromatography is preceded by the step of preparing the crude enzyme solution: crushing fermentation bacteria mud for producing T4DNA ligase, adding flocculating agent or water-soluble cationic high molecular polymer with final volume concentration of 0.1% -1%, standing, carrying out solid-liquid separation, and collecting supernatant to obtain crude enzyme liquid. In one specific example, the water-soluble cationic high molecular polymer is PEI.
Wherein the bacterium producing the T4DNA ligase is engineering bacterium carrying His-tag recombinant T4DNA ligase. The T4DNA ligase producing bacterium may be a commercially available bacterium or a self-constructing bacterium.
For either wild-type or mutant T4 ligases, the invention is applicable, for example: the gene encoding the T4 ligase may be GenBank sequence number: x00039.1. In a specific example, the T4DNA ligase is derived from Escherichia phage T4.
Wherein, the step of crushing the fermentation mud for producing the T4DNA ligase comprises the following steps: the ratio of the mass of the fermentation bacteria mud to the volume of the crushing buffer solution is 1:10, adding a crushing buffer solution into the zymophyte sludge, uniformly mixing, and crushing under high pressure to obtain a crushing solution. Specifically, the pH of the disruption buffer was 7.5 and included 10mM Tris-HCl, 50mM NaCl, 5% (by volume) glycerol, 0.1% (by volume) Tween 20. The high-pressure crushing pressure was 800bar and the crushing time was 30min.
Wherein, in the standing step, the time is 20 min-2 h, and the standing temperature is 4 ℃.
Wherein, the solid-liquid separation mode is centrifugation. Specifically, the mixture was centrifuged at 10000rpm at 4℃for 30min. The solid-liquid separation method is not limited to centrifugation, and may be another solid-liquid separation method, for example, filtration.
S120, performing hydrophobic chromatography or composite mode chromatography on the first eluent to obtain a second eluent.
Impurities such as the impurity protein and the like remained in the first eluent can be removed through hydrophobic chromatography, so that the enzyme activity of the T4DNA ligase is further improved.
In some of these embodiments, in the step of subjecting the first eluate to the composite mode chromatography, the chromatography medium used is selected from any one of Capto MMC ImpRes and Capto MMC. These media are complex hydrophobic chromatography media, and the mode of action between the complex hydrophobic chromatography media and the protein of interest includes hydrophobic interactions, electrostatic interactions, and hydrogen bonding interactions, as compared to conventional hydrophobic chromatography media, and thus complex mode chromatography and complex hydrophobic chromatography are synonymous in the present invention. The obtained T4DNA ligase is free from RNase (endoribonuclease) and Nickase (endoribonuclease) pollution by the compound mode chromatography, and the host DNA residue is low.
In some of these embodiments, the step of subjecting the first eluate to hydrophobic chromatography or to a complex mode chromatography comprises S121-S123:
s121, balancing a hydrophobic chromatographic column or a compound mode chromatographic column.
Wherein, a balance liquid is adopted to balance the hydrophobic chromatography column or the composite mode chromatography column. The pH of the equilibration buffer is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl. Further, a 5 CV-10 CV balance liquid is adopted to balance the hydrophobic chromatography column or the composite mode chromatography column.
Wherein, before the step of balancing the hydrophobic chromatography column or the composite mode chromatography column, the method further comprises the following steps: the chromatography packing was pretreated with 2CV to 3CV 2M NaCl and 2CV to 3CV0.5M NaOH and washed to pH neutrality with deionized water.
S122, after the electric conductivity of the first eluent is regulated to be below 5mS/cm, loading the first eluent into the balanced hydrophobic chromatography column or the composite mode chromatography column.
And the electric conductivity of the first eluent is regulated to be less than 5mS/cm, and then the sample is loaded, so that the T4DNA ligase in the crude enzyme solution is more favorable for being combined on a chromatographic medium, and the yield of the T4DNA ligase is improved. Specifically, deionized water or ultrapure water is adopted for conducting adjustment of crude enzyme liquid.
Wherein the flow rate of the sample is 180cm/h to 300cm/h.
S123, eluting the loaded hydrophobic chromatography column or the composite mode chromatography column by adopting a second eluting buffer solution to obtain a second eluting solution, wherein the pH value of the second eluting buffer solution is 7.0-8.0 and comprises 10-50 mM Tris-HCl and 300-500 mM NaCl.
By adopting the second elution buffer solution, the T4DNA ligase connected to the chromatographic column can be eluted thoroughly, impurities such as impurity proteins can be prevented from eluting, and the purity and activity of the T4DNA ligase are improved.
Wherein, the loaded hydrophobic chromatographic column is eluted by adopting a second elution buffer solution with the concentration of 5CV to 10CV.
S130, performing affinity chromatography on the second eluent to obtain the T4DNA ligase.
The T4DNA ligase can be further purified by adopting affinity chromatography, and the T4DNA ligase with higher activity and stability is obtained.
In some of these embodiments, the T4DNA ligase carries a His tag and the second eluent is subjected to affinity chromatography by Ni-column affinity chromatography.
Wherein the affinity chromatography medium is selected from any one of Ni Sepharose HP, ni Sepharose 6FF, TALON Superflow and Ni Sepharose excel.
Wherein the pH of the balance buffer used for the affinity chromatography medium is 7.0-8.0, and the balance buffer comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl and 10 mM-20 mM imidazole. Further, the volume of the balance column is 5 CV-10 CV.
Further, before the step of balancing the affinity chromatography column, the method comprises the following steps: 2 CV-3 CV 500mM imidazole is used for pretreatment of the chromatography medium, and deionized water is used for washing until the pH value is neutral.
Wherein the pH of the washing buffer used for the affinity chromatography medium is 7.0-8.0, and the washing buffer comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl and 30 mM-50 mM imidazole. Further, the volume of the impurity washing column is 5 CV-10 CV.
Wherein the pH of the elution buffer used for the affinity chromatography medium is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl,100 mM-200 mM imidazole. Further, the volume of the elution column is 5CV to 10CV.
In some of these embodiments, the step of subjecting the second eluate to affinity chromatography is followed by a step of subjecting the affinity chromatography eluate to ultrafiltration.
Wherein the membrane package used for ultrafiltration is 0.1m 2 10kDa ultrafiltration membrane bag.
Wherein the step of performing ultrafiltration treatment on the affinity chromatography eluent comprises the following steps: after concentrating the affinity chromatography eluent to the membrane-packed dead volume, adding a displacement buffer solution with 2 times of the membrane-packed dead volume for displacement, wherein the number of times of displacement is 3-5 times, the end point of displacement is pH 7.5+/-0.5, and the deviation between the conductance and the displacement buffer solution is not more than 1mS/cm. The pH of the substitution buffer is 7.0-8.0, and the substitution buffer comprises 20 mM-40 mM Tris-HCl and 100 mM-200 mM KCl.
Further, the ultrafiltration step further comprises the step of adopting a preservation buffer solution to preserve the ultrafiltration displacement product. The pH of the preservation buffer is 7.0-8.0, and the preservation buffer comprises 10 mM-20 mM Tris-HCl, 50 mM-100 mM KCl, 1 mM-2 mM DTT, 0.1 mM-0.2 mM EDTA and 50% (volume percentage) glycerol. The preservation buffer solution can maintain the enzyme activity and ensure the stability of the enzyme.
Further, before ultrafiltration of the second eluent, pretreatment of the ultrafiltration membrane packet is required: the ultrafiltration membrane packets were washed with 0.5M NaOH cycles for more than 30min for disinfection and pyrogen removal, followed by washing the membrane packets with ultrapure water to neutral pH, followed by rinsing the membrane packets with a displacement buffer (pH 7.0-8.0, and comprising 20 mM-40 mM Tris-HCl, 100 mM-200 mM KCl) until pH 7.5.+ -. 0.5 was filtered off.
In the preparation method of the T4DNA ligase, the crude enzyme liquid containing the T4DNA ligase is subjected to three-step chromatography, including anion exchange chromatography, composite hydrophobic chromatography and affinity chromatography, so that various impurities in the crude enzyme liquid can be removed, and the prepared T4DNA ligase has higher activity. Experiments prove that on the premise of being equivalent to the connection efficiency of the commercial T4DNA ligase, the connection efficiency of the T4DNA ligase obtained by the preparation method of the T4DNA ligase after 100-400 times dilution is higher than that of the commercial T4DNA ligase, and the T4DNA ligase has higher enzyme activity.
Further, the preparation method of the research obtains the purification process conditions of pretreatment (namely, obtaining crude enzyme liquid), three-step chromatography and post-treatment (namely, ultrafiltration and preservation) through screening and combination of various fillers, and the pretreatment can solve the problems of difficult filtration and more crude enzyme liquid impurity proteins after bacterial mud is crushed, and the step is easy for large-scale production; the three-step chromatography process comprises anion exchange chromatography, hydrophobic chromatography and affinity chromatography, and the process is easy to connect and easy to linearly amplify; and then the recombinant T4DNA ligase with high quality can be output after post-treatment. The T4DNA ligase with low host DNA residue and enzyme purity up to 95% is successfully prepared by the preparation method, the purity is high, RNase and Nickase pollution is avoided, the operation of using high-salt solution or ammonium sulfate and the like is avoided, and the enzyme activity is better protected from the influence of extreme conditions, so that the enzyme activity is higher. The preparation method of the T4DNA ligase has the advantages of simple operation method, high yield, high protein purity, high enzyme activity, easy mass production and the like. Compared with the main stream products in the market, the property evaluation result shows that the T4DNA ligase obtained by the purification of the invention has better reproducibility, thermal stability and freeze thawing stability.
An embodiment of the present invention also provides a T4DNA ligase prepared by the above method for preparing a T4DNA ligase.
The T4DNA ligase has high purity, no RNase and Nickase pollution, low host DNA residue, and better reproducibility, thermal stability and freeze thawing stability.
The following is a detailed description of embodiments.
Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples are generally carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer of the kit. The reagents used in the examples are all commercially available.
In the examples below, the chromatographic procedures were carried out on an AKTA protein purifier, unless otherwise specified.
Example 1: recombinant T4DNA ligase engineering bacteria mud collection
Recombinant escherichia coli capable of expressing T4DNA ligase with 6-His tag is subjected to induced fermentation after resuscitating, fermentation broth bacterial sludge is collected by centrifugation at 10000rpm for 30min at 4 ℃, wherein the gene for encoding the T4 ligase is shown in GenBank serial number: x00039.1.
Example 2: preparation of crude enzyme solution of T4DNA ligase
50g of the fermentation broth bacterial sludge collected in example 1 is weighed, added into 500mL of precooled crushing Buffer (10 mM Tris-HCl, 50mM NaCl, 5% (volume percent) glycerol, 0.1% (volume percent) Tween 20 and pH 7.5) according to the mass-volume ratio of 1:10, and fully and uniformly mixed, and crushed at a high pressure of 800bar for 30min to obtain a crushing solution. PEI with final volume concentration of 0.15% is added dropwise to the crushed solution at room temperature, and the supernatant is collected after standing at 4 ℃ for 2h and centrifuging at 4 ℃ for 30min at 10000 rpm. The supernatant is crude enzyme liquid containing T4DNA ligase, the supernatant is diluted by deionized water to 4.5mS/cm, and the supernatant is filtered by a 0.45 mu m filter membrane and can be used for further separation and purification.
Example 3: anion exchange chromatography of T4DNA ligase
25g of anion chromatography medium DEAE Sepharose FF was loaded into XK 16/20 column and the following chromatography was performed on AKTA protein purifier. The anion chromatography medium is firstly washed by using 2-3 CV 2M NaCl and 2-3 CV0.5M NaOH, then washed by using 5-10 CV deionized water until the pH value is neutral, and then the chromatography column is balanced by using 5-10 CV balance Buffer (10 mM Tris-HCl, 1mM DTT, pH value is 7.5). The filtrate obtained in example 2 was applied to the equilibrated column, after the application, the non-column-hung hybrid protein was washed with 5CV equilibration Buffer, the weakly-bound hybrid protein was washed off with 5 to 10CV wash Buffer (10 mM Tris-HCl, 50mM NaCl, 1mM DTT, pH 7.5), the target protein was eluted and collected with 5 to 10CV wash Buffer (10 mM Tris-HCl, 200mM NaCl, 1mM DTT, pH 7.5), the strongly-bound protein was washed off with 3 to 5CV 2M NaCl, the column was washed with 3 to 5CV 0.5M NaOH, the column was finally washed with deionized water until the pH was neutral, and the column was finally stored in 20% ethanol. The collected eluate containing the target protein was subjected to SDS-PAGE gel electrophoresis, and the detection results are shown in FIG. 1. As can be seen from FIG. 1, most of host self-impurity proteins are removed after one-step anion chromatography, and the detection result shows that the purity of target proteins in the elution buffer solution after primary separation is more than 85 percent, the recovery rate of the one-step chromatographic proteins is 30.74 percent, and the one-step protein load is 40.27mg/mL column charge.
Example 4: complex hydrophobic chromatography of T4DNA ligase
A25 g composite hydrophobic chromatography medium Capto MMC is loaded on an XK 16/20, the chromatography packing is pretreated by using 2-3 CV 2M NaCl and 2-3 CV0.5M NaOH as described in the example 3, deionized water is used for washing until the pH is neutral, the chromatography column is balanced by using 5-10 CV balance Buffer (10 mM Tris-HCl and pH 7.5), the elution component collected in the example 3 is diluted to have a conductivity below 5mS/cm and filtered, the elution component is loaded on the balanced chromatography column, after the loading is finished, the residual impurity protein on the column material is washed off by using 5CV balance Buffer, the target protein is eluted by using 5-10 CV (10 mM Tris-HCl, 500mM NaCl and pH 7.5), the chromatography packing is washed by using 3-5 CV 2M NaCl and 3-5 CV0.5 mM OH as described in the example 3, and the target protein is stored in 20% ethanol. The collected eluate containing the target protein was subjected to SDS-PAGE gel electrophoresis, and the detection results are shown in FIG. 2. As can be seen from FIG. 2, a small amount of hybrid protein still exists after the two-step composite hydrophobic chromatography, and the detection result shows that the purity of the target protein in the elution buffer solution after purification is more than 90 percent, and the recovery rate of the two-step composite hydrophobic chromatography protein is 70.92 percent by calculation, and the loading amount of the two-step composite hydrophobic chromatography protein is 6.82mg/mL column charge.
Example 5: hydrophobic chromatography of T4DNA ligase
25g of hydrophobic chromatography medium Butyl Sepharose High Performance was packed in XK 16/20, pretreated with 2-3 CV0.5M NaOH, washed with deionized water to neutral pH, and equilibrated with 5-10 CV Buffer (10 mM Tris-HCl, 1.5M (NH) 4 ) 2 SO 4 pH 7.5) the column was equilibrated and the one-step elution fraction, i.e., the elution fraction collected in example 3, was supplemented with (NH) 4 ) 2 SO 4 To a final concentration of 1.5M, add (NH) 4 ) 2 SO 4 Filtering with 0.45 μm filter membrane, loading onto balanced chromatographic column, washing off residual impurity protein on column material with 5CV balance Buffer, eluting Buffer with 5-10 CV (10 mM Tris-HCl, 0.75M (NH) 4 ) 2 SO 4 pH 7.5) and collected, and then the chromatographic packing was washed with 3-5 CV deionized water and 3-5 CV0.5M NaOH and stored in 20% ethanol. For the collected content meshThe eluate of the protein was subjected to SDS-PAGE gel electrophoresis, and the detection result is shown in FIG. 4. With reference to FIG. 4, the recovery rate of the two-step hydrophobin is 55.34%, the loading capacity of the two-step hydrophobin is 4.53mg/mL, and the detection result shows that the purity of the target protein in the elution buffer after purification>90％。
Example 6: affinity chromatography of T4DNA ligase
And loading 25g of affinity chromatography medium Ni Sepharose HP on XK 16/20, pretreating the chromatography medium by using 2-3 CV 500mM imidazole, washing the chromatography medium by using deionized water until the pH is neutral, balancing the chromatography column by using 5-10 CV balance Buffer (10 mM Tris-HCl, 500mM NaCl,10 mM imidazole and pH 7.5), adding 10mM imidazole with the final concentration to the two-step composite hydrophobic chromatography elution component which is collected in the embodiment 4 or the embodiment 5, completely dissolving the elution component, filtering, loading the elution component on the balanced affinity chromatography column, washing the residual protein on the column by using 5CV balance Buffer after loading, washing the residual protein on the chromatography column by using 5-10 CV balance Buffer (10 mM Tris-HCl, 500mM NaCl, 50mM imidazole and pH 7.5), binding weaker protein on the chromatography column by using 5-10 CV balance Buffer (10 mM Tris-HCl, 500mM NaCl,100mM imidazole and pH 7.5), eluting the target protein by using 3-10 CV balance Buffer (10 mM NaCl, 500mM imidazole and pH 7.5), washing the residual protein on the final washing the chromatography column by using 3-10 CV balance Buffer (10 mM NaCl, 500mM NaCl, and washing the residual protein on the final washing the column by using the final ethanol. The collected eluate containing the target protein was subjected to SDS-PAGE gel electrophoresis, and the detection results are shown in FIGS. 3 and 5. FIG. 3 is a SDS-PAGE of the fractions collected by the complex hydrophobic chromatography elution of example 4 for the affinity chromatography process. FIG. 5 is a SDS-PAGE of the hydrophobic chromatography elution fraction collected in example 5 for the affinity chromatography process. As can be seen from FIG. 3, the high-purity protein is obtained after affinity chromatography is carried out on the elution collection liquid of the composite hydrophobic chromatography, the detection result shows that the purity of the target protein in the elution buffer liquid after the purification is more than 95 percent, the protein recovery rate after three steps of affinity chromatography is 80.31 percent by calculation, and the protein load of the three steps of affinity chromatography is 5.38mg/mL column material.
Example 7: post-treatment of T4DNA ligase
Selecting 0.1m according to the protein amount in the three-step elution component 2 The 10kDa ultrafiltration membrane package is sterilized and pyrogen removed by washing the ultrafiltration membrane package with 0.5M NaOH circulation for more than 30min, washing the membrane package with ultrapure water until the pH is neutral, washing the membrane package with a displacement Buffer (20 mM Tris-HCl, 100mM KCl, pH 7.5) until the pH is 7.5+ -0.5, concentrating the elution components collected in example 6 to the dead volume of the membrane package, adding a displacement Buffer with a volume 2 times of the dead volume of the membrane package for displacement, wherein the number of times of displacement is 3-5, the end point of displacement is pH 7.5+ -0.5, and the deviation between the conductance and the displacement Buffer is not more than 1mS/cm. Accurately measuring a certain volume of ultrafiltration replacement semi-finished product, adding glycerol, 1mM DTT (DTT) with final concentration and 0.1mM EDTA (EDTA) with final concentration according to a volume ratio of 1:1, fully and uniformly mixing, and filtering by using a 0.22 mu m sterile filter to obtain the finished product. The finished product is subjected to SDS-PAGE gel electrophoresis, and the quality analysis is carried out on the finished product obtained by the process, and the detection results are shown in Table 1, and are shown in fig. 6 and 7. As can be seen from Table 1, FIG. 6 and FIG. 7, the T4DNA ligase RNase and the Nickase prepared by the two-step hydrophobic chromatography process of example 5 have slight contamination and low host DNA residues; the T4DNA ligase is obtained by the preparation process of the two-step composite hydrophobic chromatography of the example 4, and has no RNase and Nickase pollution and low host DNA residue. Table 1 shows the results of the detection of host DNA residues for the two end products obtained in example 7, where ERC represents the labeled sample.
TABLE 1 detection results of host DNA residues of two products obtained in example 7
Example 8: enzyme activity detection method of T4DNA ligase
T4DNA ligase Activity assay: the ligation reaction was set up as shown in Table 2 below, and a blank reaction was set up as ddH 2 O was used instead of the enzyme to be tested, the positive control was set with the addition of the amount of enzyme capable of complete ligation (15U/50. Mu.L), the molecular weight control was lambda DNA-HindIII Marker at the electrophoresis detection point, and two experiments were performed in parallel for each gradient.
The T4DNA ligase obtained in example 7 was diluted 100 to 400 times and subjected to ligation, and two experiments were performed in parallel for each gradient.
Preparing a reaction system for detecting the activity of T4DNA ligase according to the specification shown in Table 2, lightly mixing, reacting for 30min at 16 ℃, incubating for 10min at 65 ℃ after the reaction is finished, and placing on ice. And detecting connection conditions by electrophoresis, and calculating enzyme activity by multiplying the minimum volume of enzyme added according to the connection conditions by dilution times. The detection result is shown in FIG. 8. The detection conditions of the lanes in FIG. 8 are shown in Table 3, and the blank control in Table 3 is ddH 2 O replaces the enzyme to be tested. For example: lanes 1-5 are the enzyme activity detection performed by diluting the fraction collected by the complex hydrophobic chromatography of example 4 (i.e., the complex hydrophobic chromatography is used in two steps) by the affinity chromatography of example 6 and the final product obtained by the post-treatment of example 7 by 200 times, respectively adding 0,0.4,0.6,0.8,1,2. Mu.L of the enzyme volume to the reaction system, and lanes 14-18 are the enzyme activity detection performed by diluting the fraction collected by the hydrophobic chromatography of example 5 (i.e., the complex hydrophobic chromatography is used in two steps) by the affinity chromatography of example 6 and the final product obtained by the post-treatment of example 7 by 200 times and adding 0,0.4,0.6,0.8,1,2. Mu.L of the enzyme volume to the reaction system. As shown in fig. 8, on the premise of the same enzyme activity, after the finished products of the two preparation processes are diluted by the same multiple, the connection efficiency of the preparation process adopting the composite hydrophobic chromatography in the two steps is obviously higher than that of the preparation process adopting the hydrophobic chromatography in the two steps.
TABLE 2 T4DNA ligase activity detection reaction system
| Reagent(s) | Volume of |
| 10 Xbuffer | 5μL |
| λDNA-Hind Ⅲ | 15μL |
| Diluted enzyme to be tested | 0,0.4,0.6,0.8,1,2 mu L are added respectively |
| ddH 2 O | Is added to 50 mu L |
TABLE 3 detection conditions for lanes in FIG. 8
Example 9: t4DNA ligase ligation reproducibility assay
The T4DNA ligase prepared by the one-step anion exchange chromatography, the two-step composite hydrophobic chromatography and the three-step affinity chromatography is further detected. Enzyme repeatability: the T4DNA ligase obtained in example 7 and commercially available T4DNA ligase were diluted 100 to 400 times and subjected to an experiment, a ligation system was prepared as shown in Table 4, 5 parallel reactions were prepared, the reaction was performed at 16℃for 30min, and after the completion of the reaction, incubation was performed at 65℃for 10min, and the ligation was performed on ice and the reproducibility was judged by observing the ligation conditions exhibited by agarose gel electrophoresis. The detection result is shown in FIG. 9. The detection conditions of the lanes in FIG. 9 are shown in Table 5, and in Table 5, the T4DNA ligase obtained in example 7, which is a self-produced, blank control was obtained as ddH 2 O replaces the enzyme to be tested. As shown in FIG. 9, the T4DNA ligase prepared by the invention has better reproducibility by taking a commercial product as a positive control.
TABLE 4 enzyme ligation reproducibility assay reaction system
| Reagent(s) | Volume of |
| 10 Xbuffer | 2μL |
| λDNA-HindⅢ | 10μL |
| Diluted enzyme to be tested | 0.5,1,2,4,6 mu L are added respectively |
| ddH 2 O | Is added to 20 mu L |
Table 5 detection conditions for lanes in FIG. 9
Example 10: t4DNA ligase ligation thermostability assay
Enzyme thermostability: the final product obtained in example 7 and the commercial product were placed in a 37 ℃ water bath for incubation for 0, 1, 3, 5 and 7 days, respectively, a connection system was prepared as shown in table 4, diluted by an appropriate factor, reacted at 16 ℃ for 30min, incubated at 65 ℃ for 10min after the reaction was completed, placed on ice, and the thermal stability was judged according to the connection condition exhibited by nucleic acid agarose electrophoresis. The detection results are shown in FIG. 10. The detection conditions of the lanes in FIG. 10 are shown in Table 6, and in Table 6, the T4DNA ligase obtained in example 7, which is a self-produced product, the positive control is a commercially available product. As shown in FIG. 10, after incubation at 37℃for 1 day, the ligation efficiency of the commercially available T4DNA ligase was significantly reduced, whereas the T4DNA ligase prepared according to the present invention started to have a tendency of reduced ligation efficiency after incubation at 37℃for 5 days, and had relatively good thermostability.
TABLE 6 detection conditions for lanes in FIG. 10
Example 11: determination of T4DNA ligase ligation freeze-thaw stability
Enzyme freeze-thaw stability: the final product obtained in example 7 and the commercial product were subjected to repeated freeze thawing at-80℃to 25℃for 15 times, diluted by a suitable factor, and a connection system was prepared as shown in Table 4, reacted at 16℃for 30min, incubated at 65℃for 10min after the completion of the reaction, and placed on ice, and the freeze thawing stability was judged according to the connection conditions exhibited by nucleic acid agarose electrophoresis. The detection results are shown in FIG. 11. The detection conditions of the lanes in FIG. 11 are shown in Table 7, and in Table 7, the T4DNA ligase obtained in example 7, which is self-produced, is used as a blank control in ddH 2 O replaces the enzyme to be tested. As shown in FIG. 11, the T4DNA ligase prepared by the invention has better ligation efficiency after repeated freezing and thawing for 15 times under the same freezing and thawing times by taking a commercial product as a positive control.
TABLE 7 detection conditions for lanes in FIG. 11
According to the preparation method of the research, the purification process conditions of pretreatment (namely, the acquisition of crude enzyme liquid), three-step chromatography and post-treatment (namely, ultrafiltration replacement and preservation) are obtained through screening and combination of various fillers, the pretreatment can solve the problems that the bacterial sludge is difficult to filter after being crushed and the crude enzyme liquid contains more impurity proteins, and the step is easy for large-scale production; the three-step chromatography process comprises anion exchange chromatography, hydrophobic chromatography and affinity chromatography, and the process is easy to connect and easy to linearly amplify; and then the recombinant T4DNA ligase with high quality can be output after post-treatment. The T4DNA ligase with low host DNA residue and enzyme purity up to 95% is successfully prepared by the preparation method, the purity is high, RNase and Nickase pollution is avoided, the operation of using high-salt solution or ammonium sulfate and the like is avoided, and the enzyme activity is better protected from the influence of extreme conditions, so that the enzyme activity is higher. The preparation method of the T4DNA ligase has the advantages of simple operation method, high yield, high protein purity, high enzyme activity, easy mass production and the like. Compared with the main stream products in the market, the property evaluation result shows that the T4DNA ligase obtained by the purification of the invention has better reproducibility, thermal stability and freeze thawing stability.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method for preparing a T4DNA ligase, comprising the steps of:
performing anion exchange chromatography on the crude enzyme solution containing the T4DNA ligase to obtain a first eluent;
performing hydrophobic chromatography or composite mode chromatography on the first eluent to obtain a second eluent;
and carrying out affinity chromatography on the second eluent to obtain the T4DNA ligase.
2. The method for producing T4DNA ligase according to claim 1, wherein the chromatography medium for anion exchange chromatography is selected from the group consisting of Q Sepharose FF, Q Sepharose 4FF, Q Sepharose XL, Q Sepharose HP, Q Sepharose Big Beads, capto Q, QAE Sephadex A-25, QAE Sephadex A-50, DEAE Sepharose FF, DEAE Sepharose CL-6B, DEAE Sephacel, capto DEAE and Anx Sepharose 4 FF.
3. The method for producing T4DNA ligase according to claim 1, wherein the step of subjecting the crude enzyme solution to anion exchange chromatography comprises:
a counter-ion exchange column;
the electric conductivity of the crude enzyme liquid is regulated to be lower than 3mS/cm, and then the crude enzyme liquid is loaded to the balanced anion exchange column;
washing impurities of the loaded anion exchange column;
eluting the anion exchange column subjected to impurity washing treatment by adopting a first eluting buffer solution to obtain the first eluting buffer solution, wherein the pH value of the first eluting buffer solution is 7.0-8.0 and the first eluting buffer solution comprises 10-50 mM Tris-HCl, 200-300 mM NaCl and 1-3 mM DTT.
4. The method for producing T4DNA ligase according to claim 3, wherein the pH of the equilibration buffer used for equilibration of the anion exchange column is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl, 1 mM-3 mM DTT;
and/or the pH of the impurity washing buffer solution used for the impurity washing treatment of the anion exchange column after the sample loading is 7.0-8.0, and the buffer solution comprises 10 mM-50 mM Tris-HCl, 50 mM-100 mM NaCl and 1 mM-3 mM DTT.
5. The method for producing T4DNA ligase according to claim 1, wherein in the step of subjecting the first eluent to the composite mode chromatography, a chromatography medium is used which is selected from any one of Capto MMC ImpRes and Capto MMC.
6. The method of preparing a T4DNA ligase according to claim 5 wherein the step of subjecting the first eluate to hydrophobic chromatography or complex mode chromatography comprises:
a balanced hydrophobic chromatography column or a composite mode chromatography column;
after the electric conductivity of the first eluent is regulated to be below 5mS/cm, loading the first eluent into the balanced hydrophobic chromatography column or the composite mode chromatography column;
and eluting the loaded hydrophobic chromatography column or the composite mode chromatography column by adopting a second eluting buffer solution to obtain the second eluting solution.
7. The method for producing T4DNA ligase according to claim 6, wherein the second elution buffer has a pH of 7.0 to 8.0 and comprises 10mM to 50mM Tris-HCl, 300mM to 500mM NaCl.
8. The method for producing T4DNA ligase according to claim 6, wherein the pH of an equilibration buffer used for equilibration of the hydrophobic chromatography column or the composite mode chromatography column is 7.0 to 8.0 and comprises 10mM to 50mM Tris-HCl.
9. The method for preparing the T4DNA ligase according to claim 1, wherein the T4DNA ligase carries a His tag and the mode of performing affinity chromatography on the second eluent is Ni column affinity chromatography;
and/or the affinity chromatography medium is selected from any one of Ni Sepharose HP, ni Sepharose 6FF, TALON Superflow and Ni Sepharose excel;
and/or the pH of the balance buffer used for the affinity chromatography medium is 7.0-8.0, and comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl and 10 mM-20 mM imidazole;
and/or the pH of the washing buffer used for the affinity chromatography medium is 7.0-8.0, and the washing buffer comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl and 30 mM-50 mM imidazole;
and/or the pH of the elution buffer used for the affinity chromatography medium is 7.0-8.0 and comprises 10 mM-50 mM Tris-HCl, 300 mM-500 mM NaCl,100 mM-200 mM imidazole.
10. The method for producing T4DNA ligase according to any one of claims 1 to 9, further comprising the step of producing the crude enzyme solution before the step of subjecting the crude enzyme solution to anion exchange chromatography: crushing the fermented bacterial sludge for producing the T4DNA ligase, adding a flocculating agent or a water-soluble cationic high molecular polymer with the final volume concentration of 0.1-1%, standing, carrying out solid-liquid separation, and collecting the supernatant to obtain the crude enzyme liquid;
and/or, after the step of performing affinity chromatography on the second eluent, the method further comprises the step of performing ultrafiltration treatment on the affinity chromatography eluent.
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