CN106434592A - Method for rapid purification of Stoffel fragment Taq enzyme - Google Patents

Abstract

The invention discloses a method for quickly purifying Stoffel fragment Taq enzyme. After culturing overnight in LB medium, the bacterial solution is inoculated in LB medium to continue culturing until OD ₆₀₀ is 0.4, and the bacteria are resuspended in 4×Taq storage buffer Bacterial cells were placed in a boiling water bath, boiled and lysed, centrifuged, treated with an aqueous phase/organic phosphoric acid system, an equal volume of sterile glycerol was added to the upper organic phase containing Taq enzyme, and stored at -20°C for later use. The method of the invention has the characteristics of being more reliable, faster and simpler, and can provide a new purification technology for the wide application of PCR amplification technology.

Description

A method for rapidly purifying Stoffel fragment Taq enzyme

technical field

The invention belongs to the technical field of molecular biology, in particular to a method for rapidly purifying Stoffel fragment Taq enzyme.

Background technique

Polymerase chain reaction (PCR) is the most widely used DNA polymerase in molecular biology research. During the PCR process, DNA polymerase derived from Thermus aquaticus, which is stable at high temperature, is required. The development of genetic engineering strains has greatly improved the production of Taq enzyme. Lawyer et al. first reported the gene sequence of the full-length Taq polymerase, which encodes 832 amino acids and produces a 94KDa Taq enzyme. Subsequently, they truncated and improved the N-terminus of Taq to express a protein of 544 amino acids and 61KDa. Compared with the full-length Taq polymerase, the lack of 5'-3' endonuclease activity can improve the fidelity in PCR amplification; in addition, the Stoffel fragment Taq enzyme has higher heat resistance characteristics, and the half-life at 97.5°C is 21 minutes, while the full-length Taq is only 9 minutes. These two properties make Stoffel fragment Taq enzymes more widely used in biotechnology applications.

Due to the wide application of Stoffel fragment Taq polymerase, it is necessary to develop a simple and efficient purification method to obtain a large amount of Stoffel fragment Taq polymerase to meet the needs of laboratories for Taq enzymes. Utilizing the heat-resistant properties of Stoffel fragments, the applicant has developed a simple and convenient purification method. Using this method, the entire purification process only takes 1 to 2 hours, and 80 mg, about 1.6×10 ⁷ units, can be obtained from 1 L of bacterial liquid Taq enzyme. Compared with existing purification techniques, the method is not only simple, but also time-saving.

Contents of the invention

The purpose of the present invention is to provide a simple, fast and practical method for extracting and purifying TaqDNA polymerase.

The present invention is specifically realized through the following technical solutions:

A method for rapidly purifying Stoffel fragment Taq enzyme, specifically comprising the following steps:

1) Activation and expression of engineered bacteria: Inoculate engineered bacteria with Stoffel fragment Taq DNA polymerase in LB medium and culture overnight at 37°C;

2) According to the proportion, take the bacterial liquid and inoculate it in LB medium, continue to cultivate at 37°C until the OD ₆₀₀ is 0.4, add IPTG to a final concentration of 0.5mM, and continue to cultivate for 12-16 hours;

3) Centrifuge at 4000g for 5 minutes to collect the cells, resuspend the cells with 4×Taq storage buffer, place the cells in a boiling water bath, and boil to lyse;

4) After lysis, centrifuge at 16,000 g for 10 min at 4°C to remove cell residues and denatured proteins, and the supernatant is treated with an aqueous phase/organic phosphoric acid system for later use.

The LB medium of the present invention contains 100 μg/ml Kan.

The bacterial solution of the present invention is inoculated with the LB medium according to the volume ratio of 1:100.

The Taq storage buffer of the present invention is specifically: 20mM Tris-HCl pH 8.0, 10mM DTT, 0.1mM EDTA, 100mM KCl, 0.5% Nonidet P40, 0.5% Tween 20.

The boiling water bath heating condition of the present invention is: every 4min one cycle, totally two cycles, heating 8min.

The supernatant treatment method described in the step (4) of the present invention is specifically: add 10% sterile balanced potassium dihydrogen phosphate and mix, add an equal volume of dehydrated alcohol and mix, 2000g centrifugal 2min, in the upper layer containing Taq enzyme An equal volume of sterile glycerol was added to the organic phase and stored at -20°C.

The beneficial effects of the present invention are: 1) fast: in a single storage buffer, the whole purification process can be completed in only 1-2 hours, and no special equipment and no toxic reagents are used; 2) reliable and stable: after dilution After 10 times the Taq enzyme was stored at -20°C for 12 months, its activity did not change; after an overnight incubation test from 37°C to 72°C, no nuclease activity was found; 3) The aqueous/organic system is used for convenience And efficiently remove nucleic acid contamination without reducing the yield of Taq enzyme.

Description of drawings

Fig. 1 is the result of carrying out PCR amplification to aadA gene after using purified Taq enzyme to dilute at different concentrations;

Figure 2 is the result of amplifying the commercialized Taq enzyme and the Taq enzyme purified by this technology using genes of different fragment sizes;

Fig. 3 is the SDS-PAGE electrophoresis result of the purified Stoffel fragment;

Fig. 4 is the SDS-PAGE electrophoresis result of the Stoffel fragment purified by different treatment time;

Figure 5 shows the nucleic acid contamination in the boiled supernatant before and after treatment with the Aqueous/Organic biphasic system.

detailed description

The present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention to other forms. Changes to equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the solution content of the present invention fall within the protection scope of the present invention.

Example 1

1) Materials and methods

Strains and plasmids

The pBio-Taq expression vector was kindly donated by Dr. Xiongfei Ding, and its basic vector is pET-29a. Wherein, it contains T7 promoter and Taq gene sequence. Consistent with Lawyer et al.'s report, the gene expresses a 61KDa Stoffel fragment. BL21(DE3) is used as the vector host, and the bacteria carry the pLysS plasmid to make it easier to break and release the recombinant protein.

2) Purification of the truncated Taq polymerase Stoffel fragment

The plasmid for transforming BL21 bacteria was inoculated in 5 ml of LB medium containing kan (100 μg/ml), and cultured at 37° C. overnight. According to the ratio of 1:100, take 1ml of the bacterial liquid and inoculate it into 100ml of LB medium containing Amp (100μg/ml), continue to cultivate at 37°C until the OD600 is 0.4, add IPTG to the final concentration of 0.5mM, and continue to cultivate for 12-16 Hour. Centrifuge at 4000g for 5min, collect the cells, and resuspend the cells in 5ml 4×Taq storage buffer (20mM Tris-HCl(pH 8.0), 10mM DTT, 0.1mM EDTA, 100mM KCl, 0.5%Nonidet P40, 0.5%Tween 20) , the bacteria were put into the boiling water bath for different time (0-12min) and times (4min per cycle). After boiling and lysis, centrifuge at 16,000g for 10 minutes at 4°C to remove cell residues and denatured proteins, then transfer the supernatant to a new tube. In order to remove contaminating nucleic acids, the supernatant was used in the aqueous phase/organic phosphate system, and the upper organic phase containing Taq enzyme was transferred to a new tube, and an equal volume of sterile glycerol was added, stored at -20°C, and properly diluted before use.

Example 2 Protein analysis and activity determination

1) Experimental method

The concentration of Taq protein was determined by Bradford method. Take 10 μl of the sample purified in Example 1 and perform SDS-PAGE electrophoresis to estimate the protein composition.

After the purified truncated Taq polymerase was diluted, PCR amplification was performed to measure its enzyme activity, and Taq enzyme from Takara Company was used as a control. The purified truncated Taq polymerase was diluted with 1×Taq storage buffer at 1:5, 1:10, 1:20, 1:50 to amplify the cloned fragment of PU C18. In the 50μl system, use 0.2(l Taq enzyme, the reaction system is 1×PCR reaction buffer (10mM KCl, 20mM Tris-HCl (pH 8.8), 10mM (NH4)2SO4, 0.1% TritonX-100, 0.1mg/ml BSA) , 0.2mM d NTPs, 2.0mM MgSO4, 0.2(M primers (5'-atggcaccacaaacagagg-3' and 5'-ttatttgccgactaccttgg-3') amplified 800bp aadA gene (encoding aminoglycoside 3'-adenylytransferase), 25ng template DNA.

The reaction conditions were: total denaturation at 95°C for 3 minutes, denaturation at 94°C for 60 s, annealing at 55°C for 60 s, and extension at 72°C for 180 s, for a total of 30 cycles. The PCR reaction was performed on a MJ research Minicycler (USA) reactor. After the completion of the PCR program, 1 μl, 2 μl, 5 μl, and 10 μl of the PCR products were taken for electrophoresis analysis on 1.2% agarose gel, the buffer was TBE, and the molecular weight standard was DL2000. In order to test the amplification ability of Taq enzyme to fragments of different sizes, the 500bp IGF-1 gene, the 1500bp BADH gene and the 2.7kb tobacco chloroplast trnI-trnA gene fragment were respectively amplified to verify the amplification efficiency of Taq enzyme.

2) Results

Using commercial Taq enzyme as a control, serial dilution was performed, and the activity of the purified Stoffel fragment of Taq enzyme was amplified by PCR, and the activity was determined ( FIG. 1 ).

For the 0.8kb fragment amplified by 10-fold diluted Taq enzyme, take 1 μl of PCR product for electrophoresis, and its yield is equivalent to 2 μl PCR product of commercial Taq enzyme (lane 2). Therefore, use 10-fold diluted Taq enzyme for its The activity is twice that of the commercial Taq enzyme. Therefore, the activity of Taq diluted 10 times is 10 units/μl, and the concentration of the mother solution is 100 units/μl. According to this result, it can be concluded that 1.6×10 ⁷ units of Taq enzyme can be obtained from 1 L of bacterial solution. In addition, when the Taq enzyme was diluted to 20 or 50 times, only little enzyme activity was found.

In order to verify the stability of the Taq enzyme, DNA fragments of different fragment sizes were used for amplification, and the results are shown in Figure 2. The 10-fold diluted Taq enzyme and commercial Taq enzyme (Takara) amplified DNA fragments from 0.5 kb to 3.0 kb, respectively. The results showed that the purified Taq enzyme was consistent with the amplification result of commercial Taq enzyme.

The establishment of embodiment 3 purification method

1) Determination of boiling cracking time

In order to compare the effects of different boiling and cleavage times on the purification of Taq enzyme Sottfel fragments, 7 boiling times of 0, 2, 4, 6, 8, 10 and 12 min were used for analysis, and the results are shown in Figure 3.

From Figure 3, the expected Stoffel fragment (61kDa) was released into 4-fold Taq storagebuffer, boiled for 8 minutes with higher Taq enzyme yield, and no foreign protein contamination. No Taq protein was found in the unboiled control. In the control not induced by IPTG, only a small amount of Taq enzyme was produced.

2) Boiling times

In this experiment, after boiling for 4 minutes, centrifugation at 16,000g for 10 minutes at 4°C was used as a cycle. The results are shown in Figure 4. After 2 cycles and co-boiling for 8 minutes, Taq enzyme was produced in a high amount. At the same time, it was found that the production of Taq enzyme decreased after three cycles. At the same time, it can be seen from the figure that there is very little other foreign protein contamination.

3) Remove nucleic acid contamination by Aqueous/Organic biphasic system

In order to verify nucleic acid contamination, 10 μl of boiled supernatant was taken for agarose gel electrophoresis analysis, the results are shown in Figure 5.

The results showed that the samples without biophasic treatment had nucleic acid contamination (lanes 1-3). In order to eliminate nucleic acid contamination, use 2 simple steps: 1) Add 10% sterile balanced potassium dihydrogen phosphate (pH10.3), mix for a few seconds; 2) Add an equal volume of absolute ethanol into the above step 1 solution, mix for a few seconds. Centrifuge at 2,000 g for 2 min, transfer the supernatant containing Taq enzyme into a new tube, add an equal volume of sterile glycerol, and store at -20°C. Through the above steps, nucleic acid contamination can be effectively removed (lane 4).

In summary, compared with previous reports, this method has the following advantages: In a single storage buffer, the entire purification process can be completed in only 1-2 hours, and no special equipment and no Toxic reagent. 80mg, about 1.6×107 units of Taq enzyme can be obtained from 1L of bacterial solution, and the results are consistent with those reported by Desai and Lawyer’s et al. After 10-fold diluted Taq enzyme was stored at -20°C for 12 months, its activity did not change. No nuclease activity was found in the overnight incubation test at 37°C to 72°C.

In the process of optimizing the purification conditions, the yield of Taq enzyme was also taken into consideration. Experiments showed that the boiling time and times had a certain influence on the yield of the enzyme. 2 cycles, boiling for 8 minutes is the most suitable. Under such conditions, a large amount of E. coli proteins are denatured and removed, and at the same time, Taq enzyme is released into the supernatant. The production of Taq enzyme decreased when the boiling time was more than 8 minutes. In addition, the centrifugation rate also has a certain impact on protein purification. It is recommended to use centrifugation at 16,000g for 10 minutes at 4°C to remove protein.

During the purification process of Taq enzyme, if RAPD and other analyzes are used, it is necessary to remove nucleic acid contamination. In previous studies, several methods were used to remove nucleic acid contamination, such as PEI preparation and ion-exchange chromatography, 8-methoxypsoralen treatment, UV light treatment, restriction endonuclease digestion, and dialysis. These methods are not only time-consuming, but also may reduce the yield of Taq enzyme. In this paper, an aqueous/organic system is used to remove nucleic acid contamination easily and efficiently.

In summary, the present application provides a more reliable, faster and simpler method for purifying Taq enzyme. Using the boiling method, a large amount of highly active Taq enzyme can be purified within 1-2 hours, and this method can provide a new purification technology for the wide application of PCR amplification technology.

Claims (6)

1. the method for a fast purifying Stoffel fragment Taq enzyme, it is characterised in that comprise the following steps：

1) activation of engineering bacteria and expression：Take Stoffel fragment Taq archaeal dna polymerase engineering bacteria to be seeded in LB culture medium, in 37 DEG C of overnight incubation；

2) proportionally, taking bacterium solution to be inoculated in LB culture medium, continuing to cultivate to OD600 in 37 DEG C is 0.4, adds IPTG to end Concentration is 0.5mM, continues to cultivate 12-16 hour；

3) centrifuging 5min in 4000g, collecting thalline, with the resuspended thalline of 4 × Taq storage buffer, thalline is placed in boiling water In bath, boiling lysis；

4) albumen after 16000g centrifuges 10min removing cell residuum and denaturation at 4 DEG C after cracking, supernatant employing aqueous phase/ Organic phosphoric acid system is standby after processing.

2. the method for a kind of fast purifying Stoffel fragment Taq enzyme according to claim 1, it is characterised in that：Described Kan containing 100 μ g/ml in LB culture medium.

3. the method for a kind of fast purifying Stoffel fragment Taq enzyme according to claim 1, it is characterised in that：Described Bacterium solution and LB culture medium are according to volume ratio 1:100 inoculations.

4. the method for a kind of fast purifying Stoffel fragment Taq enzyme according to claim 1, it is characterised in that：Described Taq storage buffer is specially：20mM Tris-H Cl pH 8.0,10mM DTT,0.1mM EDTA,100mM KCl, 0.5%Non idet P40,0.5%Tween 20.

5. the method for a kind of fast purifying Stoffel fragment Taq enzyme according to claim 1, it is characterised in that：Described Boiling water bath heating condition is：Every 4min mono-circulation, totally two circulations, heat 8min.

6. the method for a kind of fast purifying Stoffel fragment Taq enzyme according to claim 1, it is characterised in that：Step (4) the supernatant processing method described in is specially：Add 10% aseptic balance potassium dihydrogen phosphate to mix, add isopyknic anhydrous second Alcohol mixes, and 2000g centrifuges 2min, adds equal-volume sterile glycerol, store in-20 DEG C in the upper organic phase containing Taq enzyme.