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Method for purifying recombinant escherichia coli Taq DNA polymerase

The technology of a recombinant Escherichia coli and a purification method is applied in the purification field of recombinant Escherichia coli Taq DNA polymerase, and can solve the problems of increasing the content of impurity proteins, increasing the cost, increasing the complexity of the process and the like

Inactive Publication Date: 2015-04-29
天津强微特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the subsequent Taq The article on DNA polymerase purification method also adopts the ion exchange process, but although the ion exchange process can increase the loading capacity, due to its own elution characteristics, the content of its impurity protein will increase. If it reaches a certain Purity requires additional steps of purification, which increases process complexity and cost

Method used

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  • Method for purifying recombinant escherichia coli Taq DNA polymerase
  • Method for purifying recombinant escherichia coli Taq DNA polymerase
  • Method for purifying recombinant escherichia coli Taq DNA polymerase

Examples

Experimental program
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Effect test

Embodiment 1

[0048] 3 L of fermentation broth was centrifuged to obtain 150 g of bacteria, the operating temperature was 4 °C, and 750 ml of bacteriostasis buffer was added (the ratio of bacteria to bacteriostasis buffer was 1:5), in which the bacteriostasis buffer was 20 mM Tris- HCl, pH7.5, 0.1 mM EDTA, 0.5 M NaCl, 0.5 mg / ml lysozyme, 0.02 mM PMSF, stirred for 10 min to make it evenly mixed, lysed the bacteria by high-pressure homogenization, that is, the mixed liquid was at 600 bar, Homogenize 2 times at 4 ℃ to make it no longer viscous; at 10000 g Centrifuge at 10°C for 10 min to obtain 950 ml supernatant.

[0049] Put the bacterium supernatant into a glass beaker, put it into a pre-heated water bath, stir the bacterium supernatant with a glass rod to make the temperature even, use a thermometer to detect the temperature change in real time, wait for the bacterium supernatant Start timing when the medium temperature reaches above 80 °C, and process at 80 °C for 30 min. Then put the s...

Embodiment 2

[0057] 5 L of fermentation broth was centrifuged to obtain 280 g of bacteria, the operating temperature was 10 °C, and 5600 ml of bacteriostasis buffer was added (the ratio of bacteria to bacteriostasis buffer was 1:20), and the bacteriostasis buffer was 50 mM Tris- HCl, pH8.0, 1 mM EDTA, 1 M NaCl, lysozyme 1 mg / ml, 1 mM PMSF, stirred for 10 min to make it evenly mixed, lysed the bacteria by high-pressure homogenization, that is, the mixed liquid was at 800 bar, Homogenize twice at 10°C to make it no longer viscous; at 15000 g Centrifuge at 10°C for 10 min to obtain 5700 ml of bacteriostasis solution.

[0058] Grind 1630 g with a mortar in advance, and slowly add (50% saturation) to the supernatant of the broken bacteria while stirring to ensure that the local concentration will not be too high. The precipitation temperature is controlled at 10 ℃, and the precipitation time is overnight. 15000 g Centrifuge for 20 min at a temperature of 10°C.

[0059] Grind 3920 g of ammon...

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Abstract

The invention discloses a method for purifying recombinant Escherichia coli Taq DNA polymerase. The method for purifying the recombinant Escherichia coli Taq DNA polymerase comprises the following steps: (1) breaking bacteria; (2) carrying out thermal treatment; (3) salting out; (4) carrying out Ni ion metal-chelated affinity chromatography; (5) dialyzing; and (6) obtaining preparation and carrying out split charging. The method for purifying the recombinant Escherichia coli Taq DNA polymerase has the advantages that a preparation technique is simple, easy and quick, quality of the obtained recombinant Escherichia coli Taq enzyme is high, cost is low, and the method for purifying the recombinant Escherichia coli Taq DNA polymerase is applicable to industrial large-scale production.

Description

technical field [0001] The present invention relates to a method for purifying biological enzymes, in particular to a recombinant Escherichia coli Taq Purification method of DNA polymerase. Background technique [0002] Taq DNA polymerase was obtained from a species of Thermus aquaticus ( Thermus aquaticus ) yT1 strain isolated and extracted. The yT1 strain is a thermophilic eubacterium that can grow at 70-75°C. The yT1 strain was isolated from the volcanic hot springs in the Yellowstone National Forest Park in the United States in 1969. Taq The full-length DNA polymerase gene is 2496 bases, encoding 832 amino acids, the enzyme protein molecule is 94 KDa, each enzyme molecule can extend about 150 nucleotides per second at 75-80 °C, and the elongation rate at 70 °C is greater than 60 The thermal stability of Taq DNA polymerase is a prerequisite for the enzyme to be used in PCR reactions, and it is also the reason why PCR reactions can develop rapidly and be widely use...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12R1/19
CPCC12N9/1252C12Y207/07007
Inventor 张涛田方郑春阳
Owner 天津强微特生物科技有限公司
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