Microwave-assistant extraction method of recombinant Escherichia coli Pfu DNA (deoxyribonucleic acid) polymerase

A technology of recombinant Escherichia coli and extraction methods, applied in biochemical equipment and methods, enzymes, transferases, etc., can solve the problems of protein denaturation and inactivation, few, and loss of physiological activity, and achieve easy automatic control, good effect, Effect of reducing operation steps and time

Inactive Publication Date: 2014-04-23
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the heat generated during microwave radiation, most proteins are denatured and inactivated and lose their physiological activity, so microwave radiation is rarely used to extract enzymes.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The Escherichia coli with recombinant Pfu DNA polymerase is induced and cultivated to obtain bacterial cells expressing Pfu DNA polymerase recombinantly, and then the following steps are adopted:

[0051] (1) Calculate the dry weight based on the determined water content (75%) of the wet bacteria, and add 0.03mol L according to the weight-to-volume ratio of the dry weight of the bacteria to the g / mL of the buffer solution as 1:10 -1 Phosphate buffer (pH5.0), then add 20 μg Tween20 at a mass volume percentage of 0.2% in g / mL and shake evenly until there are no bacterial clumps at the bottom.

[0052] (2) Add a magnetic rotor to the mixture, put it into a microwave reactor and start stirring at a speed of 500rpm.

[0053] (3) Select the microwave power of 700W, start microwave radiation, set the extraction temperature at 80-95°C, start timing when the temperature reaches 80°C, and continue to radiate, stop the radiation when the temperature reaches 95°C, and stop the radi...

Embodiment 2

[0056] The Escherichia coli with recombinant Pfu DNA polymerase is induced and cultivated to obtain bacterial cells expressing Pfu DNA polymerase recombinantly, and then the following steps are adopted:

[0057] (1) Calculate the dry weight based on the determined water content (80%) of the wet cells, and add 0.05mol L to the weight-to-volume ratio of the dry weight of the cells to the g / mL of the buffer solution as 1:40 -1 Tris-HCl buffer solution (pH 9.0), then add 480 μL SDS at a mass volume percentage of 1.2% in g / mL and shake evenly until there are no bacterial clumps at the bottom.

[0058] (2) Add a magnetic rotor to the mixture, put it into a microwave reactor and start stirring at a speed of 100rpm.

[0059] (3) Select the microwave power of 200W, start microwave radiation, set the extraction temperature at 70-90°C, start timing when the temperature reaches 70°C, and continue to radiate, stop the radiation when the temperature reaches 90°C, and stop the radiation whe...

Embodiment 3

[0062] The Escherichia coli with recombinant Pfu DNA polymerase is induced and cultivated to obtain bacterial cells expressing Pfu DNA polymerase recombinantly, and then the following steps are adopted:

[0063] (1) Calculate the dry weight based on the determined water content (78%) of the wet bacteria, and add 0.02mol L according to the weight-to-volume ratio of the dry weight of the bacteria to the g / mL of the buffer solution as 1:20 -1 Tris-HCl buffer solution (pH 8.0), then add 200 μL Triton X-100 at a mass volume percentage of 1.0% in g / mL and shake evenly until there are no bacterial clumps at the bottom.

[0064] (2) Add a magnetic rotor to the mixture, put it into a microwave reactor and start stirring at a speed of 300rpm.

[0065] (3) Select the microwave power of 500W, start microwave radiation, set the extraction temperature at 75-90°C, start timing when the temperature reaches 75°C, and continue to radiate, stop the radiation when the temperature reaches 90°C, an...

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PUM

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Abstract

The invention discloses a microwave-assistant extraction method of recombinant Escherichia coli Pfu DNA (deoxyribonucleic acid) polymerase. The method comprises the steps of adding a buffer solution and an assistant extracting agent into an Escherichia coli thallus cell cluster of recombinant expression Pfu DNA polymerase to ensure that thallus cells are uniformly dispersed; performing extraction on the obtained mixed solution in a microwave field of which the power is 200-1000W, and keeping the temperature of the mixed solution to be in a range of 70-95 DEG C during extraction. The method disclosed by the invention can be used for finishing the processes of cell wall breaking and thermal denaturation through one step in the same device, and compared with the conventional method, the operation time and extraction steps are reduced, the investment of operation equipment is reduced, the microwave radiation heating energy efficiency is relatively high, energy conservation and environmental protection are ensured, and the reaction process is controllable and easy to amplify.

Description

technical field [0001] The invention relates to a method for extracting recombinant heat-resistant polymerase from recombinant engineering bacteria, in particular to a method for microwave-assisted extraction of recombinant heat-resistant Pfu DNA polymerase from recombinant Escherichia coli. Background technique [0002] DNA polymerase is an important enzyme for cell DNA replication, mainly used in polymerase chain reaction (Polymerase Chain Reaction). DNA polymerases are mainly divided into six types: A, B, C, D, X, and Y, and the commonly used ones belong to type B polymerases. Pfu DNA polymerase is a high-temperature resistant DNA polymerase extracted from Pyrococcus furiosis, and it is one of the polymerases with the best fidelity. [0003] The Pfu DNA polymerase used at first was isolated and purified directly from bacteria, but it was difficult to obtain a large amount of the enzyme. Later researchers recombined its gene into other host cells to express it, and then e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
CPCC12N9/1252C12Y207/07007
Inventor 刘春朝胡建华王锋
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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