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Method for efficient expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase

A high-efficiency expression technology of uridine phosphate, applied in the direction of transferase, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of unsuitable for large-scale preparation, low protein expression, cumbersome and time-consuming operations, etc. problem, to achieve the effect of low cost, simple and fast cultivation, and high application value

Active Publication Date: 2019-02-01
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, UDP-glucose-hexose-1-phosphate uridine acyltransferase has a very low yield when extracted from wild bacteria, is difficult to separate and refine, and is not suitable for large-scale preparation
UDP-glucose-hexose-1-phosphate uridine acyltransferase is expressed in Escherichia coli, but it is expressed intracellularly in Escherichia coli, and it is easy to form inclusion bodies. At the same time, the acquisition of the enzyme protein requires the destruction of the cells. The content of miscellaneous proteins is high, separation and purification are difficult, the operation is cumbersome and time-consuming, and the protein expression level is not high

Method used

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  • Method for efficient expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase
  • Method for efficient expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase
  • Method for efficient expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase

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Experimental program
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Effect test

Embodiment 1

[0023] (1) Construction of recombinant expression vector pHT43-GalT

[0024] UDP-glucose-hexose-1-phosphate uridine acyltransferase (GalT) gene (sequence shown in SEQ ID NO: 1) is derived from Bifidobacterium longum JCM 1217, and after PCR amplification and purification, it is connected to the cloning vector pMD19, The recombinant plasmid pMD19-GalT was constructed.

[0025] The recombinant plasmid pMD19-GalT and the expression vector pHT43 were double-digested with XbaI and KpnI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-GalT.

[0026] The recombinant expression vector pHT43-GalT was transformed into Bacillus subtilis WB800N, spread on LB plates containing chloramphenicol (5ug / mL) resistance, cultured overnight at 37°C, picked transformants, extracted recombinant plasmids and verified by double enzyme digestion. like figure 2 As shown, there are two fragments after digestion, the sizes are about 8000bp (expression vector p...

Embodiment 2

[0043] (1) Construction of recombinant expression vector pHT43-GalT

[0044] The recombinant plasmid pMD19-GalT prepared in Example 1 and the expression vector pHT43 were double-enzyme digested with XbaI and KpnI as the restriction sites respectively, and ligated at 16°C overnight to obtain the recombinant expression vector pHT43-GalT.

[0045] The recombinant expression vector pHT43-GalT was transformed into Bacillus subtilis WB800, coated with LB plate containing chloramphenicol (5ug / mL) resistance, cultured at 37°C overnight, the transformants were picked, the recombinant plasmid was extracted and verified by double digestion. like figure 2 As shown, there are two fragments after enzyme digestion, the sizes are about 8000bp (expression vector pHT43) and 2637bp (UDP-glucose-hexose-1-phosphate uridyltransferase), indicating that the connection is successful.

[0046] (2) Recombinant engineering bacteria

[0047] The constructed recombinant expression vector pHT43-GalT was ...

Embodiment 3

[0053] (1) Construction of recombinant expression vector pMA5-GalT

[0054] The recombinant plasmid pMD19-GalT prepared in Example 1 and the shuttle vector pMA5 were double digested with XbaI and KpnI as the restriction sites respectively, and ligated at 16°C overnight to obtain the recombinant expression vector pMA5-GalT.

[0055] The recombinant expression vector pMA5-GalT was transformed into Bacillus subtilis 168, coated with an LB plate containing ampicillin (100ug / mL) resistance, cultured at 37°C overnight, the transformants were picked, the recombinant plasmid was extracted and double-enzyme digestion was verified. Build succeeded.

[0056] (2) Recombinant engineering bacteria

[0057] The constructed recombinant expression vector pMA5-GalT was transformed by electric shock. Bacillus subtilis 168 electrotransformed competent cells were mixed with the recombinant expression vector pMA5-GalT plasmid, added to the electric shock cup in an ice bath for 5 minutes, and elect...

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Abstract

The invention relates to a method for efficient expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase. According to the method, a recombinant expression vector is constructed fromUDP-glucose-hexose-1-phosphate uridyltransferase (GalT) gene and Bacillus subtilis by using a vector; the recombinant expression vector is transformed into Bacillus subtilis to construct a recombinant engineered strain; the recombinant engineering strain is subjected to induced culture in a liquid culture medium; and the obtained bacterial liquid is subjected to centrifugation, and the supernatant is taken. According to the present invention, the method has advantages of high yield of UDP-glucose-hexose-1-phosphate uridyltransferase, pure protein, easy recovery and purification and simple production operation, provides the convenience in the industrial large-scale production of GalT, further has advantages of yield improving, time saving, labor saving and cost saving, particularly provides the safety assurance in the application of the enzyme in the food industry, and has great significance.

Description

technical field [0001] The invention relates to a recombinant engineering bacterium capable of efficiently expressing UDP-glucose-hexose-1-phosphate uridine acyltransferase and a method for preparing UDP-glucose-hexose-1-phosphate uridine acyltransferase by improving high-efficiency expression , belongs to the technical field of genetic engineering. Background technique [0002] UDP-glucose-hexose 1-phosphaturidylyltransferase (UDP-glucose-hexose 1-phosphaturidylyltransferase, GalT), also known as hexose-1-phosphate uridylyltransferase, or galactose-1-phosphaturia Glycolyltransferase is a protease that catalyzes the conversion between UDP-glucose, galactose-1-phosphate and UDP-galactose, glucose-1-phosphate encoded by the galt gene. Among them, glucose-1-phosphate is the main energy source of most cells, and UDP-galactose is an important component of proteins and fats. This modified protein plays a role in chemical signals, building cell structures, transferring small molec...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12N9/12C12R1/125
CPCC12N9/1241C12N15/75C12Y207/07012
Inventor 李拖平李苏红佟超男孙玥
Owner SHENYANG AGRI UNIV
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